Advances in Experimental Medicine and Biology

Pharmaceutical Biotechnology
ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY
Editorial Board:
NATHAN BACK,State University ofNew York at Buffalo
IRUNR. COHEN, The Weizmann Institute ofScience
ABELLAJTHA, N.S. KlineInstitutefor Psychiatric Research
JOHND. LAMBRIS, University ofPennsylvania
RODOLFO PAOLETII, University ofMilan
RecentVolumes in this Series
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ARTERIAL ANDALLIED CHEMORECEPTORS
Editedby Constancio Gonzalez, ColinA. Nurse,and ChrisPeers
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MOLECULAR MECHANISMS OF SPONDYLOARATHROPATHIES
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Volume 650
V(D)JRECOMBINATION
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DEVEOPLMENT ANDENGINEERING OF DOPAMINE NEURONS
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INHERITED NEUROMUSCULAR DISEASES: TRANSLATION FROM
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Editedby Carmen Espin6s, Vicente Felipo, and Francese Palau
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TARGET PATTERN RECOGNITION IN INNATE IMMUNITY
Editedby UdayKishore
Volume 654
THE ISLETS OF LANGERHANS
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Volume 655
PHARMACEUTICAL BIOTECHNOLOGY
Editedby CarlosA. Guzman and GioraZ. Feuerstein
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Pharmaceutical Biotechnology
Editedby
Carlos A. Guzman, MD, PhD
Department of Vaccinology andAppliedMicrobiology,
HZI-Helmholtz Centre for Infection Research. Braunschweig. Germany
Giora Z. Feuerstein, MD, MSc, FAHA
Wyeth Research,
Collegeville, Pennsylvania, USA
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Pharmaceutical Biotechnology, editedbyCarlos A.Guzman andGioraZ. Feuerstein. Landes Bioscience I

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Library of Congress Cataloging-in-Publication Data
Pharmaceutical biotechnology I editedby CarlosAlberto Guzman, GioraZeevFeuerstein.

p. ; ern, -- (Advances in experimental medicine and biology ; v. 655)
Includes bibliographical references and index.
ISBN978-1-4419-1131-5
1. Pharmaceutical biotechnology. 1. Guzman, CarlosAlberto, 1959- II. Feuerstein, GioraZ., 1946-III.
Series: Advances in experimental medicine and biology, v. 655. 0065-2598 ;
[DNLM: 1. Technology, Pharmaceutical. 2. Biotechnology. 3. Drug Discovery. WI AD559 v.655
2009I QV 778 P53522 2009]
RS380.P4755 2009
615'.I9--dc22
2009024877
DEDICATION
To Michela Morgana and Alessia Federica
v
PREFACE
Pharmaceutical Biotechnology is a unique compilation of reviews addressing

frontiers in biologicals as a rich source for innovative medicines. This book fulfills
the needs of a broad community of scientists interested in biologicals from diverse
perspectives-basic research, biotechnology, protein engineering, protein delivery,
medicines, pharmaceuticals andvaccinology. The diverse topicsrangefromadvanced
biotechnologies aimedtointroduce novel, potentengineered vaccines ofunprecedented
efficacy and safety for a wide scope of human diseases to natural products, small
peptides andpolypeptides engineered fordiscrete prophylaxis andtherapeutic purposes.
Modembiologicals promise to dramatically expandthe scopeof preventive medicine
beyond the infectious disease arena into broad applications in immune and cancer
treatment, as exemplified by anti-EGFRreceptors antibodies forthetreatment ofbreast
cancer. Theexponential growthinbiologicals suchasengineered proteins andvaccines
has beenboostedby unprecedented scientific breakthroughs madein thepastdecades
culminating in an in-depth fundamental understanding of the scientific underpinnings
of immune mechanisms together withknowledge of proteinandpeptidescaffolds that
can be deliberately manipulated. This has in turn led to new strategies and processes.
Deciphering the human, mammalian and numerous pathogens' genomes provides
opportunities thatneverbeforehavebeenavailable-identification of discrete antigens
(genomes and antigenomes) that lend themselves to considerably improved antigens
and monoclonal antibodies, which with more sophisticated engineered adjuvants
and agonists of patternrecognition receptors present in immune cells, deliverunpre-
cedented safetyand efficacy. Technological development sucha nanobiotechnologies
(dendrimers, nanobodies and fullerenes), biological particles (viral-like particles and
bacterial ghosts) andinnovative vectors (replication-competent attenuated, replication-
incompetent recombinant and defective helper-dependent vectors) fulfill a broad
rangeof cutting-edge research, drugdiscovery and delivery applications. Mostrecent
examples ofbreakthrough biologicals include thehuman papilloma virus vaccine (HPY,
prevention ofwomen genital cancer) andthemultivalent Pneumoccocal vaccines, which
hasvirtually eradicated insomepopulations a mostprevalent bacterial earinfection (i.e.,
otitis media). It is expected that in the yearsto come similarsuccess will be obtained
in the development of vaccines for diseases which still represent major threats for
vii
viii Pre/ace
human health, suchasAIDS, aswellasforthegeneration ofimproved vaccines against

diseases like pandemic flu for which vaccines are currently available. Furthermore,
advances in comparative immunology andinnate immunity revealed opportunities for
innovative strategies for ever smaller biologicals and vaccines derived from species
suchas llamaandsharks, whichcarrytremendous potential forinnovative biologicals
already in development stages in manypharmaceutical companies. Suchrecentdis-
coveries and knowledge exploitations holdthepromise forbreakthrough biologicals,
withthecoming decade. Finally, thisbookcaters to individuals notdirectly engaged in
thepharmaceutical drugdiscovery process viaa chapter outlining discovery, preclinical
development, clinical development and translational medicine issues that are critical
the drugdevelopment process.
The authors and editors hope that this compilation of reviews will help readers
rapidly and completely update knowledge and understanding of the frontiers in
pharmaceutical biotechnologies.
Car/os A. Guzman, MD, PhD

Giora Z. Feuerstein, MD, MSC, FAHA
ABOUT THE EDITORS...
CARLOS A. GUZMAN, MD, PhD is Head of the Department ofVaccinology

and Applied Microbiology at the Helmholtz Centre for Infection Research
(Braunschweig, Germany) andAPL-Professor at the Medical School of Hannover.
He is also German Coordinator for the International Doctorate in "Experimental
Oncology" and Chair for "Vaccines & Antiinfectives " from the Indo-German
Science Centre for Infectious Diseases. He graduated in Medicine at the National
University ofRosario and obtained his Board Certification in Medical Bacteriology
in Argentina. Then he moved to the Institute of Microbiology at the University of
Genoa (Italy), as Research Fellow of the Italian Foreign Office Ministry. In Italy
he also graduated as Doctor of Medicine and Surgery and obtained his Doctorate
of Research in Microbiological Sciences. In 1994 he moved to Germany, where he
became Head of the Vaccine Research Group at the German Research Centre for
Biotechnology. He has been working in the field of vaccinology since 1989. His
work has been instrumental for the development of new adjuvants , and the estab-
lishment of Salmonella spp. as a delivery system for DNA vaccines and therapeutic
molecules. He has published more than 150 topic-related papers in international
peer-reviewed journals and is co-inventor in several international patents. He is
member ofthe Editorial Boards ofInfection andImmunity, Microbial Biotechnology,
Open Immunology Journal, Current Immunology Reviews, Open Vaccine Journal
and Open Microbiology Journal, and Associate Editor of Human Vaccines.
ix
ABOUT THE EDITORS...
GIORA Z. FEUERSTEIN, MD, MSC, FAHA is Assistant Vice President

and Head of Discovery Translational Medicine, Wyeth Research. Prior to joining
Wyeth, Dr. Feuerstein has maintained Executive Directorship positionin discovery
of cardiovascular, stroke, thrombosis and metabolic disease programs for 16 years
in SmithKline Beecham, DuPont Pharma and Merck USA. Dr. Feuerstein led the
discovery program ofCarvedilol (COREG) for heart failure, eprosartan for hyper-
tension and many development programs in stroke, anti-arrhythmics, thrombosis
and metabolicdiseases. Dr. Feuerstein servesas Editor, Biochemical Pharmacology,
editorial boardmemberofJournalofPharmacology and Experimental Therapeutics,
Journal ofCerebral Blood Flow Metabolism, Circulation Research, and Stroke. Dr.
Feuerstein is therecipient of several national andinternational awards including Award
of Excellent inCardiovascular Research,AHA; PrixGalien Award forDrugDiscovery
(endothelin antagonist), Conrad R. Lam Award for cardiovascular research, Henry
FordFoundation andWyeth R&DPresident award. GioraFeuerstein hasauthored and
coauthored over 400 peer-reviewed publications and is co-inventor on 12patents.
x
PARTICIPANTS
Shizuo Akira Cevayir Coban

Department of Host Defense Department of Host Defense
and and
21st Century COE Program 21st Century COE Program
Research Institute for Microbial Osaka University
Diseases Osaka
Osaka University Japan
Osaka
Japan Mark Day
Wyeth Research
Rafaela Argnani Collegeville, Pennsylvania
Department of Experimental USA
and Diagnostic Medicine
Section of Microbiology Thomas Ebensen
University of Ferrara Department ofVaccinology
Ferrara and Applied Microbiology
Italy Helmholtz Centre for Infection
Research
Caroline Barelle Braunschweig
Wyeth Research Germany
Foresterhill, Aberdeen
Scotland Barbara Ensoli
National AIDS Center
Aurelio Cafaro Istituto Superiore di Sanita
National AIDS Center Rome
Istituto Superiore di Sanita Italy
Rome
Italy Alberto L. Epstein
Centre de Genetique Moleculaire
Keith Charlton et Cellulaire
Wyeth Research Universite Lyon
Foresterhill, Aberdeen Villeurbanne
Scotland France
xi
xii participants
Diana Felnerova Ken llshii

Bema BiotechLtd. Department of Host Defense
a CrucellNV and
Berne Exploratory Research for Advanced
Switzerland Technology
Japan Scienceand Technology Agency
Giora Z. Feuerstein and
Wyeth Research Research Institute for Microbial
Collegeville, Pennsylvania Diseases
USA OsakaUniversity
Osaka
Epifanio Fichera Japan
Etna Biotech
Catania KewalK. Jain
Italy Jain PharmaBiotech
Basel
CarmenGiefmg Switzerland
Intercell AG
CampusVienna Biocenter RichardJ. Lewis
Vienna XenomeLimited
Austria and
Institutefor Molecular
DavinderS. Gill Biosciences
Wyeth Research The University of Queensland
Biological Technologies Brisbane
Cambridge, Massachusetts Australia
USA
Petra Lubitz
ReinhardGlueck University of Vienna
Etna Biotech Department of Medicinal Chemistry
Catania Vienna
Italy and
BIRD-C GmbH and CoKEG
CarlosA. Guzman Kritzendorf
Department of Vaccinology Austria
and AppliedMicrobiology
Helmholtz Centrefor Infection Werner Lubitz
Research University of Vienna
Braunschweig Department of Medicinal Chemistry
Germany Vienna
and
BIRD-C GmbHand CoKEG
Kritzendorf
Austria
Participants xiii
Iole Macchia GabriellaMolinari

NationalAIDS Center Environmental Microbiology
Istituto Superiore di Sanita Helmholtz Centrefor Infection
Rome Research
Italy Braunschweig
Germany
MariaTeresaMaggiorella
NationalAIDS Center MarirosaMora
IstitutoSuperiore di Sanita NovartisVaccines
Rome Siena
Italy Italy
RobertoManservigi Danilo GomesMoriel

Department of Experimental NovartisVaccines
and Diagnostic Medicine Siena
Sectionof Microbiology Italy
University of Ferrara
Ferrara EszterNagy
Italy IntercellAG
CampusVienna Biocenter
PeggyMarconi Vienna
Department of Experimental Austria
and Diagnostic Medicine
Sectionof Microbiology Rob Noad
University of Ferrara Department of Infectious
Ferrara and Tropical Diseases
Italy LondonSchool of Hygiene
and Tropical Medicine
Vega Masignani London
Novartis Vaccines UK
Siena
Italy Rino Rappuoli
Novartis Vaccines
Ulrike BeateMayr Siena
University of Vienna Italy
Department of Medicinal Chemistry
Vienna Polly Roy
and LondonSchoolof Hygiene
BIRD-CGmbHand CoKEG and Tropical Medicine
Kritzendorf London
Austria UK
Robert Mischler J. LynnRutkowski

Mibiotec Limited Wyeth Research
Worblaufen Collegeville, Pennsylvania
Switzerland USA
xiv Participants
Maria Scarselli Jean-Francois Viret

Novartis Vaccines Berna BiotechLtd
Siena a CrucellNY
Italy Berne
Switzerland
Laura Serino
Novartis Vaccines Alexandervon Gabain
Siena IntercellAG
Italy CampusViennaBiocenter
Vienna
Fausto Titti Austria
NationalAIDS Center
Istituto Superiore di Sanita
Rome
Italy
CONTENTS
1. TRANSLATIONAL MEDICINE-A PARADIGM SHIFT IN MODERN

DRUG DISCOVERY AND DEVELOPMENT: THE ROLE
OF BIOMARKERS 1
MarkDay, J. LynnRutkowski and GioraZ. Feuerstein
Abstract 1
Drug Targets-Historical Perspectives 1
Translational Medicine: Definition 2
Biomarkers-Utilitarian Classification 2
Principles of Target Selection 7
Class A-Disease Specific 8
Class B-Target Present Physiologically but in a Non-Active Form,
but Is Activated and Contributes to the Disease 8
Class C- Target Functions Physiologically but at Augmented,
Uncontrolled Fashion That Contributes to the Disease 9
Class D-Target Maintains Physiological Functions in Normaland Disease States 10
Conclusion 11
2. NATURAL PRODUCTS IN DRUG DISCOVERY: PRESENT STATUS

AND PERSPECTIVES 13
Gabriella Molinari
Abstract 13
Introduction 13
Drug Discovery Evolution 17
Natural Products Properties 17
The Urgent Need for New Drugs 18
From Microbial Diversity to Drug Discovery 19
Microbial Sources 19
Microbial Extracts 21
Chemical Screening 22
Biological Screening 22
Production, Purification and Characterization of a New Natural Product 22
xv
xvi Contents
Metagenomics for Drug Discovery ••...••.•..•......•••..••.••••••••••..•.•..•....••••......•••••••••...........•••.• 23

Natural Products Under Development•.•..•.•.•..••.•••.••••••••••........•.••.•....•.•..•.•••••••......•.••.•..••23
New Strategies in Fighting Infectious Diseases 24
Conclusion 24
3. PROTEIN PHARMACEUTICALS: DISCOVERY

AND PRECLINICAL DEVELOPMENT 28
DavinderS. Gill
Abstract 28
Introduction 28
Protein Drug Discovery 29
Novel Agents 32
Challenges and Opportunities 33
Next Generation Proteins 34
Conclusion 34
4. THE ROLE OF NANOBIOTECHNOLOGY

IN DRUG DISCOVERy 37
KewalK. Jain
Abstract 37
Introduction 37
Role of Nanoparticles in Drug Discovery 37
Role of Nanoproteomics in Drug Discovery 39
Atomic Force Microscopy for Drug Discovery 40
Role of Nanoscale Biosensors in Drug Discovery 40
Nanofluidics, Nanoarrays and Nanobiochips 41
Nanomaterials as Drug Candidates 41
Nanobiotechnology and Drug Discovery for Personalized Medicine 42
Conclusion 42
5. CONOTOXIN VENOM PEPTIDE THERAPEUTICS 44

Richard J. Lewis
Abstract 44
Introduction 44
Calcium Channel Inhibitors 45
Sodium Channel Inhibitors 45
Antagonists of Nicotinic Acetylcholine Receptors 46
Norepinephrine Transporter Inhibitors 47
NMDA Receptor Antagonists 47
Neurotensin Receptor Agonists 47
Contents xvii
6. SHARK NOVEL ANTIGEN RECEPTORS-THE NEXT

GENERATION OF BIOLOGIC THERAPEUTICS? 49
CarolineBarelle, Davinder S. Gill and Keith Charlton
Abstract 49
Introduction 49
The Rise and Rise of Antibodies 50
What Are IgNARs? 51
How Diverse Are IgNARs? 54
What Is the Function of IgNAR? 55
Developing IgNARs as Therapeutics 56
Intrinsic Therapeutic Attributes of IgNARs 56
Isolation of Antigen-Specific Clones 57
In Vitro Maturation 57
Formatting 58
Conclusion 59
7. IMMUNE INTERVENTIONS OF HUMAN DISEASES

THROUGH TOLL-LIKE RECEPTORS 63
Cevayir Coban,Ken J. Ishii and ShizuoAkira
Abstract 63
Introduction 63
Toll-Like Receptors and Their Known Ligands 64
Toll-Like Receptor Signaling 64
The Role of Toll-Like Receptors in the Human Immune System 67
TLR-Based Immune Intervention in Humans: Promise and Caution 68
TLR2 68
TLR4 69
TLR5 71
TLR7 and TLR8 72
TLR9 72
TLR3, TLRIO 73
Other Signaling Molecules 73
Conclusion 74
8. GENOME-BASED VACCINE DEVELOPMENT:

A SHORT CUT FOR THE FUTURE 81
Danilo Gomes Moriel, Maria Scarselli, Laura Serino, MarirosaMora,
Rino Rappuoli and Vega Masignani
Abstract 81
Conventional Vaccinology 81
Reverse Vaccinology 82
The Classical Reverse VaccinologyApproach 83
Comparative Genome Analysis: The Second Phase of Reverse Vaccinology 84
Subtractive Genome Analysis: Third Phase of Reverse Vaccinology? 85
Conclusion 88
xviii Contents
9. THE ANTIGENOME: FROM PROTEIN SUBUNIT VACCINES TO

ANTIBODY TREATMENTS OF BACTERIAL INFECTIONS?...... 90
Carmen Giefing, Eszter Nagy and Alexandervon Gabain
Abstract 90
Introduction 90
A New Paradigm in Bacterial Vaccine Development 92
The Advent of Monoclonal Antibodies in Disease Treatment 98
From Serum Treatment to Anti-Infective Monoclonal Antibodies 103
The Next Chapter of the Antibody Success Story: Bacterial Infections •••••••••••.•..•..••• 105
10. HSV AS A VECTOR IN VACCINE DEVELOPMENT

AND GENE THERAPY 118
Peggy Marconi,RafaelaArgnani, Alberto L. Epstein and Roberto Manservigi
Abstract 118
Introduction 118
HSV-1 Genome and HSV-Derived Vectors 120
Engineering Techniques 123
HSV-1 Based Vectors Applications 125
HSV-1 Based Vectors for Gene Therapy of Nervous System 127
Conclusion 133
11. VIRUS-LIKE PARTICLES AS A VACCINE DELIVERY SYSTEM:

MYTHS AND FACTS 145
Polly Roy and Rob Noad
Abstract 145
Introduction 145
Insect Cells and Baculovirus Expression System as Preferred System
for VLP Production 148
VLPs Produced for Structurally Simple Non-Enveloped Viruses 149
12. APPLICATIONS OF BACTERIAL GHOSTS IN BIOMEDICINE...... 159

Petra Lubitz, Ulrike Beate Mayr and WernerLubitz
Abstract 159
Introduction 159
Basic Structure of Bacterial Ghosts 160
Bacterial Ghosts as Vaccines 162
Bacterial Ghosts as Carrier of Subunit Vaccine 163
Bacterial Ghosts as Carrier of DNA 166
Bacterial Ghosts as Carrier Vehicles for Active Substances in Tumour Therapy 167
Other Medical Applications for Bacterial Ghost Packaged Active Substances 168
Bacterial Ghosts as Enzyme Reactors for Novel Probiotics 168
Conclusion 169
Contents xix
13. IMMUNE MODULATORS WITH DEFINED MOLECULAR

TARGETS: CORNERSTONE TO OPTIMIZE RATIONAL
VACCINE DESIGN 171
Thomas Ebensen and Carlos A. Guzman
Abstract 171
Introduction•.•..••..•.••••••••••.•••••••••.•.••..•.....•..............•.•.•.....................•......•.•.•..••••..•.••.••...... 171
Immune Modulators with Defined Molecular Targets 175
Bacterial Toxins and Their Derivatives 179
CDld Agonists .......•.•.•••••••••••••••••.•.....••••.•.......•...........•...............•.••..••.•••••••.••.•••••••••••••••.•• 179
Cytokines 180
Cell Wall Components 180
Co-Stimulatory Molecules 181
Bis-(3',5')-Cyclic Dimeric Guanosine Monophosphate (cdiGMP) 182
Conclusion ..................................................••.••....•.•..........................................................182
14. INNOVATIVE APPROACHES TO DEVELOP PROPHYLACTIC

AND THERAPEUTIC VACCINES AGAINST HIV/AIDS 189
AurelioCafaro, Iole Macchia, MariaTeresaMaggiorella, FaustoTitti
and BarbaraEnsoli
Abstract•..........................•.......................................•................•..•••••••••••••••••••.•....•............ 189
Introduction.............•....•..•..•..•••••••••••.••••.••••••.••••.••.•.....................•..•.........•.....•.•.•••••••••••.••189
Rationale and Roadblocks to mv Vaccine Development 191
Correlates of Protection.•....................................•.•••.•••.•••.••••••••.•.•.••............•................... 191
General Strategies Adopted to Induce Protective Immunity 198
Key Issues Relevant to mv Vaccine Development: How to Get the Right
Responses in the Right Places 205
New Particulate Delivery Systems 219
VLPs 220
Prime Boost Strategies•••••.••.•••••••••.•••••••.••••....•.................••.•.••.•••••.•.•••••••••.••.•....•.............220
International Networking to Ease and Accelerate mV/AIDS Vaccine
Development...............•....................................••................................................•......221
Conclusion 222
15. NEW STRATEGIES TO OVERCOME THE DRAWBACKS

OF CURRENTLY AVAILABLE FLU VACCINES 243
EpifanioFichera, Diana Felnerova, RobertMischler, Jean-Francois Viret
and Reinhard Glueck
Abstract 243
Introduction 243
Manufacturing of Influenza Vaccines 244
Strategies to Improve the Immunogenicity and Efficacy of Current
Influenza Vaccines 246
Conclusion 250
INDEX 253
ACKNOWLEDGEMENTS
To my daughters Michela Morgana and Alessia Federica Guzman; thanks

a lot not only for your continued support, but also for your inexhaustible
patience. Only once and half-joking you mildly complain with a "Papi, du
kummerst dich nicht urn uns" after a long business trip in 16 years . I cannot
conceive my life without my little witches.
To my family who supported me in my career development; my wife

Nili, my son Ron and my daughter Sheira Feuerstein.
We would also like to express our deep acknowledgement to all

contributors, without your engagement this book would have never come
to life. We are indebted with you for sharing your knowledge, experience
and insights. Finally, we would like to thank the staff of Landes Bioscience
for your outstanding support during this enterprise, in particular to Cynthia
Conomos, Celeste Carlton and Erin O'Brien; any mistakes are ours-you
did a terrific job .
xxi
CHAPTER!
Translational Medicine-A Paradigm

Shift in Modern Drug Discovery
and Development:
The Role ofBiomarkers
Mark Day, J. Lynn Rutkowski and Giora Z. Feuerstein"
Abstract
T
he success rate ofnovel medical entities that are submitted for registration by the regulatory
agencies and followed successful marketing hasbeen stagnating for the past decade. Failure
in efficacy and safety continue to be the prime hurdles and causes offailure. Translational
medicine is a new function within the pharmaceutical industry R&D organization aimed to im-
prove the predictability and success ofdrug discovery and development. Biomarkers are the essence
of the translational medicine strategy focus on disease biomarker, patient selection, pharmacody-
namic responses (efficacy and safety) target validation, compound-target interaction). Successful
deployment of biomarkers research, validation and implementation is adopted and embraced as
key strategy to improved the drug discovery and development towards new medical entities.
Drug Targets-Historical Perspectives

Drugs are natural or designed substan ces used deliberately to produce pharmacological effects
in humans or animals. Drugs have been part ofhuman civilizations for millennia. However, until
the very recent modern era, drugs have been introduced to humans by empiricism and largely by
serendipitous events such as encounters with natural products in search of food or by avoiding
hazardous plants and animal products. The emergence ofthe scientific era in drug discovery evolved
along-side the emergence of physical and chemical sciences at large , first as knowledge to distill,
isolate and enrich the desired substance from its natural environment, followed by deliberate at-
tempts to modify natural substances to better serve the human needs and desires .
Scientific evolution throughout the past two centuries enabled identification of biologically
active substances in humans (e.g., hormones), which were manipulated chemically to improve
(potency, duration of action and exposure), or to mitigate or abrogate undesirable actions. The
cumulative knowledge ofhuman, animal and plant biology and chemistry provided the scientific
foundation and technical capabilities to purposely alter natural substances in order to improve
them. Such evolution marked the era of"forward pharmacology" The era offorward pharmacol-
ogy is about drug design that emanates from primary knowledge of the action of the biological
target that has clear biological action.
The exponential progress in molecular biology since the mid-20th century, culminating in de-
ciphering the complete human genome in the year 2000, brought the dawn ofpharmacogenomics
·Corresponding Author: Giora Z. Feuerste in-Wyeth Research, 500 Arcola Road, COL 5230 7,
Collegeville, Pennsylvan ia 19426, USA. Email: feuersgwwy eth .corn
Pharmaceutical Biotechnology, edited by Carlos A . Guzman and Giora Z. Feuerstein.
©200 9 Landes Bioscience and Springer Sciences-Business Media.
2 Pharmaceu!iCllIBiotechnology
and the "reverse pharmacology" era.The "reverse pharmacology" eraisdefinedby the need to first
clarifythe biologyand medicalperspectives ofthe target soasto qualifyit asa drugableand phar-
maceutically exploitablefor drug discovery and developmentscheme. The pharmacogenomic era
providesvastopportunities for selectionofnew moleculartargetsfrom a gamut of approximately
30,000primarygenes, over100,000proteinsand multiplesoftheir translationaland metabolomics
products. Thus, the permutations in respect to opportunities for pharmacological interventions
are unprecedented,vast and most promisingfor innovativemedicines.
The pharmacogenomics era as a source for drug targets also posesunprecedented hurdles in
selection,validation and translation into effective and safedrugs. New technologies continue to
drive efficiency and robustness of mining the genomicdrug discovery opportunities but physi-
ologicaland integrated biologyknowledge is lagging. In this perspective, translationalmedicine
and biomarkers researchhave taken center stagein validation of the moleculartarget for phar-
maceuticalexploitation.
In this chapterweoffera utilitarian approachto biomarkers and targetselectionand validation
that isdrivenbythe translational medicineprospectof the targetto becomeasuccessful drug target.
We hereby offer classification and analytical process aimed to assess risk, innovation, feasibility
and predictability of success of translating novel targets into successful drugs. This manuscript
provides cleardefinitions on the type ofbiomarkers that are core to translationalmedicine and
biomarkersresearchin modern pharmaceutical companies.
Translational Medicine: Definition

Translational medicinein the pharmaceutical industryisa research discipline aimedto improve
the predictability of success of drug discovery and development. Translational medicineresearch
aimsto discovery,validateand implementbiomarkers in lieueof clinicaloutcomestudies,improve
the congruencyof preclinicalmodelsto clinicalrealityand establishproofof concept for efficacy
and safety based on targeted mechanismof action. In particular, translationalmedicine aims to
establishsurrogatebiomarkers to aid in earlyregistrationand promote personalizedmedicinefor
better patients selectionfor targeted mechanismof action.
Biomarkers-Utilitarian Classification
Biomarkers arethe stepping-stones for moderndrugdiscovery and development.1-4 Biomarkers
are defined as biologicalsubstances or biophysical parametersthat can be monitored objectively
and reproduciblyand used to predict drug effector outcome. This broad definition is however,
oflittle utility to the pharmaceutical processsinceit carries no qualification for the significance
and useof the biomarker. Thefollowing classes and definitionsof biomarkers arethereforeoffered
(see Fig. I}:
I. Disease Biomarkers: disease biomarkers are biomarkers that correlatestatistically with
the disease phenotype (syndrome)for which therapeuticsare developed. Correlation of
levels (in the circulation,other fluidsor tissue)or expression patterns (gene, protein) in
peripheralblood cellsor tissues should signifydisease initiation, progression, regression,
remission or relapse. When we applythese criteria to our empiricalapproach to current
strategiesto developdrugs for certain diseases, it becomesapparent that our current ap-
proachesemployedin clinicaltestingaresub-optimal.One pertinent example isprovided
by the wayindustry has approachedthe developmentoftreatments in schizophrenia.
a. Disease Initiation Misconceptions: Unfortunately in the past SO years all marketed
therapieshavebeendeveloped around the dopamineD2 receptor, eitherin the formof
full antagonismor partial agonism. Thesetreatmentsareonlyeffective on the positive
symptoms in around 70% ofpatients and are associated with treatment resistance and
poor sideeffectprofiles. Current clinicalpracticeand drug discovery is basedaround
the conceptthat the onsetof positivesymptomsrepresents the initiationof the disease.
Arguably, however, these symptomsarrivelate in the chapter of schizophrenia. The
focuson the positivesymptomshas impeded the developmentof noveltherapeutics
Translational Medicine-A Paradigm Shift in ModernDrugDiscoveryand Development 3
Biomarkers that validate the importance of the

Type 1 target in human disease and drug development
Biomarkers that define the chemical-physical

Type 2 interaction of the compound/biolog ical with Its
discrete target
Biomarkers that define consequences of

Type '3
compound/biolog ical Interaction with the target
Biomarkers that correlate with disease Init iation.

Type 4 progression. regression . rem ission. relapse or
mod ification
Biomarkers that define likelihood of pat ients to

Type 5 respond or not to compound/biological
Figure 1. Utilitarian classification of biomarkers types 1-5
driven by an under appreciation ofthe disease processes. Translational medicine focuses

on disease biomarkers and brings new focus and hypotheses to the drugdevelopment
process. For example. cognitive symptoms manifest prior to positive ones. We now
know that individuals who are at risk of becoming schizophrenic manifest. in early
adolescence. clear cognitive deficits often associated with low I Q Some. but not all. of
those individuals will go on to manifest positive symptoms (hallucinations. delusions.
paranoia).
b. Remission: A second issue is that the treatments that are used to control positive
symptoms do not improve functional outcome. In contrast. attenuation ofthe cognitive
deficits do predict functional outcome and in some cases lead to patient rehabilitation
into the work place,"
c. Relapse: Relapse is associated with thought disorder and cognitive disorganization. As
such . cognitive endpoints are seen early on in life and can be seen as an early milestone
in the initiation ofthe disease. worsens when the positive symptoms appear and remis-
sion ofpositive symptoms with improved cognitive function tracks with augmented
rehabilitation and functional outcome. Therefore, as cognitive endpoints track more
readily with initiation, progression. remission and relapse in schizophrenia it fulfills
all the criteria in the disease biomarker definition.
In addition. the duration of aberrantly expressed biomarkers could also be associated with
risk for disease even if the level of the biomarker does not change over time. In schizophrenia,
this is typified by the fact that these at risk individuals also show sensory gating deficits (e.g.,
prepulse inhibition) do not "normalize" with the majority of successful treatments of positive
symptoms," Since disease biomarkers are defined by their statistical correlation to features of the
disease it is imperative that the clinical phenotype is clearly defined. Stratification of all possible
phenotypic variables in clearly a prerequisite for accurate assessment of the discrete relationships
ofthe biomarker to the disease. Gender. age. life-style, medications. physiological and biochemical
similarities are often not sufficiently inclusive resulting in plethora of disease biomarkers claims
that are often confusing and futile.
II. Target Validation: biomarkers that assess the relevance and the potential for a given
target to become the subject ofmanipulation that will modify the disease to provide clear
4 PharmaceuticalBiotuhno/Qgy
therapeuticbenefitswhilesecuringasufficient therapeuticindexofsafetyand tolerability.

This biomarkeris intrinsicallylinked to our understandingof the disease.
a. PostmortemStutliesasSouru ofMjskaJing TargetItlmtiJiution: Often our under-
standingor employmentof our strategies to a developdrugs isoften sub-optimaland
maythereforeleadto targetsbeinginappropriatelyor incorrectlyidentified. However,
many of our approaches to target identificationare based upon receptor expression
from post mortem brain tissues. For example, post mortem brain tissues taken from
schizophrenics show heterogeneous neuropathology's ranging from ventricular en-
largement,disorganizedcelllayering [e.g.,Layers II and III of the cortex)and reduced
dendrite spinecount and arborizationin regionsof the CNS such as the DLPC and
hippocampus (e.g., ref8).
b. Anima/Models as TargetValiJation Biomarkers: However, recent preclinicaldata
has demonstratedthat chronicexposureto antipsychotictreatments(e.g., haloperidol
and olanzapine)is alsoassociated with significant decreases in total brain weight and
volume, gray matter volume, glial cell number." As such, investigation of selective
targeted "risk"genedisruption in mice,not only serveasetiologically relevantanimal
models,but by virtue of modelingthe geneticcomponent of the disease can serveas
model systems of target validation. A pertinent casein point, several of the emerging
"schizophrenia gene" disruptions are showing neuropathology that is seen in post
mortem brain tissueasdescribedin sectionlla .
m. Compound-Target Interaction Biomarkers: biomarkers that definethe discreteparam-
eters of the compound (or biological) interaction with the moleculartarget.Typifiedby
PET and SPECT, such parameters include binding of the compound to the target, its
residencytimeon the target,the specific siteofinteractionwith the targetand the physical
or chemical consequences to the targetinducedbythe compound(orbiological). Industry
needsto engageearlyin the discovery process and developSARfor TCI biomarkers early
on in the process.
IV. Pharmacodynamic Biomarkers: biomarkers that predict the consequence(s) of com-
pound (biological) interactionwith the target.Thepharmacodynamic biomarkers include
eventsthat are therapeutically desiredor adverse eventsbasedon mechanismofaction.
a. The Conupt: pharmacodynamic biomarkers can best be described by the employ-
ment of a new molecularentity [e.g., "compoundX"), that hasno PET ligand,whose
therapeutic benefitis derivedfrom indirect action upon a separatetarget system (e.g.,
dopamine) to which there exists a TCI biomarkers (Raclopride"R"). As such we can
examinethe effects of "X" (e.g., S-HT2c agonist) for the displacement of dopamine
via the useof"R".
b. Trackingthe Divergenu and Convergence ofSignaling Pathways: However, phar-
macodynamic biomarkers can be used to report on discretemoleculareventsthat are
proximal to the biochemicalpathwaythat is modified by the manipulated target or
remote consequences such as in vivoor clinicaloutcomes (morbidity or mortality).
Pharmacodynamic biomarkers arediverse and frequentlynonobvious. Advancedand
sophisticated bioinformatics tools are required for trackingthe divergence and con-
vergence of signalingpathways triggeredby compound interaction with the target.
c. "OffTarget"Effects: A subsetof the pharmacodynamic biomarkers areconsequences
inducedbythecompoundoutsideitsintendedmechanism ofaction.Suchpharmacody-
namiceffects areoftentermed"offtarget"effecrs, astheyarenot the directconsequence
ofthe compound interaction with the target. Usually, such pharmacodynamic events
are due to unforeseen lack of selectivity or metabolic transformations that yielded
metabolites not present (or detected) in the animalsused for safetyand metabolic
studiesprior to launchofthe compound into humans trailsor into human use. These
issues will not be dealt with in this chapter.
Translational Medicine-A Paradigm Shift in Modern DrugDiscoveryandDevelopment 5
V. Patient Selection: biomarkers that are used for selection of patients for clinical studies,
specifically proof-of-concept studies or confirmation Phase 3 clinical trials that are required
for drug registration. These biomarkers are important in order to help in the selection of
patients likely to respond (or conversely, not respond) to a particular treatment or a drug's
specific mechanism ofaction and potentially predict those patients who may experience
adverse effects. Such biomarkers are frequently genetic (single nucleotide polymorphism,
haplotypes) or pharmacogenomic biomarkers (gene expression), but could be any of the
primary pharmacodynamic biomarkers. Biomarkers for patient selection are now main-
stream ofexploratory clinical trials in oncology where genotyping of tumors in view of
establishing the key oncogenic 'driver(s)' are critical for prediction potential therapeutic
benefits of modern treatments with molecular targeting drugs. The success ofthe new era
of molecular oncology (as compared to the cytotoxic era) will largely depend on the abil-
ity to define these oncogenic signaling pathways via biomarkers such as phosphorylated
oncogenes, or functional state due to mutations that cause gain or loss offunction.
a. Imaging regional cerebral activation whilst patients perform tests of cognitive per-
formance can be used to dissect the discrete neural regions and substrates supporting
cognitive performance. In contrast to oncology, it is rare that there are concrete physical
matter to quantify based on the heterogeneous nature ofneuropathological abnormali-
ties (see section I). However, imaging techniques such as functional MRI (fMRI) are
bridging this gap. fMRI has the potential to be a powerful, sensitive and repeatable
tool in our armamentarium. This technology affords the potential to dissect patients
with cognitive deficits that are driven by, for example, either medial temporal lobe
or by frontal lobe dysfunction (e.g., episodic memory vs. executive function deficits)
within a clinical trial. Applied in early clinical POC studies we can, in essence, turn
our current heterogeneous clinical population into discrete, focused sub groups with
which to answer specific and focused hypothesis about the target, patient population
and ultimately increase the probability of seeing an effect with our compound whilst
improving the potential for d ifferentiation from comparators. This in turn can aid
patient selection in larger Phase III confi rm studies and can be driven by adaptive trial
design,
VI . Adaptive Trial Design: The overall objective ofadaptive trial design is to enable real time
learning. The method is based on computer modeling and simulation to guide clinical drug
development. In a first step, decision criteria and assumptions are defined and analyzed
str ateg y and stu dy designs are formulated to test competing hypotheses in one aligned
approach.
Once this framework is established, a formal scenario analy sis, comparing the fin-
gerprints of alternative designs through simulation is conducted. Designs that appear
particularly attractive to the program are further subjected to more extensive simulation.
Decision criteria steer away from doses that are either unsafe or nonefficacious and aim
to quickly hone in onto the most attractive dose -range. Response-adaptive dose-ranging
studies deploy dynamic termination rules, i.e., as soon no effect dose scenario is established
and the study is recommended for termination. Bayesian approaches are ideally suited to
enable ongoing learning and dynamic decision-making.'
The integrator role of "adaptive trials" is particularly strong in establishing links be-
tween regulatory accepted "confirm" type endpoints and translational medicine's efforts
to develop biomarkers. Search for biomarkers that may enable early decision making need
to be read out early to gain h igher confidence in basing decisions on them. A biomarker
can be ofvalue , even if it only allows a pruning decision.
These considerations highlight the importance of borrowing strength from indirect
observations and use mathematical modeling techniques to enhance learning about the
research question. For instance, in a dose -ranging study, it is assumed that there should
be some relationship bet ween the response ofadjacent do ses and this assumption can be
6 PharmaceuticalBiotechnology
1. Weak
Target Validation 2. Moderate -----.
..: 3. Strong
1. Weak No Validated Bton. , lcr
Target/Compound 2. Moderate -----, V,hurN but nol '!,1m) It
Interaction
3. Strong -----.
1. Weak
Pharmacodynam ic 2. Moderate -----. Po~ ibltbutnolpro\'Cl'l:prioran"kh~~
rmu ( .
Activ ity
3. Strong -----. Upnd a'tailb": . PO<.' .. ih ~kraKC
Unclea,; IIGas I) forllltni n ~~ ts or hunan .

1. Weak
Disease Biomarker
PO Rin'f Ikntif'itd In e-rerimental rmdclfl and hu""" but no
& Disease 2. Moderate -----,
Modification PO BID\t IIknllrlrd _ e rimmtal nude
3. Stro ng -----.
'abdated n~ hcd
Patient Stratification
Adaptive design
Figure 2. Criterion for biomarker scoring
placed to modelingalgorithm.Both safetyand efficacy considerations can bebuilt to this

model.Ideally, integration of alleffortsallthe wayfrom disease-modelingin discovery to
PKlPD modelingin earlyclinicaldevelopmentto safety/riskand business casemodeling
in late development.4•1D-12
A summaryofthe biomarkerdefinitionsproposed herein is provided in Figure 1. The utility
ofthis systemis representedin Figures 2-4,which suggesta semi-quantitativescoringsystem that
helps assess the strength of the program overall and identificationof the areasof weaknesses in
each of the biomarkers needed alongthe compound (biological) progression path. Figure5 illus-
trates the continuum ofbiomarkers research, validationand implementationalongthe complete
time-line of drug discovery and development including life cycle management (Phase IV) and
new indication (PhaseV) when appropriate.
A program for which a 'STRONG' scoreis established across all 5 biomarker'sspecifications
providesconfidencethat the likelihoodfor success from the biological and medicalperspectives
and is likelyto result in a more promisingdevelopmentoutcome. Likewise, it would be prudent
to voiceconcernsregardingprogramsthat score'WEAK', especially iflow scores are assigned to
targetvalidation, pharmacodynamic and in special cases target-compoundinteraction(e.g.,central
nervoussystemtarget).
Thisscoringsystem iscomplementary to other definitionsofbiomarkersbasedon certainneeds.
For example, surrogatebiomarkers asdefined by the Food and Drug Agency(FDA), are markers
that can be used for drug registrationin lieu of more definitive clinicaloutcome data. Surrogate
biomarkersare few and hard to establish(e.g., blood pressureand cholesterol, Fig.3).
Translational Medicine-A Paradigm Shiftin ModernDrugDiscovery and Development 7
Class A Target highly specific to the

disease
Target normally present In humans bu

Class B activated largely in disease state
Target present and functions in normal

Class C physiology but excessively active In
disease state
Target known to be present and

Class 0 functions Indiscriminately In normal
or disease states
Figure 3. Type 1 biomarkers: target validation translational medicine perspectives
Principles ofTarget Selection

Two keyguiding principlesare essential in the earlyselectionprocessof moleculartargets:
A. That modulating the target carries the prospect of unequivocal medicalbenefit (efficacy)
to patients beyond standard of care.
B. That benefit can be garnered while mainraining a sufficient levelof safety that can be
realizedwithin the attainablecompound exposure.
Sucha missionis frequentlyunachievable and hence, esrablishing an acceptable "Therapeutic
Index"isthe practicalgoalfor most drug developmentschemes. Commonly,a therapeuticindexis
establishedbycalculationof the ratioof the MaximumToleratedDose (MTD) and the Minimum
Effective Dose (MED) in animalefficacy and safetystudies.In this light, targetsselectedfor drug
developmentcan be classified with respect to risk assessment based on the following categories
(Fig. 3):
ClassA: Targetonly present and contributing to the disease process.
ClassB:Targetpresentphysiologically but in a nonactiveform; but isactivatedand contributes
to the disease.
Class C: Target functions physiologically but at augmented, uncontrolled fashion that con-
tributes to the disease.
ClassD: Targetfunctions in normal statesand indiscriminately in disease (e.g., no difference
in target expression, functions and distribution can be identified in disease as compared to the
normal physiological stare).
................
..............
..............................
Experi- Discovery Pre
mental Development
Figure 4. Building translational medicine via biomarkers research
Class A-Disease Specific

A disease specific moleculartarget should be a moleculethat operatesonly in the disease state
and is not participating in physiological (normal) functions. Drug interaction with such targets
shouldprovideefficacy with the lowestchancefor mechanism-based adverse effects when manipu-
lated by drugs. Examples ofsuchtargetsaregeneticdisorders, whichresultin eitherover-activity or
lossofactivityofthe target.Suchisthe casein Chronic Myelogenous Leukemia(CML) that results
from aberrant recombination of DNA from chromosome22 into chromosome9 (Philadelphia
Chromosome), fusing the Bcr and Abl genes into an overactingtyrosine kinase, which drives
oncogenic transformation. To cure the disease, potent and selective inhibitors of this aberrant
kinasehad to be discovered, a task that took over a decade to accomplished." Such targets have
the potential for a high safetyprofile. It is however, important to note that this example maydoes
not necessarily represent the ultimate approach for this disease since the activityof the kinase
(Bcr/Abl) is drivenby the Abl kinasecatalyticsite,which ispreservedin its physiological format.
Thus, inhibitors of this target/kinase by drugs such as Gleevec maystill carry the potential for
interferencewith in tissue/cells where the Abl kinaseisphysiologically active.
Another example that is applicable to this category is a disease such as Myasthenia Gravis
wherespecific antibodiesthat blockthe acetylcholine receptorscause progressive muscle weakness.
Specific neutralizingagentsto these antibodies are likelyto provide high efficacy in treating the
disease with the likelihoodof feweradverse effects 14 sincesuch antibodiesare not physiologically
present in human beings. Theseexamples are typicalfor "Type 1 class A:' target validation. The
biomarkers that need to beestablished for this "Type1class A"categoryshouldfocuson validating
the specificity of the target to the disease state.
Class B-Target Present Physiologically but in a Non-Active Form, but

Is Activated and Contributes to the Disease
This class of targetshas little or no discernible physiological activityin the normal states,yet
in certain pathophysiological situations, the target is presented, activated and plays a role in a
Translational Medicine-A Paradigm Shift in ModernDrugDiscovery and Development 9
Pre- Dev
Exp Discovery Phase 2 3 4
Dev Track
CR&D
L
C
M
Early biomarkers team

Strategy & Initiatives
Discovery
Figure 5. Translational medicine: biomarkers implementation across the pipeline
pathophysiological event. Example ofsuch targets in the "Type 1 class B" category is the P-selectin
adhesion molecule. This adhesion molecule is normally cryptic within platelets and endothelial
cells. Upon activation of these cells, P-selectin is presented on the surface of the cell and medi-
ates adhesion interaction with its ligand, a mechanism believed to playa role in thrombosis and
inflammation. Inhibitors of P-selectin binding to its ligand, the P-Selectin Glycoprotein ligand
(PSGL-l), are expected to provide clinical benefit with a lower likelihood of adverse events. To
validate this situation, biomarkers that confirm the preferential role of the activated target in a
pathophysiological process while maintaining little physiological function is essential.
However, one must be aware ofpotentially serious limitations to this approach where cryptic
targets in the physiological state that are activated in the pathological condition and where inhibi-
tion of the target may not only provide for significant therapeutic benefit, but where inhibition
ofsuch a target may also expose the patient to some other risk, such as loss of host defense from
injury. Such is the case ofthe platelet adhesion integrin molecule, GPlIb/IIIa, which serves as the
final common pathway for platelet aggregation . Interfering with activated G PIIb/IIIa binding to its
ligand (e.g., fibrinogen) provides effective and often life-saving therapy in patients with acute risk
for thrombosis; however chronic treatment with G PIIB/IIIa antagonists have not been particularly
effective in providing benefit due to the relatively high frequency ofsignificant adverse effects due
to bleeding since platelet adhesion to matrix protein is essential to seal bleeding sites in trauma and
disease conditions. Thus biomarkers for this class must establi sh the full physiological significance
of the target in order to assess the therapeutic index oftolerability (benefit as well as risk).
Class C-Target Functions Physiologically but at Augmented,

Uncontrolled Fashion That Contributes to the Disease
This classoftargets includes molecules that play an active role in normal physiological processes,
some of which may be critical to health. Such is the neurotransmitter glutamate in the central
nervous system, which is essential to cognition, memory, thought processes and state ofarousal.
However, in ischemic stroke or asphyxia, glutamate release is uncontrolled and reaches to highlevels
over prolonged periods that are believed to be neurotoxic and likely contribute to neuronal death
10 Pharmaceutical Biotechnology
following a stroke. Inhibitors of glutamaterelease or antagonistsofits action at various receptors

are believedto carry the potential for effective treatment for stroke provided the inhibition of
excess release of the neurotransmitter can be achieved in a timelymanner and only to an extent
that preserves the physiological need of this transmitter and overshort periods(onlythat limited
period where excess glutamate is neurotoxic).Such targets may be pharmaceutically exploitable
when their manipulation is carefully tuned to the pathophysiological context).
Another exampleof a target in this categoryincludesthe Human Epidermal Growth Factor 2
(HER2) inhibitor,Herceptin,whichin certaincancers (e.g., breastcancer) isconstirutively activated
and participatesin the oncogenicdrive. Inhibition ofthe HER2, whileclearly oftherapeuticvalue
in breast cancer,has alsobeen associated with heart failuredue to the physiological roleofHER2
in the cardiacmyocytesurvival-signalingpathway. 15Thus, the biomarkerchallenge in modulation
of class C targets of this nature is in identifyingbiomarkersthat assess the needed 'titration' for
inhibition of the target activityonly to an extent that is necessary for the maintenanceof normal
physiological function.
Class D-Target Maintains Physiological Functions in Normal

and Disease States
This class of targets encompasses the largest group of molecular targets exploited so far by
modern drugs. Many members of this class have yielded highly beneficial therapies. This class
consistsof molecular targets that are known to have important physiological functions, which
cannot be differentiatedwithin a disease context; that is to saythe target is not different in its
expression levels (gene, protein) or signalingpathwayin the normal and disease states.A priori,
such targetsharbor the greatestrisk for mechanism-based adverse effects, as there is no apparent
reason to expect that modulation of the target in the disease state will spare the normal physi-
ologicalfunction of the target.
Examples of suchtargetsincludethe coagulationfactor inhibitors [e.g., FIX, FXa and throm-
bin), which are critical to maintain a physiological level of homeostasis; hence, inhibition of
these targetscarries inherent bleedingliabilities. Likewise, all current anti-arrhythmicdrugs [e.g.,
amiodarone,lidocaine,doferillde), whileeffective in treatinglife-threatening arrhythmias,allcarry
significant liabilityfor mechanism-based pro-arrhythmic effects and thepotentialforsuddendeath.
The biomarkerchallenges for this class aredefiningthe finebalanceneededbetweenefficacy in the
disease context and expectedsafetylimitations.Biomarkers that definethe acceptable therapeutic
indexare keyto the successful utility of drugs that modulate suchtargets. However, targetsin this
class do not necessarily exhibit narrow safety margin for clinically meaningful adverse effects.
Significantexamples are the L-typeCa2 + channelblockers.The L-typeCa2+ channelisan essential
conduit of Ca2+ needed for 'beat by beat' Ca2+ fluxes that securepreciserhythm and contractility
ofthe heart, skeletalmuscle, neuronalexcitability and hormone and neurotransmitter release. Yet,
L-typeCa2+ channelblockers areimportant and sufficientlysafedrugs that are usedto treat hyper-
tension, angina and cardiacarrhythmiaswith undisputable medicalbenefits. However, inherent
to the L-type Ca2+ channel blockers in this class of targets are mechanism-based adverse effects
associated with rhythm disturbances, hypotension, edemaand other liabilities.
Probablythe best examplefor system specificity of physiological targets that provide major
medicalbenefitswith ahigh safetymarginisthe Renin-Angiotensin-Aldosterone (RAAS)system.
The RAAS is an important blood pressure, blood volumeand blood flow regulatorysystem, yet
its manipulation by several different pharmacological agents (rennin inhibitors, angiotensin I
convertingenzymeinhibitors, angiotensinII receptorsantagonists)has yieldedhighly beneficial
drugs that reduce risk of morbidity and mortality from hypertension, heart failure and renal
failure, despite the fact that the system does not demonstrate significant operational selectivity
between normal and disease states (especially hypertension). However, mechanism-based hy-
potension and electrolytedisturbancescan limit the therapeuticbenefitofthesedrugs and elicit
significantadverse effects when the RAASis excessively inhibited.16 The biomarkerchallenge for
these targets is to definethe relative or preferentialrole ofthe target in its variousphysiological
Translational Medicine-A Paradigm Shiftin Modern DrugDiscovery and Development 11
activities, whereminor manipulation in one organ might providesufficient therapeuticpotential

while providing a low likelihoodfor adverse effects that result from more substantial inhibition
of rhe sametarget in orher organs.
Conclusion
The analysis and classification offeredin rhis chapter regardingbiomarkers in drug discovery
and developmentaim to highlight the need to carefully study and analyze rhe significance of rhe
targetselected fur rherapeutic interventionasrhefirstcross roadforsuccess or failure in rhedevelop-
ment of effective and safedrugs.F'Ihe analysis and utility of biomarkers alongrhe process of drug
discovery and development has become an integral part of rhe "learnand confirm" paradigmof
drug discovery and developmentin leadingpharmaceutical organizations suchasWyerhresearch.
Suchanalyses are useful to guide rhe "learnphase"in searchfor biomarkers that can better assess
the benefitsand risksassociated wirh manipulation of rhe moleculartarget.
Thescopeof this paperdoesnot allowfor adetailedreview of rhe"learnand confirm" paradigm.
for which rhe readers are directed to recent references.P:'?Various technological and strategic
activities are needed to establishrhe biomarkerstrategiesfor rhe various targets described. The
need to addressrheseissues viabiomarkers research, validationand implementationcommencing
at rheveryearlystages of rhe drug discovery and developmentprocess isemphasized. In rhe phar-
maceutical setting. it meanscommencingeffons to identify biomarkers for all 5 categories listed
above. Sucheffortscould commenceevenbeforea tractablecompound (biological)is in hand, a
timewheretargetvalidationisaclearfocusof rheprogram. Ascompoundbecomesavailable. com-
pound-target interaction. pharmacodynamic (efficacy and safety) biomarkers and strategies for
patient selectionand adaptivedesignneedsmust beexplored. At the onset of rhe "firstin human"
studies.allstrategies.plansand biomarkers research shouldbewell workedout (asmuchaspossible).
We believe that fundamental changes in the structure. function and interfaces of Pharmaceutical
R&D isurgentlyneeded to provideforakeyroleof translationalmedicineand biomarkers research
towardsmore successful discovery and developmentof innovative medicines.
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CHAPTER 2
Natural Products in Drug Discovery:

Present Status and Perspectives
Gabriella Molinari*
Abstract
atural products and their derivatives have been and continue to be rich sources for drug
N discovery. However, natural products are not drugs. They are produce in nature and
through biological assaysthey are identified as leads, which become candidates for drug
development. More than 60 % of the drugs that are in the market derive from natural sources.
During the last two decades , research aimed at exploiting natural products as a resource has seri-
ously declined. This is in part due to the development ofnew technologies such as combinatorial
chemistry, metagenomics and high-throughput screening. However, the new drug discovery ap-
proaches did not fulfilled the initial expectations. This has lead to a renewed intere st in natural
products, determined by the urgent need for new drugs, in particular to fight against infect ion s
caused by multi-resistant pathogens.
Introduction
H isto rically,chemical substances . derived from animals , plants and microbes have been used to
treat diseases. Plants and microorganisms produce unique bioactive substances, providing access
to very different types oflead compounds, the natural products.
Natural products have played and continue to play an invaluable role in the drug discovery
process, particularly in the areas of cancer and infectious diseases. In fact , more than 60% of the
approved drugs are of natural origin. In the modern drug discovery era there are three major
sources ofnew compounds: original natural products, structures derived semi-synthetically from
natural products and combinatorial synthetic compounds based on natural products models. '?
Bioactive natural products are mostly low-molecular weight organ ic compounds known as second -
ary metabolites.t The producer organisms can growth without synthesizing the se metabolites and
produce them in response to environmental cues. These compounds could be produced in nature
as "weapons" that organisms use to fight each other.S.6
In this chapter will be described the role ofnatural products in the evolution ofdrug discovery,
giving emphasis to the natural products which provide candidate s to be used in infectious disease
therapy: the antibiotics. Antibiotics are biologically active molecules with different structures and
mode of action made by microorganisms, which are active against other microorganisms at low
concentrations. Many antibiotics are made chemically by modifi cation ofnatural products through
a process called semi-synt hesis and the effective compound is a semi-synthetic derivative. In Table
1 are sum marized different antibiot ics from natural origin actually used in the clinic. The majority
ofantibiotics inhibit targets involved in essential microbial fun ctions: protein synt hesis (30S and
50S subunits of the ribosome and RNA polymerase), DNA replication (D NA gyrase) and cell
*Gabriella Mol inari-Environmental Mi crobiology, Helmholtz Centre for Infection Research,

Inhoffenstrasse7, D-38124 Braunschweig, Germany. Email: gabriella.molinari@helmholtz-hzi.de
Pharmaceutical Biotechnology, edited by Carlos A. Guzman and Giora Z. Feuerstein.
©2009 Landes Bioscience and Springer Science+ Business Media .
...
~
I
Table 1. Microbial secondary metabolites or derivatives used as antibiotics
Antibiotic Class Target Derived From Produced By
Am ikacin Semisynthetic aminogly- Protein synthesis: bind ing to the Kanamyci n

coside 30S ribosomal subunit
Amoxycillin Semisyntheti c hydro xyam- Cell-wall synthesis: penicill in-bind - Ampicillin
pici llin ing protein (PBP)
Amphotericin B Natu ral polyene macrolide Fungal membrane Streptomyces nodosus
Amp icillin Semisynthet ic aminobenz yl- Cell-wall synthesis: PBPs Penicillin
penicil lin
Az ithromycin Semisynthetic 15 membered Protein synthesis: bind ing to the Erythromycin
azalide 50S ribosomal subunit
Azt reonam Sem isynthetic monocyclic Cell-wall synthesis: PBP3 SQ-26180 Chomobacterium violaceum
B-Iactam
Bacitracin Natu ral thiazolpeptide Peptidogl ycan synthesis: lip id - Bacillus licheniformis
pyrophosphorylase inh ibitor
Caspofungin Semisynthetic lipopeptide Fungal wall: Pneumocandin B Glarea lozoyensis
glucan synthesis
Cephalosporin Natural cephem Cell-wall synthesis: PBPs - Cephalosporium acremonium
~
Chloramphenico l Natural phenicol Protein synthesis: binding to the Streptomyces venez uelae "~
50S ribosomal subunit
~
"
Ii:
Clavulanic acid Natura loxa-l -penem B lactamase inhibitor - Streptomyces clavuligerus ~"
l;:
Clindamy cin Semisynthetic thiooctopy- Protein synthesis: bind ing to the Lincosam ine Streptomyces lincolnensis b;,
-
ranoside 50S ribosomal subunit t"
~
So
lS
<>
continued on next page I~
~
i:
Table 1. Continued I ~
"1::l
<l
Antibiotic Class Target Derived From Produced By l}
....
~
Dalfopristin-Quin- Semisynth etic streptogramin Protein synthesis: bindi ng to the Pristinamycin Strep tomyces p ristinaspiral is s
upristin 50S riboso mal subunit b
Daptom yci n Semisynthetic lipopept ide Bacterial membr ane A-21978C Strep tomyces roseoporus ~
b
:;:.
Erythromycin Natural 14-membered Protein synthesis: binding to the Saccharopolyspora ery thrae <l
~
macrol ide 50S ribosomal subunit Streptomyces eryth reus
~
Fosfomycin Natu ral phosphon ic acid Cell wall: synthesis of peptidogly- Streptom yces fradiae
can precursors
Fusidi c acid Natural fusidane Protein synthesis: translation stage Fusidium coccineum
Gentamyci n Natural aminoglycoside Protein synthesis: bind ing to the Micromonospora purpurea
30S ribosom al subunit
Griseofulv in Natural benzohydrofuran Fungal cell w all synthesis Penicillium griseofulv um
Imipenem Semisynth etic carbapenem Cell-wall synthesis: PBPs Thienam ycin
Josamycin Natural 16-membered Protein synthesis: binding to the Streptom yces narb onensis subsp .
macrol ide 50S ribosomal subuni t josamyceticus
Kanamycin Natural aminoglycoside Protein synthesis: bind ing to the Streptomyces kanam yceticus
30S ribosom al subunit
Meth icill in Sem isynthet ic penicill in Cell- w all synthesis: PBPs Penicill in
Mu pirocin Natural pseudomo nic acid Protein synt hesis: blocking isol eu- Pseudomo nas fluorescens
ci n incor poration
Netilmi cin Sem isynth etic Protei n synthesis: bindin g to the Sisomicin
ami noglycoside 30S ribosom al subunit
continued on next page I ...

V.
....
0-
I
Table 1. Continued
Antibiotic Class Target Derived From Produced By
Novobiocin Natural coumarin DNA synthesis: DNA gyrase - Streptomyces spheroids S. niveus
Nystatin Natural polyene macrolide Fungal membrane - Streptomyces noursei

Penicillin Natural B-Iactam Cell-wall synthesis: PBPs Penicillium chryseogenum
Polymyxin Natural lipopeptide Bacterial membrane Baciflus po/ymyxa
Rifampin Semisynthetic ansamycin RNA transcription: Rifamycin Nocardia mediterranei
RNA polymerase
Sisomicin Natural aminoglycoside Protein synthesis: binding to the Micromonospora inyoensis
Spectinomycin Natural aminocyclitol Protein synthesis: binding to the Streptomyces flavopersicus
Spiramicyn Natural 16-membered Protein synthesis: binding to the - Streptomyces ambofaciens
macrolide 50S ribosomal subunit
Streptogramin Natural macrocylic Protein synthesis: binding to the Streptomyces diastaticus
peptolides 50S ribosomal subunit
Streptomycin Natural aminoglycoside Protein synthesis: binding to the - Streptomyces griseus
~
III
~III
Teicoplanin Natural lipoglycopeptide Cell-wall synthesis: peptidoglycan - Actinop/anes teichomyceticus ~
l::
Tetracycline Natural polyketide Protein synthesis Streptomyces aureofaciens ~.
16SrRNA !l
b:I
Thienamycin Natural carbapenem Cell-wall synthesis: PBPs Streptomyces catt/eya l::'
~
S-
:s
Vancomycin Natural glycopeptide Cell-wall synthesis: peptidoglycan Streptomyces orientalis
'"
~
Natural Products in DrugDiscovery 17
wall synthesis. Many other cellular functions which are essential for microbes are also present in
mammalian cells making them unsuitable targets .
Drug Discovery Evolution

The time frame between the initial discovery of a potential drug candidate and the market
launch of a new therapeutic agent usually is 10-14 years (Fig. 1). For this reason it is difficult to
measure the real trend in the drug discovery programs during the last two decades. However, the
worldwide pharmaceutical natural product patents trend show a decline from 1990 to 1999 and
a recent increase ofpatenting up to 2001 ? The interest in discover new natural products declined
when new technologies became more attractive for the pharmaceutical industry to be imple-
mented in their search for new drug candidates. The evolution ofmolecular techniques allowed
the identification and isolation of purified protein targets . The use of defined molecular targets
combined with automatization, modern robotic instruments, sensitive detectors, data processing
and control software allowed the development of tests using microvolumes for the screening of
libraries of chemical structures: the high-throughput screening (HTS). The definition "hit" and
"lead" development were then incorporated to the drug discovery process. Through the HTS mil-
lions oftests could be automatically performed, testing compounds for their activity as inhibitors
or activators of a specific biological target [l,e., hit). Active structures could be then identify in a
short period of time (Le., leads) and they are the starting point for drug design? Combinatorial
chemistry provided the possibility to obtain large collections or libraries by synthesizing combina-
tions ofa set ofsmaller chemical structures," Further development ofmolecular biology, cellular
biology and genomics allowed the discovery of new molecular targets, which were introduced
in the HTS screens. This determined that industries concentrated their efforts in the generation
and exploitation ofsynthetic chemical libraries, abandoning the search for new natural products.
However, in the target-specific approach only one target can be screened at a time and during
target-based discovery preclinical and clinical studies resulted in high failure rates. In addition ,
despite the speed of synthesis, combinatorial chemistry has not yielded an increased number of
leads,"It is common knowledge in medicinal chemistry that the removal ofchiral centers, introduc-
ing additional flexibility into molecules and decreasing its size,generally leads to a lessspecific and
weaker acciviry.iThe greater flexibility ofcombinatorial compounds is likely to have detrimental
entropic consequences for the binding of these compounds and could affect also their ability to
induce conformational changes in the receptor required for biological function. Finally, com-
mercial trends shifted the interest ofindustry from the infectious diseases research field to others
with more economical success (e.g., drugs to treat autoimmunity and/or chronic cardiovascular
or neurodegenerative diseases).
Natural Products Properties

Natural products structures have the characteristic of high chemical diversity, biochemical
specificity and high binding affinities to their specific receptor,":" As they are produce in nature,
it is easy to suppose that all of them have a function and they have naturally evolved to be more
effective. The generation ofnatural product diversity has occurred within the constraints ofavail-
able biosynthetic reaction and precursors, but also in the context ofbiological utility. The synthetic
routes for the natural product generation have coevolved with the functional requirements oftheir
ligands.' Natural products interact with a wide variety of proteins and other biological targets,
acting also as modulators ofcellular processes when they inhibit protein-protein interactions?
Different chemical and molecular characteristics from active structures can be investigated
to compare diversity between compounds ofdifferent origin. Molecular properties such as mass,
number of chiral centers, prevalence of aromatic rings , molecular flexibility, distribution of
heavy atoms and chemical properties are evaluated. In an analysis performed by Lipinski" were
estimated the solubility and permeability of compounds by experimental and computational
approaches. This work introduced "the rule offive" which defines the properties relevant for the
characterization of small molecules for medical use. These properties are: molecular mass «500
Combinatorial
Plant
chemistry
extracts
Extracts
libraries
Target
identification
Clinical
Drug
evaluation
10,000 - - - - - - -... 1,000 - - - - - - - - -... 1
10-14 years
Figure 1. Stages in the drug discovery process.
Da) , number of hydrogen-bond donors «5), number of hydrogen-bond acceptors «10) and a
calculated octanol-water partition coefficient to indicate the ability to cross biological membranes
«5). Other studies characterized the molecular frameworks and substituents,13.14 the statistical
analysis ofdifferent drug databases" and the introduction ofa drug-like index. 16The work from
Feher and Schmidt? analyzed the molecular diversity of natural products in comparison to that
ofcompounds obtained from combinatorial chemistry and synthetic drugs derived from natural
products. After the analysis of several databases they counted: 10,968 drug molecules, 670,536
combinatorial compounds, 3,287 natural products and 27,338 molecules considered as natural
and semi-natural. Many molecular properties, such as number ofchiral centers, rotatable bonds,
unsaturations, atom types, rings and chains were evaluated and results showed that natural
products and combinatorial libraries have different properties, demonstrating that combinatorial
compounds are substantially less diverse than either drugs or natural products. Furthermore, the
diversity of combinatorial compounds is confined to an area where appears to be little diversity
for natural products, thereby raising the question of the real significance of this diversity in the
context of biological processes (Le., why this type ofdiversity was not positivdy selected during
evolution). In conclusion, products obtained from natural origins show more diversity, contain a
greater number ofchiral centers and higher steric complexivity.
The Urgent Need for New Drugs

Microbial infections are responsible for approximatdy 26% ofdeaths worldwide. Thus, there
is an urgent need for new drugs , particularly to fight against the multi-resistant infectious agents.
Particularly bacteria show an outstanding capacity to develop resistance against common used
anti -bacterial drugs through a wide variety ofmechanisms . This has lead to a practical exhaustion
in the repertoire of active antibiotics, including compounds such as carbapenems, tetracyclines,
fluoroquinolones and aminoglycosides, The last resource drugs , as imipenem for gram-negative
and vancomycin for gram-positive , are no more effective against the "superbugs" This is the new
term for infectious agents which have developed resistance to almost all commercial drugs. These
multiple resistant bacteria are now one of the most challenging problems for modern medicine.
There are limited and rather expensive therapeutic options for infections caused by Staphylococcus
aureus, particularly for the methicillin-resistant S. aureus(MRSA) strains. which show an incidence
exceeding 40% in some European countries. 17 The new drugs on the market (e.g., linezolid, quinu-
pristin-dalfopristin, daptomycin) have limitations, such as high rates ofside effects or low efficacyin
pulmonary infections.'?Other problematic gram-positive pathogens are the vancomycin-resistant
enterococci and penicillin-resistant Streptococcus pneumoniae. Another class of superbugs, the
opportunistic gram negative pathogens [e.g.• Pseudomonas aeruginosa, Burkbolderiacepacia and
Stenotropbomonas maltopbilia], which are frequently from environmental origin and generally infect
compromised patients, are intrinsically resistant to many antibiotics. IS Acinetobacter baumanii, a
pathogen which is responsible of7% ofall cases of pneumonia and kills 40% ofinfected patients,
has successfully developed resistance against all common antibiotics including colistin (polymyxin
E), the last universally active drug against this pathogen.19,20Mycobacterium tuberculosis, a pathogen
considered under control, is now re-emergingas problematic due to its resistance and persistence."
In addition. the emergence of new fungal and viral diseases poses the need of rapid discovery in
response to new infectious diseases [i.e., we cannot afford to wait 10-15 years).22.23
From Microbial Diversity to Drug Discovery

The battle against microbial resistant pathogens demands new and better drugs , which are more
difficult to find after decades ofhard work on discovery programs. However, natural products and
their derivatives, continue to be rich sources for lead discovery.The question is if classicalapproaches
will succeed in discovering new leads after more than 60 years ofnatural products search. Classical
natural products drug discovery programs are based on extract collections obtained from natural
resources, followed by screening. bioassay guided isolation, identification of new compounds.
structure elucidation and large scale production (Fig. 2). Microorganisms are capable to carry out
a tremendous variety of reactions, adapting to a large array ofdifferent environments, allowing a
culture to be transplanted from nature to the laboratory flask and later to a fermentor, in order to
produce active compounds in large quantities under tightly controlled conditions.
Microbial Sources
Microorganisms. bacteria and fungi were and still are a rich source of natural products.
Over 6.000-12,000 compounds of microbial origin with anti-microbial activity have been
isolated. However. only five phyla (Actinobacteria. Bacteroidetes, Cyanobacteria, Firmicutes and
Proteobacteriat include species that produced bioactive molecules which were developed to drugs."
In the past , drug discovery programs focused only in group oforganisms, as Actinomycetes (mostly
represented by Streptomycetes) and fungi isolated from easily accessible environmental soil samples
and cultivated under standard conditions. Approximately 3,500 bioactive compounds have been
recognized from the genus Streptomyces. In particular, strains from Streptomyces griseus and S.
hygroscopicus produce over 50 and 200 different compounds with anti-bacterial activity, respec-
tively," Other known producers ofbioactive compounds are Bacillus spp. and Pseudomonas spp.
Different strains from Bacillussubtilis produce over 60 different active compounds. Other novel
groups ofmicroorganisms, such as Myxobacteria, resulted to be rich sources of new strucrures .P:"
However. despite the huge amount ofbioactive compounds characterized each year from microbial
sources, only a relatively small number ofcompounds, approximately ISO, have been commercially
developed as antibiorics. YThe most important are listed in Table 1. Most of them are produced
by Streptomyces raising the question if they are the only microorganisms able to produce active
compounds with low toxicity.
Nevertheless. the microbial world represents 90% of all biological diversity and less than 1%
has been explored." Mining this microbial diversity is the key for obtaining high compound
diversity. A huge source for new natural products remains unexplored and the microbiological
efforts are actually concentrated in the study ofunexplored niches to access unknown uncultured
Microorganisms from
unexplored environments
sampling
•
Cultivation
•
Isolation
Extracts library Microbia l extracts Microbial cultures
UV & MS analys is
active tractions
•
Database search
Screens tor detection
ot biological activities
HPLC profiling of
extract fractions •
Lead identification
• Yields optim ization

• Large-scale production
• Isolation and purification
• Chemical characterization
• Biological characterization
Figure 2. Discovery of natural products of newly isolated microorganisms.
microbial diversity (e.g., marine environments or tropical forests). In addition, the development of
new cultivation technologies could let to identify and characterize new forms ofbiodiversity. 23,24,29
Good examples are the probes obtained from the deep-sea hyper saline anoxic basins of the
Mediterranean sea, which harbored uncharaeterized biodiversity.30-32 Novel lineages ofmicrobes
have been found and several novel enzymes exhibiting exceptionally catalytic activities ofindustrial
Natural Products in Drug Discovery 21
relevance have been recovered and characterized, indicating that new groups of microbescould
exhibit new capacities.
The selectionof the environmentalniche to exploreis matter of continuoussearch.New com-
paniesareableto providematerial fromexoticenvironments. However, microbiological expertise
isrequiredfor the isolationof newmicroorganisms.New methods,ashigh-throughputcultivation
usingmicrotitre dishes, microencapsulation of singlecellsand automated processes wererecently
developedto increase the percentageof strains culeured.P'" Once the environmentalstrains are
isolated, antagonist methods could be used for the detection of substances with anti-microbial
activity.Thecocultivation ofa targetand a newisolatedmicroorganism in solidmediaallowa rapid
identificationof antagonism.However, the observationof antagonismdoes not implysuccess in
the discovery of the activestructure. The antagonist compound could be produced only in pres-
ence of the target or in low amounts makingdifficultthe characterization process. Furthermore,
this method limits the exploitationof a new isolateto anti-microbialcompounds.
Microbial Extracts
Bioactive natural products are secondarymetabolitesproduced and secretedin nature by mi-
croorganisms duringcompetition in microbialcommunities. In the laboratory, duringcultivation,
microorganisms may also produce many compounds as a result of their secondarymetabolism.
The extraction of the secondary metabolitesproduced during growth will let to the generation
of a library of extracts, which could be stored and screenedin parallelscreens using a varietyof
assays (Fig.2).
Cultivation is a crucial process since microorganisms could show the capacity to produce
bioactive compounds only under specific growth conditions. Fewgeneralrulescould be applied
for the determination of the optimal cultivationconditions for production of bioactivities. First,
should be takeninto considerationthe requirementsof the group of microorganisms under study
(e.g., the addition of seasaltsor the useof sterilizedseawater for marineisolates). A widerangeof
differentmediahavebeensuccessfully usedfor the production ofbioactivemetabolites. Literature
search, once the species of the producer strains are known, could provide usefulinformation for
the choice of the appropriate medium and growth conditions (e.g., temperature and aeration).
Optimization of production yields should be investigated before shifringto large-scale produc-
tion. During the initial cultivationof a largenumber of unknown environmentalstrains, a general
mediumin which organisms grownin formerstudiesshowedproduction ofbioactivecompounds
could be applied.However, when strainsareselectedthrough the primaryscreens for their capac-
ity to produce bioactive substances, different cultivation strategies should be further explored.
Conditions that influence the yields of secondarymetabolitesproduction are:variations ofgrowth
substrates(e.g., carbon sourceand concentration),variation of growth parameters (e.g., effectof
time of incubation, temperature and aeration) and the addition of supplements or precursors"
[e.g., Mn, Mg and DMSO). Afrercultivationand filteringof the biomass, wholebroth extraction
is the primary step of the isolationprocess. In addition to the classical extraction usingdifferent
solvents (e.g., chloroform,acetoneand ethylacetate), several newapproaches weredevelopedover
the last years to increase the efficiency of extraction.37 Alternative, cultivationcould be performed
in the presenceof a resin.38.39 The resinwilladsorbthe secondarymetabolitesproduced during the
whole incubation time, stabilizingthem and reducing the risk of degradation. In addition, this
approach facilitates the biomass separation processby collectingthe resin on a filter or a sieve.
Finally, the adsorbedcompounds areeluted (e.g., methanol) and the extracts areconcentrated by
rorary evaporarion."
Crude extracts arecomplex, containingfrom 10to 100metabolites. Thequantityofcompounds
present in an extract is not know, many of them are present at low concentrations, limiting the
possibility to useHTS technologyfor identification ofbioactivities.Therisksin performingscreens
usingcrudeextracts are: (i) the concentrationlevels ofcompoundsmight not be enoughfor detec-
tion of activityand (ii) the presenceof other compounds could inhibit the test. Moreover, new
HTS assays arebasedon fluorescence reactions and manycrude extractscontain compoundsthat
could affectthe readout of the assay byautofluorescence or absorbtion at the samewave lengths?
However,interferences couldbe overcome using"lifetime discriminatedpolarization',a technique
that reducepotential interferences byrejectionofsignals from short-lifetimesources" or by using
red-shifted wavelength dyes.42 Once available a library of extracts,two main strategies could be
pursued for the discovery of new compounds: the biologicaland the chemicalscreenings.
Chemical Screening
During the chemicalscreeningeach extract is analyzedby high performanceliquid chroma-
tography (HPLC). This analysis coupled to ultraviolet(UV) photodiode arraydetection and to
massspectrometry(MS)usingeleetrospray ionization(ESI)and atmospheric pressure lonlzarion,"
will providethe massand UV spectradata of eachpeak,which will be usedto comparewith data
from databases to recognize new compounds. Liquid chromatography (LC) combined with
MS, UV and nuclear magnetic resonance (NMR), using techniques such as LC/UV, LC/MS,
LC/MS/MS and LC/MS/NMR quicklyprovidestructureinformationwith minimalamountsof
compounds.r'Thisenablesthe differentiationbetweennovelcompoundsand known compounds
present in crude extracts. Isolationof known compoundscan be avoidedand onlysubstances with
novel structures are isolated to generate a natural product library. Theselibraries contain new
molecules that need to be incorporatedin biological screens in the searchfor possible applications.
The chemicalscreeningis recommendedparticularlywhen extractsobtained from newgroupsof
organismsare analyzed.
Biological Screening
During the biological screening,parallel assays (e.g.,detection of anti-bacterial, anti-fungal,
anti-yeast and anti-mycobacterial activities; inhibitionofenzymatic processes; effects on eukaryotic
cells) areperformedwith the crude extractslibraries. Assay interferences, particularlybythe enzy-
matic tests,could be reducedbyscreeningfractionatedsamples obtained from crude extracts.45.46
Thesesamples contain less complexmixturesand the relative concentration of the compounds is
increased. therebyincreasing the chancesof detectingbiological activities. However, this strategy
requiresa first fractionation/purificationstep (through HPLC) beforeperformingthe screening
for biological activities and multipliesthe number ofsamples to be screened.
During the primaryscreens bioactive extractsareselectedfor further analysis.The compound
contained in the crude extractand responsible for the bioactiviry should be recognizedand iden-
tified. One or more rounds of chemicalpurificationand biological assay might be necessary for
identifyingand isolatingthe activecomponent in the complexmixture.At this point of the drug
discovery processcombined expertise from microbiologists and chemistsis required. A strategy
that allows a rapid identificationof the activeprincipleis the HPLC-activity profiling.The active
extract is separatedon an analytical HPLC and fractions are collectedin a microtitre plate.The
fractionsare tested in the biological assay that allows the identificationof the activefraction (Fig.
2).The activepeak is further analyzedby LC coupledto UV detection and MS.47.48The massand
UV data allow recognition of known compounds through databases49.5o (e.g., Chapman & Hall
Dictionary of Natural Products"). When no matches are found. the new moleculeshould be
identified.New activities for known compoundscanbe discovered byimplementingnewscreens.
Then, the activeprinciple need to be isolatedfor characterization.
Production, Purification and Characterization

ofa New Natural Product
Largeramount of compoundsisrequiredfor the isolationand purification.An important and
complicatedaspectin the drug discovery programsis the transferof the production processfrom
the small-scale laboratorycultivation or 51fermentorto large-scale equipmene.f Rarely abiological
processbehaves the samewayin large-scale fermentorsasin smalllaboratorycultivation. Mixing
and aerationarethe main difference betweenthe twogrowthconditions.Bioengineeringexpertise
is required for fermentation surveillance.F'The cloningof the completebiosynthesis cluster in a
Natural Products in Drug Discovery 23
heterologoushost isindicatedwhen the producingorganismisdifficultto handle, growthsslowly

or produce lowyieldsof the activecompound.Thepurificationof a new moleculefrom a complex
crude extract is a task that limits the speed of the drug discovery process. Different chromatog-
raphy systems are employedfor purification.F" Identification of compounds usually involves a
combination of various techniques including NMR, MS, UV and infrared (IR) spectrometry.
New advances in NMR and MS spectroscopy allowthe structure elucidationwith smallamount
of compound (from 0.5 to 1 mg).29.43 However, largerquantitiesof purifiedprincipleare required
for the chemical and biological characterization, aswellasfor the studyof the mechanismof action
and in vivoactivity. Structure/activitystudiescontribute to understand the interaction between
the new moleculeand its target. As mentioned at the beginning, an activenatural product is not
a drug. It is a lead which should be further developed to obtain a drug. Activecompounds with
high toxicity, poor pharmacokinetic propertiesand questionablein vivorelevance are not further
exploredor need to be modifiedin the laboratory. Combinatorialchemistrycouldyieldmoreuse-
ful derivatives through derivatisation or biotransformation.54Nature often represents the starting
point for the developmentof semisynthetic compounds with improvedstabilityand/or activity
(Table 1). Furthermore, the total chemicalsynthesis of new compounds could be an alternative
method to microbialproduction or provideinsightsabout the structure/activity relationships of
the compound.
Metagenomics for Drug Discovery

Most of the microbial world remains uncultivated; many potentially activecompounds are
unknown. The number of microorganisms cultured from soilsamples represents 1%of the total
microbial community.28The analyses of 16S rRNA genes amplifiedfrom soil discovered novel
phyla.55.57 Marine microbial communities studies using cultivation-independent genomic ap-
proaches resulted in the characterization of many new phylogenetic lineages suggesting that the
majorityof the microbialdiversityhas to be discovered." Metagenomics, a strategyto access the
geneticpotential of uncultured microorganisms, could allowthe discovery of novelcompounds
through a culture-independent process.57.59,60 DNA isolated directly from soil or other samples
is cloned into a bacterial artificial chromosomevector to construct genomic libraries, thereby
accessing the metagenomes from microbial communities. However, this new technology has
limitations for obtaining novelcompounds with complexstructures. The probabilityof finding
a biosynrheticcluster encoding a new active metabolite in a random DNA library is reduced.
Furthermore,Escherichia coli isan inappropriatehost for functionalscreening, because genes may
be not expressed or the metabolicprecursorsare not present. However, new suitablehosts (e.g.,
Streptomyces, Actinomycetes and Pseudomonas) areactuallyunder characterization. Theseexpres-
sion hosts could provide a more appropriategenetic,biochemicaland physiological background
to produce bioactive molecules from genes that arederivedfrom uncultivatedbacteria.However,
the cloning and expression of genes from membersof differentor unknown lineages could fail.
The useof metagenomics in drug discovery is a strategyadopted byseveral companies which are
developinghigh-capacity vectors, suitable transformation protocols and engineeredhost to ac-
commodate largegeneclusters for production of complexsecondarymetabolites.60•61
Natural Products Under Development

Thereareat least127naturalproductsor naturalproduct-derivedcompoundsthat arecurrently
under development,of them,only a fewhaveanti-bacterial propertles.f Leedset alhavereviewed
the most important naturalproducts underdevelopmentwith anti-bacterial activity." Seven anti-
bacterialcompoundsthat targetlipid II, a membrane-anchoredcell-wall precursor,areactuallyon
preclinical developmenr.s' TheyareproducedbyLaaococcus.Actinomycetes,Streptomyces,Cytophaga
and Pseudomonas and showactivityagainstmulti-resistant gram-positive bacteria. Because lipid II
production is restrictedto bacteria,these compounds should havelow toxicityin humans.
Other recentlydescribedcompoundsare activeagainstmulti-drug-resistantstrainsof staphy-
lococci and enterococci. They are examples of new compounds found in well-known sources
(e.g., a collection of soil isolates) by using new screens or by sampling previously unexplored
environments.
Platensimycin has a novd chemical structure and targets an enzyme involvedin fatty-acid
synthesis. This compound was discovered by screening250,000 extractswith a new screen.64.65
Samplingof unexplored environmentsfrom Indonesia also allowedthe discover of a new vari-
ant of a macrolactin produced by B. subtilis, which show activity against MRSA and VRE.39
Interestingly, this new macrolactin seems to havea novel mechanismof action by targeting the
celldivisionprocess.
New Strategies in Fighting Infectious Diseases

During drug discovery, extractsshowingpowerful biological activities in paralld screens are
continuouslyinvestigated. Moreover, newscreens areimplemented,accordingto new therapeutic
needs.66 In addition, the recent availability of complete microbialgenomesfacilitates structural
approachesaimed at the identificationof new molecular targets.67•70 However, natural products
are produced in nature in response to competition in microbialcommunities. Therefore, the mi-
crobialproducer should be resistantto the action of the compound.Thisimpliesthat a resistance
mechanism pre-exists in nature which is coupled to the production pathway.7I,72 Furthermore,
microorganisms can rapidlyevolve resistantmechanisms due to their genomics plasticityand the
existenceofmechanisms for horizontal genetransfer. Thus, the fight againstmicrobialresistance
could be a battle lost from the beginning.Therefore, it is necessary to developnew strategies to
combat infectionsagents.
Virulenceor pathogenicity refers to the abilityofan organismto establish an infectionand cause
disease in a hose,"Productsthat aid the pathogenicityofa bacteriumaretermedvirulence factors,?4
Molecules that inhibit the production of virulencefactors,affectthe growth,block the spreador
interfereduringhost-pathogen interactions represent alternative therapeuticapproaches.Y" Rather
than kill the pathogen, once the pathogen is weakened, the host immune system could take over
and clear infections. Deeper knowledge on host-pathogen interactions and the molecular basis
ofimmunoclearance couldyieldnew targets and new strategies for successful therapies.Another
focus of innovation is drug discovery through system biology, a biology-driven approach that in-
volves screeningcompoundsbyautomated response profilingin disease modelsbasedon complex
human-cell system.66.77.78 Infection biology represents the emergingfidd where the efforts from
microbiologists, immunologists, cellbiologists, system biologists, chemists, pharmacologists and
cliniciansare combined to fight infection.
Conclusion
The decline in the discovery of natural products during the last decades was a consequence
ofthe shift of interest when scientists concentratetheir effortsin chemicaland geneticcombina-
torial methods, as well as in the devdopment of HTS for testing existingcompounds libraries.
Nevertheless, nature still contains a treasureof diversitywhich it is essential to tap, particularly
considering the meageroutcome from combinatorial approaches. Hopefully, the environment
offers us its treasure of unexplored resources, allowing the discovery of new weapons against
emerginginfectious.
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CHAPTER 3
Protein Pharmaceuticals.
Discovery and Preclinical Development
Davinder S. Gill·
Abstract
P
roteins are natural molecules that carry out important cellular functions within our bodies.
Their preciserole is crucialto the maintenance of good health. Malfunctioning proteins or
those not produced optimally result in disease. The foundation ofbiopharmaceutical drug
therapyhas thereforebeento modularecellular function bytargetingspecific proteinsexpressed on
or outsidethe cell. Because most biopharmaceuticals arenatural in origin,theyare biologically and
chemically verydifferent from conventional medicines. In addition to differences in mechanism of
action,biopharmaceuticalsdiffer in theprocess bywhichtheygetmanufactured andddivered.Because
of their large, complex structure,they must often be produced by culturingcells and then purified
from a host of cellular components. Thiscanbe time-consuming and costly. Also, most biopharma-
ceuticals aregiven by injectionunder the skinor by infusioninto the veins. This creates significant
limitations to their utility. Nonetheless, biopharmaceuticals can be verypowerfuland sdectivein
disease applications such as in rheumatoid arthritis or cancer. This chapter describes methods by
whichproteins drugs arediscovered, optimizedand developed, It alsocovers novd agents and next
generationproteinsaswellassomeof the challenges and opportunitiesin the area.
Introduction
Most biopharmaceuticals today can be broadlyclassified into one ofthree major categories-
monoclonal antibodies, fusion proteins or native biologics. Monoclonal antibodies are a grow-
ing class of Biopharmaceutical products that includes 21 FDA approved drugs to date.1 The
list includes products such as Bevacizumab (Genentech), Panitumumab (Amgen), Rituximab
(BiogenIdec/Genentech) and Adalimumab (Abbott). In 2006, this class of drugs generated
greater than $19 billion in annual salesworldwide, a figurethat is expectedto growat the rate of
30-40%for the next S years,"? Fusion proteins are a class of drugs exemplified by Eeanercept, the
extra cellulardomain of the TNFa receptor (p7S) fused the Fcdomain ofinununoglobulin IgG.
Since its launch in 1998, Etanercept is used for the treatment of chronic inflanunatory diseases
such as Rheumatoid arthritis, plaquepsoriasis and alkylosing spondylitis. Other examples of this
class of drugs include Alefacept(BiogenIdec) for treatment of multiple sclerosis and Abatecepr
(Bristol-Myers Squibb) also for treatment of rheumatoid arthritis. Fusionproteins are an estab-
lished class of drugs with medicinessuch as Etanercept projected to reach $S billion in global
salesby 2010.3 Native biologics is a class of drugs that are also referredto as secretedproteins or
replacementtherapiesand it represents the earliestcategoryofbiotherapeutic drugs approvedby
the US FDA. It includeserythropoeitins, interferons,insulins,clottingfactors, growthhormones
and interleukins.Bymarket size, this class of Biopharmaceutical products dominates the global
portfolio of protein-baseddrugs. Erythropoeitins alone drew closeto $10 billion in worldwide
salesin 2004 and the number is stillgrowing.'
*Davinder S. Gill-Wyeth Research, BiologicalTechnologies, 87 CambridgePark Drive,
Cambridge, Massachusetts 02140, USA. Email: dgill@wyeth.com
PharmaceuticalBiotechnology, edited by CarlosA. Guzman and Giora Z. Feuerstein.
©2009 LandesBioscience and SpringerScience+Business Media.
ProteinPharmaceuticals: Discoveryand PreclinicalDevelopment 29
Protein Drug Discovery

Screening
Discovery ofBiopharmaceuticals, aswith anymodern medicine, beginswith the identification
of a molecular"target", one that is closely associated with disease. The target can be either a miss-
ing or an overproduced or a malfunctioningprotein or its derivative. A therapeutic hypothesis
is formulated around the function of the target protein. Using recombinant DNA techniques,a
series of candidate therapeutic proteins are then generated to test the hypothesis. The strategyto
screencandidatesdependson the type of protein beingdiscovered. For fusionproteins and native
biologics, screens tend to be relatively smallwith evaluationof 10-20 different proteins for lead
identification. Thesecandidatesgenerally includevariantswith small(1-2 amino acid) modifica-
tions. In the caseof receptor fusionproteins changes are madeeither in the receptor ectodomain
or in the hinge region and in the more rare casein Fcportion of the protein. In the caseof native
biologics, the variants can be singleor double amino acid changes introduce to improveexpres-
sion or stabilityeither asit relatesto product formulation or to pharmacokinetics. In somecases,
variantscanalsoincludeengineeringof the carbohydratecompositionof the protein for improved
pharmacokinerics.' The smallpanel of proteins is then evaluated in a number of in vitro assays to
selectthe most suitable leadprotein.
Screens for monoclonal antibodies can be much larger. While monoclonal antibodies have
beenaround nowfor more than threedecades, their developmentastherapeuticagentsisrelatively
recent however. Screeningfor monoclonalantibodies has traditionallyrelied on the hybridoma
processdevelopedbyKohlerand Milsteinin 1975.Theprocessinvolves somatically fusingrodent
B-cells derivedfrom spleenor lymph node with mousemyeloma cellssuch asSp2/0. Successfully
fused cells can be screenedfor antibodies that selectively bind target antigen. Typically, primary
screens are based on ELISA or a FACS assay. Increasingly however, the trend has been to carry
out functional assays upfront wherepossible. As an example, a reporter geneassay can be used as
a primary screen to identify hybridomas producing ligand-neutralizing antibodies or in the rare
case a receptor-activating antibody. However since any initial screeningis done using pools of
typically 500 hybridomacells per well,cloning by limited dilution must be carriedout to isolate
single hybridomasfrom the pooL This is typically done over one or two rounds of subcloning
before the monoclonality of the hybridoma is established. Further, not all hybridomasgrow at
the same rate. This requiresconstant monitoring of the pool to ensure all possible hits from the
fusion screenare isolatedand rescued.
Little has changed in the hybridomatechnique sincethe daysof Kohler and Milstein. While
being overall robust, the hybridoma process is not very efficient. Typically only 1-2%ofB-cells
in the spleen or lymph node of an immunized rodent are actuallyantigen specific," The somatic
cellfusion technique used to produce hybridomas is blind to the specificity of the B-cell. Thus,
a number of irrelevantB-cells fusealongwith relevantclones. Moreover, cellfusions conducted
usingthe classic PEG reagentareinefficient with less than 1 in 20,000 B-cells resultingin a viable
hybridoma,"Techniques suchaselectricfield-induced hybridization techniqueshavebeenreported
to improvefusionefficiency. Regardless, hybridomas can sometimes be unstableresultingin a loss
of antibody producing genesovertime.
To overcome these limitations new techniques such as the Selected LymphocyteAntibody
Method (SLAM) havebeen developed.! The aim of these approaches is to bypass the somaticcell
fusion bydirectlysamplingB-cells and to rescue antibody-encodinggenes from a cells ofinterese,
The SLAM approach consists of two steps. In the first step, biotinylated target antigen is cova-
lentlycoupledwith streptavidincoatedsheepred blood cells. Next antibodyproducingB-cells are
mixedwith antigen coated sheep blood cells followed by addition of anti-IgG rabbit anti-serum
and guinea pig serum as source of complement. Formation of plaquesis then visualized under a
microscope. In the second step, a desired B-cell, identified from a largepool oflymphocytes by
its ability to form hemolyticplaque,is isolatedusinga micropipette. Antibody producing genes
from the single B-cell are then isolated using RT-PCR followed by cloning of the VH and VL
30 PharmaCl!UhCil/Bioltchn%gy
domains.The advantage of the SLAM approach is that it directlysamples antibody producing

B-cells bypassing the hybridomafusion step.This allows screeningof a much higher number of
B-cells (500,000)versus typically 2000-4000wells in a hybridomafusion.RareB-cellclonescan
thereforebe more efficiently isolatedfrom a vastpool ofirrelevant clones.
An approachdifferentfrom the screeningofhybridomasisbaseda completely newtechnology
calledphagedisplay that doesnot requireimmunizationof animalsor screening ofantibodypro-
ducingcells.Thisapproachisbasedon firstdirectlyisolatingantibodyencodinggenesfromB-cells
and then cloningthem asrepertoiresofindividualantibodiesin the form ofbindingfragments."
Thesecan be either single-domain antibodies, single-chain FvofFab fragments. Theserepertoires
can then be displayed on hosts suchasfilamentous bacteriophage. Theadvantage ofthis system is
that it not only obviates the need to carryout lengthyimmunizations but it alsoremoves any in
vivo biases around immunodominant epitopes on target antigens. Further,since bacteriophage
can be cultured more easily than antibodyproducing mammalian cells, the system lends itselfto
automation that can be integratedinto high-throughput screeningmethodologies.
The recombinantantibodyapproachbeginsby masscloningof antibodygenes derivedeither
from an immunizedsourcesuchas a rodent or evena human exposed to pathogensor infectious
agents. With sequencing of the human genomenow complete, it is relatively straightforwardto
designsetsofdegenerate primersto amplifyby PCR genesencodingantibodyvariable domains.
Once the individualgene fragments are cloned the actual format by which they are assembled
can vary. The most widelyused format is that of the single-chain Fvwherein the heavyand the
light chainvariable regions of the antibodyare linked through a 15-20amino acidflexible linker.
However, several labs havealso reported the successful use of the Fab format. The scFvis a less
stablemoleculeand one that ismoreprone to aggregation than the Fab.1o However, its singlegene
constructmeansit iseasier to manipulate. Also, scFv's in generalexpress better than Fabs.'! Smaller
fragments such as single-domain antibodies havealso been successfully cloned as recombinant
repertoireshowever biggerfragments particularlythose that contain the Fc portion haveproved
to be challenging.
Cloned ensembles of antibodygenes canthen be propagatedin bacteriaor in yeast. 12 Bycreat-
ing a geneticfusionbetweenthe antibodybindingdomain (scFv or Fab)and host surface protein
(e.g., phage pIlI or yeast Aga2) a link between genotype and phenotype is created. This allows
massscreeningofantibodyrepertoirethrough affinitydrivenselections. Only thosephageor yeast
that display bindersget selected whilethe rest get washedaway. Selectedphageor yeastare then
rescuedand amplified for successive roundsof selectionand amplification. At the end of3-4 such
cycles, phageor yeastarethen sampledindividually for bindingactivity. Thosephageor yeastthat
displayusingbinding propertiesare rescued and antibody genes contained within them isolated
for further manipulations.
One ofthe key advantages ofsuchinvitroscreening methodsisthat the antibodyselectivity can
be drivenbasedon the desiredoutcome. Forexample, if antibodiesagainsta particularepitopeor
domainofthe antigenaredesiredthen counter-selections againstundesiredregions canbe carried
out. Or if antibodieswith slowoff-ratesare to be selectedthen phage or yeastcan be incubated
in bufferat incubation times beforeelution. In cancerapplications, antibodiesthat bind surface
antigensand get internalizedmaybe of value. In vitro approaches allowdirect selection of these
types of antibodies.13 Perhaps the ultimate screening approachis one whereantibody repertoires
are directlyselectedin vivo. Thishasbeen successfully demonstrated." However, its utility to the
drug discovery process remains to be proven.
Optimization
Proteins,likesmallmoleculedrugs,frequently requireoptimizationbeforeleadcandidatescan
beidentifiedforfurther development. Often,the optimizationisfocused towardsimprovement of
binding affinityor selectivity. But it can alsoinclude reduction of potential for immunogenicity
or improvementof solubilityof stability characteristics. For fusion proteins or nativebiologics
ProteinPharmaceuticals: Discovery and Preclinical Development 31
since the initial screens are small, sometimes optimization in included upfront as in the case of
darbopoetin alfa.s
Over the past decadeseveraldifferent optimization technologieshavebeen developed. It isnot
possibleto reviewthem exhaustively here.However,it isimportant to highlight a fewkeytechnolo-
giesthat havehad the most impact in the protein optimization process.Most protein optimization
technologies haveessentiallyrelied on two keysteps. The first step has been to generate chemical
diversity within the protein through a variety of mutagenesis techniques. Depending on the size
ofthe diversity created, the second step is to useanyone ofa number of "Display"technologies to
sort through the libraries and isolate important variants. There are also semi-rational approaches
based on protein structural modeling that generate small and focused chemicaldiversity that can
be screened using standard methodologies.
One of the earlyapproaches used to generate diversity was the use oferror-prone PCR.lsThe
approach reliedon the lowfidelityofthe polymerasesused in the PCRprocessto randomly create
mutations across a stretch of DNA. When the low fidelity of an enzyme like Taq polymerase is
combined with certain PCR conditions such as high Mg2+ concentrations, the error rate can be
as high as 0.01 mutation/bp/PCR cycle. The advantage of the error-prone PCR processis that it
is simple, it introduces mutations acrossthe gene of interest and can therefore be very useful for
identify structure/function "hotspots". The disadvantage is that biaseswith certain polymerases
have been reported and newer polymeraseshave been engineered to overcome the problems but
still the overallmutational rate achievedislow.A different approach, targeted mutagenesis,works
better when there is knowledge ofwhere mutations need to be introduced as in the caseantibody
CDR regions.P This approach generates far more chemical diversity than error-prone PCR but
in a more restricted portion of the sequence. Targeted mutagenesis can involve any number of
oligonucleotide-mediatedstrategies.Thiscan include spikingwild type nucleotidesor codons with
mutants or replacingstretchesof sequences with nucleotidesor codons that arecompletelyrandom-
ized such that each nucleotide is substituted by every other possibility. Thus if three nucleotides
are substituted with four possibilitiesat each position (G, A, T or C) then this generates 34 or 81
possiblecombinations. For larger sequence stretches the permutations are even larger.
Yetanother approach that combinesthe breadth oferror-prone PCR and the depth oftargeted
mutagenesisis Directed Evoludon." Here, codons aredesigned to introduce all possiblemutations
at a given position in linear sequence. However, rather than carry this through in a combinato-
rial fashion all along the sequence, parallel synthesis is conducted to construct small pools of
mutagenic sequence.Thesepools are then assayed using traditional screens to isolate functionally
important variants.
Two more mutagenesis approaches are worth mentioning. One involves randomly shuffling
DNA to createchemicaldiversiry.'<Iheprocessbeginsby firstdigestingDNA into smallfragments
usinga restriction endonucleasesuch asDNase I. Digested fragmentsarethen randomly combined
and amplifiedusingPCR to recreatethe full length gene. The diversityof sequencescreated bythis
process is sorted for functionally important clones. The process is then repeated until the protein
is optimized, for example,until a certain improvement in binding affinityis achieved.
For some of the mutagenesis approaches discussed above there is the limitation that the
chemicaldiversitygenerated can only be partiallysampled. That isbecausethe number ofpossible
permutations rise exponentially with linear sequence sampled whereas the maximum number
of variants that can tested is around 1010 due to limitations in bacterial transformations. Some
of these limitations are alleviated by the use of cell-free systemsas described below. But another
approach has been to use semi-rational or computational methods to sample protein sequence in
silico before any experimental works begins.P The process begins with construction ofa protein
structural model followed by analysis of all possible sequences that permit the fold predicted by
the model. Using computational resourcesa large number of possiblesequencesare screened and
those predicted to retain the protein functional foldgiventhe highest score.Only the most optimal
sequences predicted by the algorithm are synthesized and experimentally tested. Thus, sequence
space is reduced to fewerfunctionally relevant sequences.
As discussed abovein those cases wherevery largechemicaldiversityis generated one needs

a selection tool to sort through the enormous collection of variants. Three types of "Display"
technologieshave been developedand used successfully in the drug discovery process. All three
technologies are basedon a simplebut a powerfulprinciple: a robust link betweensequenceand
function. In the caseof phage display, fusing genesof interest with anyone of the genesencod-
ing a phage coat protein creates this link. Several proteins havebeen used successfully including
pIlI, pYIII, pYI and pIX although pIlI remainsthe most widelyused,"The number ofdifferent
classes of proteinsdisplayed on phageisprobablythe widest.Thisincludespeptides,single-domain
antibodies, scFv's, Fabs, growth factors, cytokinesand receptor ectodomains. Thus, as a library
selectiontool, phage displayis probablythe most versatile.
Ribosomedisplayisa cellfreesystemin whicha geneofinterest istranscribedand translatedin
vitro in acellfreeenvironmentusingE. coli, rabbitreticulocyte or wheatgermenracts ," Translated
polypeptidesare folded in vitro and them made to remain anchored to the ribosomeusinglong
polypeptide tethers that are part of the translated sequence. By doing so, the folded protein is
linked to the mRNA sequenceencoding it. Theseprotein-ribosome-mRNA complexes are then
sorted through stepsof affinityselectionfollowed by rescueof functionallyimportant complexes.
RescuedmRNA is then reverse-transcribed to produce cDNA which can then be amplifiedby
PCR followed by iterative rounds of in vitro translation and selection.The main advantage of
ribosome displayis that it is cell-freewhich meanssequencediversitylarger than phage libraries
can be sampled (_10 14) . Further, steps to carryout bacterialtransformationsand preparation of
viralstocksareobviated.Therefore, multipleparallelproteins canbe optimized.Thedisadvantage
is that the systemis not as robust due to presenceof ribosomes and mRNA. Therefore, selection
conditions have to be extensively optimized.
A relatively new entrant into the displaytechnologyfieldis yeastdisplay.22 In this approach a
the genetic linkage between structure and function is created by fusinga gene of interest to the
Aga2p gene which encodesthe adhesion subunit of the yeast agglutinin protein. Unlike phage
or ribosomes, yeastis a eukaryote which means that folding and posttranslationalmodification
is different and closerto that in mammaliancells. Further, through the fusion process described
above10,000-100,000copiesof the protein of interestaredisplayed per cell.Thismeansquantita-
tivescreeningof variantscan be carriedout byfluorescence activatedcellsortingpermitting both
equilibriumand kineticselections. A limitation ofyeastdisplayissmallerlevels oftransformations
than permissible in phageor ribosomedisplay therebylimitingthe amount ofsequencespacethat
can be sampled.
Novel Agents
As mentioned in the introduction, most biopharmaceutical products can be classified as
monoclonal antibodies, receptor fusion proteins or native biologics. However, there are some
products that do not fallin anyof thesecategories. Two of them are worth mentioning under the
categoryof novelagents.
The firstof this novelset of proteins areantibodydrug conjugates.Thesenovelagentsare part
protein and part smallmolecule drugs.Thenoveltycomesfrom the roleplayedbyeachcomponent
in producing a safe, pharmacological effect. It is well recognizedin fields such as Oncology that
if chemotherapeutic drugs could be made safersuch that they more selectively kill cancer cells
than normal cells, it would greatlyimprovetheir safetyprofile. Antibody drug conjugates do just
that. An excellent example of this mechanism is Gemtuzumab Ozogamicin which is approved
as monotherapy for treatment of relapsedacute myeloid leukemia." This drug consists of three
components: a humanizedmonoclonalantibodydirected againstthe CD33 antigen expressed on
myeloidcells, a hydrolysable bifunctional linker and calicheamicin, a potent cytotoxicdrug. By
itself,calicheamicin is too toxicand cannot be usedwithout serious sideeffect. Without the drug
the antibody by itself has no effect. However, couplingthe two allows the drug to be selectively
deliveredto targetpositivecells minimizingnonhematologictoxicity. Upon binding to CD33, the
conjugategets internalizedinto endosomeswhereuponthe linker holding the calicheamicin gets
Protein Pharmaceuticals: Discovery and Preclinical Development 33
hydrolysed. Calichaemicin gets released resulting in cell death. Since the approval ofCemtuzumab,
this type ofapproach hasbeen used for the development ofseveral conjugates for both hematologic
malignancies as well as for solid tumors, In such casesa variety ofcell surface antigens such as ErbB2
or CD30 have been targeted using chimeric or hwnanized monoclonal antibodies conjugated to
other cytotoxic drugs such as maytansinoid or aurastatin.24-2S
Another class ofbiopharmaceutical drugs that are novel agents is one composed of proteins
that exert their pharmacologic effect through the implantation ofa device. An example ofthis class
of products is recombinant hwnan bone morphogenetic protein - 2 (rhBMP2).26 rhBMP2 is a
member ofthe TGF~ superfamily that has strong osteogenic properties. This protein is too potent
to be delivered systemically..But when administered along with absorbable collagen sponge (ACS)
as a matrix , rhBMP21ACS is effective at inducing de novo bone formation. The drug has been ap-
proved by the FDA for three distinct indications in the orthopedic area-interbody spinal fusions,
open tibial fractures and for autogenous bone grafts. The choice ofthe delivery device for rhBMP2
has a strong effect on its clinical activity. It was the development ofa suitable carrier that took up
a significant time in the clinical development of rhBMP2.26 Given that several other members of
the TGF~ superfamily are candidates as drugs in the muscle, tendon and bone repair areas, lessons
learnt from the development of rhBMP2 would help these programs move forward.
Challenges and Opportunities

Perhaps, where protein drug discovery most differs from discovery ofconventional drugs is the
attention that is paid to maintenance ofhwnan composition ofthe protein drug, to efficientprotein
synthesis in cultured cells and to the preparation of the drug for delivery by injection . Because
conventional drugs are synthetic organic molecules , these considerations normally do not apply.
Another difference is that certain tests carried out to verify potential side effects ofconventional
medicines are not necessary for Biopharmaceuticals. This is because Biopharmaceuticals do not
inhibit the function of normal hwnan proteins found in the liver [e.g., cytochyeme p4S0) or in
other critical organs such as the heart (e.g., hERG). Outside ofthis , criteria to establish safety and
efficacy are quite similar between protein and conventional drugs.
The early monoclonals made using the hybridoma process were murine in origin. When tested
in clinical trials these proteins were quickly recognized as 'foreign" by the hwnan immune system
and eliminated. There was thus the need for approaches to reduce the potential immunogenic-
ity of proteins by reducing their nonhwnan content. Early attempts in this regard were oriented
towards development of chimeric antibodies. These molecules possessed the minimal binding
domains derived from mouse origin with the remainder of the constant domain derived from
human IgG. Chimerization reduced the risk associated with immunogenicity and successful
product launches were made possible-Rituximab for treatment of non -Hodgkins lymphoma
and Cetuxirnab for head and neck cancer. However, chimeric antibodies did not completely rule
out the risk for immunogenicity as there still remained significant amounts of mouse protein in
the molecule. Three new technologies arose to fulfill this need and all three have now resulted in
successful products.
In a seriesofadvances, the murine content ofantibodies was reduced using protein engineering-
techniques. The first development was to "humanize" mouse antibodies. In an approach pioneered
by Winter and colleagues complementarity-determining regions (CDRs) of murine antibodies
were grafted on hwnan framework region s such that the CDRs were the only mouse protein in
the antibody," However, a problem with this method was the observation that simply grafting
CDRs of mouse antibodies on hwnan antibody framework regions resulted in a loss of binding
affinity. This is because the CDRs fold in the form ofloops which must be correctly positioned
by the frameworks for optimal binding. In a second approach pioneered by Queen, problems
associated with CDR grafting were successfully solved." The Queen approach required changes
at key framework position s using sequence alignment between the mouse donor sequence and
the hwnan acceptor sequenc e and also by the use ofcomputer models. This approach was vastly
successful in overcoming the problems associated with antibody hwnanization. Indeed, the first
34 PharmaceuticalBiotedmology
generation of antibodies approvedby the FDA washumanized antibodiessuch as Trastuzumab,

Omaluzumaband Natalizumab.
However. the earlynineties sawthe emergence of two noveltechnologies that shaped the dis-
coveryofthe currentgenerationof monoclonalantibodies.Thefirstwasthe developmentofphage
displaytechnologydiscussed above. While the earlyproof-of-conceptexperiments conducted to
validate this technology were based on construction of peptide librariesof single-domain anti-
bodies, the fieldrapidlyexpanded to included phagedisplaylibrariesof scFvand Fabfragments.
Construction oflargediverse phagerepertoiresallowedscientists to bypass the hybridornaprocess
and obtain antibodiesthrough completely in vitroapproaches. However,antibodiesisolatedusing
this approach were not always of the highest affinityas they had not gone through the somatic
hypermutation processthat antibodies from immunizedsourceshad. Largerrepertoiresor those
derived from autoimmune sourceswere made to mitigate the problem but the affinityproblem
wasnot completelysolved.
A secondtechnologythat arosewasthe development of transgenic mousestrains harboringloci
ofhuman Iggenes. In this technology, genesencodingendogenousmouseIgwerefirstinactivated
usinggene-targetingtechniques.Thiswasfollowed bythe systematic introduction oflarge chunks
ofthe human Ig loci (H , K and A) usingyeastartificial chromosomes into the mousegermline."
Reconstituted mice stablyexpressed human immunoglobulins at normal levels and had normal
B-celldevelopment. Most importantly, when challengedwith a human protein asan immunogen,
these mice mounted a strong immune response of fully human antibodies which included class
switchrecombinationaswellassomatichypermutation.Thus,creationofsuchtransgenicanimals
obviatedthe need to firstcreatea mousemonoclonalantibody and then to carrythrough human-
ization. The fact that transgenicmice mounted both a primary and a secondaryresponse meant
that high affinity, fully human antibodies of the IgG class could be readilyobtained. However.
sincethe processofmakingsuch antibodiesreliedon immunizationand screeningof hybridomas
limitations to the traditional hybridomaapproachstill applied.
Next Generation Proteins

The nature ofbiopharmaceuticaldrug discovery has undergone a fundamental changein the
past 5-7years. Drawingupon the success of firstand second-generation protein pharmaceuticals,
namelysecretedfactorssuch as erythropoetin and receptor fragments such aseramecept, the in-
dustry has increasingly shiftedtowardsthe developmentofmonoclonalantibodiesas therapeutic
agents.Recent product launches by majorbiotech companiesas wellaslate stagebiopharmaceu-
tical pipeline candidatesare now mostlymonoclonal antibodies. Genentech's Bevacizumab and
Wyeth'sBapineuzumab are excellent examples of this trend. However, the industry has alsofaced
several challenges.
While hugely successful. monoclonal antibodies are still very large, complexmolecules that
require significantengineeringat the molecularlevel to be effective. Enabling technologies to
carry out this type of engineeringare often held by smallbiotech companies whereaccess can be
restructured. More importantly however, in the recent months, BigPharmahas serially acquired
biotech companiesthat pioneered the developmentof these technologies.
Another trend that hasaffectedthe biopharmaceutical industryhasbeen the rapid advances in
proteinsequence, structureand functioncoupledwiththe commercial needfor newer,cost-effective
protein therapies.This trend has led to the developmentof technologies to exploitnovelprotein
scaffolds as therapeutic precursors. Strategies includeengineeredfragments of monoclonal anti-
bodies as well as nonantibody scaffolds. Thesestrategies have recentlybeen reviewed elsewhere
and are referenced here.
Conclusion
The US Food and Drug Administration approvedthe first protein drug developedusing re-
combinant DNA technology(human insulin) in 1982.Thus, the protein drugindustryis relatively
youngwhen comparedwith the traditional drugindustry.As discussed in this chapter, significant
Protein Pharmaceuticals: Discovery and PreclinicalDevelopment 35
new technologies developedin the recent years promise to allow rapid advancementof protien
drugs. Biopharmaceuticals have enjoyed tremendous success in the recent years judged by the
surgeof approvals by the FDA. This success drawsupon technological advances madein the field
of protein therapeutics but also on a greater realization that there are significant opportunities
within pharmaceuticaldrug development to exploit the power of biopharmaceuticals. Indeed,
we are now witnessing the emergence of protein drug developmentopportunities in areassuch as
metabolicdisorders,Alzheimer'sdisease and osteoporosis. Traditionally,theseareaswerereserved
for conventionaldrug discovery. As we moveforward into the new millennium,it is hoped that
the synergybetween biopharmaceuticals and conventional medicines can be further leveraged
for saferand more cost-effective treatments. If successful, these therapies will addresssignificant
unmet medicalneedswhoseaggregate cost to the healthcaresystems worldwideruns into tens of
billionsof dollarseachyear.
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4. Pavlou AK, Reichert JM. Recombinant protein therapeutics-success rates, market trends and values to
2010. Nat Biotechnol2004; 22(12):1513-9.
5. Elliott S, Lorenzini T, Asher S et al. Enhancement of therapeutic protein in vivo activities through
glycoengineering. Nat Biotechnol 2003; 21(4):414-21.
6. Kodituwakku AP, Jessup C, Zola H et al. Isolation of antigen-specific B-cells. lmmunol Cell Bioi 2003;
81(3 ):163-70.
7. Pirofski L, Casadevall A, Rodriguez Let al. Current state of the hybridoma technolog y.J Clin Immunol
1990; 10(6 Suppl) :5S-12S.
8. Babcook JS, Leslie KB, Olsen OA et al. A novel strategy for generating monoclonal antibodies from
single, isolated lymphoc ytes producing antibodies of defined specificities. Proc Nad Acad Sci USA 1996;
93(15) :7843-8.
9. Marks JD, Hoogenboom HR, Bonnert TP et al. By-passing immunization. Human antibodies from
V-gene libraries displayed on phage. J Mol Bioi 1991; 222(3 ):581-97.
10. Rothlisberger 0, Honegger A, Pluckthun A. Doma in interactions in the Fab fragment : a comparative
evaluation of the single-chain Fv and Fab format engineered with variable domains of different stabilit y.
J Mol Bioi 2005 ; 347(4 ):773-89.
11. Hust M, jostock T, Menzel C et al. Single chain Fab (scFab) fragment .BMC Biotechnol 2007; 7:14.
12. Feldhaus MJ, Siegel RW; Opresko LK et al. Flow-cytometric isolation of human antibodies from a
nonimmune Saccharomyces cerevisiae surface display library. Nat Biotechnol2003; 21(2 ):163-70.
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targeted medicine. Curr Opin Chern Bioi 2002; 6(3):399-404.
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CHAPTERS
Conotoxin Venom Peptide Therapeutics

Richard}. Lewis"
Abstract
enom peptides offerenormous opportunity for the discovery of peptide drug leads. This
V reviewfocusses on the potential of conesnailsthat havedevelopedarrays ofsmallpeptides

aspart of highlyevolved venomsusedfor preycaptureand defence. Manyof thesepeptides
selectively modulateion channelsand transporters,makingthem avaluable sourceof newligands
for studyingthe rolethesetargetsplayin normal and disease physiology. A number of thesecono-
peptidesreducepain in animalsmodelsand several arenow in preclinicaland clinicaldevelopment
for the treatment of severe pain often associated with diseases such ascancer.
Introduction
Venomous animalshavedevelopedrich cocktails of pepndes they deliverthrough specialised
envenomation apparatus into the soft tissueof animals. Thesevenom peptides havediverse and
selective pharmacologies.' Their evolved bioactivitymakes venom peptides a unique source of
bioactives fromwhichnewtherapeuticagentsand research toolscanbedeveloped. To-date, alinear
peptide from the saliva of the gilamonster lizard(Exendin-4), adisulfidebonded globularpeptide
from a cone snail (ro-MVIIAor Prialt) and a peptide mimetic (captopril)of a snakeangiotensin
convertingenzymeinhibitor (teprotide) have reach the clinic to changeclinicalpractice for the
treatment ofdiabetes,pain and hypertension.' This chapter focusses on the therapeuticpotential
ofthe smallbioactive peptides conotoxin or (conopepcides) produced by marine moluscs ofthe
familyConidae.
Conotoxins are amongst the most interesting of the venomous species. They have evolved
hundreds of highly selectivity peptides that help immobilise their prey of either including fish,
molluscs or worms.Theirsmallsize, relative easeof synthesis, structuralstabilityand target speci-
ficitymake them ideal pharmacological probes (Table 1). Somewhatsurprisingly, manyof these
classes of conotoxins act on pain targets,allowingthe specific dissection of keyion channels and
receptorsunderlyingpain and providingimportant new ligandsfor developing pain therapeutics.
It is estimated that in excess of 50,000 conopeptideshaveevolved for prey immobilisation, with
<0.1% characterised pharmacologically. A surprising number are highly selective for a diverse
rangeof mammalianion channelsand receptorsassociated with pain pathways. In this chapter,we
discuss how differentclasses of venompeptides from marine cone snails(Table 1) can be used to
improveour understandingand treatmentofpain.Conotoxinsactat manyof the ion channelsand
a smallernumberof receptorsfound in pain pathways (Fig. I). Ofparticularinterestareconotoxins
that inhibit pain pathways byblockingcalciumand sodium channels, the nicotinic acetylcholine
receptor,the norepinephrine transporter, the NMDA receptorand the neurotensin receptor.
*Richard J. Lewis-Xenome Ltd, Indooroopilly 4068 and Institute forMolecular Biosciences,

The University of Queensland, 4072, Brisbane, Australia. Email: r.Jewis@imb.uq.edu.au
Conotoxin Venom Peptide Therapeutics 45
Table 1. Amino acid sequence and pharmacology of analgaesic conopeptide classes
Class' Name Sequence" Pharmacology
X M rlA NGV CCGYKLCHO C Norepin ephrine transport inhibitor

a Vcl.1 GCCSDPRCNYDH PEIC* Neuronal nicotinic AChR inhibitor
11 PillA RLCCGFOK SCRSRQCKOHRCC* TTX-sensitive sodi um channel
inhibitor
110 MrVIB ACSKKW EYClVPII GFIYCCPGLICGPFVCV TTX-resistant sodi um channel
inhibitor
OJ MVIIA CKGKGAKCSRLMYDCCTGSCRSGKC* N -typ e calci um channel inhibitor
CVID CKSKGAKCSKLMYDCCSGSCSGTVGC*
Conan - Co nan- GEyyLQyNQyLlR yKSN NMDA antago nist
tokin s tokin-G
Contul- Contul- pESEEGGSNATgKKPYIl L Neurotensin receptor agonist
akins akin-G
' Sequences of representat ive co nopeptides of each class are shown . Conopepti des w ere isolated
from fish hunt ers C. geographus (G) C. magus (M), C. catus (C), C. purpurascens (P), or mollusc
hunters C. marmoreus (Mr) and C. victoriae (Vc).
bB is 6- bromotr yptophan, 0 is trans-4 -hydro xyproli ne, y is y-carboxyglutamic acid, Tg is glycosylat-
ed threo nine and *am idated C-termini. Cysteines invol ved in disulfide bo nds (bolded) that connect
is discrete patterns depending on sequence.
Calcium Channel Inhibitors

It has long been established that Ca 2+ influx into nerve terminals through calcium channels
initiates the release of neurotransmitter which allow signalling to other nerves and muscle. In
recent years, much has been learned about th e nature of these channels wh ich open in response
to cell depolarisation. The se chan nels have been classified into six groups according to the ir elec-
trophysiologi cal and pharmacological properties, termed L-, N -, P-, Q-, T- and R-types. Given
this d iversity, there is considerable opportunity to develop selective inhibitors ofcalcium channels
playing a key role in pain pathways.
tu-Conoroxins from cone snails are unique tools with which to identify and determine th e
physiological role of different neuronal calcium channels. Since N-type calcium channels play a
key role in pa in transmission (see Fig. 1), it is no t surp rising th at to-concroxin s specific for N-type
channels are potent analgesics.t " Extensive stru ct ure-activity relation ship studies have allowed th e
development ofa pharmacophore model for ui-conotoxins' that allows the rational development
of further specific N -type inhibitors, including macrocylic peptides and pepeidomimmetics,"
Sub-nanornolar dose s of ru-conotoxin MVIIA (w-MVIIA) or w-CVID delivered directly to the
spinal cord (intrathecally) produce analgesia for up to 24 hr in rats." w-M VIIA (Prialr, Elan)
recently gained FDA approval for th e treatment ofotherwise unmanageable severe chr onic pain.
However, development w-CVID (AM3 36, AMRAD) has not progressed beyong a Phase IIIla
clinical trial in severe cancer pain sufferers, where is produced clear signs ofefficacy.Unfortunately,
both ui-conotoxins produced unwanted side effects at therapeutic doses. The discovery of new
ui-conoroxins with selectivity profiles that produce fewer side effects may lead to the development
of better analgaesics in th ese classes.
Sodium Channel Inhibitors

Like th e structurally related calcium channels, sodium channels playa key role in controlling
neuronal excitability, but in th is instance they are critical for initiation and transmission ofsignals
along ner ve projections. Based on their suscep tibility to block by the puffer fish toxin tetrodo-
toxin (TTX ), sodi um chan nels can be divided into TTX-sensitive (TTX-S) and TTX-resistant
cold, hot.
acid. mechanical
Figure 1. This figure illustrates targets associated with acute and chronic pain states. "Pain"
signals arising in the periphery (e.g., the skin, a wound, or in bone cancer) travel through
nerves to a key excitatory synapsesin the spinal cord . Depending on the strength of this signal
and other activating or inhibitory infl uences, this signal can enter the brain and be perceived
as pain of varying intensities. The complexity of the different inflammatory and neuropathic
pain types and their associated specific pathways, has frustrated attempts to rationally de-
velop new classes of pain therapeutics. The specificity of conotoxins may circumvent this
problem, especially if they can be discovered or developed to act selectively at specific
targets expressed by nociceptive primary afferent nerves. Attractive targets include TTX-R
channels (Nay1.8), the TTX-S channels (Na,1.7), capsaicin sensitive channels (TRPV1)and the
pH sensitive channels (ASICs), as well as neurokinin, neurotensin and glutamate (NMDA)
receptors and the descending inhibitory pathway associated w ith norepinephrine release and
reuptake (N ET). Bolded targets are inhibited by conotoxins. Several classes of conopeptides
(OJ-, X-, a-conopeptides and the contulakins, see Table 1) have entered clinic development as
novel analgaesic for chronic pain. OJ-MVIiA(Prialt, Elan) is now approved by the FDA to treat
otherwise unmanageable severe chronic pain (figure modified from ref. 1).
(TIX-R) classes. A number of these sodium channel subtypes are implicated in clinicalstates
such as pain (seeFig.1)7 as wellasstrokeand epilepsy. Giventheir criticalrole in the central and
peripheral nervoussystem, it is not surprisingthat conesnailshaveevolved a number of different
ways to target thision channelclass. However, despitesubtypeselective sodiumchannelinhibitors
havingconsiderable therapeuticpotential, little progress hasbeen madetowardsthe development
of sodium channel inhibitors from cone snails. This may changewith the recent discovery that
J.l0-conotoxins MrVIA and MrVIB, selective inhibrors of a keyTTX-R sodium channelin pain
pathways, is analgesic in animalswithout affectingmobility!
Antagonists ofNicotinic Acetylcholine Receptors

The nicotinic acetylcholine receptors(nAChRs) areafamilyof pentamericligandgatedcation
channelsthat playa keysignalling roleat synapses and neuromuscular junctions.Thea-conotoxins
are a largeand growingclass of smallpeptidesfrom cone snailsthat competitively inhibit specific
subtypesof the nAChR.9 Muscle selective a-conotoxins (e.g., GI, seeTable 1) may representan
alternativeto the use of smallmoleculecurare-mimeticmuscle relaxants, which are used during
surgerybut haveslower than ideal recovery periods.Interestingly, the novel n-conoroxin, Vel.I ,
has been identified as havingpotential analgesic properties following peripheral administration
to rats.'?This result contrasts the analgaesic effects usually attributed to agonistsof the nAChR
Conotoxin Venom Peptid« Therapeutics 47
actingcentrallyand appearsto be mediated bya specific subtypeof the receptorwith a previously

unrecognisedrole in pain. Unfortunatley, Metabolichas discontinued clinicaltrialsofVcl.1 due
to an anticipated lackof efficacy in humans (affinityfor the human form of the receptoris report-
edly much lowerthan for the rat form).
Norepinephrine Transporter Inhibitors

Thenorepinephrinetransporterplays a keyrolein reducinglevels of neuronally released norepi-
nephrine (also known asnoradrenaline).Thetricyclic anti-depressants inhibit NET and appearto
produceanalgaesia byenhancingthe descending inhibitory pathwaycontrolledbynorepinephrine
release. Unfortunately, this class ofdrugsalsohaveanti-depressant effects and significant off-target
pharmacologies that limit there usefulness in the treatment of pain. X-Conopeptides firstisolated
from Conus marmoreus are highlyspecific, noncompetitiveinhibitors of norepinephrine uptake
through the NET!! that producepotent analgaesia in rats.A syntheticvariantof the X-conotoxins
(Xen2174)iscurrentlybeingdeveloped asa novelanalgesic byXenomeLtd,12 Xen2174 is currently
being evaluated intrathecally in a Phase VIla safety trial in cancer patients suffering otherwise
unmanageable pain. Initial resultsarepromisingboth in termsof safetyand efficacy. Interestingly.
the binding site for X-conopeptides on the NET partially overlaps the tricyclic anti-depressant
binding site but not the NE binding site,providingcluesto the developmentof noncompetitive
smallmoleculeinhibitors."
NMDA Receptor Antagonists

Conantokins are specific inhibitors of the N-methyl-n-aspartate (NMDA) receptor. These
peptides are helicalin structure and competitively inhibit glutamate activationof this receptor.
especially at NR2B subtype.Malmberger al14 showedthat intrathecalconantokinG or T alsohave
analgesic activityin pain modelsof tissuedamage, nerveinjuryand inflammationin miceat doses
that were-20-fold lowerthan thoseaffecting motor function.Thus,subtypespecific inhibitors of
the NMDA receptor alsohavetherapeutic potential in the managementof pain.
Neurotensin Receptor Agonists

Cone snailsproduce a glycosylated neurotensin analoguenamed contulakin-G" that is a po-
tent analgesic in a wide rangeof animalmodelsof pain." Interestingly, contulakin-G is 100-fold
less potent that neurotensin for NTRI. but -IOO-foidmore potent as an analgesic. Basedon its
potency and wide therapeutic window, contulakin-G (CGX-1160) went into earlystageclinical
development by Cognetix Inc. for the treatment of pain. Development of the conantokins and
contulakinsis now on hold with the demiseofCognetix.
Acknowledgements
Aspectsof thisworkweresupportedbyNHMRC Programand Projectgrants.ARC Discovery
grants and Auslndustry.
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with neuropathic pain. Pain 2005; 118:112-124.
13. Paczkowski FA, Sharpe lA, Dutertre S et al. X-Conopeptide and tricyclic anti-depressant interactions at
the norepinephrine transporter define a new transporter model. J Bioi Chern 2007; 282:17837-17844.
14. Malmberg AB, Gilbert H, McCabe RT et al. Powerful antinociceptive effects of the cone snail venom-de-
rived subtype-selective NMDA receptor antagonists conantokins G and T. Pain 2003; 101:109-116.
15. Craig AG, Norberg T, Griffin D er al. Contulakin-G, an O -glycosylated invertebrate neurotensin, J Bioi
Chern 1999; 274:13752-9.
16. Allen]W. Hofer K. McCumber D et al. An assessment of the antinociceptivc efficacyof intrathecal and
epidural contulakin-G in rats and dogs. Anesth Analg 2007; 104:1505-13 .
CHAPTER 6
Shark Novel Antigen Receptors-

The Next Generation ofBiologic
Therapeutics?
Caroline Barelle, Davinder s. Gill and Keith Charlton"
Abstract
ver recent decades we havewitnessed a revolution in health care as new classesoftherapeu-
O tics based on natural biological molecules have become availableto medical practitioners.
These promised to target some ofthe most serious conditions that had previously evaded
traditional small molecule drugs , such as cancers and to alleviate many ofthe concerns ofpatients
and doctors alike regarding adverse side effects and impaired quality oflife that are often associ-
ated with chemo-therapeutics. Many early ' biologics' were based on antibodies, Nature's answer
to invading pathogens and malignancies, derived from rodents and in many ways failed to live up
to expectations. Most of these issues were subsequently negated by technological advances that
saw the introduction ofhuman or 'humanized' antibodies and have resulted in a number ofcom-
rnercial 'block-busters. Today, most ofthe large pharmaceutical companies have product pipeline s
that include an increasing proportion ofbiologic as opposed to small molecule compounds. The
limitations of antibodies or other large protein drugs are now being realized however and ever
more inventive solutions are being sought to develop equally efficaciousbut smaller, more soluble.
more stable and less costly alternatives to broaden the range of drug-able targets and therapeutic
options. The aim of th is chapter is to introduce the reader to one such novel approach that seeks
to exploit a unique antibody-like protein evolved by ancestral sharks over 450 M years ago and
that may lead to a host ofnew therapeutic opportunities and help us to tackle some ofthe pressing
clinical demands ofthe 21st century.
Introduction
The world of pharmaceutical development and the treatment of disease were revolut ion-
ized in 1982 when the first recombinant protein drug, insulin, was approved for human use.
This event was the dire ct result of previous momentous achievements, notably in 1972 when
Paul Berg at Stanford produced the first recombinant DNA (rDNA) and the following year
when Herbert Boyer first transformed E. coli bacteria with a rDNA plasmid that included a
gene encoding a human protein. Boyer went on to establish Genentech, the world's largest
Biotechnology Company. The reason that this was possible at all is that the same genetic code
is shared by all living organisms, reflecting their common ancestry. Initially clinical develop -
ments con centrated on generating recombinant versions of human proteins for the treatment
ofdeficiencies , such as Factors VII, VIII and IX for blood clotting disorders and human growth
hormone (hGH). Other popular products were cytokines, ego interleukins, interferons and
*Corresponding Author: Keith Charlton-Wyeth Research, Cornhill Road, Foresterhill,
Aberdeen AB25 2ZS, Scotland, UK. Email: charltkeswyeth.corn
PharmaceuticalBiotechnology, edited by Carlos A. Guzman and Giora Z. Feuerstein .
©2009 Landes Bioscience and Springer Science-Business Media .
tumor necrosis factor-a. In the latter cases, it was fortuitous that these proteins exhibited the
desired therapeutic effectsdespite being produced in bacterial expressionsystems that do not
allow for the inclusion ofposttranslational modifications,specifically glycosylation. A notable
exception to this waserythropoietin (EPO), which initiallyhad poor in vivoefficacy due mainly
to its pharmacokinetic profilewhen not glycosylated. Thisproblem wasovercomesubsequently
by production ofEPO in mammalian cell culture.
It was perhaps the emergence of antibodies as therapeutic reagentsthat have reallyhad the
most profound and excitingeffecton the developmentof new treatments for diseases that had,
until recently, evadedthe best effortsof the medicalcommunity.
The Rise and Rise ofAntibodies

The history oftherapeuticantibodiesdatesbackmore than a century,when Emilvon Behring
first demonstrated that anti-seracould be used to treat conditions from snakebites to diphtheria
and tetanus. Thesetreatmentswerelimited howeverto acuteindicationsand it wasn'tuntil nearly
100 yearslater when Cesar Milstein's group generatedthe first monoclonalantibody producing
cellline (Hybridoma) that antibody therapiesbecamea realpossibility.
Despite the ability now to generate antibodies of known and predetermined specificity in
unlimited quantities from these immortal celllines,other limitations quicklybecameapparent.
The principal problemslaywith the murine origin of these antibodiesand indeed werethe same
as those encountered a century before. The immune systems of patients recognized these early
antibody therapeutics as being 'foreign' proteins and responded accordingly by generatinghu-
man anti-mouseantibodies,the so-calledHAMA response. This restrictedapplications to acute
indications where a single-dose could be administered. It required the development of several
new technologiesto gradually overcome theselimitations.Without goinginto details,thesetech-
nologiesprovided the means to isolatethe antibody-encodinggenes, to manipulateand clone the
DNA and to gain a greaterunderstandingof structure-functionrelationships and which elements
of the protein sequences define its species origin. The immunogenicityof antibody therapeutics
wasfirst tackledbycloningthe variable domain genes from desirable rodent antibodies(that part
of the antibody that defines its antigen-bindingcharacteristics) onto the constant region genes
(definingsecondaryeffectorfunctions) of human origin,resultingin chimericantibodies. Further
refinementwas introduced by engineeringthe variabledomains, for example cloning the actual
antigen-bindingloops, or complimenrariry determining regions (CD Rs) onto a human V-region
scaffold that is structurally similar, a processknown as CDR-grafiing. The success of these ap-
proaches is illustrated by the fact that the majorityof antibodiesapprovedfor therapy currently
on the market are of rodent origin.
Whilst additional techniqueshavebeen developed sinceto further humanize,or de-immunize
proteins, the drive to generatemore precisely human antibodiesfrom the outset progressed sig-
nificantlyin the 1980sand 1990s with the advent of two remarkable advances. The first wasthe
ability to clone entire repertoiresof human antibody genesdirectlyfrom volunteer donors into
phage displaysysrems.P This allowedverylargepools (up to 1 x lOll) of antigen binding sitesto
be reproduced on the surface of bacteriophage, with one antigen specificity per phage particle,
which could then be selectedin vitro againstany antigen of choice. The secondwas to introduce
sectionsofhuman antibodygenelociinto micein which the correspondinglocihad beendeleted.
This resulted in transgenicmousestrainsthat, upon immunization, would respondby generating
human antibodies in vivo.' A fundamentallyimportant aspect of both technologies is that they
enablescientists to generatehumanantibodiesagainsthuman antigens.Again,it istestament to the
power of these technologies that the overwhelming majorityof antibodiescurrentlyundergoing
clinicaltrials (-400) are derivedusingone or other of these approaches.
Antibodies havenow emergedasthe majorclass of biologictherapeutics. An intrinsically high
levelof specificity for cognatetarget antigen and amenabilityto in vitro molecularengineering
make them extremely attractivecandidatesfor clinicaldevelopment. Moreoverthey are nature's
answeramongst the jawedvertebratesto combat infectiousagentsand malignantcells. However,
Shark Novel Antigen Receptors-The Next Generation ofBiologic Therapeutics? 51
there are a number ofapplications for which the whole antibody was not generally held to be the
ideal molecule as they have a number oflimitations. IgG antibodies are the most common serum
form and the one favored for almost all therapeutic development programs. They are multi-domain,
four chain molecules of -1 SO kDa with two identical antigen binding sites, each formed by the
association ofone heavy chain with one light chain. Their size and to a lesserextent their Y-shaped
configuration restricts their mobility and targeting to more accessibleextra-cellular and cell-surface
antigens . This limitation is exemplified by the number of approved antibody drugs and those
currently under investigation that target the same narrow range of antigens and clinical indica-
tions. The ability to access more cryptic , intra-cellular or recessed epitopes currently intractable
to conventional antibodies, will undoubtedly afford the opportunity to exploit a new range of
clinically relevant targets.
Accordingly, there is a continual drive within the immuno-therapeutic and diagnostic com-
munities to develop smaller, more stable, highly soluble, high affinity (nM) binding domains.
To this end an extensive array of recombinant antibody formulations have been developed,
reducing antibodies to minimal binding domains, modulating effector functions, increasing
valency and avidity and conjugating to other protein domains to improve pharmacokinetic
or in vivo efficacy.An ideal biologic therapeutic will possess all of these attributes. In order to
generate promising candidate products, it is also necessary to couple ease of manufacturability
i.e., high functional expression yields, low aggregation and consistently homogenous protein
production with freedom to operate in a complex intellectual property landscape. One very
exciting discovery to have emerged recently and that could have a significant influence on
future therapeutic generation is the discover y of a new antigen binding molecule from sharks,
the IgNAR. This chapter aims to introduce the reader to this remarkable protein, to describe
some of its unique characteristics and to discuss how these might be exploited to lead us into
the next generation of biologic therapeutics.
What Are IgNARs?

The immunoglobulin novel (or new) antigen receptor (IgNAR) was first identified in the
lab of Martin Flajnik and coworkers in the early nineties. The somewhat serendipitous route of
discovery originated from the work ofAndrew Greenberg who was studying nurse shark immu-
noglobulins. He observed cross-reactivity of a nurse shark anti-IgM polyclonal antibody with an
unknown band on a Western blot with mobility slightly greater than that ofmonomeric IgM and
which was subsequently shown to be a novel single domain 'heavy chain' previously unknown to
sclence.v' Serum IgNAR exists as a hornodimer devoid oflight chains, with independent variable
domains that exhibit structural flexibiliry." The levelsof circulating nurse shark IgNAR have been
measured at approximately 0.1 -1.0 mg/ml which is 20-40 x less than IgM.7•R
Nurse sharks are member s of the order Orectolobiforme. Collectively there are eight order s
based on gross anatomical and molecular criteria." Diverging from a common ancestor approxi-
mately 450 million years ago, they are the oldest vertebrate taxon to have all the components of
an adaptive immune system, namely major histocompatibility complex (MHC), T-cell receptors
(TCR), immunoglobulins (Ig), RAG recombinase activity,somatic hypermutation and specialized
primary and secondary lymphoid tissue.10 They belong to the classChondrichthyes or Cartilaginous
fish which can be divided into two subclasses:the elasmobranchs (sharks, rays and skates) and the
holocephalans (chimaeras and ratfish). IgNAR has also been identified in other members of the
Orectolobiformes, including the spotted wobbegong (Orfctolobus maculatus) and bamboo sharks
(see ref. 11,S. Nuttall personal communication) aswell as two speciesofdogfish, the smooth hound
(Mustflus canis) and spiny or spurdog (Squalusacanthias) (Fig. 1) from the orders Carcharhiniforme
and Squaliforme respectively.1 2.1 3IgNAR sequences from the horn shark-a member of the order
Heterodonris, have also been deposited in sequence databases. As spiny dogfish and nurse shark
are separated phylogenetically by approximately 200 million years, it is tempting to surmise that
IgNAR evolved prior to this split and may be ubiquitous amongst elasmobranchs.
Figure 1. The spiny dogfish Squalus acanthias, one of the smaller and easier to keep shark
species currently being evaluated as a source of novel IgNAR-based immuno-therapeutics.
The first IgNAR cD NA clones to be sequenced were isolated from a nurse shark spleen library
and were shown to consist of a variable domain and five constant domains consistent with im-
munoglobulin superfamily V and CI-SET domains.t" As with classical B-cell antigen receptors,
IgNAR exists as both membrane-bound and soluble forms . In higher vertebrates the former ac-
tivates lymphocytes while the latter serves to bind serum antigen and provide secondary effector
functions. There are two distinct splice-variants ofthe membrane-bound form , with three and five
domains respectively. The soluble form also comprises fivedomains." Differential amplification of
these has shown that the secretory form exhibits a much greater degree ofmutation, indicative of
antigen-driven selection. IS What, if any,effector function is provided by the secretory form has yet
to be determined. The sequence ofIgNARshows greater homology with IgLk and TCR-V chains
than IgH and shows very low levels of sequence homology to human VH.4.s From a structural
perspective it falls somewhere between cell adhesion molecules, antibodies and T-cell receptors,
perhaps providing some insight into its evolutionary origins . It is conceivable therefore that a recep-
tor which evolved independently ofantibodies was subsequently adopted by the immune system."
The most profound differencesbetween IgNAR and conventional antibodies lie at the hydrophobic
interface between VH and VL domains. IgNAR lacks many of these residues and replaces others
with hydrophilic ones resulting in a greatly truncated CDRl region and high solubility in the
absence of light chains . The lack of CDRl makes IgNAR-V the smallest independent antigen
binding domain in the animal kingdom with a molecular mass of approximately 12 illa.
To date, there are three defined types ofIgNAR known as 1, II and III (Fig. 2). These have
been categorized based on the position ofnoncanonical cysteine residues which are under strong
selective pressure and are therefore rarely replaced .6.17.18
All three types have the classical Ig canonical cysteines at positions 35 and 107 that stabilize
the standard immunoglobulin fold, together with an invariant tryptophan at position 36. There
is no defined CDR2 as such, but regions of sequence variation that compare more closely to
TCR HV2 and HV4 have been defined in framework 2 and 3 respectively," Type I has germline
encoded cysteine residues in framework 2 and framework 4 and an even number of additional
cysteines within CDR3. Crystal structure studies ofa Type I IgNAR isolated against and in com-
plex with lysozyme" enabled the contribution of these cysteine residues to be determined. Both
the framework 2 and 4 cysteines form disulphide bridges with those in CDR3 forming a tightly
Shark Nove/Antigen Receptors-The Next Generation ofBi%gic Therapeutics! 53
Type I
Type II
Type III
Figure 2. Structure of rearranged IgNAR genes showing positions of canonical (0) and non-
canonical (e) cysteine residues, disulphide bonds (connecting lines), conserved tryptophan
(W) and hyper-variable (CDR/HV ) regions.
packed structure within which the CDR3100p is held tightly down towards the HV2 region."
Interestingly, to date Type I IgNARs have only been identified in nurse sharks-all other elasmo -
branchs, including members of the same order have only Type II or variations of this type.
Type II IgNAR are defined as having a cysteine residue in CDRI and CDR3 which form
intra-molecular disulphide bonds that hold these two regions in close proximiry.P:" resulting in
a protruding CDR3 (Fig. 3) that is conducive to binding pockets or grooves. Type I sequences
typically have longer CDR3s than Type II with an average of21 and 15 residue s respectively. This is
believed to be due to a strong selective pre ssure for two or more cysteine residues in Type I CDR3
to associate with their framework 2 and 4 counterparts." Studies into the accumulation ofsomatic
mutations show that there are a greater number of mutations in CDRI of Type II than Type I,
whereas HV2 regions ofType I show greater sequence variation than Type II. This evidence cor -
relate s well with the determined positioning of these regions within the antigen binding sites." A
third IgNAR type known as Type III has been identified in neonates. Thi s member ofthe IgNAR
family lacks diversity within CDR3 due to the germline fusion ofthe D 1 and D2 regions (which
form CDR3) with the V-gene . Almost all known clones have a CDR3length of 15 residues with
Figure 3. Backbone structures of Type I and Type II anti-lysozyme IgNAR compared w ith a
human VH domain .
little or no sequence diversity. Tissue expression patterns show an initial high level in the spleens
of newborn nurse sharks that then declines after 2-4 months, leaving the epigonal organ as the
primary tissue ofexpression throughout adulthood." It is hypothesized that this type ofIgNAR
may have evolved to protect young pups from early stage exposure to a common pathogen, or to
be involved in regulating immune system development.
How Diverse Are IgNARs?

Sharks have three heavy (IgNAR, IgM and IgW) and four light chain (I/NSS, II/NS4, III/NS3
and a) isotypes identified to date .8,21 All shark antibody genes are arranged in a "cluster" format, in
contrast to the "translocon" organization exhibited in mammalian systems" (Fig. 4).
Each cluster comprises one V segment, one or more D segments, one] segment (that can be
partially or fully germ-line joined) and a single set ofC segments which are rearranged exclusively
within individual clusters by the activity of RAG recombinase.P'f" Key to the heterogeneity of
the shark naive repertoire is the junctional diversity achieved through N-additions, trimming and
D-region re-arrangement (all three reading frames and possible D-region inversion). As in higher
vertebrates, terminal deoxyribonucleotide transferase (TdT) is the enzyme responsible for the
nucleotide addition 5' to the D-regions and is highly homologous to that in mammals.'? Recent
work by Malecek and coworkers studying the IgM loci in nurse shark has shown that allelic exclu-
sion does occur in B-Iymphocytes but probably not via chromatin condensatjon, the mechanism
adopted in translocon configurations. ~I Currently, there is no evidence to show that recombination
events occur between clusters and it is commonly believed that isotype switching does not occur
in cartilaginous fish. More probably, isotype is fixed at an early stage ofontogeny.
As IgNARs are devoid of light chain, they lack the combinatorial diversity of convention
VH-VL antibodies. This deficiency is compensated for by an increased number of recombina-
tion events through the presence of additional D -regions and the introduction of extensive
junctional diversity (Fig. 4). IgNAR conforms to the cluster arrangement of V and C domain
gene organization and in nurse shark, the gene family is composed offour loci.' Diversity ofthe
naive IgNAR repertoire is achieved by four recombination events (between the V region, three D
regions and] region) as well as extensive Nand P nucleotide addition which results in consider-
able CDR3 heterogeneity. Somatic hypermuration, although not responsible for generating the
naive repertoire, is evident in the secretory form ofIgNAR. High levels ofmutation that share
Translocon
H
Higher [
vertebrates
Cluster
Ig MIW H
Cartilag enous IgNA R H

fish ~=~~~::':"'--==:"'-~~~~~~====...J
Figure 4. Translocon and cluster arrangements of immunoglobulin genes in higher vertebrates

and cartilaginous fish respectively. Adapted from: Flajnik ME Nature Rev Immunol 2002 ;
2:688-698;23 with permission from Nature Publishing Group.
Shark NovelAntigen Receptors-The Next Generation ofBiologic Therapeutics? 55
similar mutational patterns and serine codon hotspots (AGC /T) as those seen in mammalian
systems have been identified in addition to strand bias and a bias in favor of transitions over
transversions. Unusually, base changes occur in tandem (doublets and triplets), particularly in
"hotspots" and palindromic repeats."
What Is the Function ofIgNAR?

Higher vertebrates possess an adaptive immune system (AIS) that is mediated primarily by
Band T-Iymphocytes. In contrast to innate immunity (the first line of defense against invading
foreign pathogens) which is based upon restricted germline encoded receptors, the receptors of
the adaptive immune system are generated by recombinatorial processes. The levels ofhypermuta-
tion seen in IgNAR are indicative of a secondary immune response . Tailoring a binding domain
through mutation accumulation to increase affinity for target antigen is a classic hallmark of an
adaptive immune response. The mechanism by which V(D)J immunoglobulln gene segments are
rearranged is such that the repertoire of receptors has sufficient diversity to potentially recognize
any pathogen or toxin. Importantly, adaptive immunity also results in memory. Secondary exposure
to a pathogen previously encountered results in a much more rapid and aggressive response.
It has been known since the mid-sixties that elasmobranchs are able to respond in an anti-
gen-specific manner when challenged.? During this period, more detailed immunization studies
were carried out in smooth dogfish (Mustelus canis), spotted dogfish (Scyliorhimus canicula) lemon
shark (Negaprion breoirostrisi and nurse sharks (Ginglymostoma cirratum) characterizing the puri-
fied serum fractions responsible for this response and identifying them as 19S and 7S IgMp3-37
Both forms of IgM were present at similar high levels, representing approximately 50% of total
serum protein. Upon antigen challenge, there was a temporal response ofIgM subtypes with some
studies reporting an initial increa se in 19S followed by an increase in 7S and another suggesting
a secondary enrichment of 19S.38,39
Evidence that IgNAR is part of the adaptive immune response in sharks was demonstrated
conclusively in the lab of Martin Flajnik by following the antigen specific IgNAR fraction in
the sera of immunized animals." Antigen-specific IgNAR and monomeric IgM showed similar
expression profiles , plateauing at approximately 4-5 months in response to multiple boosts ofhen
egg lysozyme (HEL). Increased levels of antigen-specific IgNAR were preceded by an increase
in antigen-specific monomeric IgM. Interestingly, sharks are the only known vertebrates to have
both pentameric and monomeric form s ofIgM. The inter-relationship of the two forms remains
to be fully understood, however early studies suggest that the two forms are fully independent and
not merely splice variants ofthe same anribody.":" The current model, though not yet proven, is
that the B-cells commit at an early stage to express a specific heavy chain isotype. Class switching,
which is observed in higher vertebrates, is not seen in sharks . This is thought to be due to the ar-
rangement ofheavy chain genes which is not conducive to recombination between clusters. Based
on the immunization studies carried out to date it is believed that the first line ofimmunological
defense in elasmobranchs is low affinity pentameric IgM-with ten binding sites, this large protein
benefits from increased avidity enabling it to bind strongly to antigens despite imprecise specificity
and poor affinity. There follows a delay before the detection in sera of both monomeric IgM and
IgNAR. By ceasing immunization ofthe animals for an extended period until titers had dropped
significantly, Flajnik's group effectively demonstrated that re-immunization with the same antigen
elicited a much mor e rapid response , indicating that sharks generate anrigenic-rnemory.'"
In higher vertebrates, B-cell maturation takes place in the primary or generative lymphoid
organs e.g., bone marrow. Lacking such tissue and indeed a true lymphatic system, elasmobranchs
have developed two specialized tissues where lymphopoiesis takes place : the epigonal and Leydig
organs. All sharks po ssess on e or both of these primary lymphoid organs, which exhibit similar
cellular composition containing large numbers of developing granulocytes, blast cells, plasma
cells and lymphocytes. As its name implies the epigonal is attached to the gonads, whereas the
Leydig is a mass oflymphomyloid tissue situated in the sub-mucosa of the oesophagus.v" Both
organs exhib it expression of RAG recombinases, TdTs and B-cell specific transcription factors
confirming their function as primary lymphoid tissues.27•44 In adult sharks, many secretory B-cells
can be located in the epigonal organ suggesting a role as a reservoir for activated antibody secret-
ing Bvcells, similar to the role ofbone marrow in mammals."
Spleen and thymus have been identified as the secondary lymphoid organs in sharks. The
spleen, which is believed to be the site ofantigen driven B-cell activation , changes histologically
as sharks mature . Studies in nurse shark pups show the presence ofIgM positive but no IgNAR
positive cells-it takes approximately five months before the latter are detectable." The spleen is
highly vascularized and compartmentalized into red and white pulp zones, although "classical"
germinal centers where B-cell activation and antigen-specific affinity maturation occurs in higher
vertebrates are not seen. However, the fact that IgNAR does undergo somatic hypermutation and
there is white zone compartmentalization of B-cells, T-like cells and dendritic cells, is strongly
indicative ofthe spleen being the site ofB-cell activation."
Developing IgNARs as Therapeutics

So far, this chapter has discussed the what, the how and the why ofIgNARs in its natural bio -
logical context. We now need to explore how all ofthese physical and biochemical attributes can
be exploited in an applied fashion to generate efficaciousimmunotherapeutics.
Intrinsic Therapeutic Attributes ofIgNARs

In the absence of interventive in vitro manipulation, IgNARs V-regions naturally exhibit
many of the desirable properties ofbiologic therapeutics. In its natural context IgNAR is found
in the serum of sharks, which contains 350 mM urea (a protein denaturant) as a means of
osmo-regulation. It may be the need to retain functionality in this harsh environment that
contributes to the extraordinary stability exhibited by IgNAR . In addition to tolerating high
concentrations of denaturants, IgNAR have remarkable folding properties. When exposed to
extremes of temperature, they 'melt' as expected . However, on return to physiological condi-
tions they refold correctly, restoring full functional binding to most of the protein.12.46.47 They
also exhibit high solubility presumably due to their hydrophilic surface and so produce high
functional expression yields in bacterial culture. Whilst interesting in themselves, these features
are insufficient to have significant implicat ions on future therapeutic development. There are two
additional key characteristics that may confer such a contribution. Firstly, as mentioned earlier,
IgNAR are the smallest known naturally occurring antigen binding domain at 12 kDa (Fig. 5).
This lends them both to greater mobility and the opportunity to target epitopes otherwise ac-
cessible only to small molecules.
(e) (d)
38 12 kf).-
Figure 5. Molecular models illustrating the structures and relative sizes of examples of the
many antigen-binding formulations currently being developed for therapeut ic applications;
(a) whole IgG antibody, (b) scFv-Fcfusion (SMlp®), (c) bi-specific trivalent camelid nanobody
and (d) shark IgNAR single domain.
SharkNovelAntigenReceptors-The Next Generation ofBiologic Therapeutics? 57
It is the opinion ofthe authors however, that the unusual topographyofthe IgNAR binding
site, particularlyasa resultof the lackof a true CDR2 and the lengthybut conformationally con-
strained CDR3 (Fig. 3), are paramount to its potential. Theseenable IgNARs to be targeted to
recessed epitopes that conventionalantibodiescannot reach, evenwhen that part of the antigen
is sufficiently exposed. Evidence for this is borne out by the nature of epitopes recognized and
targeted by IgNARs,e.g., the active sites of enzymesY·46.48-50
Isolation ofAntigen-Specific Clones

IgNARbindingdomainsareamenable to phagedisplay technologywhichhas beenfundamental
in revolutionizing recombinantantibodyengineeringand hasdriventhe developmentof antibod-
ies and other novel protein scaffolds as therapeutic agents. Phage displaycouplesphenotype to
genotypeand facilitates the isolationof antigen-specific binding domainsand enables subsequent
molecularengineeringto achieve greateraffinity, solubilityand stability. A detailed description
of this technologyand its contribution to drug developmentisbeyond the scopeof this chapter,
howeverthere are numerous excellent reviews coveringthe topic.2.51.52The first antigen-specific
IgNAR that had undergone natural in vivoselectionwasisolatedfrom a nurseshark immunized
with hen egg lysozyme." Bycomparing the sequences of HEL specific clones isolated from an
immunized phage library, the degree of somatic hypermutation and key residue changes that
had occurred during the in vivo maturation processto increase affinitywere determined.53 The
relationships between progressive mutation accumulationand increased antigen-antibodyinter-
actions were identified by comparingthe crystalstructures of an in vivo matured HEL specific
binding domain and its putativegermlineancestorin complexwith antigen.54 Thisstudy isolated
both Type I and Type II IgNAR binding domains with affinities of approximately 20 nM and 1
nM respectively. The isolationof antigen-specific IgNAR binding domainsfrom a phage display
library was published by Nuttall et al." This group built a semi-synthetic IgNAR library using
natural frameworks isolated from wobbegongsharks. As the diversityof the naive repertoire is
basedpredominantly on the sequencewithin CDR3, this group applied random mutagenesis of
this regionand succeededin selecting recombinant IgNARspecific for the Gingipain K protease
from Porphyromonas gingivalis.11 The study was extended by increasingthe library size via the
introduction of naturally occurring IgNAR CDR3 's from a naiveshark repertoire and interest-
ingly resulted in the isolationof two proteins not derivedfrom the synthetic CDR3library but
from natural proteins." A group in the USA were the first to isolate IgNAR binding domains
from spinydogfish" usingboth naiveand semi-synthetic libraries. Theyisolatedbinders to SEB,
cholera toxin and ricin that had similaraffinities (10-300 nM) to those isolatedby Nuttal et al
from similar wobbegong libraries. This works demonstrates that high affinity antigen-specific
IgNARcan be successfully generatedfrom both syntheticand immunizedphagedisplay libraries
derivedfrom a varietyof shark species.
In Vitro Maturation
During the antibody drug developmentprocess, isolatinga leadbinder isjust the beginningof
a lengthyprocess. In the majority, if not all cases, there is a need to modify aspectsof the protein.
This might involve increasingaffinity, fine-tuning specificity, increasingfunctional expression
yield/solubility, resolving potential immunogenicityissues or modulatingpharmacokinetics. The
precisealterations will varydependingon the inherent characteristics of the leadprotein and the
application for which it is intended. An interesting exampleis targeting of solid tumors, where
experience hasshownthat improvements in bindingaffinitydo not necessarily equateto improved
efficacy. A predominant reasonfor this in manycases isthat antibodieswith veryhigh affinities are
likely to localize on the tumor surface and not achieve the desiredpenetrarion.P-" In this instance,
a more moderate affinity, combined with reduced molecularweight and associated increases in
mobility/diffusion might contribute favorably to efficacy. Indeed it has been demonstrated that a
loweraffinitydiabody (two linked singlebindingdomains)exhibited a greatercoverage of tumor
targetsthan the ten fold higher affinitysingledomain."
58 PharmaceuticalBiottchnology
Engineeringisolatedclonesto increase affinityand specificity had been achievedby random

in vitro mamratlon/" CDR randomized mutagenesis47•50 and by targeted residuechanges based
on structural alterationsto the CDR3loop to increase flexibility and antigen cleftbinding.59The
latter examplebroadenedthe specificity to encapsulate a greaterbreadth ofspecies cross-reactivity.
Asthe binding domainin questionwasisolatedagainsta malarialtarget,it washoped that broader
specificity would provide an advantage with respect to treating the disease in a varietyof states
of progression. The ability to readilyconfer such characteristics to new drugs in development is
essential in order to assess efficacy in industrystandard (though far fromideal)nonhuman animal
models.
Formatting
Leadantibodiesareusually isolatedinitiallyassingle-chain fragments and typically do not have
the pharmacokineticsuitablefor therapeutic administration. In addition, there is the complica-
tion of effectorfunctions which mayor maynot be desirable, dependingon the target and mode
of action required. For imagingpurposes there is no requirement for effectorfunction, indeed
this would be undesirable. Further more,such reagentsshould ideallybe of minimalsize, both to
maximize tissuepenetrationand to minimizeserumhalf-life.Antibodiesdeveloped for therapeutic
purposesoften require a much longer half-life to increaseefficacy and reducethe need for repeat
dosing. Where effectorfunction isdesirable, it istypicallyachievedbyengineeringthe bindingsite
into an IgG-I format.Forcertainapplications an IgG-4formatispreferred,providingan extended
half-lifebut not recruiting complementactivation. Alternativetechnologies to increase half-life
ofsingledomains include conjugation to polyethylene glycol, so-calledPEGylation,60 by target-
ing human serum albumin (HSA) in a bi-specific format61.62or byengineeringsingle-domain-Fe
fusion proteins. For manyindications,the bindingofan antibody isnot sufficient to generatethe
desiredcellkilling. In suchcases, it isoftenpreferredto 'arm' the antibodywith a toxin,enzymeor
pro-drug. Thereis little doubt that antigen binding singledomains afforda number ofpotential
opportunities that are not found with conventional antibodies or antibody fragments. Chief
amongst these is the ability to designand build multi-valentconstructs e.g., diabodies, triabod-
iesetc. Not only can such molecules increaseapparent affinitydue to avidityeffects, but can also
be multi-specific enablingsimultaneous binding to more than one target. Such approaches can
be used to modulate pharmacokinetics, increase tissue specificity or to deliver payloads. Our
current knowledgeon the structure and function ofthe variousdomainsof antibodies,together
with protein engineeringadvances mean that such modificationsare readilyachieved. There is
currently little published researchdescribingthe formatting ofIgNAR, one exceptionbeing the
dimerization to createbivalentIgNARs.63
One veryimportant facetof any biologic therapeuticthat has been mentioned brieflyalready
is that of host immune responses. It is well known and accepted that whole antibodies from
nonhuman species are likelyto elicit a degreeof anti-protein response in some if not allpatients.
There are now a number of wellestablishedtechnologies that havebeen developed to overcome
this problem, particularlywith respectto antibodiesof rodent origin.The past two decadeshave
seen a drive to isolate antibodies from sources that are as human as possible from the outset.
There is howevergrowingacceptance that evena 'fullyhuman' antibody can been seen asforeign
by a proportion of the patient population. In these cases it is the variable domains that form the
binding site that provideimmunogenicepiropes, rather than the constant domainswhich are far
more immunogenicwhenderivedfrom nonhuman origins. This has someveryimportant implica-
tions for the bio-pharmaindustry. On one hand, it isnow consideredan essential part ofthe drug
development processthat any biologicbe analyzed for indications of potentially immunogenic
epitopes and that appropriate measures are taken to engineer these in such a wayas to alleviate
the problem without adversely affectingdrug function. On the other hand, it alsomeansthat the
perceiveddisadvantage of starting with a nonhuman protein is perhaps not such a handicap as
wasonce thought.
SharkNovelAntigenReceptors-TheNext Generation ofBiologic Therapeutics? 59
Conclusion
Does shark IgNAR representa suitableplatform to produce the next generationof therapeu-
tic biologic molecules? We have witnesseda whole new sector of the pharmaceutical industry
emergearound antibody therapeuticsover recent decades. There wasinitially some disappoint-
ment that early antibodies derivedfrom rodents did not, on the whole, deliveron the possibly
over-ambitious expectationsof many commentators that "magic bullets"would lead to a major
break-throughin the treatmentof manydiseases, not leastcancer. Sincethosedaysthe industryhas
matured considerably and our expectations are perhapsmore measured, though in manyways no
less ambitious. The principaldriversbehind the recent resurgence must surelybe advances in the
generationof 'fullyhuman' antibodiesthrough phagedisplayand transgenicmiceand the ability
to de-immunizeboth human and nonhuman proteins. Thesetechnologies havehad a profound
effecton the therapeuticantibody landscape- ten years ago only two monoclonal antibodydrugs
were FDA approved. Todaythat number is 21, including 8 'blockbusters', with several hundred
at variousstages of clinicaland preclinicaldevelopment.Todaybiologics representan increasing
proportion of pharma companies'pipelines, a trend that is also expected to continue. The time
when antibody based therapeuticswereconsideredthe answerto our all of our problemsis long
overhowever-"onesizefitsall"simply doesn'twork.Greaterunderstandingof humanbiologyand
in particular the biologyof disease processes has highlighted the manydeficiencies of antibodies.
We are now entering a new era,wheredesignerbiologics willplayan increasingrole.
Single domain proteins are ideallysuited to these new approaches in many respects. The sim-
plicity of a receptor that does not require the correct association and alignment of two separate
domainsto generatetarget recognition,e.g.,Vh-Vl, has obviousadvantages in relationto produc-
tion of functional material.Thisisevenmore sowhen consideringmulti-specific molecules where
mis-pairingofbinding domain partners resultsin lossoftarget recognition.The ability to target
multipleepitopesor antigens withasingle molecule presents numerousadvantages, includingbetter
celltargetingor tissuespecificity in cases whereone antigen isoverexpressed on target cells but is
not unique to these cells. It can alsobe used to bring together differentcelltypesto improvedrug
efficacy, e.g., havingone domain that targetsa cellsurfaceantigen and a secondthat has agonistic
binding to T-cellreceptors. Other possibilitiesinclude targetingHSA to increase serumhalf-life,
or directing an enzyme, radio-label or toxin to a desired tissueso reducingdosagerequirements
and minimizing off-site toxicity. Of course not all designerbio-therapeuticsrelyon the antigen
recognitionelementof an antibody. A good example of this Enbrel,wherethe solubleform of the
tumor necrosis factor (TNF) receptor is fused to an antibody Fc region.
Where then doesIgNARfit into the equation?Asantigenbindingsingle domainsthey fulfill all
of the attributes and opportunities describedabove. Theyare readilyexpressed at high functional
levels in a varietyof systems and are amenableto both in vivoand in vitro display systems. The
antigenbindingloopscanaccommodate extensive modifications to modulateantigenbindingand
they can easily be engineeredasfusionsto additional functional protein domains.
Howeverthere a number of other attributespossessed bythis unique class of receptorsthat are
generating the realexcitement and openingthewayfor newopportunitiesto beexploited.At a mere
12 kDa comparedto -25 kDa for the minimalfunctionalbindingunit of aconventionalantibody,
the V-regions of IgNARare the smallest naturallyoccurringantigenbindingdomain.Thisin itself
reducespotential immunogenicityissues and providesfor rapid renal filtration and short serum
half-life which can be readily extended if required as describedearlier. The smallsizeaffordsthe
possibilityto localize therapiesveryquicklyto target tissues and to improvedeliveryasa resultof
increasedpenetration of solidtumors.Theyarealsoableto access antigensthat are unavailable to
conventional antibodies dueto stericconsiderations. Comparedto conventional antibodies, IgNAR
exhibit a remarkable degreeof stability. Theyareableto resisthigh concentrationsof denaturants
and after exposure to temperaturesof 90'C they will refold to their original conformation, so
restoringbinding function. This property probablyresultsfrom the adverse environment found
in the blood of sharks,which useureaasan osmo-regulator. Froma clinicalperspective it not only
meansthat IgNAR basedtherapeuticsarelikely to havean extendedshelf-life, particularlyuseful
in many areasof the developing world where refrigeration is not always available, but may also
open up opportunities for novelroutesofadministrationrather than parenteral.Thereisa second
class ofnaturallyoccurringsingle domainantigenreceptors found in camelids and termedVHH.64
UnlikeIgNARtheseareclearly evolved fromantibodyheavychainsand sharesignificant sequence
homology. Theysharemanyof the propertiesofIgNAR suchassolubility, stability, smallsizeand
an extendedprotruding CDR3 and there areseveral noveltherapeutics in development basedon
this molecule. It issomewhat remarkable that, despitesharingverylittle sequence homologyand
beingseparatedbysome400 millionyears,sharkIgNARisStrUcturally and functionally incredibly
similarto camelVHH-a true case of convergent evolution. VHH sharethe samenoncanonical
cysteineasTypeII IgNAR.Thereishowever no TypeI equivalentin camels and asa resultIgNAR
has the potential to provideevengreatercoverage of otherwiseunavailable epitopesbyvirtue ofa
seconddistinct bindingsitetopography. Wehave described earlierthe issue ofimmunogenicity and
the impact that this has on the development of biologic therapeutics. Byanalyzing the structure
ofcrystallized IgNARproteins we can see that they adopt a conformationthat isverysimilarto
that of somehuman VH domains (Fig.3). Whilst they appearintrinsically to showverylow im-
munogenicityin mammals (unpublished), it mayneverthe less be possible to engineerthem into
a more human formatbyreplacing componentsofthe framework regions with thosefrom human
antibodies. Conversely, it maybe equally plausible to engineersomeof the usefulcharacteristics
ofshark IgNARinto human VH domainsby takingthe reverse approach.
Overall, there areclearly immense opportunities for future therapeuticdevelopment basedon
this novelprotein-harnessing a receptorthat predatesthe ageof dinosaurs to tacklesomeof the
urgent clinicalchallenges of the 21st century.
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CHAPTER 7
Immune Interventions of Human

Diseases through Toll-Like Receptors
Cevayir Cohan, Ken J. Ishii and Shizuo Akira*
Abstract
T
oll-like receptors (T LRs) are the immune sensors for infections, triggering robust innate
immune activation followed by protective adaptive immunity against various infectious
diseases. Recent evidence, however, has suggested that TLRs are involved in the pathogen-
esis ofmany diseases, including not only infectious diseases but also autoimmune diseases, allergy
and atherosclerosis. Therefore, prophylactic or therapeutic application of TLR-based immun e
interventions should be potent, but their safety must be demonstrated using experimental animal
models as well as human resources , including analysis ofsingle nucleotide polymorphisms. Here,
we focus on recent advances in understanding of the protective and pathogenic roles ofTLRs in
human di seases.
Introduction
An important role of the innate immune system in the first-line defense against pathogens,
and the underlying molecular and cellular mechanism(s) involved not only in infectious diseases
but also in cancer, allergy, autoimmune disea ses and atherosclerosis, has recently been unveiled.
When the Toll pathway in Drosophilameltlnogaster was discovered in the mid-1990s, it was initially
thought to be important in embryonic patterning but was then found to be a component of the
host defense against fungal and Gram-positive bacterial infections. I Subsequently, its mammalian
homologues, evolutionarily conserved Toll-like receptors (TLRs) have been discovered. '
Th e mammalian TLRs are a class of pattern recognition receptor (PRR) molecules consist-
ing of at least 11 members that recognize microbial compo nents known as pathogen-associated
molecular panerns .v' Similar to the other PRRs, mammalian TLRs are widely distributed on/in
the cells of the immun e system and are capable of discriminating among a varie ty of invading
pathogens such as protozoans, fungi , bacteria and viruses ,' Direct interaction ofTLRs with the
cognate ligand triggers intracellular signalin g pathways through multiple adapters, transduction
and transcription molecules, leading to robust immune responses characterized by production of
inflammatory cytokines, chemokines and immunoglobulins, and up-regulation ofcosrimulatory
molecules. In particular, the activation of dendritic cells via TLRs is critically important because
of their ability to prime the adaptive immune responses linking innate immune responses to
adaptive immuniry. -?
In addition to their 'primary function' offighting invading microbes, TLRs are also involved in
the pathogenesis ofman y diseases. In particular, TLR recognition ofself-molecules derived from the
host (e.g., nucleic acids) may be linked to autoimmune diseases and po ssibly to other immunological
' CorrespondingAuthor: Shizuo Akira-Professor, Department of Host Defense, Research

Institute forMicrobial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
PharmaceuticalBiotechnology, edited by Carlos A. Guzman and Giora Z . Feuer stein.
©2 009 Landes Bioscien ce and Springer Science + Business Media.
disorders.In human. TLRs and their mutations (e.g.•singlenucleotidepolymorphisms (SNPs))

have recently been linked to susceptibility. not only to infectious diseases. but also to chronic
inflammatorydiseases. atherosclerosis and asthma(for review, seerefs. 8-10).
Such a potential impact ofTLR-mediated immune regulationon human diseases has stimu-
lated a wide range of clinicalfields. Agonistsand antagonistsfor TLRs. and inhibitors ofTLR
signalingmolecules are presentlyunder development for a varietyof therapeutic applications.
Choosing a highly 'effective and proper' but 'safer' TLR-agonist/antagonist is critical for the
development of improvedvaccine adjuvant and immune-stimulatory agents. TLR antagonists
or inhibitors ofTLR-signalingmolecules, on the other hand. mayprovideanother opportunity
for the developmentof drugs to prevent and/or treat diseases in which TLRs areinvolved in the
etiologyor pathogenesis. Effort should alsobe made to understand their mechanism of action.
and to evaluate their safetyrelying not onlyon animalmodelsbut alsoon human resources suchas
SNPs. In this chapter. we discuss recentadvances and understandingof the TLR-relatedresearch
field. in particular the molecularand cellularmechanisms underlying TLR-mediated innate im-
mune responses and their impact on human diseases. in the hope of helpingfuture development
ofmore efficient and saferTLR-basedimmunotherapies.
Toll-Like Receptors andTheir Known Ligands

Themammalian TLRfamilyconsists ofatleastII members.TLRscomprise ahorseshoe-shaped
extracellular domain with dozensofleucine-richrepeatmotifs. a transmembrane domain (except
for TLR3). and a cytoplasmic Toll/IL-IR (TIR) domain similarto that of interleukin-I (IL-I)
receptors(IL-IRs ).5TLRI , 2.4. S. 6 and possibly II areexpressed on the cellsurface. whileTLR3,
7.8 and 9 arebelieved to reside insidecells (e.g., endoplasmic reticulumand/or endosomes). Each
TLR is expressed in a varietyof immunecells. includingmacrophages, dendritic cells and B cells.
and somestromalcells suchasendothelialcells and epithelialcells.TLRs can recognize molecular
pattern(s)within lipids.proteinsor nucleic acids that areconserved amongbut uniqueto microbes
that usually do not existin the host (TableI). Forexample.TLR2 aloneor togetherwith TLRI or
TLR6 detectsbacteriallipoproteinsand lipoteicoic acids. whichareuniqueand essential compo-
nentsof the bacterialcellwall;TLR3 recognizes double-stranded RNA.an intermediate generated
duringviralreplication; TLR4 recognizes lipopolysaccharide (LPS). atypicalcellwallcomponent
of Gram-negative bacteria; TLRS recognizes flagellin, a protein necessary for flagellated bacteria;
TLR7 andTLR8 recognize single-stranded RNAderived fromRNAviruses;andTLR9 recognizes
unrnethylatedCpG motifsof DNA observed in certain bacteriaand viruses."
New ligands for TLRs are continuously increasing in number and seem to become more
diverse and complexthan wasinitiallythought (Table I). EachTLR now has avarietyof ligands
derived not only from microorganisms. but also from host cells that are particularlydamaged
or dyingfor various reasons. For example, TLR9 recognizes a host chromatin-DNA complexas
wellas hemozoin, a malarial metaboliteof the host heme molecule.'!How such structurallyand
chemically unrelatedligandscan be recognized by the sameTLR is not fullyunderstood.While
the precisemolecularmechanism(s) ofTLR ligand recognitionthus need further investigation.
one such mechanism is that the TLRs are not alone; rather. they orchestratewith other PRRs.
therebyenablingthe recognitionof such a diverse rangeof molecules.P'" Interventionbetween
TLR-mediatedprotectiveimmuneresponses and the pathogenesis of a varietyof human diseases
is thereforea double-edged sword.
Toll-Like Receptor Signaling

Another important key to understanding TLR-mediated immune responses is differential
intracellularsignalingdownstream of the TLR initiated by the TIR domain in its cytoplasmic
portion. which is shared with a member of the IL-IR superfamily. Upon stimulation. adaptor
moleculets) that also havea TIR domain are recruited to the TLR-TIR domain. Myeloid dif-
ferentiationprimaryresponse gene88 (MyD88)isan essential adaptor molecule for most TLRs
exceptTLR3 (Fig. I).1he other adaptormolecule. Toll-IL-IRdomain-containingadaptor protein
Immune InterventionsofHuman Diseases through Toll-LikeReceptors 65
MyD88 dependent .-~---~ MyD88 independent
TLRS, 10 , 11 TLR116 TLR2
Cytoplasm
TLR3
TRIF
Figure 1. MyD88, Myeloid differentiation primary response gene 88 ; TIRAP, Toll-IL-1R

doma in-containing adaptor protein ; TRIF, Toll/IL-1Rdomain-containing adaptor inducing IFN-beta;
TRAM, TRIF-related adaptor molecule; TRAF6, Tumor necrosis factor receptor-associated factor
6; TAK1, Transforming growth factor-beta-activated kinase 1; IRAK4, IL-1 R-associated kinase 4;
TANK /TBK1 , TRIFand TRAF-family-member-associated NF-KB activator/binding kinase 1; IRF3,
Interferon regulatory factor 3. (Sightly mod ified from ref. 12.)
(TIRAP) specifies MyD88-dependent TLR2 and 4 signaling. TLR3 and 4-mediated activations
Signalthrough ToUIlL-l R domain-containing adaptor inducing interferon-beta (IFN~) (TRIF),
the so-called MyD88-independent pathway, leading to the induction ofIFN~ and IFN-inducible
genes. The TRlF-related adaptor molecule (TRAM) further specifies the MyD88-independent,
TRIF-dependent Signaling pathway ofTLR4, acting as a bridging adaptor between TLR4 and
TRlF (Fig. I).
Upon TLR-stimulation, MyD88 as well as TRIF subsequently associate with tumor necrosis
factor receptor-associated factor 6 (TRAF6), culminating in the activation of nuclear factor
(NF)-KB and /or the mitogen-activated protein kinase (MAP-Kinase) pathway, resulting in the
production ofpro-inflammatory molecules including cytokines and chemokines (reviewed in ref.
4).TRAF6 forms a complex with ubiquitin-conjugatingenzymes (Ub) such as Ubcl3 and activates
transforming growth factor-beta-activated kinase I (TAKI),which in turn activates transcription
factors NF-KB and activator protein-I (AP-I) through the canonical IKBkinase (IKK) complex
and MAP-Kinase. The IKK complex is composed of two catalytic subunits, IKKa and IKK~.
NEMO (also known as IKKy) encodes the regulatory component ofthe IKK complex, which is
responsible for activating the NFICB signaling pathway. IKK phosphorylates IKBand targets it for
degradation. The removal OfIKBenables NFKB to translocate into the nucleus, where it activates
the transcription ofvarious target genes.
In addition to these signaling pathway s controlled by IKK complexes, TLR7/9 mediated
MyD88-dependent signaling has a distinct signaling pathwa y for Type I IFN production. MyD88
Table 1. Toll-like receptors and their diverse ligands (based on ref. 12)
Ligand Ligand
(Non Hostderived = (HostDerived =
TLR Exogenous) Endogenous) Source
TLR2 Peptidoglycan Bacteria

(TLR1+TLR2) Triacyll ipopeptides Bacteria
(TLR6+TLR2) Diacyl lipopeptides M ycoplasma
Lipoteichoic acid Gram -po sitive bacteria
Hemagglutinin prote in Mea sles viru s
GPI Trypanosoma cruzi,
Plasmodium falciparum,
Glycolipid Treponema maltophi/um
Zymosan Fungi
Heat-shock protein 60 Helicobacter pylori
Heat-shock protein 70 Host
TLR3 dsRNA West Nile virus

MouseCMV
Schistosoma mansoni
siRNA Synthetic
mRNA Host
TLR4 Lipopolysaccharide Gram-negative bacteria

Taxol Plants
Fusion protein F Respiratory syncytial virus
Envelope protein Mous e mammary tumor virus
Antrolysin 0 Bacillus anthracis
Phosphorylcholine Filarial nematode
Glycan Helm inth
Heat-shock protein 60 Chlamydia pneumoniae
Helicobacter pylori
Heat-shock protein 60 Host
Heat-shock protein 70 Ho st
fl-Defensin 2 Host
Fibrinogen Host
Fibronectin Host
Hyaluronic acid Host
Heparan sulphate Host
Fatty acids Host
Tamm-Horsfall Host
glycoprotein
Surfactant protein A Host-Lung
Modified LDL Host
TLRS Flagellin Flagellated bacter ia

TLR7 ssRNA Influenza, HIV-l
Parechovirus 1
Imidazoquinoline Synthetic compounds
UlsnRNP autoantigens Host
continued on next page
Immune Interventions o/Human Diseasesthrough Toll-Like Receptors 67
Table 1. Continuted
Ligand Ligand
(Non Hostderived = (Host Derived =
TLR Exogenous) Endogenous) Source
TLRB ssRNA Viruses

Coxsackie B virus
Parechovirus 1
Imidazoquinoline Synthetic compounds
TLR9 CpG DNA Bacteria, synthetic ODN

DNA viruses
Hemozoin Plasmodium falciparum
Chromatin complex Host
TLR10 Not determined
TLRll Profilin-like molecule Toxoplasma gondii
formsacomplex with IL-l R-associated kinase 4 (IRAK4) and lRAKl . Dependingon the typesof
cells or stimuli,TLR-MyD88-dependentsignaling requires lRAK4 or lRAKl.I4-16While lRAK4
isnecessary for mostTLR-mediatedpro-inflammatory cytokineproduction bymacrophages and
dendritic cells, lRAKl was shown to be essential only for TLR7- and TLR9-mediated Type I
IFN production byplasmacytoid dendriticcells. I?Recently, TRAF3 and IKKa werefound to be
indispensable for TLR7/9-induced IFNa induction.18.20 TLR3 and TLR4 also mediateType I
IFN productionviaanothermajoradaptormolecule, TRIF and TRAF-family-member-associated
NF-KB activator (TANK) bindingkinase 1 (TBKl).TBKl comprises a family with inducible IKB
kinase(IKK-i, alsoknown as IKK-E), and thesekinases directlyphosphorylateinterferonregula-
tory factor 3 (IRF3) and IRF7.21,22TBKlIKK-i-mediated TypeI IFN induction is not restricted
to TLR3 and 4, but is alsoinvolved in TLR-independent virus-, RNA- and DNA-inducedType
I IFN produceion.P'"
Transcriptionfactorssuch as IRF5 and IRF7 werealsofound to be important mediatorsfor
TLR-dependent and -independenr signaling pathways. In particular, IRF5 was found to be in-
volved in mostTLR-mediatedpro-inflammatory cyrokineproduction but not IFNa production,
independentlyofNF-KB or the MAP-Kinase signaling pathway.v In contrast, the transcription
factor IRF7 wasshown to playa criticalrole in TLR7- and TLR9-mediated IFNa production
by direct interaction with MyD88as wellas TLR-independent TypeI IFN production induced
by viruses.lv'"
The Role ofToll-Like Receptors in the Human Immune System

In vitro and in vivo experimental modelselucidatingTLR function and physiological roles
in infectiousdiseases and associated immune disorders haveincreased our understandingof the
importance ofTLRs in both protectiveand pathological immune responses. However, animal
studies do not always reflect human physiology. In particular, differences between each mouse
and human TLR in termsof ligandspecificity, celltypes on which the TLR is expressed and the
associated diseases (model) mayhamper further development ofTLR-basedpreventive and/or
therapeuticapplications in human. Forexample, taxol,an anti-cancer drugderived fromplants,isa
TLR4ligand in mice, but isnot aTLR4ligand in humans.ln contrast,hexa-acylated LPSderived
from Pseudomonas aeruginosa is a ligand for human TLR4, but not for mouseTLR4.26Another
example is that CpG DNAs (ligand for TLR9), which stimulate mouse cells, do not stimulate
human cells well.27.28 MurineTLR9 isexpressed in widerangeof dendritic cells (DC) , important
antigen-presenting cells including conventional and plasmacytoid DC; however, human TLR9
is expressed in plasmacytoid DC only and not in conventional (myeloid) DC. suggesting that
TLR expression patterns in certain immune cellsdiffer between murine and human cells.and that
careful human studies should be carried out to determine their potency.
In humans. definitive and suggestive evidence has accumulated by large-scale analyses ofSNPs
in TLRs and several human diseases such as infectious diseases. asthma. atherosclerosis. and
autoimmune diseases (see ref. 10). The idea is that naturally occurring variation in the innate im-
munity genes has an important role in human susceptibility to a variety ofdiseases. Identification
and functional characterization ofpolymorphisms in innate immunity-related genes may provide
insight into genetically determined susceptibility to disease that might allow us to understand the
nature and outcome ofthe disease. and evaluate diagnostic and therapeutic strategies." Knowing
the TLR SNP genotype of a patient suffering a severe infectious disease may allow clinicians to
tailor treatment and evaluate the prognosis. In the sections below. we discussthe relevance ofTLR
SNPs in human diseases and future manipulation tactics.
TLR-Based Immune Intervention in Humans: Promise and Caution

TLR agonists have been used or are under clinical development; the best examples are
adjuvants in vaccine formulations. CpG DNA as a TLR9 agonist induces strong T helper
1 (Thl) immune responses. and its efficacy as a vaccine adjuvant has been demonstrated in
nonhuman primates and humans. P-" Monophosphoryllipid A (contains lipid A. a com-
ponent of LPS. a TLR4 agonist). and R848. an agon ist ofTLR7. are also being developed
as vaccine adjuvants. 32-34 Evidence obtained from animal experimental models suggest that
TLR agonists are very potent in eliciting an innate immune response. followed by an adap-
tive. especially cellular, immune response; they are therefore promising candidates as vaccine
adjuvants. Accumulating evidence ofTLR polymorphism in humans suggest the need for a
careful evaluation ofgenetic as well as functional variation in the target population. The first
caution came from the Lyme disease vaccine. which is based on the outer-surface lipoprotein
A (OspA) ofthe pathogen Borrelia burgdorj'eri. Some individuals with very low antibody titers
after vaccination were found to have low responsiveness to TLRI and 2 ligands. and to have
lower macrophage expression of Tl.Rl ."
The other caution with TLR-based immune interventions is TLR-mediated innate immune
activation is so strong that the outcome may be deleterious to the host. For example. with CpG
ODN. a promising TLR agonist . it has recently been shown that robust TLR9-mediated innate
immune activation can cause multifocalliver necrosis and hemorrhagic ascites.36In addition. TLR
recognition of self-molecules of the host [e.g.• nucleic acids) might be linked to autoimmune
diseases and other immunological disorders. For example. the TLR9ligand CpG ODN has been
implicated in triggering autoimmune diseases such as systemic lupus erythematosus (SLE) and
rheumatoid arthritis. 37.38
Thus. to develop potent and safeTLR agonists for immune intervention in humans. we need to
have knowledge ofhuman Tl.Rfuncrions, human-specific variations such astarget TLR-expressing
cellsand SNPs. and, more importantly. the deleterious effects ofTLR(agonist )-mediated immune
activation.
TLR2
TLR2 is a major mammalian TLR that can recognize various components ofbacteria . viruses.
fungi and parasites by forming heterodimers with TLRI or TLR6.39•40 Studies in animals have
shown that TLR2-deficient mice are more susceptible to infection with Gram-positive bacteria (i.e.•
Staphylococcus aureus.Listeria monocytogenes.Streptococcus pneumoniae).Mycobacterium tuberculosis
and B. burgdorferi. 41 In humans, several SNPs were identified in TLR2 and an Arg753Gly poly-
morphism was found to be associated with reduced pro-inflammatory responses to Staphylococcus
infection (Table 2),42Another TLR2 polymorphism. Arg677Trp, wasassociated with susceptibility
to certain infectious diseasessuch as tuberculosis and leprosy. suggesting the importance ofTLR2
genetic variants in the response to infections in humans,42-46
Immune Interventions ofHuman Diseases through Toll-LikeReceptors 69
It is clear that TLR2 plays an essential role in the recognition of several microorganisms, and
promotes defense; thus, manipulation of TLR2 function could contribute to the design of new
therapeuticstrategies for preventionand/or vaccine development againstinfectious diseases. For
example, the TLR2 agonistMalp-2(TLR2/6ligand) has already been investigated experimentally
in mice as a mucosal adjuvant,47.48 The TLR2 agonist Pam3Cys-S~ (TLR211 ligand) has been
examinedexperimentally to treat established Inflammarion." On the other hand, TLR2 agonists
can result in experimentalasthma," but a polymorphismin TLR2 reduced the risk of asthma."
A TLR6 polymorphismwasalsofound to reducethe riskof asthma." An additional cautionwith
TLR2 agonistsis their link to atherosclerosis. Accumulatingevidence suggests the involvement
of multiple microorganisms such as Chlamydia pneumonia, Helicobacter pylori and cytomegalo-
virus in the inflammatory etiology of atherosclerosls." Most TLRs, including TLRl, 2, 4 and
5, are expressed in atherosclerotic plaquesby several cell types, and can triggerTLR2- or other
TLRlMyD88-dependent activation, includingthe production of pro-inflammatory cytokines and
chemokines in aeherogenesis.b" However, an Arg7S3GlnpolymorphismofTLR2 in humanswas
found to be closely related to coronaryreseenosis." suggesting that the TLR2 pathwaymayhave
a dual role in the pathogenesis of atherosclerosis. Nevertheless, the aboveinformation should be
consideredcautiously in the caseof usingTLR2 agonises/antagonists.
TLR4
TLR4 recognizes LPS,a majorcomponent of the Gram-negative bacterialcellwallthat plays
criticalrolesin the pathogenesis of Gram-negative bacterialinfectionssuch assepsis.57'59It is im-
portant to searchfor new pharmacological interventions, through manipulationofTLR4, due to
the increasing antibiotic resistance of Gram-negative and Gram-positive infections, and bacterial
product-relatedcomplications suchassepticshockthat cannot be treatedwith antibiotics. Bycon-
trollingor modifyingTLRs and their signaling, the entire septicprocess maybe modified.6O•61
In a mousemodel,TLR4- and MyD88-deficient micewerefound to be resistant to LPS chal-
lenge. 57.62 A similarphenomenon wasobservedin humans, in that some people with the TLR4
polymorphisms Asp299Glyand Thr399llewerehyporesponsive to inhaledLPS.59 Furtherstudies
sought the link betweentheseSNPsand susceptibility to several infections, and revealed that such
TLR4 polymorphisms increased susceptibility to respiratorysyncytial virus(RSV) infection, bru-
cellosis, severe malaria, and candidalbloodstreaminfections(refs. 63-66,and seeTable2 for TLR4
SNPs and relation to human diseases). Thesedata are in accordance with previousreports that
TLR4 recognizes not onlyLPS, but alsoRSVfusionprotein," indicatingconsiderable correlation
between functional mutation(s) and human infectiousdiseases causedby a varietyof microbes.
TLR4 and several other TLR polymorphisms werefound to be relatedto chronicinflammatory
diseases such as Crohns disease, ulcerative colitisand sarcoidosis. Indeed, TLR4 wasconsidered
to trigger the development of Crohn's disease in mice through microbial recognition in the
intestine and following inflammatory responses/" In humans, TLR4 expression was found to be
elevatedin patients with ulcerative colitisor Crohns disease, and the Asp299Glyand 1hr399Ile
polymorphisms ofTLR4 havebeenlinkedto the etiologyof both of thesediseases and the chronic
course of sarcoidosis.f"?' Moreover, TLR4 has been suggested to play an important role in the
recognition of H pylori in the gastric mucosa," though TLR4-independent detection is also
possible," probablydue to TLR2 recognitionofatypicalH pylori LPS,74.75or NOD-dependent
recognition of peptide glycan." In addition, H pylori HSP60 wasdemonstrated to activateboth
TLR2 and TLR4.77 Further investigation ofTLR2 and TLR4 polymorphisms in H pylori infec-
tion in humans would be necessary to elucidatethe precisemolecularand cellularmechanismis)
in H pylori-induced pathogenesis.
In addition,studieshavesuggested associations ofTLR4 polymorphism withchronicobstructive
pulmonarydisease and severe asthma,78.79 whileother studieshaveobservedno association between
TLR4 and TLR signaling molecule polymorphisms and the riskof asthma'" Clearly, more studies
will open new avenues to understandingthe roleofTLR4 in asthmaticand allergic patients.
Despitethe pathogenicrolesdescribedabove, LPSand its purifiedproducts havebeen usedas
pharmaceutical agentsdue to their potencyin elicitinginnate immuneresponses. Initially, purified
Table 2. Toll-like receptors and their relation to human diseases
TLRor SNP(s)
Signaling or
Molecule Genes Effect on the Disease Reference
TLR2 Arg753Gln Susceptibilityto Staphylococcus infection 42

Susceptibilityto tuberculosis 44
Resistance to Lymedisease 137
Increased risk for coronary restenosis 56
Associated with severe atopic dermatitis 138
Arg677Trp Susceptibilityto leprosy 43
Susceptibilityto tuberculosis 45
GT-repeat Susceptibilityto tuberculosis 46
polymorphism in
intron II
-16934 Reduced risk for asthma 51
TLR1,2,6 Nonsynonymous Extensionof inflammatory bowel diseases 139
variants
TLR4 Asp299Gly Hyporesponsiveness to LPS 59
Susceptibilityto meningococcaldiseases in 140
infancy
Susceptibility to brucellosis 64
Susceptibilityto osteomyelitis by Gram-negative 141
bacteria
Hyporesponsive to Porphyromonas gingivalis 142
Increased risk for bacterial vaginosis 143
Association with Crohn's disease 69
Risk factor for Crohn'sdisease 144
Association with ulcerative colitis 69
Lower incidence of carotid artery stenosis 145
Lower incidence of acute coronary events 146
Lower incidence of myocardial infarction 147
Resistance to chronic obstructive pulmonary 78
disease
Associated with the severityof asthma 79
Reduces the risk of developing late-onset 148
Alzheimer's disease
Associated with gastricMALT lymphoma 149
Asp299Gly and Susceptibility to septic shock 150
Thr39911e Susceptibilityto severe RSV infection 63
Resistance to legionnaire's disease 151
Susceptibilityto Candida bloodstream infections 66
Association with chronic sarcoidosis 71
Decreased susceptibility to RA 152
Lower incidence of allograft rejection 153
Thr39911e Susceptibility to severe malaria 65
Association with ulcerative colitis 70
Increased risk of severe acutegraft versus host 154
disease
Immune Interventions ofHuman Diseases through Toll-Like Receptors 71
Table 2. Continued
TlR or SNP(s)
Signali ng or
Molecule Gene s Effe ct on the Disease Reference
C119A Risk for ischemic stroke 155

11381G/C Increased risk of prostate cancer 156
Variant alle les Lower risk of prostate cancer 157
Rare coding variants Susceptibility to meningococcal diseases 158
TLR5 392STOP Susceptibility to legionnaire's disease 93
Association to Crohn's disease 98,100
Resistance to SLE 94
TLR6 Ser249Pro Dec reased risk for asthma 52
TLR9 T-1237C Association with Cro hn's disease 159
Increased risk of po uchitis 160
TLR10 c.+1031G>A and Association with asthma 161
c.+2322A>G
Haplotype Association with risk for nasopharyngea l 132
GCGTGGC variant carcinoma
IRAK4 IRA K 4 deficiency Susceptibility to pyogenic bacterial infections 135
IKBKG IKKy (N EM O) Susceptibility to pyogenic bacterial infections 133
deficiency and atypical mycobacteria
NFKBIA IkBa-deficiency Susceptibility to pyogenic bacterial infectio ns 162
and atypica l mycobacteria
Association with sarcoidosis 163
Association with multiple myeloma 164
IRF5 Rs2004640 T Increased risk of SLE 136
TLR6-1-10 Synergistic effect of Increased risk of prostate cancer 165
gene cluster several alle les
and IRAK4
LPS(containslipid A) wasthought to provideprophylactic protection againstsubsequent bacterial

or viralchallenge in animals; however. its extremetoxicitypreventedits use." Effortsto eliminate
the toxicity of lipid A led to the developmentof monophosphoryllipid A (MLA).81.82 MLA has
been in human clinicalstudies as a new-generation vaccine adjuvant against infectious diseases
and seasonalallergic rhinitis, and was proved to be safe and effective.83.84 Lipid A analogs such
as E5531, E5564 (n-D-glucopyranose) and CRX_52685·87have been demonstrated to act asLPS
antagonists by blocking the effects of endotoxin. and have been in Phase II clinical trial against
sepsis and the complicationsof coronary artery bypass surgery.'" TAK-242 is another promising
compound that actsthrough blockingTLR4-mediated signalingand hasbeen in PhaseIII to treat
severe sepsis (pressrelease, http ://www.takeda.com/press/05072701.htm). Thus,TLR4ligands
are apparentlya double-edged sword.requiringattention to safetyconcernsin order to makeuse
of their potency for therapeutic applications.
TLR5
TLR5 recognizes the bacterialprotein flagellin that is found in the flagellar structure of many
bacteria." TLR5 is expressed in epithelialcells in the lung and gut, but is alsohighlyexpressed in
residualdendritic cells, such as in the lamina propria of the intestine." Signaling of flagellin via
TLR5 enhancesthe diversityof the response, probablybyengagingMyD88-independentadaptors
to activate the interferonpathway, whileTLR5-independent recognition machinerywasreported
recently'" Flagellin isa potent immuneactivator, stimulatingdiverse biologiceffects that mediate

both innateinflammatory responses and the development ofadaptive immunity. Theproteinnature
offlagellin is consideredan advantage for manyimmuno-therapeuticapplications, mainlydue to
its easeof manipulation; for example, a DNA vaccine encoding chimericprotein or the protein
itselfwith antigenicprotein and flagellin has been studied.91.92
Humans with a common stop codon polymorphismat the ligand-bindingdomain ofTLR5
(392STOP) werefound to be highlysusceptible to legionnaire's disease causedbyLegionelL:zpneu-
mophila93 (Table2). Interestingly, the samestopcodon polymorphismwasfound to protect people
from developingSLE, indicatinga possible connection ofTLR5-mediated immune responses to
infectiousdiseases with the developmentof autoimmune diseases,"
In addition to TLR2 and TLR4, TLR5 hasbeenfound to playa criticalroleagainstSalmonelL:z
typhimurium infection of the gastrointestinalsystem. TLR4 is important in the earlydetection
of S. typhimurium infection,whereas TLR2 plays important rolesaftercellularinvasion." More
importantly,it appearsthat TLR5 recognitionofS. typhimurium flagellin isthe majordeterminant
ofS. typhimurium infection in the gut.96•97 TLR5-deficientmicewereresistantto S. typhimurium
infection, suggesting that TLR5 maybe the sensor, not only for the pro-inflammatoryresponse
to flagellin, but alsofor bacterialinvasion ofthe host via Hagellln."
Several studies haveshown strong antibody responses to the flagellin of commensal bacteria
in Crohns disease patients but not in ulcerative colitispatients,98.99 suggesting that flagellin can
stimulate both innate and adaptive immune responses, triggered possibly through TLR5, that
maypromote the pathogenic response in Crohn's disease. Consistently, TLR5 stop codon poly-
morphism was found to be negatively associated with Crohn's disease.'?" Taking these findings
together,useof flagellin or a modifiedversionofit asan immunostimulatoryor adjuvantagent in
vaccineformulationsmaybe of interest because ofits protein nature, though the safetyconcerns
with TLR5 again remain to be elucidated.
TLR7 andTLR8
Single-stranded RNA genome or oligoribonucleotides with sequences derivedfrom HIV or
influenzavirus, and small synthetic compounds known as imidazoquinolins, are recognizedby
TLR7 in miceand TLR8 in humans,activatingvarious immunecells that elicitTypeI IFNsaswell
as cellularimmune responses.P':'?' Several TLR7 agonistshavebeen approvedfor clinicaluse in
variousviralinfections.105The TLR7 agonistimiquimod (5%cream)has been shown to be effec-
tive for externalgenitalwarts,basalcellcarcinomaand actinic keratosis,I06-108 and is in a Phase I
clinicaltrial against human paplllomavirus.f Several other syntheticTLR7 agonist compounds
havebeen in PhaseI or PhaseII trialsagainsthepatitis B virus,hepatitis C virus, and cancer.'"
TLR7 also recognizes autoantigens complexed with RNA, such as UlsnRNP (nuclear
self-antigen) in mice,I09-112 that could play important roles in the pathogenesis of SLE. These
recent reports raisedsafetyconcernsabout the syntheticTLR7 agoniststhat arealreadyin usein
humans,in that they mayact in asimilarmannerto the autoantigens, activatingaTLR7-rnediared
immune cascade and triggeringand/or worseningcertain autoimmune diseases such as SLE.In
turn, TLR7 antagonists could be important pharmaceutical agents for the treatment of SLE. A
carefulevaluationof dose,formulation and route of administration should be made to overcome
these issues.
TLR9
TLR9 recognizes unmethylatedCpG (cytosinephosphate guanosine) motifs found in bacte-
rial and viral DNA.30 Syntheticoligodeoxynucleotides (ODNs) that contain these CpG motifs
triggerTLR-mediated, MyD88-dependentsignalling ofmacrophages, dendritic cellsand B cells
to produce pro-inflammatorycytokines, chemokines and immunoglobulins. The robust innate
immune response to CpG ODNs skews the host'simmune milieuin favourofThl cellresponses
and pro-inflammatory cytokineproduction,an effectthat underlies their useasimmunoprotective
agents,vaccine adjuvantsand anti-allergens. Preclinical studiesprovideevidence that CpG ODNs
Immune Interventions ofHuman Diseases through Toll-Like Receptors 73
are effective for each of these uses and can modulate the immune response to co-administered
allergensand vaccines." CpG ODNs have had promising results in human use and haveentered
PhaseIII clinicaltrialsagainst several typesofcancer,including melanoma,lymphoma, and nons-
mall cell lung cancer, either alone or in combination with chemotherapy.r' The other promising
application of CpG ODN under clinicaldevelopment is a vaccinecomposed ofallergen antigen
conjugated with CpG ODN to either treat or prevent allergicdiseases.113•114 CpG ODN skews
the Th2 allergicimmune milieu to a protective Th 1 immune milieu, whereby allergicsymptoms
are diminished or reduced.us
Recent evidence,however, suggests that CpG ODN should be categorizedinto severaltypes
and is not the only ligand for TLR9. There are at least severaltypes of CpG ODN that activate
innate immune responses differentially and that maybe usedin differentapplications.116Moreover,
ODN-based TLR9 agonists that do not require unmethylated CpG motifs have been demon-
strated,117.118 suggesting that TLR9-mediated immunotherapeutic applications can be tailored
for respective applicarions." ? Although these TLR9 agonists are very potent and promising in
eliciting innate and adaptive immune responses, and can be used in many immune-therapeutic
applications, there are recent reports suggestingthat TLR9 alsorecognizesother molecules, such
as heme metabolites derived from malaria-infected red blood cells,120 and self-DNA-chromatin
complexes.V!" These results raised safety concerns for DNA-based TLR9-mediated immune
intervention, though the roleofTLR9 in the etiologyand pathogenesisofSLE iscontroversialin
animal models and human SNP studies.122.124 It was previously shown that TLR9 ligand-in-
duced arthritis was reduced by guanine-rich ODN (suppressive ODN or inhibitory ODN), an
antagonist ofTLR9Ys In addition, the sameODN has been found to reduce the pathogenesisof
collagen-induced arthritis, a naturally occurring autoimmune disease, I 26.127suggestingthat such
TLR9 antagonismmayblockor reducethe pathogenesis ofautoimmunedisease and mayovercome
the deleteriouseffectsofTLR9 ligand therapies.
TLR3,TLRIO
TLR3 recognizes double-stranded RNA derived from synthesis (e.g., poly-I:C), the viral
genome, or the intermediates generated during viral replication,all ofwhich havebeen shown to
playan important rolein anti-viralresponses. Poly-I:C wasone of the first therapeutic agentsused
to treat HIV and leukemiapatients, but wasabandoned due to its toxicity.l28 Severalstudies have
been undertaken to reducethe toxicityof poly-I:C and there areclinicaltrialsagainstbreastcancer
and ovarian cancer.!" To date, there havebeen few studies investigatingthe possibleinvolvement
of TLR3 in the pathophysiology of human diseases. Pirie et al reported that polymorphisms in
the TLR3 gene may be associatedwith Type 1 diabetes, though studies of a larger population
seem to be needed.l"
TLRIO isfound in humans but has no functional homologue in rodents,and to date no ligands
havebeen identified. However, TLRI 0 sharesa common locuswith TLRI and TLR6 that makes
hetero/homodimers with TLR2.Thus, it hasbeen speculatedthat TLRI 0 couldact asa coreceptor
with TLR2 .131Fewstudies haveinvestigatedpolymorphismsof the TLRI 0 genein relation to hu-
man diseases,especially in the lungand nasopharyngealregion,becauseTLRI 0 isexpressed mostly
in the pDC of those regions.'?' Recent studies havefound a relationship between variousTLRI 0
polymorphisms and increasedrisk of asthma and nasopharyngealcarcinoma (Table 2).29.132
Other Signaling Molecules

Recent evidencehas suggested that primary immunodeficiency diseases of humans could be
attributed at least in part to abnormal TLR signaling (for recent review, see ref 9). In humans,
primary immunodeficiency diseases due to natural genetic mutations ofgenes involvedin intra-
cellularsignalingfor innate immune responses havebeen reported. Patients with X-linked hypo-
hydrotic ectodermal dysplasia, characterizedby increasedsusceptibilityto pyogenicand atypical
mycobacterial infections, were reported to possess a mutation in the IKBKG gene that causes
defectiveproduction of IKKy(N EM 0) protein.!" This is not surprising, since NEM a encodes
74 PharmaceuticalBiouchnology
the regulatorycomponent of the IKK complex responsible for activating the NF-KB signaling
pathway, which is critical for the TLR-mediated NF-KB activation that regulates immediate
transcriptionalresponses in innate immunity. Similarly, lRAK4 wasfound to be a keysignaling
moleculethat affects mostTLR signaling. Supportingthe resultsobtained from lRAK4-1- mice,
which developed defective responses to bacteriallnfections.P' patients who havefrequent pyro-
genicinfectionswerefound to haveautosomalrecessive amorphicmutationsin lRAK4. 13s More
recently, IRFS,which isimportant for the transactivation ofvariouspro-inflammatory genes, has
polymorphisms in humans that increase the riskof SLE.l361he intracellular signaling molecules
involved in innate immuneresponses arethereforepotential targetsfor immuneintervention,and
attention should be paid to their polymorphisms.
Conclusion
Investigations ofTLR-mediatedinnateimmuneresponses haveopenednewperspectives to the
understandingof the pathophysiology of disease. Mousestudieshaverevealed that TLR agonists
or antagonistscanbe usedto preventor treat immunedisorders. In humans,searchingfor SNPsin
TLR genes and their relationto diseases will leadto better understandingand control of disease,
and future development ofnewtherapeuticapproaches. However, TLR-mediatedinnateimmune
activationin response to infectionor tissuedamagemayresult in a deleterious outcome such as
sepsis, autoimmunedisease, or atherosclerosis, dependingon the TLR and the signaling pathway.
Furthermore, inhibition ofTLR signaling maycauseinnate and/or adaptive immunedeficiency,
which couldbelethalforcertainpatient populations[i,e., patientswith SNPs).1hus, the risksand
benefitsof the manipulationofTLR-mediated immuneresponses need to be balanced.
Acknowledgement
Wethank the members oftheAkiraLaboratory forvarious discussions. This workwas supported
bygrants from the MinistryofEducation•Culture, Sports,Science and Technology inJapan,and
from the 21st Century Center ofExcellence (COE) ProgramofJapan.
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Biomarkers Prev 2006 ; 15:480-485.
CHAPTER 8
Genome-Based Vaccine Development:

A Short Cut for the Future
Danilo GomesMoriel,Maria Scarselli, Laura Serino,Marirosa Mora,
Rino Rappuoli* andVega Masignani
Abstract
acterial infectious diseases remain a major cause of deaths and disabilities in the world.
B Although conventional vaccinology approaches were successful in conferring protection

against several diseases, they failed in providing efficient vaccines against many others.
Together to the sequencing of the first genome, a new chapter in the vaccinology history started
to be written. Reverse vaccinology changed the way to think about vaccine development, using
the information provided by the microorganisms' genome against themselves. Since then, reverse
vaccinology has evolved and helped researchers to overcome the limits ofthe conventional vaccinol-
ogy approaches and led to the discovery and development ofnovel vaccines concerning emerging
diseases, like Neisseria meningitidis B and Streptococcus agalactiae. A lot ofwork must be done, but
deciphering the information provided by genome sequences and using it to better understand the
host-pathogen interactions has proved to be the key for protection.
Conventional Vaccinology
Vaccine development followed the same basic principles for more than two centuries. When
Edward jenner inoculatedJame s Phipps with a bovine poxvirus to induce protection against the
closely related human pathogen smallpox virus in 1796 and then , almost a century later, Pasteur
developed a live attenuated vaccine against rabies, the basic principles for vaccine development were
established 1 These approaches served as guidelines for the development ofvaccines throughout
th e twentieth century, allowing the protection against many once lethal infectious diseases. In face,
all existing vaccines were developed using at least one of the following approaches: killed (inac-
tivated), live attenuated or subunit (includin g the protein-conjugated capsular pol ysaccharides,
toxoids, cell-free extracts, recombinant proteins and stand-alone capsular polysaccharides)vaccines.
Because of those basic principles, several infectious diseases can be prevented by vaccines. Table
1 shows the vaccines licensed for immunization and distribution in USA , all of them obtained
basically by conventional approaches.' Conventional approaches led to great achievements such
as the eradication of smallpox and the virtual disappearance of diseases like diphtheria, tetanus,
poliomyelitis, pertussis, measles, mumps, rubella and invasive Haemophilus influenzae B, increas-
ing the life quality and expectan cy.' It was also important to pro vide the basis of vaccinology
but showed to be time-consuming, leading to years or even decade s of research. Inactivation and
attenuation were the first choice for many years, but the difficulty in cult ivating some microorgan-
isms in vitro and the fact that even att enuation may result in detrimental or unwanted immune
respon ses showed that these approaches are impractical in some instances," Even the purification
of specific antigens failed in many cases in providing a protective vaccine candidate, since the
·Correspondi ng Author : Rino Rappuoli - Novarti s Vaccines, Via Fiorentin a 1, 53100 Siena,
Italy. Email: rino .rappuoli @novartis.com
Pharmaceutical Biotechnology, edited by Carlos A. Guzman and Giora Z . Feuerstein.
©2009 Lande s Bioscience and Springer Science-eBusiness Media.
Table 1. Vaccines licensed for immunization anddistribution in the USA
Vaccine or Target Type
Anthrax Subunit
Diphtheria Subunit
Haemophilu5 influenzae b Subunit
Hepatitis A Inactivated
Hepatitis B Subunit
Human papillomavirus Subunit
Influenzavirus Inactivated, live
Japanese encephalitis Inactivated
Measles Live
Meningococcus Subunit
Mumps Live
Pertussis Subunit
Plague Inactivated
Pneumococcus Subunit
Polio Inactivated
Rabies Inactivated
Rotavirus Live
Rubella Live
Smallpox Live
Tetanus Subunit
Tuberculosis Live
Typhoid Live, subunit
Varicella Attenuated
Yellow fever Live
Herpeszoster Live
methods usuallyused led to the identificationof the most abundant but also most variable and
lesssuitablevaccinecandidates,'Although successful for manypathogens,conventionalvaccinol-
ogy still left many diseases uncontrolled by vaccination. Considering that even new diseases are
sure to emergethrough evolutionbymutation and geneexchange, interspecies transferor human
exposure to novelenvironmentsf afasterand morereliable approachmustbe available to promptly
respond to those threats.
Reverse Vaccinology
The sequencingof the-first bacteriumgenomein 1995 led the vaccine developmentto enter a
new era and to open a new chapter in the vaccine developmentguidebook.Suddenly, allproteins
encoded bya microorganismwereavailable and for the firsttime,aftermore than two centuries, it
waspossibleto identifyvaccine candidateswithout usingthe conventionalvaccinology principles.
This new approach, named reverse vaccinology, gives full access to allthe proteins that a microor-
ganismcan encode and, by computer analysis, it is possible to identifypotential surface-exposed
proteins in a reverse manner,starting from the genomerather than the microorganism. Problems
related to noncultivablemicroorganisms and also to antigens that are not expressed in in vitro
conditions, conferringthe most important obstacles for vaccine development, could be avoided
by the reverse vaccinology approach. The feasibUity of this approach relies on the availability
of a high-throughput system for protective immunity screeningand also on good correlates of
protection (Fig. 1).The greatestlimitation of the reverse vaccinology approach is the inabilityin
identifyingnonproteic antigenssuch aspolysaccharides, componentsof manysuccessful vaccines
Genome-Baud Vaccine Development: A ShortCutfor theFuture 83
+
Figure 1. The reverse vaccinology equation: the availability of n genome sequences, associated
to advanced bioinformatic tools and reliable high-throughput systems for protective immune
screening have proved to be a balanced equation for vaccine development.
and glycolipids, a new promising group ofvaccine candidares.' Nevertheless, reverse vaccinology
seemed to be a powerful tool that could help researchers to overcome the obstacles ofconventional
vaccinology and lead to the discovery and development ofnovel vaccines for the most concerning
emerging diseases.
The Classical Reverse Vaccinology Approach

For many years great efforts were concentrated in the development ofa vaccine against serogroup
B N. meningitidis (meningococcus), a bacterium that causes about 50% ofmeningococcal cases of
sepsis and meningitis and has a mortality rate of 5-15% despite the advances in therapeutics. N.
meningitidis is a Gram-negative capsulated bacterium that can be classified in 13 serogroups on the
basis ofthe chemical composition ofthe capsule polysaccharide. Among them, only 5 serogroups
have been associated with meningococcal meningitis and sepsis: A, B, C. Y and W135. For sero -
groups A, C, Y and W135 the use of capsular polysaccharides as vaccine components has shown
to confer protection in adults and infants. especially when the polysaccharide is conjugated with a
carrier protein. eliciting a T-cell dependent immune response and conferring long-term protection.
Unfortunately, this approach was not feasible for serogroup B, since its capsular polysaccharide is
a polymer of a(2-8)-linked N-acetylneuraminic acid that is also present in mammal tissues and
its use in a vaccine could lead to autoimmune responses,"
When many other conventional approaches failed to produce an effective vaccine against
meningococcus B, the reverse vaccinology approach appeared as a logical and promising alterna-
tive to deliver a vaccine. While the N. meningitidis genome sequence was still been assembled,
computer analysis allowed the prediction of proteins that could be surface-exposed or homolo-
gous to known factors associated with virulence and pathogenesis, leading to the selection of 570
potential vaccine candidates. Successful cloning and expression was obtained for 350 proteins in
Escherichia coli, which were then purified and used to immunize mice." lmmune sera were then
tested in ELISA, FACS and Western Blotting analysis in order to confirm their surface-exposed
localization and also in complement-mediated bactericidal assays to test their immunogenicity
and protective activity, since this assay correlates with protection in humans. Out of 81 proteins
found to be strongly positive in at least one of the mentioned assays, 28 showed to be positive in
all ofthem. To confirm the possibility ofusing these candidates in a vaccine able to protect against
heterologous strains , their presence and conservation were tested in a panel of 31 strains of N.
meningitidis isolated worldwide and over many years. Out of the 28 proteins tested, five showed
to be strikingly conserved in the panel, a result not quite expected since they are surface-exposed
proteins. In less then two years, reverse vaccinology achieved what the conventional vaccinology
approaches pursuit by decades: surface-exposed proteins in N. meninyitidis B, able to induce
protection and cross-reactivity among distantly related strains and serot ypes, suitable to be used
in a universal vaccine against this microorganism. Simple by extracting the benefits from genome
information and applying them in the development of novel vaccine s.
Comparative Genome Analysis: The Second Phase

ofReverse Vaccinology
The success obtained with-the N. meningitidis B experienceprompted the applicationof the
reversevaccinologyto other pathogens, such as Streptococcus pneumoniae, Porphyromonasgingi-
va/is, Chlamydia pneumoniae, Bacillus anthrads, Streptococcus agalactiae, Streptococcus pyogenes,
ExPEC and many others, becoming a routinely classical approach for vaccine development
(Table 2). But something unexpected happened when the completegenome of a virulent isolate
ofS. agalactiae (group B streptococcus)was sequenced. S. agalactiae is one of the leading causes
of bacterial sepsis, pneumonia and meningitis in neonates in the USA and Europe and also an
emerging causeof infection in the elderly. Conjugate vaccines based on the fivemajor capsular
polysaccharides were currently under development,but they were not able to cover all available
serorypes, Since conventionalapproachesfailedin providinga universaland efficient vaccinefor
the most affectedgroup and sincethe completegenomesequenceof two S. agalactiae strainswas
available in 2002, the classical reverse vaccinology approach sounded logical.But the S. agalactiae
experience wasgoingto be more challenging. In order to verifythe diversity ofS. agalactiae genome
and provide information for a future universal vaccineagainst this microorganism,comparative
genomic hybridization was applied, using the sequenced strain as reference. It was found that
approximately18%of the genesencoded in the sequencedstrain wasabsent in at leastone ofthe
other nineteen S. agalactiae tested.Theproblem is that comparativegenomehybridizationis able
to provide information only for the genomesequencethat isshared among the strains. Therefore,
specific genesin the other strains that are absent in the sequencedgenomecould not be detected.
It could be a problem for a universalvaccineachievement, leadingthe classical reversevaccinology
to evolve. Sequencingthe genome of only one strain could not be enough anymore to provide
the information needed for a universal vaccine development, especially when a high variability
is observed To provide the information requested, another additional six genome sequencesof
S. agalactiae were determined. This new information showed that 1806 genes are shared by all
strains of S. agalactiae, representing the 'core genome' that corresponds to approximately 80%
ofthe average number ofgenesencoded in each strain. The core genome mainlyencodesfactors
for functions that contribute to the major metabolic pathways, the so calledhousekeepinggenes
that usuallydefinethe identity of a species. The complementaryset of genes,absentin at leastone
strain, correspond to the 'dispensable genome', probably related to the adaptation of strains in
specificenvironmentalconditions byconferringselective advantages. Mathematicalextrapolation
predicted that, no matter how manystrainshavebeensequenced,eachnewgenomeshouldprovide
genesthat havenever been found before.Sequencingadditional genomesallowedthe estimation
Table 2. Examples of vaccines that havebeen developed usingreverse

vaccinology-based approaches
Statusof
Pathogen Disease Vaccine Development Ref.
Neisseria meningitidis B Meningitis and septicemia Phase II 7

Streptococcus agalactiae Septicemia, pneumonia and Preclinical 9,10
meningitis
Streptococcus pneumoniae Pneumonia Preclinical 11
Bacillus anthracis Anthrax Preclinical 12

Chlamydia pneumoniae Pharyngitis, bronchitis and Preclinical 13
pneumonitis
Porphyromonas gingivalis Periodontitis Preclinical 14

Genome-Based Vaccine Development:A ShortCutfor theFuture 85
s.
of agalactiae pan-genomesize(the set of genes that will be observedat leastonce if an infinite
number of differentstrainswould to be sequenced), corresponding to 2713 genes, of which 907
belong to the dispensable genomeand alsoallowedthe prediction that the pan-genomeisgoingto
growabout 33 newgeneseverytime a new strain issequenced. Thisprofileiscompletelydifferent
of those observed for other microorganisms, such as Bacillusantbracis. Eight genome sequences
weredetermined for this microorganism, but after the fourth it wasverifiedthat the number of
new genesadded to the pan-genomerapidlyconverged to zero.15•16
Comparative genome analysis also provided the information necessary to face the quest of
providing a universal vaccine against S. agalactiae. Computational algorithms predicted 589
surface-associated proteins, of which 396 belong to the core genome and 193 were absent in
at least one strain. Each of this protein was tested for protection and four antigenswereable to
elicit protective immune responses in the animal model, not only by passive immunization and
challenge in newborn mice but also active maternal immunization and challenge of offspring
within the first48 hoursoflife." None of theseprotectiveantigenscouldbe classified asuniversal,
because three of them wereabsent in a fraction of the tested strainsand the fourth, belongingto
the core genome, had a deficientsurface accessibility in some strains.17 The cocktailcombining
the four best candidatesconferred59-100% protection against a panelof 12 S. agalactiae isolates,
includingthe majorserorypes, aswellas two strains from a less common serotype." Without the
determination of additional genome sequences, a universal vaccine against S. agalactiae would
havebeen compromised. The comparative genome analysis provided new concepts in delivering
universal vaccines by the reverse vaccinology approach,evenfor microorganisms in which a high
variabilitycan be observed, opening the pan-genomicreverse vaccinology era.
Although the classical reverse vaccinology approach was efficient in providing protective
candidatesagainst serogroup B N. meningitidis, subsequent analysis showed that NadA, one of
those best candidates,ispresentin only 50%of strainsfrom patients with meningococcal disease
and only 25% of strains carried by healthy people, indicating that also the pan-genome of this
microorganism could havethe samefeatures of S. agalactiae pan-genome." It ispossible that the
availability of novelgenomesequencesof N. meningitidis couldprovideevenmorecandidatesthat
could increase the coverage and efficiency alreadyachieved.
Subtractive Genome Analysis: Third Phase ofReverse Vaccinology?

After the sequencingof the firstgenomein 1995,great effortshavebeen concentrated in the
determination of the genomesequences of other pathogenic bacteriathat could lead to the devel-
opment of novelvaccines basedon the reverse vaccinology approach such as the N. meningitidis
B and S. agalactiae experiences. Comparativegenome analysis of pathogenic strains (the second
generationof reverse vaccinology) haveconcentratedtheir effortsin comparingpathogenicstrains
of a species looking for the identification of antigensthat could lead to a maximumcoverage in
a universal vaccine. But what about nonpathogenic strains? It could sound strange to sequence a
nonpathogenic strain genome in order to obtain a vaccine against those pathogenic ones, but a
nonpathogenicgenomecouldprovidethe information necessary for the identificationofantigens
that really couldmakethe difference inpathogenesis, responsible for the moststrictly host-pathogen
interactions. In a subtractivecomparative genomeanalysis, genesconservedbetween pathogenic
and nonpathogenic strains of a same or even related species could be discarded, reducing the
number of candidates and, consequently, reducing the time for the delivery of a vaccine. Since
the abilityof causingdiseases isfrequentlyrelatedto the integrityof somegenes, algorithmsmust
take into account somegeneinactivationprocess such as frameshifiing. Ofcoursea whole set of
factorsare usually responsible for pathogenicity, therefore,alsoin this casethe sequencingof only
one nonpathogenic genomecould not be enough for a complete understandingof pathogenicity.
In some cases, lookingfor candidateswhere they do not existcould provide the answers that the
comparisonbetween pathogenic strains could not give.
The concept of commensal strains must be also clarified for the success of this approach,
since the term 'commensal' is usually associated with nonpathogenic strains or even probiotics.
Table 3. Sequencing genome projects of infectious bacterial speciesassociated with

human diseases
Organism Name GenomeProjects
Borrelia afzelii, Clostridium difficile, Fusobacterium nucleatum, Helicobacter 3

pylori, Mycobacterium bovis, Mycoplasma genitalium, Neisseria
meningitidis, Rickettsia rickettsii, Shigella dysenteriae, Shigella flexneri,
Vibrio parahaemolyticus
Acinetobacter baumannii, Borrelia burgdorferi, Burkholderia thailandensis, 4
Salmonella typhimurium, Shewanella putrefacien, Shewanella sp.
Burkholderia cenocepacia, Chlamydophila pneumoniae, Legionella 5
pneumophila, Ureaplasma parvum
Streptococcus suis 6
Pseudomonas aeruginosa 7
Clostridium perfringens, Mycobacterium tuberculosis, Streptococcus 8
agalactiae, Ureaplasma urealyticum
Clostridium botulinum 9
Burkholderia mallei, Coxiella burnetii 10
Bacillus anthracis, Campylobacter jejuni 11
Streptococcus pyogenes 13
Streptococcus pneumoniae 14
Francisella tularensis, Vibrio cholerae 15
Bacillus cereus, Staphylococcus aureus, Yersinia pestis 16
Haemophilus influenzae 19
Burkholderia pseudomallei 21
Listeria monocytogenes, Salmonella enterica 24
Escherichia coli 25
Commensalbacteriaarethosethat usually colonizean individualwithout causingdisease. Within

the commensalflorawecanfind the 'pathogeniccommensals' that havethe abilityto causedisease
when the organismis vulnerable. It is important not to confusethose 'pathogenic commensals'
(like S. pneumoniae and H. injluenzae B) with the 'nonpathogenic' ones {like some species of
Lactobacillus).18
The structure and composition of the flora is a result of selectionat both microbialand host
level, leadingto a mutual cooperation and stabilityof this complexsystem, allowingthe flora to
form a natural barrier that has numerousprotective (pathogen displacement, nutrient competi-
tion, receptorcompetition,etc),structural(barrierfortification, induction ofIgA,immunesystem
development,etc) and metaboliceffects {metabolization dietarycarcinogens, synthesis ofvitamins,
ion absorption, etc.)." Hence the delivering of vaccines against microorganisms that belong to
the commensalflorais not an easytask,evenfor the 'pathogenic commensals'. It has been shown
that polysaccharide heptavalentvaccine againstS. pneumoniae can lead to the replacement with
nonvaccine serotypes, but still able to cause infection and that the polysaccharide-conjugated
vaccineagainst H. injluenzae B led to the increase of serotypeF incidence. 2O•21 The consequences
of a vaccineagainstcommensal floracomponentsare usually unexpected,sincereducingthe level
ofcompetition could promote the emergingofother 'pathogeniccommensal." Consideringthat
Genome-BasedVaccine Development: A Short Cutfor theFuture 87
A) C1asalcalnwerH vKdnology
B) Pan.genome reverse vaeelnology
C) Subtractive roverse vaeelnology
Figure 2. Reverse vaccinology approaches. A) The classical reverse vaccinology approach

consists in min ing a genome sequence for the ident ification of putat ive surface-exposed
antigens that could be used as vaccine candidates. B) The pan-genome reverse vaccinology
approach compares different genome sequences of different strains to increase the cover-
age and to avoid the escape of the microorganism by antigen variabili ty. C) The subtractive
reverse vaccinology approach consists in comparing pathogen ic and nonpathogen ic genome
sequences in order to select those antigens that could be directl y involved in pathogenesis.
virulence , fitness and colonization factors could be overlapping and dependent of the niche and
environmental conditions, understanding the 'nonpathogenic' commensals could prevent th e
development ofvaccines with a minimum impact to the commensal flora, especially for microor-
ganisms that possessboth phenotypes, such as E. coli.
88 PharmaceuticalBiotechnobJgy
Conclusion
To date, 1,327bacterial genomes have beensequenced, of which 520 genomesequences have
alreadybeen completed. Table3 shows the number of genomesequencing projects of bacteria
that could be associated to human diseases." For most of them, no efficient vaccine is available.
In those cases, a comparative genome-based analysis would permit not only the better under-
standing of strain variability and pan-genome estimation, but also the delivering of a single or
combined-antigen universalvaccines in afield where conventionalapproaches have failed asproved
byN. meningitidis BandS. agalactiae experiences (classical and pan-genome reverse vaccinology).
Even forvaccines already available, theseapproaches couldpermitthe improvement andincrement
of protection byinclusion of novelantigens, when required. Moreover, the knowledge of antigen
genevariability coulddrivestrategies of rationalengineering to improvethe coverage with proven
efficacy. Consideringthe fact that nonpathogenic strainsarepresentfor manygenera, sequencing
of nonpathogenicgenomes couldbe alsoimportant in highlightingthe antigens that really could
make the difference in pathogenesis. Comparative genome analysis between pathogenic and
nonpathogenicgenomes would prompt straight to the genes responsible for pathogenesis and,
in most cases, responsible for the closest interactions betweenpathogenand host and, therefore,
good vaccine candidates (Fig. 2).
In conclusion, manydiseases can not be still controlledbyvaccination and more diseases are
expected toemerge.Aftertwohundredyears ofvaccine research anddevelopment, genome sequenc-
ingprovidedcrucialinformationrequiredto defeatinfectious microorganisms. Nevertheless, we
need to learn to decipherthe informationgeneratedin order to convertit in protective vaccines.
Classical reversevaccinology approach madethefirstmoves, beensuccessful forthedevelopment of
avaccine againstN. meningitidis B. In spiteof that,it had to evolve in orderto besuccessful against
amorecomplex microorganism likeS. agalactiae. And it stillneedsto evolve,providingnoveltools
that will avoidthe continuingescape of microorganisms from host defense mechanisms.
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CHAPTER 9
The Antigenome:
From Protein Subunit Vaccines to Antibody
Treatments of Bacterial Infections?
Carmen Oiefing, EszterNagy and Alexandervon Gabain*
Abstract
ew strategies areneeded to masterinfectiousdiseases.Theso-called"passive vaccination",
N i.e.,preventionand treatment with specific antibodies,hasa provenrecordand potential

in the managementof infectionsand entered the medicalarenamore than 100yearsago.
Progress in the identificationofspecific antigenshas becomethe hallmarkin the developmentof
novelsubunit vaccines that often contain only a singleimmunogen,frequentlyproteins, derived
from the microbe in order to induce protective immunity. On the other hand, the monoclonal
antibodytechnology has enabledbiotechnology to produceantibodyspecies in unlimitedquantities
and at reasonable coststhat are more or less identicalto their human counterparts and bind with
high affinityto only one specific site of a givenantigen. Although, this technologyhas provided
a robust platform for launching novel and successful treatments against a varietyof devastating
diseases, it isup tillnowonlyexceptionally employed in therapyof infectious diseases. Monoclonal
antibodies engagedin the treatment of specific cancersseemto work by a dual mode; they mark
the cancerous cellsfor decontamination by the immune system, but also block a function that
intervenes with cell growth. The availability of the entire genome sequence of pathogens has
stronglyfacilitatedthe identification ofhighlyspecific protein antigensthat aresuitabletargetsfor
neutralizingantibodies,but alsooften seemto playan important role in the microbe's life cycle.
Thus, the growingrepertoire of well-characterized protein antigens will open the perspective to
developmonoclonal antibodiesagainstbacterial infections.at leastaslast resort treatment, when
vaccinationand antibioticsare no options for prevention or therapy. In the following chapterwe
describeand comparevarioustechnologies regardingthe identificationof suitabletarget antigens
and the foundation ofcognatemonoclonal antibodies and discuss their possible applications in
the treatment ofbacterialinfectionstogether with an overview of current efforts.
Introduction
Infectiousdiseases remain a major threat againsthuman life.Microbialinfectionsare stillout
ofcontrol in manyparts of the less developedworldwhere they count for most of the deaths,but
alsocausean often underestimatedtoll ofdeath [e.g.,communityacquiredPneumococcal diseases
and Pseudomonas infectionsin patients in intensivecare), life-longmutilation (infertilitydue to
Chlamydia trachomatis), medicalcomplicationdue to nosocomialinfectionscausedmostoftenby
Staphylococcusaureus, Enterococcusfaecalis,Klebsiella sspand fungi.It isestimatedthat nosocomial
infectionsannuallyadd US$5-10 billionto the costof the nationalhealthcaresystem in the United
States,' Apart from infections causedby viruses and protozoa that only in specific instancescan
*Corresponding Author: Alexander von Gabain-Intercell AG, Campus Vienna Biocenter 2,
A-l030, Vienna, Austria. Email: agabain@intercell.com
Pharmaceutical Biotechnology, edited by CarlosA. Guzman and Giora Z. Feuerstein.
The Antigtnome 91
be treated with suitable pharmaceuticals, the emergence of antibiotic-resistant strains of nearly

all kinds ofbacterial pathogens in the community and in hospitals is occurring at an increasingly
alarming rate. 2,3 The increase of nosocomial infections, the comeback of bacterial infections in
immune suppressed individuals, e.g., TB in AIDS patients," and the lately appeared scenario of
bio-terrorism, e.g., in the context of anthrax,S,6 are reminders that new strategies are needed to
master infectious diseases in prophylactic and therapeutic settings.
Vaccination is undeniable the most successful medical intervention in the control ofinfectious
diseases. However, since vaccine-induced immune protection against specific microbes takes more
than a couple of weeks to develop and postexposure vaccination is only exceptionally a useful
tool, combination with passive immunization is indispensable (e.g., treatment against the rabies
virus. reviewed in ref 7). when instant protection or treatment is required. Therapeutic vaccines
are still in the exploratory stage of development and more prone to find their application in the
treatment ofchronic infectious diseases,8.9rather than to become an immediate measure against a
sudden infectious threat. On the other hand. most vaccines seem to confer protective immunity
to the vaccinated individuals by the means to induce specific antibodies that capture the invading
microbe. prior it had an opportunity to colonize in the exposed host. The so-called "passive vac-
cination" i.e., prevention and treatment with specific antibodies, has a proven record and potential
in the management of infections.
Already the pioneers of early microbiology and immunology in the late 19th century, led
by their prominent proponents. Emile Roux and Emil von Behring, have realized the concept
of "passive vaccination", namely that sheep and horses inoculated with filterable toxin extracts
derived from Corynebacterium diphteriae cultures were able to mount an "anti -toxin" in their
blood. Serum derived from the animals' blood was able to rescue children in the lethal stage
of the infection caused by the same pathogen. Revisiting this historical landmark therapy of
diphtheria, it was realized that the "anti-toxin" in the serum ofinoculated animals is synonymous
with a protein species coined today antibodies and the "toxin" with a virulence factor secreted
by the pathogen during infection. Thus , the remarkable and groundbreaking therapy concept
explored more than 100 years ago, has paved the way to "passive immunization". i.e., all kind of
serum-treatments that have found their broad medical applications in prevention of e.g.•viral
infections or in emergency treatments against e.g., snake venoms." :" Serum antibodies against
microbes and even isolated antigens, like the diphtheria toxin. are polyclonal, meaning that they
bind-in case of a specific antigen molecule-to a variety of sites or-in case of a microbe-to
multiple surface structures.
The advent of the monoclonal antibody technology launched by Georg Kohler and Cesar
Milstein nearly 30 years ago, has enabled biotechnology to engineer specific antibody species
that bind with high affinity to only one specific site of a given antigen and can be produced in
unlimited quantities. Follow-up technologies made it possible to produce monoclonal antibodies
that are more or less identical to their human counterparts, employing microbial and tissue culture
resources for manufacruring." During the last decade, monoclonal antibodies have infiltrated the
therapeutic arena with great success and thereby provided a plethora of novel treatments against
a variety of typically devastating diseases including specific cancers, autoimmune diseases and
other pathological conditions." The common denominator of all monoclonal antibodies used
in therapy is to bind to highly specific sites of typically well characterized protein targets and
thereby intervene with biological functions involved in the pathogenic condition; e.g., to growth
hormone receptors expressed at the surface of malignant cells. 14•l s Interestingly, monoclonal
antibodies engaged in the treatment of specific cancers seem to work by a dual mode; they mark
the cancerous cells for decontamination by the immune system , but also block a function that
intervenes with cell growth. 16
Progress in the identification ofspecific antigens has become the hallmark in the development
of novel subunit vaccines that only contain single specific structures derived from the microbe
in order to induce protective immunity. The first viral subunit vaccine on the market that has
become a great success is directed against Hepatitis B virus and based on recombinant protein
92 Pharmaceutical Biottchnology
technology. Alsopathogen-specific glycosides coupledto carrierproteins aresuccessfully usedin

so-calledconjugatedvaccines directedagainstbacterialinfections; an example is"Prevnar" a regis-
tered vaccine againstPntumococcus.17The successful development ofsubunitvaccines comprising
isolatedmicrobialcomponentsas antigens has supported the notion that antibodiesper se, may
suffice to neutralizepathogensin the body evenin a setting of"passive vaccination". So far only
one anti-infective monoelonalantibody,whichisdirectedagainstthe Respiratory Syncytial Virus
(RSV) (Palivizurnab), has entered the therapeuticarena." A number of anti-infective antibodies
based on specific antigensagainstbacterialinfectionsare in the stageof clinicaland preclinical
development(Table1).
Theavailability of the entiregenomesequence ofpathogensand subsequently the application
ofproteome and genomebasedtechnologies havefacilitatedthe identificationofhighlyspecific
protein antigens suited for the development of novel bacterial subunit vaccines," One of the
recentlydescribedmethods designedto comprehensively mine bacterialgenomesfur protective
antigens, has taken advantageof antibodies derived from humans who have encountered the
target pathogen with positiveoutcome. The sum of all protein antigens that are recognizedby
cognateantibodiesfrom individuals exposed to the pathogenhasbeendefinedasantigenome.2ll-22
Typicallythe antigenome comprises100 to 200 antigens. Applyinga number ofselective filters
and criteria to the antigenome, in vitro validation makes it possibleto reduce the number of
best-suitablecandidateantigensfor vaccinedevelopmentto about IS to 30 (unpublished data).
Such antigensare presentlytested in advancedpreclinicaland earlyclinicaltrials (Kuklin et al23
and unpublished data).The availability of bacterialprotein antigenswith promisingprofilesfor
vaccinedesign,but also the identification of specific host targets,haveprovided novelgates to
developmonoelonalantibodiesfor protection and treatment againstspecific infectiousdiseases.
In the followingchapter we willdiscuss the impact of discoveryand characterizationofspecific
antigens on the development of novelvaccines and antibody treatments.
A New Paradigm in Bacterial Vaccine Development

The capabilityof the human immune system to identify and eliminatepathogensand path-
ogen-infected cells is the cornerstone of immunization, the most effective strategy to prevent
infectious disease. However, vaccines are still not available against major pathogens including
Meningococcus serogroup B, Gonococcus, Helicobacter pylori and Shigtlla. Traditional vaccines
are mainly based on inactivated or attenuated microbesor more recentlyon polysaccharides of
a particular pathogen. Due to the fact that such vaccines cannot prevent numerousdiseases, or
evenworse,inducesevere sideeffects,noveland definedvaccines arebeingdeveloped to overcome
these limitations. Improvedvaccines are needed to combat diseases for which current vaccines
are inadequate (e.g., tuberculosis) or against pathogens that had not been on the rarget list fur
immunization,such as Staphylococci and Enterococci both with an enormouspotential to develop
drug resistance.24,2SThe recentlyemergingthreat ofbioterrorism booststhe need for newvaccines
further.
Most of the newgenerationvaccines comprise subunitsofpathogens(purifiedprotein, toxoid,
polysaccharide with or without conjugation) and havemademajorheadways in controllingserious
diseases. At present, thereareonlytwovaccines basedon recombinantproteins(againstHepatitis
B and Lyme disease) that areshownto be effective in preventinghuman infections. Nevertheless,
protein based recombinantvaccines are considered to be the most promisingapproach to meet
the demandsoffuture vaccinology.
In order to designnovelsubunit vaccines, the proper antigenshaveto be identifiedand sub-
sequentlyevaluatedin experimental animal models mimickinghuman diseases. While vaccine
developmentfor obligatepathogenswith well-defined virulence mechanisms has progressed well,
those bacteriathat are in the focus of current vaccine efforts (e.g., opportunistic pathogensand
those with multipleserorypes) havemore complexpathogenesis.
Vaccinologists are witnessinga remarkable revolution in technologies that now contribute
to rapid identificationof novelvaccinecomponentsagainstmanyImportant human pathogens.
~
Table 1. Therapeutic antibody approaches against bacterial infections ~
~.
Drug Pathogen Antigen Target Type of Antibody Highest Phase Originator Indication ~
;;I
"
Aurograb '" S. aureus A BC transporter Huma n-de rived single III NeuTec Pharm a M ethi cill in -resistant
chain variab le S. aureus infections
fragment (scFv) the rape utic
antibody
Altastap h' S. aureus S. aureus Type 5 Polyclonal (5% Ig prepa red II Nabi Staphylococca l
and Type 8 from donors imm unized Biopharmaceuticals infections
po lysaccharides wi th StaphVAXTM)
Pagibaxi mab Staphylococcus Lipoteic hoic Chimeric mAb II Eli Lilly/Bio synexus Staphylococcal
spp. acid (LTA) infect io ns
S. epidermidis
Aurexis'" S. aureus S. aureus ClfA Hum anised mAb II Inhibitex Treatment- in
co mbi nation wit h
standard-of-care
antib iotics-of serio us
S. aureus infec tions in
hospitalized patients
Veronate'" S. aureus Staphylococca l Human polyclona l Preregistario n Inh ibitex Staphylococca l
fib rinogen-binding infectio ns in VLBW
protein s SdrG (very low birth w eight)
and C1fA infants: Prevention of
(MSCRAM M) hospitaI-associ ated
infections in pre mature
infa nts we ighing less
than 1,250 grams
~
~
Table 1. Continued
Drug Pathogen Antigen Target Type of Antibody Highest Phase Originator Indication
ETI211 S. aureus S. aureus protein A Antibody conjugate: Precl in ical Elusys Therapeu tic s Methicillin-resistant
anti-human comp lement S. aureus infecti ons
receptor Type-1 mAb
chemically cross-linked
w ith an anti-So aureus
protein A mAb
S. aureus S. aureus IsdB Fully human mAbs Precl in ical MerckJlntercell Staphylococca l
mAb infections
SdrC mAb S. epidermidis SdrC -fibrinogen- Human polyclon al Prec lin ic al Inhibitex S. epidermidis infecti o ns
binding MSCRAMM
prote in
Enterococcal Enterococcus MSCRAMM«> Fully human mAb s Precl ini cal Inhib itex/Dyax Drug-resistant
mAb proteins entero co ccal infect io ns
IC47 S.pneumoniae Surface proteins Fully human mAb s Preclini cal Kirin/lntercell Pneumo co ccal infect ions
therapeuti c in immunocompromi sed
anti bodies patient s
KBPA 101 P. aeruginosa Directed against Fully human mAb s II Kenta Biote ch Pseudomonal
pseudomon al infecti o ns
~
serotypes
Anti-P. P. aeruginosa Natural human Fully human mAbs I Berna Biotech Pseudomonal
'~"
~
aeruginosa immune response infect ions 11:
mAbs ~.
......
~
Anti-P. P. aeruginosa Undisclosed Fully human mAbs Precl ini cal Millenium Biologix Pseudomonal t·
aeruginosa Corporation infect ions s..~
mAbs :s
"
continued on next page ~
~
Table 1. Continued I "~
~.
Drug Pathogen Antigen Target Type of Antibody Highest Phase Originator Indication
MDX 066 Clostirium Toxin A Fully hum an mAb s II M edarex, Universit y C. difficile infection s,
II
difficile of M assachu sett s Di arrh ea
M edical Schoo l
MDX 1388 C. difficile Toxin B Undisclosed Preclinical M edarex, MB L C. difficile infec tions
DiffGAMTM C. difficile C. difficile toxins Bov ine immu noglobuli ns I Imm uCell C. difficile infect ions
Anti-botul ism Clostridium Type A-b otuli num mAbs Preclin ical XOMA Biological agent s used in
neurotoxin botulinum neurotoxin bioterrorism
mAbs
Urtoxazumab Shiga-l ike B-subunit of Recom bi nant II Teijin Pharm a haem olytic uraem ic
tox in -pro du cing shiga-like toxin hum anised mAbs syndro me
E. coli II (SLT II)
Raxibacum ab B. anthra cis B. anthracis Fully human mAbs I Hu man Genome Anthrax
protective antigen Sciences
Valortirn' B. anthra cis B. anthracis Fully human mAbs I M edarex A nthrax
protective antigen
Anthirn" B. anthracis B. anthracis Heteropolymer chemica lly I Elusys Therapeuti cs Anthrax
protect ive antigen linked mAb
Afelim om ab Septic shock human TN Fa. Murine mAbs III Ab bott GmbH & Co. sept ic shoc k
KG
Ant i-TNFo. Sept ic shoc k human TN Fo. Sheep pol yclonal II Protherics septic shoc k (amo ng
(amo ng ot hers) others)
--
Data w ere co llec ted fro m the Ad is R&D Insight database and homepages of listed companies in July 2006 using the wo rld w ide web. Antibodies were sorted
according to antigen target: first, antibo dies targeting pathogen surface structures are listed, fo llowe d by anti -tox in anti bodies and last antibodies against TNFo..
M onoclonal Antibo dies are abbreviated with mAbs. This is not a co mplete list.
~
96 PharmauutiaJBiotechnology
The availability of complete genome sequences of pathogens has dramatically changed the
perspectives for developing improved and novel vaccines by increasing the speed of target
identification. Genomics-based technologieshave many advantages compared to conventional
approaches.which are time-consumingand usuallyidentify only abundant antigensexpressible
under in vitro culture conditions. Strategiesbasedon genomicshavemade major contributions
to the identification and selection of novel vaccine candidates to combat bacterial infections
by exploiting genome sequence information in alliance with adjunct technologies. including
in silico prediction (bioinformatics). expression analyses (random mutagenesis. microarrays,
in vivoexpressiontechnologies). or protein/peptide based selection methods (proteomics and
immune-selection usingpeptide expression libraries). Although. most technologiescan be read-
ily applied to most pathogens. certain strategiesare more suitable than others due to distinct
advantages and limitations.
The most promising candidate antigens haveto be (1) expressed during human disease; (2)
accessible (surfacebound or secreted) for functional antibodies or effector immune cells; (3)
conservedamongstrains; (4) essential for in vivosurvival in order to avoidcounter selection;and
(5) protective in animal models mimickingthe relevant human disease. There is no technology
available today that can selectantigen candidatesfulfilling all five attributes.However. a compre-
hensiveselectionprocedure meetingthe keycriteriacan be combinedwith a validationscreening
that addresses the remainingrequirements.
To date. approximately 300 pathogen genomesequences havebeen determined (http://www.
tigr.orglcmr). Genome sequences of bacterialpathogens contain an average of 2700 genes. thus
appropriate selectioncriteriahaveto be applied to reduce the number of antigen candidatesfor
empiricaltesting.Bioinformatics has been successfully employedfor the prediction ofcandidate
antigensofextracellular pathogens.due to the specific features easingthe predictionof cellsurface
and secreted proteins and/or the identification of genes that show sequenceand/or structural
homology to known virulencefactors. 26 This type of genome-based systematic searchfor vaccine
candidateswastermed"reverse vaccinology".The validityof thisapproachwasfirstconfirmedbythe
identificationofprotectiveantigensfrom Meningococcus serogroupBand laterfrom Pneumococcus
(reviewedin ref 27). "Reverse vaccinology in silicoprediction" typicallytargetsup to 25%ofall
genome-encoded proteinsand. thus,necessitates subsequenthighthrough-putcloningand recom-
binant protein expression.Inclusionof more restrictive selectioncriteriabecamepossiblethrough
the availability of several genomesfor individual pathogenic species. Comparative genomics is
another suitabletool to identifygenes sharedamongspecies of relatedpathogensor. alternatively.
to identify genespresent only in pathogenic, but not in attenuated or narurallynonpathogenic
strains or species. Such approaches havebeen successfully applied to Group A Streptococcus and
Mycobacterium,28-30
Thehallmarkofeffective vaccine antigensistheir abilityto induceantibodiesand/or to activate
immune cells. Regardingthis feature, in silicoprediction of antigenicityis stillin infancy. It is an-
ticipatedthat with the wealthof knowledge currentlybeinggenerated. it will bepossible to develop
prediction algorithmsto pinpoint proteins likely to be immunogenicand/or protective." More
advanced is the strategyto mine genomicsequencedatabases of intracellularpathogensfor pre-
dicted T-cellepitopesand validatethem experimentally basedon immunerecognition.32J 3 Despite
all successes. the bioinformaticgenomemining approach has limitationsdue to the inaccuracy of
available algorithms. regardingthe prediction of (1) open-readingframes that encode proteins;
(2) surfaceand secretedproteins; (3) genefunction basedon homologysearches. Moreover. it is
almost impossible to predict the conditions under which candidateantigensareexpressed. unless
the genesare equipped with well-definedregulatorysequences and promoters.
The availability of complete pathogen genome sequences stimulated the development and
wide-spread application of high density DNA-arrays. Comparative microarray analysis identi-
fies genomic diversityand conservationpatterns among bacteria.The developmentof vaccines
cross-protective among serotypes and variantsof pathogenicspecies specifically profitsfrom this
The Antigenome 97
analysis, as it was demonstrated by the identification of common genes and protective antigens
from major serotypes of Streptococcus agalactiae.34,35
Profiling of genomic expression with microarrays has revolutionalized the analysis of genes
involved in microbial pathogenesis (reviewed in ref. 36) . Considering its value in vaccine devel-
opment, the emphasis is focused on pathogen-host interactions. In several studies novel vaccine
candidates were identified, based on requirement for infectious state and dissemination, adhesion
or evasion of innate defense mechanisms.Fr'?This approach-that heavily relies on genome an-
notation and bioinformatics-is most powerful in providing a global view on integrated cellular
processes active during infection. Again, it has to be followed by combined application of gene
cloning, recombinant protein technology and in vitro functional assaysto validate target selection
for vaccine development.
Proteome analysis has rapidly developed in the postgenome era and is now widely accepted
as a complementary technology to genetic profiling (reviewed in ref AI). The most direct way
of using proteomics technologies for antigen identification is the combination of conventional
proteome analysis with serology. There have been a number of recent studies investigating the
"immunoproteome" ofirnportant human pathogens (for an example see Haas et al42) . Combining
"reverse genomics" and proteomics isespeciallyuseful for confirmation ofbioinformatic prediction
of0 RFs and surface location. Moreover, a strong asset ofproteomic studies is the identification of
surface located proteins that cannot bepredicted by bioinformatic means.43,44 Serologicalproreome
analysis of enriched membrane and cell wall fractions from several pathogens, such as S. aureus,
Bacillus antbracis and S. agalactiae has indeed demonstrated to identify novel surface antigens
and protective vaccine candidates without sequence features that could have been recognized by
in silico prediction algorirhms.v"
The design ofproteome-based studies has to be carefully performed, since there is an inherent
risk to preferentially detect abundant proteins and to miss those that are expressed only under
in vivo conditions and have lower solubility (e.g., membrane and surface proteins). Another need,
not necessarilymet by proteome analysis,is that protective vaccine components have to be derived
from proteins expressed under disease conditions against which prevention is directed. As many
virulence factors and antigens are only expressed in vivo, approaches that solely rely on in vitro
grown bacteria are likely to miss important protective antigen s.
Evaluation of immune responses against any candidate antigen is a crucial validation task
and cannot be circumvented. Therefore, techniques using human immunogenicity as their pri -
mary screening and selecting parameter on a genome-wide basis seem to be especially valuable
for vaccine development. Recently a novel approach combining the advantages of full genome
coverage and serological antigen identification was published. The method was first applied to
the genome-wide identification of in vivo expressed antigens from S. aureus by using antibod-
ies from human serum and comprehensive small-fragment genomic surface display libraries .P
Subsequently, the technology was extended with an integrated approach for antigen validation
as selected clones are directly subjected to generation ofepirope-specific immune sera for surface
localization and in vitro functional assays. This feature allows the analysis of antigens without
the demanding task ofhigh through-put recombinant protein production. This method, named
antigenome technology, has been extended to many important human pathogens and validated
by the discovery ofnovel and highly protective antigens, in addition to the identification ofthe
majority of the one s that have been previously described.P Since the antigenome technology
provides a subset of all genome-encoded proteins, which are expressed by the pathogen in vivo
and induce antibodies in humans, the identified antigens fulfill major requirements ofvaccine
candidate antigens. It is interesting to note that the antigens confined by the antigenome seem
ofien to be involved , as secreted and surface bound proteins, in virulence functions and , thus ,
being attributed to the "parhosphere" that has been defined as the growing gene pool in which
pathogens meet and mingle to cause diseases.48lt is observed that many ofthe identified antigens
from various pathogens were not or only very weakly expressed under in vitro growth conditions,
indicating that a proteomic approach that preferentially selects abundant proteins would likely
fail to identify them. As the bioinformatic genome mining approach depends on the accuracy
of available algorithms, potential vaccine candidates can be missed due to a misleading or not
existing annotation. Based on the analysis of the antigenomes of fifteen pathogens. approxi-
mately 25% ofall identified antigenic proteins can only be assigned to hypothetical proteins or
proteins with unknown function. Many ofthe identified antigens would, thus. be not be found
by a bioinformatic approach. The cumulative data obtained fur the fifteen antigenomes showed
that a large fraction ofthe antigens identified by this method represents cell surface or secreted
proteins. Nearly fifty percent of all antigens fell into four cellular role categories: cell wall, cel-
lular processes, transport and binding proteins and determinants of protein fate. In order to
pinpoint candidates for vaccine development, a comprehensive and rapid validation strategy
to retrieve the most promising antigens from the 100-200 antigens was implemented. Clones
selected from peptide display libraries are directly subjected to generation of epitope-speci6c
immune sera used fur testing of surface localization and in in vitro bactericidal assays.The hu-
man immunogenicity of identified antigens is evaluated with synthetic peptide epitopes. The
application ofthese major selection criteria combined with traditional gene conservation studies
reduces the antigenome to a small number ofcandidate proteins that can be rapidly expressed in
recombinant form fur subsequent in vivo studies. The re-idenrification ofmost ofthe previously
identified protective antigens of Staphylococci and Streptococci. such as PspA. M1 protein. Sip
and ClfA gives further supports the power of the antigenome technology. Most importantly.
novel protective proteins yielding animal protection in animal vaccine models. were found in
the prioritized groups of antigens derived e.g., from S. aureus23 and Streptococcus pneumoniae
(unpublished data) , respectively. Thus, the utilization ofprotective antigens-included in sub-
unit vaccines-as targets for monoclonal antibodies, provides an attractive strategy to develop
novel treatments against life threatening infections. Such a notion is supported by recent data
showing that protection can be conferred to naive animals, using serum directed against target
antigens that have been validated in vaccine models (Nagy et al, personal communication).
The Advent ofMonoclonal Antibodies in Disease Treatment

The renaissance of antibody therapy since the mid-1990s was mainly possible through sig-
nificant improvements in antibody generation and purification (Fig. 1). The first step towards
nowadays production technologies was the description ofthe unlimited generation ofmonoclo-
nal antibodies by Georges Koehler and Cesar Milstein in 1975,49for which they were awarded
the Nobel Prize in 1984. They fused mou se myeloma cells with normal antibody-producing
splenic B-cells isolated from mice that were immunized with sheep red blood cells as antigen.
The resulting hybridoma cells possessed the immortal propagation potential of the myeloma
cells and secreted anti-sheep red blood cell antibodies. Selected clones could then be cultured
indefinitely and secreted large quantities ofmonoclonal antibodies.
Despite their success as research tools, mouse monoclonal antibodies as human therapeutics
are limited fur various reasons. The main problem is the high immunogenicity of these foreign
proteins in humans resulting in fast clearance (short halflife) and toxicity by human anti-mouse
antibodies (HAMAs).50 Moreover, mouse antibodies have a reduced effect in human recipients
due to their nonoptimal interactions with human complement and F, receptors,"
In the early 1980s strategies for chimerization and humanization were ensued to overcome the
limitations ofmouse monoclonal antibodies. Chimerization demands the joining ofthe variable
regions of mouse antibodies with the constant domains of human immunoglobulins that takes
advantage ofrecombinant DNA techniques resulting in chimeric antibody derived from mouse and
human antibody genes." Although being less immunogenic than murine monoclonal antibodies,
human antichirneric antibody responses have even been reported for chimeric antibodies. 53 To
further reduce the undesirable immune response and confined inactivation, the mouse segment
within the humanized monoclonal antibodies has been restricted to the complementarity deter-
mining regions (CDR) in CDR-grafted "humanized" anribodies.Yln order to humanize a mouse
monoclonal antibody, the closest matching human immunoglobulin allotype is first identified by
structural comparison.55.56Then recombinant approaches are used to graft the CDRs from mouse
hybridomas to the corresponding selected human immunoglobulin framework. As a result, the
~
~
~.
~
<>
~
COR·grafted Snllbodles '"
(Jonese/a/ ., fOOB)
Transgenic
hlm8n
srclbodles
(Gnlen et et;
Mouse monoc Ionel f99f)
ertibodles (Kilhler
Spednc & Ws/ein, fP75) I
InltOducllon 01 Chimer1c recombinent PIlege displ8'i
ertibodies
preted against ettirricroblsl flnlIbodies(Mooison el
~f flnlIbodies
ch&lOOlherspy a/., fOOf) (McC8trel1y el a/.,
b8cteri8l1o>dns
(Behring &
Kilaulo, f89lJ)
! 1
- - - - - - - - - -............ 'C
I j _.....
r
1890 1935 1975 1980
'---v--"
1890-193S: Senm therapy
lor Ire8lmentlor \'Ilrious
Inledious diseeses
Figure 1. Evolution of antibody therapy and application in the c1inic.49 ,s 2,s 4,61,71 After extensive use of anti serum therapy at the beginning of the last
century it was almost abandoned until the advent of monoclonal antibodies. In general new monoclonal antibodies were approved by the FDA 10
years after the development of a new technique was reported in the literature. Palivizumab is the only momodonal antibody currently used in the
'0
clinic that targets an infectious disease. '0
100 Pharmaceutical Biouchnology
antibody only contains the antigen binding regionfrom mouseorigin.whilethe remainderof the
variableand constant regionsis derivedfrom a human source.
While routine mousemonoclonal antibody production has been established. human mono-
clonal antibodies cannot be generated by conventionalhybridoma technology, sinceit was not
possibleto found human celllines that secreteconstantlyhigh levels of antibodiesand, further-
more. humans cannot be challengedwith all kind ofantigens,due to ethical and safetyreasons.
Nowadays. phagedisplaytechnology(reviewed in refs. 57-59) and transgenicmicewith a human
antibody locus (reviewed in ref 60) represent established, widespread and robust technologies
that allowthe generationof potent human antibodies.
Phage displaytechnologies enable in a simple to use and highly versatile procedure for the
selectionof antibodiesagainstknown or novelantigens.The phage displaylibrary (firstdescrip-
tion by McCaffertyet al61) represents a collectionof independent clonescarryinga foreignDNA
sequence encoding an antibody domain expressed as a fusion with the coat protein of mainly
filamentous bacteriophages. as M13 or Fd (reviewed in ref 62). Monoclonal antibody libraries
can be recruited from immune fragments that are already biased towards certain specificities
(encoded in the genome of immunized or infected animalsor humans),or naiveunbiasedfrag-
ments that can be derivedfrom nonimmune natural or semi-synthetic sources, bypassing the need
for previousimmunization. Byapplyingthe best suitableselectionprocedures, those phagesthat
bind to the target antigen with highest affinityare retained.The phagesareenriched by selective
adsorption to an immobilizedantigen ("panning") (reviewed in ref. 12); howevervariousspecial-
ized screening techniques exist.57.6~5 Phage display provides the opponunity to mimic human
immune response, alsobecauseof the high degreeof natural variationsfound in the replicationof
the phagegenomes.66 B-cellmaturation in vivorequiresrecombinationofgermlinegenesegments
accompaniedwith changes and mutations that can be imitated in vitro by DNA random cloning
ofVH and VL chain genes. 67The somatichypermutation processthat naturally contributes to
the affinitymaturation of antibodiescanbe achieved artificially byinsertingpoint mutations into
genesegmentsofcomplementaritydetermining regions. 68.69
A method to circumvent the laborious steps of founding humanized and to obtain directly
human monoclonal antibodies wasdevelopedbyengineeringtransgenicmicewith a human im-
munoglobulin locusassourcefor antibodyproducing hybridomacelllines.(reviewedin ref. 60).
Already in 1985 Alt et al proposed to exploit transgenicmice for the generation oftherapeutic
antibodies." and as soon as 1994 the Xenolvlouse" (Abgenix, Inc.FI and the HuMAb Mouse"
(Gen-Pharm-Medarex}" were reported to be the first mice carryingboth the human VH and
VL repertoire createdvia pronuclear microinjectionor yeastprotoplast fusion with embryonic
stem cells, respectively. For monoclonal antibody generation B-cells are isolated from immu-
nized mice and fused to hybridomas,in a similarmanner to the traditional mouse monoclonal
antibody production. By employing rnicrocell-mediared chromosome transfer-a technique
capable to transfer very large fractions of the human germline-Tomizuka et al generated a
chimeric mouse-TransChromo Mouse" (TC Mouse™) carryinghuman chromosomes2 and
14 regionscontaining the human K -light-chainand heavy-chain loci?3,74 In order to increasethe
lowefficiency of hybridoma production due to instabilityof the IgKlocus,the KM MouscTM was
created by cross-breeding the Kirin TC Mouse™ with the MedarexYAC-transgenic mouse."
These mice possess the capability to carry out VDJ recombination. heavy-chain class switch-
ing and even somatic hypermutation of human antibody genesin a normal mode to generate
high-affinityantibodies with completelyhuman sequences.f The resultingantibodies exhibit a
half-lifesimilar to natural human antibodies" and show only differences in glycosylation pat-
terns, thereby representinga major improvement in hybridoma technology." Although human
monoclonal antibodies derived from transgenic mice havenot yet paved their wayup to FDA
approvaland registration.so far clinicaltrialswith them havenot revealed adverse immunogenic
sideevents in patients.71l•w in contrast to chimeric,CDR graftedor phagedisplayderivedmono-
clonal antibodies." However there is still a need for confirmingthese promisingdata by testing
transgenic mousederivedantibodies in largersubject cohorts.
The Antigenome 101
On the other side,the success of phagedisplaytechnologies in mimickingthe in vivoantibody

selectionprocessin essence has led to intensive explorationof possible improvements, mainlyin
the fieldof new display techniques. All these new selectionplatformssharefour major steps: (1)
the creation of genotypicdiversity; (2) the linkagebetweengenotypeand phenotype; (3) the ap-
plication of a screeningprocedure; and (4) the amplification of the selectedbinding sites.
In the Ribosome and mRNA display method,82 the antibody and its encoding mRNA are
linkedbythe ribosomewhichismadeto stop without releasing the polypeptide.8lo85 Theuseofe.g.,
nonproof-readingpolymerases providesadditional diversitybetween generations and therefore
represents a verysuccessful technique in the fieldof antibody affinity maturation.f
Theattempt in displaying antibodieson the surface ofdifferentmicrobes hasonlybeensuccess-
ful so far, when employing the yeastSaccharomyces cerevisiae.87 Antibodiesaredisplayed viafusion
to the a-agglutinin yeastadhesionreceptoron the cellwalland selectioncan be accomplished via
flowcytometriccellsorting.Besides yeast display, alatelydescribed.&cherichia coli basedapproach
is currentlyunder development/"
Recently developedantibodyplatformtechnologies includeretroviraldisplay,"protein-DNA
display,90 microbead displayby in vitro cornpartmenralizarion," in vivo growth selectionbased
on protein fragment complementation'?and other techniques." However, their advantages over
more establishedsystems remainto be demonstrated.
One problem in the applicationof monoclonal antibodies lies in their restriction to a single
specific epirope,limiting their ability in eliminatingdynamicand evolving targets and retaining
activityin the event of antigen mutation. A new generation of therapeutic antibodies that may
overcome the restriction of monoclonal antibodies is the development of a recombinant poly-
or oligoclonal antibody technology," For the generation of "Syrnphobodies"-fully human,
antigen-specific recombinant polyclonal antibodies-antibody producing cells are isolatedfrom
naturally immune donor blood. cDNA encoding human heavyand light chains are amplified
and linked together by Symplex PCRTM; pooled PCR products are then inserted into an expres-
sion vector and screenedfor antigen binding. Constructs expressing the selectedantibodies are
cloned into Chinese hamsterovarycells wherethey are site-specific integratedinto the genome."
Thus, such adevelopmentof a human antibody repertoiremirrorsthe human polyclonalimmune
response againstspecific antigens.
Besides the fact that the recombinant expression of antibody genesis often difficultbecause
of their large size, the usage of whole immunoglobulins sometimes causes undesiredside effects
that are mediated by the Fcpart of the antibody.To overcome such problemsantibodyfragments
such as Fab, scFv, diabodiesand minibodieshavebeen engineeredby removing either the entire
constant regionor the Fcportion.'! Advantages sharedby theseantibody fragments includetheir
better clearance fromwholebody, better tissue/tumor penetration characteristics and their simple
and straightforward production in bacteria bypassing mammalian cell based production. The
smallestfragments are singlechain fragment variables (scFv) formed by tandem arrangementof
the VH and VL domains joined by a flexible linker peptide exhibitinga comparable affinityof a
Fab.95.96 Their biological effects can be enhanced through linker length reduction that generates
noncovalent scFv dimers "diabodies'j'" by further shortening trirners" or even tetramers can
be formed." ScFvs havealso been modified to delivertoxins and chemotherapeutics to various
tumors by binding to cancer-associated antigens, e.g., by coupling the Pseudomonas aeruginosa
exotoxin A to scFv. 1OO Linking of scFvs of different specificity creates bispeclfic antibodies that
bind two different structures on single or different cells.'?' Other truncated antibody variants
are Minibodies-homodimers of scFv-CH3 fusion proteins-and Flexminibodies-scFv-IgG 1
hinge region fusion proteins.102
Whole antibody molecules can be modified as well by coupling with anti-microbial drugs.
Antibodies possessing specificity to microbial antigens can be simultaneously linked to toxins,
acting as immunotoxins that way. For example, Human Immunodeficiency Virus (HIV) and
Cytomegalovirus specific antibodies have been linked to the ricin A chain or the Pseudomonas
Exotoxin A.103.105 Unfortunately toxins can elicit immune responses limiting their repeated
102 Pharmaceutit:lllBiotechnology
therapeutic use. An alternative represents the linking of radionuclides to specific antibodies

that do not need to be internalized. liketoxinsand are unlikely to producesignificant immune
responses. Radionuclide-labeled antibodieshave been testedagainst Cryptococcus neoformans and
pneumococcal infections in mice.I06.107 Anotherdevelopment in modifyingthe antibodymolecule
wasthe creationofbispecific antibodiescarrying two different Fabfragments and recognizing a
microbialepitope for pathogen binding and at the same time a host immune component. This
strategywasshown to be successful in animalmodels for the clearance ofbacteriophages'P and
p.aeruguinosa. I 09
The applicationof humanizedand evenfullyhuman antibodies-is associated with low tox-
icity and high specificity. The benefit of high specificity is that only disease-causing pathogens
are targeted and therefore the host flora should not be altered or resistant microorganisms be
selected. A caveat is that pathogens with high antigenic variation may require more than one
monoclonalantibody for therapyand mutants lacking the antibodydeterminant could emerge
during treatment. Antibody molecules are highlyversatile; by binding to a single determinant
theycanmediatevarious biological effects includingtoxinneutralization. microbial opsonization,
complementactivation and antibody-directed cellular cytotoxicity (Fig. 2). Antibodiescan also
be usedto targethost cells and enhanceimmunefunctionsespecially desirable againstinfectious
diseases and tumors or to suppress immuneresponses by reducing the number of immune cells,
neutralizingcytokines or blockingreceptors.
antibody-dependent
cell-mediated cytotoxicity
~~
toxin x~\
neutralization ~
.~.>
~*
virus *-J
neutralization \
protein function
blocking
Figure 2. Biological effects of antibodies in infectious disease. Antibodies neutralize viruses

and toxins, block protein functions important for microbial adherence or growth, activate
complement and microbial opson ization and are a prerequisite for cell-mediated cytotoxic-
ity. All these functions together facilitate the host to combat the invading pathogen (Adapted
from Casadevall et al.a)
1heAntigenome 103
The major disadvantages of antibody based therapies are high costs associated with produc-
tion, storage and administration. Since antibodies have to be produced in live expression systems,
the risk of contamination with prions or viruses requires continuous monitoring and testing.
Additionally, antibodies have to be administered shortly after infection to be efficient, requiring
rapid microbiological diagnosis . Additionally manufacturing ofSymphobodies, mimicking poly-
clonal antibodies in human immune response , may still have to prove that they can be obtained
without chance in their composition under stable GMP conditions.
From Serum Treatment to Anti-Infective Monoclonal Antibodies

In the late 19th century Behring and Kisato discovered the efficacyofimmune sera in treating
infectious diseases, such as diphtheria and tetanus yo In 1891 the Klemperers already protected
rabbits against S. pneumoniae with immune sera showing the potential usefulness of passively
administered antibodies for the treatment of pneumococcal infections.'!' However reliable
anti-pneumococcal therapy was not availableuntil the mid 1920s, since the development ofsuccess-
ful serum therapy required the discovery that pneumococci are genetically diverse and only type-
specific sera provide protection. Improved vaccination schedules for serum donors to generate good
immune responses and advanced antibody purification techniques, as well as the standardization
ofserum potency were necessary steps in the introduction ofserum therapy (reviewed in ref. 112) .
The high death rate associated with meningococcal meningitis lead to fast developments also in
this sector; a significant reduction of the case fatality rates was already achieved with horse sera
in the early 20th century. ll2·m
Serum therapy reached its heyday in the 1920s to the mid 1930swhen it was standard clinical
practice in the treatment ofa variety ofinfectious diseases caused by S. pneumoniae, C. dipbteriae,
Neisseria meningitidis, Haemopbilus influenzae, Streptococcuspyogenes and Clostridium tetani. The
broad application ofserum as treatment for pneumococcal disease can be estimated regarding ad-
vertisements ofthat time in medical journals (Fig. 3). However, with the advent ofanti-microbial
chemotherapy passive immunization with serum was largely abandoned for the treatment of
bacterial infections due to major advantages in being less toxic, more effective and cheaper. Serum
th erapy was often associated with severe side effects including fever, chills and allergic reaction s
and delayed toxicity called "serum sickness" a syndrome associated with rash, proteinuria and
arthralgia. Moreover, for satisfying efficacyprecise diagnosis , appropriate and nondelayed dosage
was necessary asking for physicians with considerable experience . The production of horse or
rabbit therapeutic sera was very expensive because of the need for animal facilities, purification
techniques, adequate storage and standardization. Nevertheless lot-to-lor variation could not be
fully eliminated (reviewed in ref. 114).
Upon the arrival ofthe antibiotic era, anti-sera were still used for toxin -mediated disease such
as botulism, tetanus and diphtheria in addition to anti -toxin therapy in the treatment ofvenomous
snake bites. I I The lack ofefficient anti -viral treatments also stimulated the use ofantibody prepara-
tions as postexposure prophylaxis in e.g., rabies or hepatitis B (reviewed in ref. 9).
In spite ofthe previously experienced shortcomings, long-time neglected antibody based thera-
pies face a renaissance today. The description ofhybridoma technology in 1975 49 fired researcher's
imagination in developing new therapies against cancer, autoimmune or infectious diseases. As
early as at the dawn ofthe 20th century Paul Ehrlich already dreamed about the use ofantibodies
as "magic bullet" for the treatment of cancer. Indeed, in the mid' 1980s th e first efficient use of a
monoclonal antibody for the treatment of refractory lymphoma was reported. II S The anti-tumor
effect was only temporary, since murine monoclonal antibodies have only short in vivo halflife and
are immunogenic in humans; moreover the y don 't kill target cellsforcefully due to low efficiency in
complement activation and antibody dependent cell cytotoxicity. The first FDA approved murine
monoclonal antibody for clinical use was OKT3 targeting CD3 in 1986 and was designed for
prevention and treatment oforgan rejection .' !"
Fortunately, monoclonal antibody techniques underwent continuous and tremendous im-
prov ements in reducing the mouse derived portion ofthe protein and enabling the production of
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SERUM· TYPE I SERUM fJederle
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du.•• Im pna\'cil met hod, of manufacturE permit uf a reduction in the
lobar pneumonia is dearly iudi<::&1cd by t:ak:ina the toa k of ticura llrilT'liouf:
reported dwinr: the put ... yca.n by PAlKo SULLOWA and ROIl ... Rt:nxt-I) .......t CONC t:NT RATf. O
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b ll, :lnd be"n<-licial effect . are e vident.
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of treatment, Antipneumococcic Serum Type I (Lederk) 0/ treatment, Antipneumococcic Serum Type I (Lederle)
deserves your consideration deserves your consideration
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early 20th century. ~
The Antigenome 105
chimeric,humanizedand nowadays evenfullyhuman antibodies. In the 30 years sincethe inven-

tion of hybridoma technique 21 monoclonalantibody therapieshavebeen approvedby the FDA
(source: Adislnsight, 07.07.2006); only a singleone targets an infectiousdisease-Palivizumab
(Synagis') against respiratorysyncytial virusinfections.
In spite of the incredibleeffortsundertaken to developnovelantibody basedtreatmentswith
hundredsof monoclonalantibodiesbeingcurrentlyunder preclinical developmentor clinicaltest-
ing,onlythe minorityof theseeffortsaredirectedagainstinfectious targets.Amongviralinfections,
AIDS is far the most exploredarea(Table2). Due to the extremevariabilityof neutralizingHIV
epitopes,in addition to those combatingthe virusparticleitself, 117.118 manymonoclonalantibody
approaches target host molecules (such as CTLA-4, CD4, LFA-1 , CCRS) to hinder viralentry
(reviewed ref 119). Emergingviral infections causedby the SARS corona virus and West Nile
Virusalsoattracted the attention of monoclonalantibodydevelopers and several preclinical efforts
are expectedto enter clinicaldevelopment (Table2).
Due to the widespread appearance of multi-drug resistant bacterial pathogens and the in-
creasing population of immuno-compromisedpatients, more and more efforts are focusedon
antibody-basedstrategies againstpathogenic bacteriaand fungi. Especially considerable effortswere
and arestillundertakento treat septicshockcausedbygram-negative bacteriavianeutralizationof
endotoxin and of TNF-a induced earlyin the disease, unfortunatelywith no successful outcome
sofary o.123The mostfrequentmicrobial targetsof newdevelopments areopportunisticnosocomial
pathogens, suchasS. aureus and epidermidis, P. aeruginos« and Candidaspecies (Table 1and Table
3).The moleculartargetsfor thesemonoclonalantibodiesaresurface structuresof thesepathogens,
includingcapsular polysaccharides, cellwallglycolipids and surface proteins.Theprimaryaimisto
increase opsonophagocytic eliminationof the respective organisms with the help of the host'sim-
munecells. Unfortunately, in immune-compromisedpatients (under anti-tumor treatment,organ
transplantation, old age), the number of effective phagocytic cells is significantly lower than in
healthypeopleand relying onlyon opsonophagpcytosis maynot besufficient for cure.Monoclonal
antibodies that target surface proteins and that also have essential functions in in vivo survival,
multiplication(celldivision, nutrient acquisition) and pathomechanisms (adhesion, cytoroxiciciry,
immuneevasion), offeranother opportunity to reducebacterialgrowth and ameliorateinfectious
damageto the host.124. 127 A single chain anti-fungalantibody that wasselectedby the hsp90pro-
tein of Candida albicans from antibody cDNA librariesof patients who recovered from invasive
candidiasis isbeingdeveloped(Mycograb"). It consists of the antigen-bindingvariable domainsof
antibody heavyand light chainslinked together to a recombinant protein that is expressed in E.
coli. Mycograb" is not dependent on recruitment of white blood cells or complement,but simply
acts by binding and inhibiting hsp90 of Candida.!"
Current fearof bioterrorismusingbiological weapons encourages the development of antibody
therapiesagainst anthrax, botulism, ebola or smallpox virus infections and aims to provide im-
mediate immune protection through antibodies that either neutralize the pathogensand toxins
themselves,or targetthe host byblockingcorrespondingreceptorsto preventinfectionor toxicity.
Recently Cohen and coworkers demonstrated the inhibition of the lethal effectof anthrax toxin
viablockingof its human coreceptor, LRP6 with LRP6 specific antibodies.F'
The Next Chapter ofthe Antibody Success Story: Bacterial Infections

In spite of the historicallandmark therapyagainstdiphtheria, antibody therapyagainstbacte-
rial infections,only exceptionally, has entered the medical arena in the last 70 years. The advent
of antibioticsduring the fortieshascertainlydiscouraged the developmentof further serumtreat-
ments againstbacterialpathogens.
Antibiotics haveseemingly become a relatively cheep and mostlyreliable weapon to control
most bacterial infections and epidemics. Alongside with the increase of hygienic standards, the
penetrations of mandatory childhood vaccinations and antibiotic treatment, bacterialinfections
seemed to be a medical problem confined only to less developed parts of the world. The cost-
efficient availability, the seemingly evergrowingpipelineof novel antibiotics with increasingefficacy
.....
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Table 2. Therapeutic antibodies against viral infections
Highest
Drug Pathogen Antigen Target Type of Antibody Phase Originator Indication
Paliv izum ab RSV RSV F glycoprotein Humanised mA b IgGl Launched Medlmmune RSV infections
Motavizumab RSV RSV F glycoprotein Hu manised mA b IgGl III Eli Lilly/ RSV infecti ons
(derived from Medlmmune
Palivizumab)
2G12,2F5, HIV HIV gp120, HIV gp41 Human mAb II Polymun HIV infecti o ns
4El 0 Scientific treatment & pr evention
hNMOl HIV V(3) region of the HIV- Humanised mA b I SRD HIV infectio ns treatmen t
1 envelope protein gp120 Pharmaceutical s
HIVgp41 HIV H IV gp41 surface Fully human mAb Precl inical MedarexiPfizer HIV infectio ns treatment
mAb glycoprotein
HEB/GTM Hepatit is B Und isclosed Poo led plasma of individuals Launched Nab i Hepati tis B
with high ti ters of anti body to Biopharmaceutic als
the hepatiti s B surface antigen
Ctvecit" Hepatitis C Undisclosed Plasma of infected patients w ith I Nabi Hepati tis C
high levels of anti-hepatitis C Bioph armaceuticals,
virus (HCV) anti bodies Novartis
XTL 6865 Hepatit is C HCV envelop e E2 Fully human (derived from I XTL Hepatiti s C ~
prote in human immune cells of Biopharmaceuti cals '~"
convalescent patie nts) ~
Ii:
Ant i-SARS Severe acute SARS coronavirus Fully human mAb Preclinical Medarexl Coronav irus infectio ns ~.
mAb respirator y glycoprotein S Massachusetts -
syndro me Biologic Laboratories ~
~
(SARS) s..
;,.
..
~
....
~
Table 2. Continued
Highest
Drug Pathogen AntigenTarget Type of Antibody Phase Originator Indication
CCR5mAbOO4 HIV Human CCR5 Fully human Human Genome HIV infections treatment
Sciences
mAb 1F7 HIV Idiotype common to Murine mAb IgMlC Preclinical Immune Network HIV infect ions treatment
anti-HIV and anti-SIV
antibodies
mAb B4 HIV HIV receptor complex Murine mAb Preclinical United Biomedical HIV postexposure
prophylaxis
mAbA3D8 HIV CD44 Murine mAb Preclinical Duke University HIV-1 infections,
Medical Center myeloid leukaemia
CFY 196 Rhinovirus ICAM-l(receptor for Tetravalent humanised Fab Preclinical Perlan Rhinovirus infections
HRV) fusion protein Therapeutics
Anti-IFN-y Herples Interferon-y (IFN-y) Polyclonal antibody Advanced HSV infections
polyclonal Simplex virus Biotherapy treament, among other
antibody (HSV) applications
Bavituximab Viruses Phospholipid Chimeric antibody Peregrine Cancer, Hepatitis C
abnormally exposed on Pharmaceuticals treatment, Influenza ~
vascular endothelium virus infections, Viral
of tumor blood vessels infections ~
i
~.
Data were collected from the Adis R&D Insight database and homepages of listed companies in July 2006 using the world wide web. Data were grouped into: b;
first, antibodies targeting the virus particle itself and then second, those targeting host structures. Monoclonal Antibodies are abbreviated with mAbs. This is not t·
~
a complete list.
:s
..s,
P:f
S:l
~
~.
s
<>
~
Table 3. Antibody based therapies against fungalinfections
Highest
Drug Pathogen Antigen Target Type of Antibody Phase Originator Indication
Mycograb" Candida spp . Heat shock protein Huma n genet ica lly Preregistration NeuTec Treatment of system ic
(c. elbicens, 90 (hsp90) recomb inant antibody Pharm a candidiasis, Cancer
C. krusei, (e.g., Breast cance r)
C. parapsilosis,
C. tropica lis)
Candida MAb Candida spp . MSCRAM M" Undisclosed Precl inica l Inhibitex Treatment of cand id iasis
proteins
ACE 5033 As pergillus A . fumiga tus Fully hum an mAb Precl inical ACE Asperg illosis, M ycoses
fum igatus surface protei n Bio 5ciences,
Ge nmab
Candida mAb C. alb icans Corixa antigens Fully hum an mAb Preclinica l G laxoSmi thKline Fungal sepsis caused by
and Amgen C. albicans
Data were collec ted from the Adis R& D Insight database and homepages of listed companies in July 2006 using th e world w ide web. M on oclo nal A ntibodies are
abbreviated w ith mAbs. This is not a complete list.
....
~
invadingthe market has establishedthe attitude in the medicalcommunityup into the 1970sof
the last century that bacterialdiseases maybelong to the past. However. the emergingpattern of
rnuleidrug-resisrant strains of an increasing number of pathogens in hospitals and communities
has quicklyended the optimisticbeliefthat the repertoireof anti-bacterial treatments will suffice
the challenges in the infectiousdisease arena (for reviewseeref 130). Also the discovery, develop-
ment and registrationof novelantibioticshavenot fulfilled the too optimistic expectationsthat
new registrations of treatments maybounceoff the threat ofuntreatablebacterialinfections (see
commentariesby Clarke!" and in Blocenrury'Pj.The infiltrarionofgenomics.P' intelligentdrug
designand molecularstudiesofbacterialhost interactionsin antibioticdevclopmenr'" has rather
ledto the soberingrecognitionthat the numberof suitabletargetsfor newanti-bacterial drugsmay
be ratherlimited.135•137Furthermore. the oftensevere sideaffects, includingallergic reactions against
specific antibiotics.isrestrictingtheir applications, sometimes in criticalmedicalconditionswhen
they are most needed. Last, not least antibioticsoften lead to lysis of bacterialcellsand thereby
freeingendotoxins at high levels, therebycausingovershootingimmunity includingsepsis. 138
On the other hand before the advent of the monoclonal antibody technology. treatments of
bacterialdiseases with antibodieshavebeenout of the reachof economicalfeasibility. Production
of antibodies by immunizinganimalsas resorts to obtain serum is not a trivialprocessregarding
quality, reproducibilityand unwanted contaminations. Also as one has experienced with whole
cellbacterialvaccines. immunization with in vitro grown pathogens maynot lead to the type of
specific antibodies that neutralize them, since they may not display the proper antigens at the
surface. Thus,the progress madein definingdisease specific antigensfor vaccinedevelopmenthas
provided noveltools to raisehighlyspecific antibodiesthat mayprevent or block bacterialinfec-
tions or at leastsupporting the recovery process.
Theskepticism in the medicaland scientificcommunitytowardsthe paradigmof anti-infective
anti-bacterialmonoclonalantibodiesis nurtured by multiple linesof thoughts:
1. Existingtreatments are sufficient to control bacterialdiseases.
2. Monoclonal antibody therapymaynot find its wayinto treatment schedules that would
justify the costs.
3. A singlemonoclonalantibody directed against a specific antigen per se maynot be able
to counteract the pathogeniccourseof a bacterialinfection.
While all three argumentsare widdy accepted, a closerlook into the paradigmdiscloses that
they are not necessarily substantiated. if one considers the medical need, the progressmade in
identificationofsuitableantigenictargets and the positiveexperience of usingmonoclonal anti-
bodies againste.g., malignantdiseases (reviewed in ref 139).
The medical need is given, wheneverconventionaltreatment and prophylaxis are not avail-
able. S. aureus in context with nosocomialinfections is equallya target for antibody treatments
as Pneumococcus, both representing problem germsin intensivecare (Table4).
Moreover, costsfor antibodytreatmentsin connectionwith abovedescribedinfectiousdisease
outbreaksoften missingadequatemedicaltreatmentsappearto be not too dramatic,if one relates
them to the hospital conditions and the underpinning economical efforts spent. Last but not
least, the increasingly wide usageof monoclonalantibodiesoutside of the infectionsdisease area
has certainlyaided in loweringthe costsof developmentand manufacturing,therebypavingthe
wayto noveltreatments.!
Thequestion remainswhat kind of features form the prerequisites for a monoclonalantibody
in order to be able to counteract a bacterialinfection?The answerto this problem lies-to our
opinion-in the sdection of the bestsuitableantigenictargetsfor the developmentofmonoclonal
antibodies. Theantigensshouldbeexpressed on the pathogensurface duringthe infectiousprocess;
preferredthroughout the mostimportant stages ofdisease manifestation: i.e., duringcolonization,
spreading and invasion. Also the antigens of choice should have a proven record to be a target
of antibodies from individuals who haveencountered the pathogen with positiveor protective
outcome. In addition, the selectedantigensshould be conservedamong all clinicalstrains of the
germ causingthe underpinning infections.
Table 4. Medical need for novel therapeutics against nosocomial pathogens
S:l
Pathogen Disease Target Group Incidence Medial Need ~
.g.
No socomial Sepsis, bacteremia, Ho spitalized Nosocomial infections: High incidence High s.
pneumonia, wound and surgical (esp. elderly, 2 million/yr/US; medical costs (20 Bn$/yr in ::l
....
site infection, osteomyelitis, immunocompromized) 1 of 20 hospital admissions; dev. World) Increasing
urinary tract infect ions 100.000 death/yr/US antibiotic resistance
s. aureus Sepsis, bacteremia Premature newborns, < 34t h 1-2/1000 life birth Support the premature
pregnancy week immune system
S. aureu s Sepsis, bacteremia, Hospitalized, esp. surgery 25% of nosocomial infections High mortality
pneumon ia, wound infection, patients High incidence
osteomyelitis High medical costs
Multi-drug resistance
S. pneumoniae/ Bacteremia Community acquired, eld erly - 75.000/yr/ US High mortality
Pneumococcus Pneumonia - 500.000/yr/ US High incidence
Meningitis - 5.000/ yr/ US High medical costs
Antibiotic resistance
Group B Strep/ Bacteremia, pneumonia, Premature newborns, <34th 1-2/1000 life birth Support the premature
S. agalactiae meningitis pregnancy w eek immune system,
increasing ab resistance
P. aeruginosa wound infection, bacterem ia, Intensive care, burn pati ents 80% of 2nd and 3rd grade burn High mortality
S. aureus sepsis patients High medical costs
Multi-drug resistance
P. aeruginosa Pneumonia, sepsis Intensive care, artifici ally 15-20% of HAP and 80 % of Multi-drug resistance,
ventilated VAP high mortality, high
medical costs
E. faecalis and faecium Sepsis, bacteremia, Abdominal surgery pati ent s 10% of nosocomial infections Mortality, multi-drug
endocarditis resistance (E. faecium)
Klebsiella pneumoniae Nosocomial pneumon ia Hospitalized (esp. elderly, 10% of nosocomial infections Multi-drug resistance
immunocompromized)
Hospital associated pneumonia (HAP) makes up 5-10/1000 hospital admissions and ventilation associated pneumonia (VAP) accounts for 80% of all HAP. Hos-
pitalized burn patients average out 50.000 per year in the US. Of all approximately 9 million life births in the US and Europe per year premature birth before the
36th week accounts for 25% and premature birth before the 34th week for 10%. ....
....
....
All up to here listed features of target antigens may suffice the need to detect the intruder
with a monoclonal antibody. particularly ifthe bound antibody funnels the bacterium into the
immunological decontamination program, e.g.• opsonization. On the other hand, the lesson
learnt from antibiotics is that they have to kill the pathogen or at least disable bacterial growth
in the host. In the light of the notion that prevention or treatment ofa bacterial infection with
monoclonal antibodies may be restricted to a single antibody. one would aim the target antigen
also to exert a function needed for bacterial survival in the host . Thus, the antibody will neutralize
a virulence factor or an enzyme needed in the infections life cycle of the pathogen. Such a dual
mode of action resembles the features of monoclonal antibodies employed in cancers therapy:
these antibodies seem to block cancerous cells by marking them for the immunological destruc-
tion, but also by blocking their growth. Thus. monoclonal antibodies need to be directed against
carefully selected antigenic targets in order to achieve an optimum ofinterference with bacterial
survival in the host.
The recently invented antigen identification procedure that is designed to establish the "antig-
enorne" of pathogens has been instrumental in the development of novel bacterial subunit vac-
cines.2()'22 Characterization ofas. aureus antigen derived from the antigenome-that is presently
used in preclinical and clinical programs-has indeed revealed its involvement in virulence and
survival function.23,128.14O
The feasibility of antigens to serve as targets for monoclonal antibody treatments can
be pretested in vaccine models where protection of pathogen-challenged animals is ac-
cessed,t06,107.109.124.129.141.152 There is no doubt in mind that antigens giving the wanted protection
in a vaccine model may not be sufficient when employed for the development ofanti-infective
antibodies. However. the potency of an antigen in providing protective immunity as vaccine
may be a positive and sufficient selective criterion, alongside with all the other features that have
been described for antigens selected for subunit vaccine development.
References
1. Patti JM. Immunotherapeutics for nosocomial infections. Expert Op in Investig Drugs 2004;
13(6):673-9.
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CHAPTER 10
HSV as aVector inVaccine

Development and Gene Therapy
Peggy Marconi, * Rafaela Argnani, Alberto L. Epstein
and Roberto Manservigi
Abstract
T
heverydeepknowledge acquiredon the genetics and molecularbiologyofherpessimplex
virus(HSV), majorhuman pathogenwhoselifestyle isbasedon a long-termdualinterac-
tion with the infected host characterized by the existence of lytic and latent infections,
has allowedthe development of potential vectorsfor several applications in human healthcare.
Theseincludedelivery and expression of human genes to cells of the nervoussystem, selective de-
struction of cancercells, prophylaxis againstinfectionwith HSV or other infectious diseases and
targetedinfectionofspecific tissues or organs. Threedifferentclasses ofvectorscanbederivedfrom
HSV-l: replication-competent attenuatedvectors, replication-incompetent recombinant vectors
and defective helper-dependent vectorsknown asamplicons. Thischapterhighlightsthe current
knowledge concerningdesign, construction and recent applications, aswellas the potential and
current limitations of the three differentclasses of HSV-l-basedvectors.
Introduction
Thehumanherpesviruses arean impottantfamily of viruses,whichbecomeestablished invarious
tissues for the life of the host.1,2 Herpes simplex virus (HSV), is a complexhuman neurotrophic
virus that afrerinitial infectionand lyticmultiplicationat the bodyperiphery. generally at oral or
genital epithelialcells, entersin sensorynerveendingsinnervatingthe siteof multiplicationand
it is retrogradetransported to the nucleus of sensoryneurons.v'
The genome is a linear double stranded DNA of 152 kb encodingat least80 geneproducts.
Thegenomeisreplicating bya rollingcircle mechanism forminghead-to-tailconcatamers, During
the replication, the presence of inverted repeats sequences flanking the two unique segments of
the genome (unique long UL and unique short US) are causing the formation of four isomers
equallyinfectious(Fig. 1).3,5
The infectious virus particle comprehends an icosahedral capsid, which contains the viral
DNA genomein association with core proteins. Around the capsidthere is an amorphouslayer
knownasthe tegument,containingsome20different proteinswithstructuralandregulatoryroles,
surroundedbyan externalenvelope containingdifferentglycoproteins involved in differentfunc-
tions,amongwhich the firststepsof bindingand entry into the host cell. Once the de-enveloped
particlehasentered the cytoplasm, it istransported through association with microtubules to the
nuclearmembranewhere the viral DNA is released into the nucleus through the nuclearpores."
all the viral replication, from the transcription to the assembly of a new capsid, takesplaceinto
*Corresponding Author: Peggy Marconi-Department of Experimental and Diagnostic

Medicine-Section of Microbiology, University of Ferrara, Via Luigi Borsari 46, Ferrara-44100.
Italy. Email: mcy@unife.it
©2009 LandesBioscience and Springer Science+Business Media.
HSVas a Vector in Vaccine Development and Gene 1herapy 119
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120 PharmaceuticalBiotechnou,gy
the nucleus. Following the release of the viral DNA into the nucleus, the viralgenome circular-
izesand a cascade is initiated with the transcriptionof five immediateearly(IE) genes (infected
cellprotein ICP4. ICP27. ICPO. ICP22, ICP47) through the binding of a viralprotein present
in the tegument (VP16). in combinationwith cellular factors. to the enhancerelementpresent
in all IE promoters (TAATGARAT)?The products of the abovegenes are responsible of the
transactivationof the earlygenes (E) which are encodingenzymes and DNA binding proteins
required for the viralsynthesis; the IE and E are followed by expression oflate genes (L) which
products are principally structural proteinsof the capsid. tegumentand envelope (primarilyviral
structural components).3.5
Following natural infection the virus is know to be axonaltransported from the peripheryto
the cellbodyof the sensory ganglionwhereestablishes alyticor alatent infection.8 HSV-1persists
in the latent statein the nervoussystems of the host for a lifetimewherethe viralgenomepersists
in an epichromosomal state associated with histoneswithout integratinginto the host genome,"
During a latent infectionthe virusisin a relative quiescentstatewherethe transcriptionislimited
to a singleregionofthe viralgenomeand only a groupoflatency-specific RNAsaredetectablein
the nucleiofneuronalinfectedcells. Due to several stimuli. the viruscan be reactivated from the
latency and usually by anterogradetransport gets back to the site of primaryinfection where it
starts a newlyticcycle. Only in fewcases the viralparticleis retrogradetransported to the central
nervous system (CNS) and starts a latent or a lytic infection, which evolves in encephalhis.'?"
The newlyreplicatedvirustransported anterograde. usually to a siteat or near the portal of entry.
maycausea localized coldsoredisease lasting2-10dayswith subsequentremission when the cold
soresdisappear. Over time. periods of remission generally increase in length and the duration of
cold soresdecrease. until the person rarelyhas active disease. Thisprocess is regulatedby specific
immunity developed by the patient againstthe virus." The virusinfection is. however, life-long
and can be retriggered in some individuals by specific events. such as sunburn. stress or other
infections. 13,14
HSV-l GenomeandHSV-Derived Vectors

Thecompleteknowledge of the HSVsequences andprogress ofmolecular techniques has leaded
to the development ofHSV asavectorforseveral potentialapplications in humanhealth.15.19These
include (i) delivery and expression of human genes to the nervoussystem cells.20,21 (ii) selective
destruction of cancercells,22,23 (iii) prophylaxis and immunotherapyagainsttumors 24,25 and (iv)
prophylaxis againstinfections with HSV and other infectious diseases,z6.27
In the viralgenomethere areapproximately 80 geneproductschatcanbe classified asimmedi-
ateearly(IE or a). early(E or ll) and late (Lory) dependingon their kineticsofexpression during
replication. Theviralgenes can alsobe categorizedaccording to whetherthey areessential or non
essential for virusreplication (Fig. 1).Essential genes are required to producenewinfectious viral
particlesin permissive cellculture infections. Non essential. or accessory, genesencodeproduces
that are not absolutely requiredin cellculture but are important for optimum lyticreplication or
affectthe natural lifecycle ofthe virusin vivo. contributingto host range. pathogenesis. or latency.
The viral DNA contains at least 37 essential genes. The USregionof the genomecontains only
one essential gene encoding the glycoprotein D. which offers the opportunity to replace large
segments ofviralsequenceswith foreign DNA.4.28,29 The modifiedHSV genomeshould be ableto
accommodate up to 40-50 kbof exogenous sequences. However. the modification oftheseviruses
to reducepathogenicityand increase safery often resultsin the lossof viral activities. which are
required for efficient genedelivery and lifelong association with the host.
In recentyears, newtechnologies haveallowed researchers to get deeperinto these problems."
The challenge for manyresearch groupsis to developthe toolsto rendertheseorganisms harmless
yet effective for targeted gene transfer and appropriategene expression. Vectors based on HSV
Type 1 are currently (a) arnplicon vectors, (b) replication-defective viruses and (c) genetically
engineeredreplication-competent viruses with restrictedhost range. 19.31
HSVas a Vector in Vaccine Development and Gmt!1ht!rapy 121
Amplicon vector
Amplicon plasmid
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Figure 2. Structure and mechanism of packaging of the amplicon vector.
Amplicon Vectors
Amplicon vectors are HSV-l particles identical to wild type HSV-l from the structural, im-
munological and host-range points of view, but which carry a concatemeric form of a plasmid
DNA. named the amplicon plasmid. instead of the viral genomeY ':l4 Amplicon vectors possess
several advantages as gene delivery vehicles: (a) a large transgene capacity (150 kb), (b) the repeti-
tive character ofthe genome carried by the amplicon particle ensures the introduction ofmultiple
copies ofthe transgene per infected cell; (c) the ability to infect a wide variety ofcell types. includ-
ing dendritic cells; (d) the ease ofvector construction; (e) the limited toxicity due to the lack of
viral coding sequences.
Amplicons are bacterial plasmids that contain one or more transgene cassettes and two non
coding viral sequences, an origin ofDNA replication (ori) and a D NA cleavage/packaging signal
(pac) and they require a helper system to be produced. In the presence ofHSV-l helper functions.
a circular amplicon can be replicated and amplified as head-to-tail concatemers and packaged
into HSV-l particles as approx 152 kb linear DNA (Fig. 2).35 Classically. amplicon vectors were
prepared in cells transfected with the amplicon plasmid and superinfected with helper HSV-l.
As the helper virus was generally a replication-defective mutant ofHSV-l , the amplicon stocks
were produced in transcomplementing cell lines. However. the use of standard HSV-l as helper
resulted in the production of helper -contaminated vector stocks. 36•37 The contaminant helper
particles. even if defective. induced significant cytotoxicity and inflammatory responses. prevent -
ing their use in gene therapy or vaccination protocols." To overcome these obstacles. different
helper systems that produce essentially helper-free vector stocks have been recently developed ."
The last generation of helper system consists of the entire HSV-l genome. without pac signals.
cloned as a bacterial artificial chromosome (HAC) in E. coli supplying the full set of transacting
HSV-l functions.39,40 Another different helper system recently developed is based on the deletion.
by Cre/loxP-based site-specific recombination. ofthe packaging signals ofthe helper virus in the
cells that are producing the arnplicons."
Replication-Defective vectors
The replication-defective viruses are viral vectors where "essential" genes for in vitro viral
replicationare either mutated or deleted.Therefore, these mutants cannot growexceptin trans-
formed cell lines, where they are complemented in trans. To date, several replication-defective
vectors havebeen constructed in which the IE genes, expressing infected cellproteins (ICP) 0,
4, 22, 27 and 47, havebeen deleted in various combinations. IE genes are expressed shortly a.tter
viral entry into the host cell and are required for initiation of a cascade of E and L viral gene
transcription. ICP4 and ICP27 are essential for replicationand the deletion of one or both of
thesegenesrequiresadequatecomplementing celllinescapable of providingin trans the proteins
encoded bydeletedviralgenes.42-44 ICPO, 4 and 27 areresponsible for Eand Lgeneexpression.45.46
Beside its transcriptionalfunctions,ICP27 alsoaffects the splicing, polyadenylation and stability
of mRNAs. ICPOis a promiscuous transactivator actingon ICP6 gene,which encodesthe viral
ribonucleotidereductase largesubunitand possesses a hybridpromoter,whichisactivated asan IE
and an E fUnction.47.48 ICP22, the viralproduct that might be involved in sequestering of cellular
DNA polymerase 49 is phosphorylatedby two accessory genes ULl3 and US3-encoded proteins
50-52 and isrequiredfor the optimalexpression of the ICPOprotein. Deletionof the ICP22 IE gene
can be responsible for an overexpression OfICPO.53 ICP47 inhibits MHC class I antigenpresen-
tation contributing to the virusescape from the immune surveillance.54-S6The "firstgeneration"
of replication-defective HSV-l basedvectorsconsistedof mutants deletedin the single essential
IE gene encoding ICP4, namelyd120.4s Although these vectors show reduced pathogenicity
and can be used to efficiently transfer and transiently express reporter genes in brain, they are
nonetheless cytotoxicfor neurons in culture. Cell lines that complementICP4 and ICP27 have
permitted the construction of a "second generation" of highlydefective mutants.43,S7.S9 To date,
several replication-defective vectorshavebeenconstructedin which ICPO, ICP4, ICP27, ICP22
and ICP47 geneshavebeendeletedinvarious cornbinations.w" Deletionof allfive IE genes(ICP
0,4,22,27 and 47) preventsvirustoxicityfor cells at high multiplicityof infection, allowing the
vector geneto persistin cells for longperiods 60-62 demonstratingthat the residual cytotoxicityof
the "firstgeneration" of replication-defective HSV-l basedvectorsresultsfrom the expression of
the other four IE genes.28The multiplydeleted mutants showan unusually prolongedtransgene
expression from the ICPOIE promoter or the HCMV IE promoter in neurons.v The advantages
of these second generations of replication-defective vectorsare characterized by absence ofearly
and lateviralgeneexpression and provideenoughspace to introducedistinct and independently
regulatedexpression cassettes for differenttransgenes.?
AttenuatedHSVvectors
Deletion of somenon essential viralgenes resultsin viruses that retain the abilityto replicate
in vitro, but are compromised in vivo, in a contextdependent manner.64•6SAmongthe limitations
to the useofHSV is the fact that wt virusishighlypathogenicand cerebralinjectioncauses fatal
encephalitis. Toxic and/or pathogenicproperties of the virus must, therefore, be disabledprior
its useas a genedelivery vector.
Several genes involved in HSV replication, virulence and immuneevasion, whichare non es-
sentialforvirallifecycle in vitro,havebeenidentified. Thesegenes areusually involved in multiple
interactions with cellular proteins,which optimize the abilityof the virus to growwithin cells.
Understandingsuch interactionshas permitted the deletion/modification of these genes, alone
or in combination, to createHSV mutants with a reducedabilityto replicate in normalquiescent
cells, but that can replicate in tumor or dividingcells. These attenuated viruses harbor further
modificationso they alsoserveas therapeuticgenedelivery vehicles.6S.66
ManyHSV-l and HSV-2 genes that arenon essential in culturealtervirulence in animal models.
Among these genes, the ones encoding thymidine kinase (TK), ribonucleotide reductase (RR),
the virion-host shut off (Vhs)67and the ICP34.S proteins havebeen extensively studied.68 TK
is involved in optimizingnucleic acid metabolism for virusgrowth and is necessary for efficient
replicationin neurons. RRis necessary for the conversion of rNTPs to dNTPs in neurons,which
HSVas a Vector in Vaccine Development and Gene1herapy 123
are otherwise lacking but necessary for the synthesis ofnew viral DNA during virus replication/"
The Vhs function of HSV causes rapid destabilization of host RNAs and translational arrest."
Vhs also destabilizes viral messages, resulting in over accumulation of IE and E genes during
lytic infection?Q-72The ICP34.5 neurovirulence factor has been found to be essential for HSV
pathogenicity?' It appears to provide multiple functions to the virus life cycle, one ofwhich is to
block the arrest in translation, which usually occurs in virus-infected cells as an anti-viral response
preventingvirus replication. This effect is mediated through the cellular PKR kinase, which phos-
phorylares the translation initiation factor eIF2a, thereby stopping translation. ICP34.5 recruits
protein phosphatase Ia, to rephosphorylare eIF2a, allowing protein translation and continued
virus replication. Tumor cells often display an impaired PKR pathway and/or elevated levels of
eIF2a, that allow replication ofICP34.5-deleted viruses, as the inactivation ofthe PKR response
is less critical in this contest.V" Secondly, ICP34.5 seems to be involved in allowing new virions
to become packaged and leave infected cells in a cell type-specific fashion . Consequently, in non
permissive cells in the absence of ICP34.5 the nucleo capsids are retained in the nucleus and a
productive infection cannot ensue.
Use of non replicating viruses or non viral systems as vectors can limit the maximum achiev-
able efficiency ofgene transfer. In contrast, use ofreplicating vectors to allow replication ofgenes
delivered initially to a small number of cells and their subsequent transfer to neighboring cells,
as infection spreads, can significantly increase the efficacy of gene delivery?6-78Attenuated HSV
vectors have been tested as live viral vaccines, as oncolytic viruses and as gene therapy vectors to
deliver transgenes to the nervous system.
Engineering Techniques
Alterations of the HSV genome can be achieved in a number ofways. These usually require a
two-step process (named: marker transfer/marker rescue) in which portions ofthe herpes genome ,
which have been cloned into a plasmid vector, are first modified in vitro. The plasmid DNA is
then cotransfected into cultured cells with infectious viral DNA and recombinant viruses are
selected . Several methods have been described to insert DNA sequences into the viral genome.
Efficient recombination into specific sites within the viral genome has been achieved in vitro using
a recombination system der ived from phage Pl.79 It is also possible to enhance the frequency of
recombination.59 The initial requirement is the insertion ofa reporter gene such as ~-galactosidase
(lacZ) cassette flanked by Pac! or Pmei restriction enzyme sites not otherwise found in the viral
genome. The second phase is the substitution of the reponer gene with other foreign cDNAs by
digestion ofthe vecto r DNA with Pac! or Pmei to remove the lacZ gene and subsequent repair of
the vector genome by homologous recombination with a transgene expression plasmid. Potential
recombinant identified by a "clear plaque " phenotype after X-gal staining arose at high frequency
(80-100%) (Fig. 3).59 A different procedure involves transfection ofcells with overlapping cosmids
containing appropriate insertion or deletions. Expression of genes contained in cosmids leads
through recombination to the con struction of full-length viral genome. f -" To select recombi-
nant vectors it is critical to have a system by which to identify successful recombinants. The viral
TK gene is particularly useful site for insertion since its inactivation does not affect in vitro the
replication ofthe virus. TK mutants can be easily selected in the presence ofbromovinyl deoxyu-
ridine or acyclovir.82.83Another marker system involves disruption of nonessential viral envelope
glycoprotein genes, such as the ones encoding gC or gE. Recombinant viruses are identified by
loss ofan antigenic determinant ofthe glycoprotein using specific monoclonal antibodies (black
or white plaques staining).
Traditionally, recombinant HSV vectors have been generated through homologous recombi -
nation between the HSV genome and a recombination plasmid, which usually requires laborious
screening or selection and can take several months. Recent advances in bacterial artificial chromo-
some (BAC) technology have enabled cloning ofthe whole HSV genome as a BAC plasmid and
subsequent manipulation in E. coli. Thus, we sought a method to generate recombinant HSV
vectors more easily and qu ickly using th e bacterial recombination machinery. 84.85
~
a b UL b' a' a' c' Us a c
./'
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virus encoding the marker gene
(marker transfer)
/'
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with the transgene carrying plasmid
a b UL
! b' a' a' c' Us a c
./'
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carrying the transgene
(marker rescue) ~
l:.
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Figure 3. Mechanism of construction of recombinant viruses by classical homologous recombination (marker transfer/marker rescue) and using the .,;s
"Pad system".
s-
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HSVas a Vector in Vaccine Development and Gene Therapy 125
BAC cloning requires insertion of mini F plasmid sequences and antibiotic resistance genes
into the viral genome and the length of these BAC backbone sequences is usually greater than 6
kb in total. Insertion ofBAC sequences into the wild-type HSV genome (152 kb) will increase the
genome length to -1 58 kb and there will be no space left for the insertion ofadditional sequences.
To avoid deleterious effects ofthe BAC sequences, includinggrowth defects and potential transmis-
sion between bacteria and man , some herpesvirus BAC clones have been constructed with 10xP
site-flanked BAC sequences , which can be removed by Cre recombinase.86-88
HSV-l Based Vectors Applications

HSV-I Based Vectors for Vaccination
Many of the HSV based vectors have been used in gene therapy stu dies and some of them as
experimental vaccines against HSV-l infection.26.89.90 However, studies related to the evaluation of
the potential ofthese vectors, as foreign gene or protein delivery systems for immunological stud-
ies are very limited. The use of HSV vectors requires the development ofmutated viruses that are
genetically stable, incapable of replicating in the CNS and ofspreading in immunocompromised
individuals , not transmissible from immunized individual by contacts and , at the same time, capable
of inducing protective immunity against the disease. Recent major breakthroughs in the field of
HSV-l technology authorize and support the use ofHSV-l as vaccine vectors for the delivery of
foreign antigens.89.91'95 In particular, HSV vectors show several advantages for prophylaxis against
viral infections. They have been shown: (i) to elicit strong and durable immune responses byvarious
routes of inoculation;96.97 (ii) the viral DNA persists inside the host's cell nucleus as an episomal
element, thus eliminating the safety concerns deriving from the random integration of the viral
genome into the host's DNA; (iii) the y carry the tk gene, that, in case of undesired effects, can be
used, in combination with specific anti-viral drugs, to kill the virus-harboring cells.
The efficacy ofall ofthese vectors might potentially be affected by the preexisting immunity to
viral ant igens in host. The effect ofpre-existing immunity on HSV-l vectors remains controversial,
with some studi es showing strong immune response in the face ofanti- HSV-l immunity,96.98 whereas
another stu dy showed a reduction in the immune response to a transgene, with the intensity of
the redu ction depending on the route ofinocularion."
Amplicon Vectors
Amplicons were studied as vaccines against HIV 96 or intracellular bacteria, " They show unique
advantages over other viral vectors. " Firstly, amplicon particles are absolutely apathogenic for
infected cells since their genome is devoid from HSV-l genes. Secondly, the repetitive character
ofthe genome carried by the amplicon particle ensures the introduction of multiple copies ofthe
transgene transcription unit per infected cell, likely resulting in strong expression. Lastly, the pan-
tropic properties ofHSV-l particles in experimental systems, which are conserved in amplicons,
should allow these vectors to infect a large range ofcells, including dendritic cells.
Moreover, amplicons could also allow antigens to be presented by both MHC pathways during
the same immunization protocol. This could be achieved (i) by introducing the transgene both
in the amplicon genome and in the helper genome or, (u) by inducing the in vivo production of
empty virus-like capsids ofselected viruses (e.g., HIV-l, HCY, or HPV-16). Concerning this last
property, it has been shown that HSV-l amplicons encoding Moloney murine leukemia virus gag,
pol and env genes can ind uce the synthesis of retrovirus-like particles in cultured cells. Amplicons
have been used to efficiently transduce the full set ofproteins ofMoMLV retrovirus vectors, thus
rescuing integrated retrovirus vectors,lOO.lOl as well as the nonstruceural'P" or structural'?' proteins
ofHCV. 1M An interesting remark is that amplicon expressingcytokine genes have been found to
be a promising strategy for the development of tumor vaccines. 105.106
Replication-Defective Vectors
Until recently, it was believed that, to be effective, viral vaccines must cons ist of a live, repli-
cation-competent virus or a large do se of inactivated virus. Replication oflive virus was believed
to be essentialto provide sufficient immunogen to induce a strong immune response. However,

several non replicatingvaccines, including replication incompetent HSVs, have been shown to
induce an immune response.92.107 TheseHSV mutants show a reduced cytotoxicity, due to their
inability to replicateand to spreadin the host, but maintain the capabilityto infect a wide range
oftissues and host species.
HSV replication-defective viruses with mutations in essential genes that failto form progeny
virionsand DISC viruses with mutationsin structuralproteingenes that formuninfectious progeny
virionshavebeen usedasvaccines againstHSV infectionsand asvaccine vectors.I08•II O It has been
shownthat an HSV-2 doublemutant (dl5-29) doesnot causeanydisease in immunodeficient mice
indicatingthat the viruswouldbesafeevenin immunocompromised individuals. I I I DISC-HSV-2
has been shown to be an efficient vector for eytokinegene delivery into tumor cells and that the
expression of mGM-CSF or hIL-2 enhancesthe immunogenicityof whole-cell vaccines.I12
Theappealingpropertiesof replication incompetentHSV-I-basedvectorsinducingstrongCTL
response, both in murineand in simianmodels, againstforeign genes delivered byviralparticles have
madethem verypromisingcandidatesfor potential anti-HIV-l and alsoother viralor intracellular
bacterial pathogens vaccine development.91,1l3,114It has been shown that a mutant HSV-l virus
deletedfor the ICP4, ICP22 and ICP27 genes and expressing ovalbumin (OVA) asa modelantigen
elicited protection in mice against a lethal challenge with a recombinant Listeria monocytogenes
expressing OVA.91 A similarvector, expressing HIV-l Tatprotein,hasbeendemonstratedto induce
long-term Tat-specific immune responses in the Balb/c murine model.!" Moreover, vaccination
of Rhesus macaques with a HSV-l mutant virus that contains a deletion in ICP27 and expresses
SIV Env and Nef antigensshowed partial protection against mucosalchallenge with the highly
pathogenic SIVmac239.115 In the sameanimalmodel,usinga prime-booststrategyof vaccination,
recombinantHSV-l vectorsdeletedfor ICP4, ICP22,ICP27 and ICP47 and expressing Gag,Env
and a Tat-Rev-Neffusionprotein of SlY,elicitedrobust anti-Gagand anti-Envcellularresponses
and induced partial protection againstintravenous challenge with SIVmac239.27,92
Due to their abilityto acceptmultipleheterologous genes, the IE replication defective vectors
could be usedfor innovative and synergistic strategies ofimmunization. Forexample, it ispossible
to engineervectorsto express specific chemokines and cytokines, together with antigenstargeted
to MHC-I or II molecules, in order to attract monocytes to the sitesof infection,to induce their
differentiationinto dendritic cellsand to favor antigen presentation.
Replication-Competent Vectors
Attenuatedlivevirusesarethemosteffective to serve asvectorsforvaccination. However, a major
concern exists about attenuated HSV asa vector.In fact, in addition to the problemof genotypic
stabilitythere are other safetyissues includingquestionsregardingthe potential ofvaccine vector
to establishlatency, reactivate or recombinewith virulent wild type strain. To overcome someof
theseproblems,an approachbasedon definingand eliminatinggenesinvolved in neurovirulence,
latencyor reactivation wasdeveloped.I 16
The firstand to date the onlyone, attenuated HSV-l virusto be constructed and analyzedasa
viralvaccinein humans,wasthe NVl 020 (formerly R7020) strain.I 17 Thisvirus,basedon HSV-l
strain F, has a portion of the unique short region of the viral genome,encoding glycoproteins
G, D, I and E, replacedby the homologous region from HSV-2 and possesses only one copyof
ICP4. This virus is verystronglyattenuated in rodents and primates. In a dose escalation study,
localreactionswerenoted in HSV-l-infected persons.A dose-dependentinduction of antibodies
occurred in HSV-l seronegative subjects, but the developmentof this mutant has been stopped
sinceit resulted too over-attenuatedand it wasconsequently poorly immunogenic.
Thegoalto construct a safe, less attenuated vaccine candidate,lead to the construction ofRAV
9395 mutant.us RAV9395 isbasedon HSV-2,strain G, which carries deletionsin the UL55 and
UL56 genes, encoding proteins with unknown functions, the deletion ofwhich causes attenua-
tion and deletion in both copiesof the y34.5 gene (encoding ICP34.5 protein). Concomitant
with this deletion, both copiesof the open readingframe (ORF) P havealso been deleted. The
tk gene was left intact and functional, conferring acyclovir sensitivity to the recombinant virus.
When used as a live viral vaccine in a guinea pig model of HSV-l infection, it was shown to be
protective and it was also demonstrated that the immunologic answer depended on the route of
administration of the virus.
AD472 is an evolution ofRAV 9395 , in which deletions in UL43.5 and in the US 10-12 region
were added to obtain an additional safety level by increasing the genetic and phenotypic stability
ofthe virus. I 19 In a guinea pig model , AD472 administered intramuscularly did not prevent infec-
tion and viral replication in the vaginal tract, but reduced lesion development and severity in a
dose-dependent manner after HSV-2 wt challenge. Moreover, it generally precluded establishment
oflatency by the challenge virus.
Mutations in TK, especially for HSV-2 , do not attenuate the virus sufficiently for human
vaccines.12o•12l Other attenuated HSV-l and HSV-2 viruses with single deletion in vhs or in RR
respectively,122.l23 were shown to determine a protective immunity when tested in animal models,
but still they are too neurovirulent to be used for human trials.
A further improvement to antigen presentation to the immune system could involve the dele-
tion , from the viral DNA backbone, ofthe genes that codify for the vhs and the ICP47 proteins. In
fact, two mechanisms have been described by which HSVinhibits antigen presentation by MHC
class I and class II molecules. The first is related with the ability of the Vhs protein to accelerate
the degradation of cellular mRNA molecules'? and has also been shown to block dendritic cell
maturation and thus to inhibit the immune response against the vector -delivered rransgene."
The elimination of the UL41 locus from the viral genome was reported in the same paper to
allow dendritic cell activation and also to stimulate the antigen specific T -cell response in vitro.
The second is based on the abilit yofICP47, one ofthe immediate early proteins, to bind to Tap,
the transporter associated with ant igen processing and to prevent peptide translocation into the
endoplasmic reticulum. \ 4.\ 6
HSV-l Based Vectors forGene Therapy ofNervous System

The Neurotropic Properties ofHSV-l
H SV-l presents several outstanding adaptations to the nerve system and each ofthem can be
rationally exploited in the design ofgene therapy vectors with regard to neurological applications.
HSV-l contains genes that control neuroinvasiveness and neurovirulence; this virus can move
both in the retrograde and anterograde directions and disseminates transynaptically from neuron
to neuron. The virus envelope contains several glycoproteins that mediate entry to neurons due
to the recognition of specific receptors (nectins), In most neurons, HSV-l will establish a latent
infection, a situation in which the viral genome will persist as a stable chromatinised episomal
element and in which all lytic genes are silenced. Recent studies indicate that, during latency, the
viral genome generates a chromatin structure that allows it to behave much like a mammalian
minichromosome, with very sophisticated regulation ofgene expression. The LATsdo not encode
proteins and there is increasing evidence suggesting that they can playa major role in the inhibition
of the apoptotic response of neurons to virus infection, thereby preventing cell death and favor-
ing eventual reactivation of the virus. The latent virus genome can be reactivated by stress, fever,
or immune suppression and , through anterograde traveling along axons, the virus is transported
back to the sites ofinitial epithelial infection , causing recurrent mucocutaneous infections which,
in most cases, remain asymptomatic. Recent data indicates that HSV-l anterograde movement
along the axons is dependent upon the interaction ofvirus proteins with plus-end microtubule
motors that move the capsids toward the axon terminals. Many studies indicate that most ofthese
neurotropic features are retained in defective and attenuated HSV-l vectors, including the abilities
to been efficiently transported along axons in both directions and to establish latent infections
with prolonged gene expression, both in sensitive and in motor neurons.
Amplicon Vectors
Amplicons havebeen used to deliverand express transgenes in neurons in vitro and in brain
in vivo.They have been used to deliverneurotrophins,like nerve growth factor (NGF)124.125 or
brain-derivedneurotrophicfactor (BDNF),124.126 antiapoptoticgenes,127.128 heat-shockproteins 129
or antioxidant enzyrnes.P" in attempts to protect neuronsagainsta varietyof neurological insults,
in many different experimental settings.Ampliconsexpressing genesaffecting neurotransmitter
expression or neuroreceptorsynthesis havebeenusedto studybehavioral features, likelearningand
memory.131'134 Amplicons havealsobeenusedto deliver tyrosinehydroxylase and other genes to the
nigro-striaralsystemor to culturedstriatalcells, in studiesaimedto treat Parkinson's disease.135•139
More recently, ampliconswereshown to be ableto delivergenesto the retinalpigment epithelial
cellsofthe rat retina, but not to the adjacentphororecepeors.l'? In this study,ampliconsallowed
rapid and efficient, but transient, gene transfer, following subretinalinjection.
The limitation in the ampliconssafetyprofilewas the presenceof helper virus panicles that
resultedin somecytotoxicity.Thisproblemhasbeencircumvented recently byusingaplasmid-based
BAC transfection system to providethe helper functions, although the particleyieldis relatively
low.39.40 Geneexpression in vivousingamplicons hasbeen reponed to persistaslongasone month.
However,it cannot be excludedthat long-termexpression from amplicons maybe relatedto per-
sistent low-level replicationby contaminating recombinant wild-typevirus in the brain. In fact,
in contrast with other ampliconpreparations,"helper-free" stocksproduce only transient expres-
sion of reporter transgenein vivousing the samepromoter reported previously to remain active
long-term.Another limitation of the amplicons wasthat theycannot accommodate insertslonger
than 10kb. Wade-Martins and coworkers havedevelopedan efficient viraldelivery and expression
systembasedon the HSV-l ampliconvector,termed the iBAC, or infectiousBAC that can carry
largegenomiclocuswith surrounding sequences.r'"!"
Replication-Defective Vectors
Major advances have recently been made to improve the characteristics of these vectors, in
particular to reduce their toxicity, to modulate the greamess and the time-course of transgene
expression, to precisely target specific cell populations and to transfer multiple genes. 17.21.144-147
NonreplicativeHSV vectors havebeen tested in many different gene therapy animal modelsof
various neuropathies, Parkinson's disease,148-150Alzheimer's disease,'!' chronicpain 152,153or lysosomal
storagedisorderswith neurological involvemenr.P'
Therapyoflysosomalstoragedisorderswith neurological involvement such asTay-Sachs (TS)
disease requiresproduction and distribution of the missingenzymeinto the CNS. Several thera-
peutic approaches allowrestoringthe enzymaticactivityin manykeytissues (kidney, liver, spleen,
etc.) but the reduction of the GM2 ganglioside depositsin the CNS is difficultto achieve since
CNS, is kept in a privileged environment separatedfrom the blood systemby the blood-brain
barrier (BBB), which represents an obstacleto therapy.154 Martino et al havedemonstrated that a
nonreplicatingHSV vectorencodingfor the hexosaminidase (Hex)A a -subunit (HSV-TOaHex)
and deliveredinto the internal capsule of the TS brain animal model was able to re-established
the Hex A activityand removedthe GM2 ganglioside storagein both injected and controlateral
hemisphereand in the cerebellum one month of treatment.Thestudiesconcerninglysosomal stor-
agedisordersareparticularlyimportant because theyrepresentthe firstevidence ofthe distribution
ofa therapeutic viralvector throughout the entire CNS and suggest that the anatomic structure
ofthe brain maybe a usefultool in therapyfor geneticneurodegenerative dlsorders.P'
Among them, vectorsexpressing multiple trophic factorsseemto be verypromisingas a side
treatment for neurodegenerative diseases. Motor neuron disease (MND) isa groupof neurological
diseases characterized by degenerative process of the upper and lowermotor neuronl'" in differ-
ent pans ofthe motor system includingthe spinalcord, brain stem and motor cortex. One of the
major breakthroughs in the fieldof CNS regeneration is the concept that neurotrophic factors
(NTFs), which are endogenoussolubleproteins regulatingsurvival, growth,morphologicalplas-
ticity, or synthesis of proteins for differential functions of neurons, governthe processes involved
HSVas a Vector in Vaccine Developmentand Gene1herapy 129
in brain and spinal cord repair. 1S6 Experimental evidence indicates that treatment with multiple
neurotrophic factors can significantly increase motor neuron survival in comparison with the
delivery ofa single factor alone.IS7.IS8 HSV-l vectors that have been engineered to express multiple
neurotrophic factors have been used to deliver these molecules to specific neuron populations.IS9
Nonreplicative vectors containing basic fibroblast growth factor (bFGF), ciliary neurotrophic
factor (CNTF) and EGFP (as a reporter gene) have already been shown to induce proliferation
and differentiation in 0-2A oligospheres obtained from newborn rat brains in vitro and also in
the rat hippocampus in vivo. ls8
Replication-Competent Vectors
One of the potential target organs of replication competent HSV vector applications, the
peripheral nervous system (PNS), seems likely to promise the most successful results. In fact,
inoculation ofHSV vector by peripheral routes can take advantage ofthe natural life cycle ofthe
virus, which usually infects axonal nerve terminal at peripheral sites before retrograde transport to
neuronal cell bodies where latency is established. It is well known that viral replication is necessary
to cross the synapses among neurons and for efficient establishment oflatency.l60 In the PNS there
are a number ofpotential applications for HSV replication competent vectors capable ofperipheral
replication and axonal transport, including the stimulation of regrowth of damaged nerves, the
study and treatment of various pain states, the protection of neurons from further degeneration
in motor neuron disease, the study and treatment ofvarious neuropathies, the study of neuronal
development and the screening of the relevance of genes implicated as being important in any
of these processes by a gene delivery approach. Thus, viruses mutated in either gC or TK or RR
have been extensively used, which, while being somewhat attenuated compared to wt virus, are
also replication-competent. The data obtained to date show the potential ofsuch vectors for gene
transfer. Attenuated vectors in fact demonstrated to be highly efficient in driving proenkephalin
A (PA) gene expression in dorsal root ganglia (DRG),161 to deliver genes into monkey eyes162 and
to rodent visual system l63 and to express active nerve growth factor beta subunit (~-NGF) in
latently infected DRG .
HSV-I Based Vectors for Cancer Gene Therapy

HSV vectors have wide-range natural hosts and have been proven to efficiently infect numerous
human tumor cell lines in vitro. A number of new therapies have been developed for treatment of
cancer and the knowledge ofthe basic defects that occur in malignant tumors has lead to the conclusion
that the association ofdifferent therapeutic approaches is the method to eradicate these malignan-
cies. Potential genes should induce a selective anti -tumor response that attacks the primary tumor,
inhibits metastasis, prevent recurrence and does not promote drug resistance. Another important
feature of HSV-l-based vectors for cancer gene therapy is their capacity to express the autologous
tk gene, encoding the TK enzyme, a well characterized suicide gene, widely used in gene therapy of
different experimental tumors l64-166 and which has already been tested in clinical trials.167-169 Another
advantage of the use ofTK/GCV system is that it is capable of killing both vector-transduced and
neighboring cells,owing to the effect. 164,170,l7I
Recent efforts at modifying the envelope of the HSV-l virion to target specific receptors, e.g.,
replacement ofthe heparan sulfate binding domain in gC in the envelope with a receptor ligand or
single chain antibody, indicate that it is possible to selectivelyincrease infectivity oftumor cells bearing
corresponding receptors. m .m Infection of normally non Wettable cells has been achieved using a
soluble adapter fusion protein consisting ofthe HSV-l envelope gDand a single chain antibody for
the epidermal growth factor receptor (EGFR), which is enriched on many tumor cells.'?'
Amplicon Vectors
Most anti-cancer amplicon vectors used to date have employed standard amplicon vectors, which
efficiently deliver genes to the cell nucleus but are lost with successive cell division. Therapeutic
transgenes used in the context ofamplicon vectors have included anti-angiogenic factors , immune
enhancing agents, proapoptotic proteins and RNAi.17S
Stunting of tumor growth canbe achievedby inducinghypoxiathrough inhibition of neovas-

cularization. HSV ampliconvectorshavebeen usedto attenuate angiogenesis and therebyinhibit
pancreatictumor growthbyexpression ofadominant-negative solublevascular endothelialgrowth
factor (VEGF) receptor, sFlk-l under control of a promoter induced in hypoxic conditions.176,177
HSV amplicon vectors have also been evaluated as cancer vaccines by expressing combina-
tions of cytokines and immunomodulatory proteins for treatment of a variety of experimental
tumors.24,17S.178.181
A promising, new approachto canceris the selective degradationof mRNA by RNA interfer-
ence (RNAi)182 or interferencewith microRNAsthat support tumor growth.'!' HSV amplicon
vectors expressing siRNAs have been used recentlyto mediate posmanscriptional silencingof
EGFR, which is frequentlyactivatedin human glioblastoma cells'" and to inhibit the expression
ofBKV T-Agand tumorigenicityofBKV-transformedcells. 18s
Ofthe widerangeof prodrug activatingenzymes testedfor cancertherapy,186,187 onlya fewhave
been deliveredvia HSV ampliconvectors. One of these is HSV TK , which convertsnucleoside
analogues, such as ganciclovir, into toxic analogues which incorporate into replicating DNA
and lead to celldeath.188A chimericfusion protein between cytochrome P450 4Bl and GFP in
combination with the prodrug 4-ipomeanolwas found to confer toxicityto glioma cells with a
bystandereffect.189Ampliconvectorshavealsobeengirdedwith twosynergistic pro-drugactivating
enzymes, TK and cytosinedeaminase (CD) for tumor therapyand imaging.I90.191
The useofa replication-conditional virus to package ampliconvectorcanimprove theefficacy of
cancertherapybycombiningdelivery of therapeuticgene(s)viathe ampliconvectorwith selective
viraloncolysis of tumor cells bythe replication-conditionalvirus.It has beendemonstratedlocaland
distant immune-mediated controlof coloncancergrowthwith fusogenic membraneglycoproteins
in combinationwith viraloncolysis.192.193Moreover, an ampliconvectorthat expresses an essential
viral gene, such as ICP4, can complement ICP4' recombinant viruses to efficiently replicate and
causelysis in prostate cancercells.194 Furthermore, it hasbeen shownthat the immunostimulatory
effects of amplicon vector-mediated cytokine expression enhance direct viral-induced oncolysis
in a syngenic squamouscellcarcinomaflankmodel,1os.19S.196
Targetingproliferatingtumor cells viathe transcriptionalcontrol of therapeuticgenescan po-
tentiallyimprove the safetyand efficacy ofcancergene therapy. It has been shown that transgene
expression couldbetargetedto proliferatingcells whencellcycle transcriptional regulatory elements
are incorporated into amplicon backbone vectors.197•199 For example, transcriptional regulation
can be rendered specific to human hepatocellular carcinomacells by insertingthe chimericgene
Gal4/NF-YA under the regulationof a HCC-specific hybrid prornorer.P?
Tumor necrosisfactor-related apoptosis-Inducing ligand (TRAIL) has been shown to induce
apoptosis in neoplasticcells. It has been reported the efficacy of amplicondelivered TRAIL and
its secretedform (S-TRAIL)in treatingtumors in vivoand in monitoring both genedelivery and
efficacy of TRAIL-mediated apoptosisby dual-substrate bioluminescence imaging.201.203
Replication-Deftctive "Vectors
Multipleimmediateearlygene-deleted nonreplicative HSV-l vectorsarecharacterized byhigh
efficiency of transduction of several different host species and celltypes,both dividingand non
dividing,includingvarious tumor aswellasendothelialcells. 23,204-207 Differentreplication-defective
HSV vectors have been produced that deliver anti-cancer transgenes to tumor cells such as
melanoma." gliosarcoma,2OS,208.209 or glioblastoma.i'" Two or more therapeutic molecules, acting
additively or synergistically, can thus be expressed at comparablelevels bycellstransducedwith a
combination vector, which is clearly an advantage in comparisonwith co-administrationof two
or more vectors encoding a single transgeneand also in comparisonwith co-expression of two
molecules, separatedby IRESsequences, bya unique vector.
Thesemutant vectors express, in association with the autologous HSV-l tk gene acting as a
suicidegene when accompanied by its pro-drug ganciclovir, further transgenes chosen for their
potential to synergize in tumor cell killing and induction of anti-tumor immunity with genes
HSVas a Vector in Vaccine Development and GeneTherapy 131
encoding for solublehuman cytokines(IL-2, GM-CSF and IFN-y),the human B7.l geneencod-
ing a costimulatorysurface antigen (CD80);43 rat connexin 43 geneimprovingthe HSV-l TK/
GCV killing of glioma cells by increasing the bystander effect 211 or rat connexin and human
TNFa. 21O,212 Recently, an HSV-l-derivedreplication-defective vector (TO- IFI16)wasdeveloped,
204 which has been shown to efficiently transduce an interferon-inducible gene (IFIl6), into pri-
mary human umbilicalvein endothelialcells(HUVEC), which are usually poorly transfectable.
It has also been possible to infect HUVEC cells with similar HSV-l-based vectors expressing
anti-angiogenic fusionproteinsendostatin::angiostatinand endostatin::kringleS.Theexpression
ofanti-angiogenic proteinsbydirectlyinfectedHUVEC cells has beenshownto inducecytostatic
effects in proliferationassays in vitro.Also,by addition of gancyclovir to the cellculture media,a
majorcellkillingeffectwasobserved.P' In vivo,the expression of autologoustk genein association
with GCV was shown to be highly efficient in both reducing small tumor masses growth rates
and also in inhibiting tumor cell engrafiment. The expression by tumor cells of vector-encoded
angiostaticproteins was also extremely efficient in inhibiting the tumor establishment, both in
presenceor in absence of GCV.206
The wide spectrum of dividingor nondividing cell types that can be easily infected by non-
replicative HSV-l vectorsand amongthem endothelialand dendritic cells, alongwith their large
exogenous DNA accommodatingcapacity, makesthese vectors very attractivedelivery systems.
These uniquefeatures mightbeofextreme importanceforcombinedtherapeutic strategies requiring
the simultaneousexpression of high levels of multiple foreigngenes, likesuicidegenes, cytokines
or other immunomodulatorymolecules, anti-angiogenic proteins,solublegrowthfactorreceptors
and so forth. As varioustypes of tumors present differentcharacteristics, the high manageability
of large,wellcharacterized HSV-I genomemight permit the combination,in a unique backbone,
of the most appropriateexogenous genes for treatment of eachparticular tumor.
Replication Competent Vectors

Oncolytic viruses, which selectively infect or replicate in cancer cellswhile sparing normal
cells, havebeen exploredas alternative cancertherapies in both preclinicaland clinicaltrials.77.78
The optimal strategy might be to derive a replicatingvector from a highly prevalentbut weakly
pathogenic human virus,213 so the reversion to wt would then be of no serious risk to the patient
or to the population.
Construction of oncolyticviruses that cannot only target cancercells, but can alsoretain their
abilityto infect,usurphost replicationmachinery, then release newlymadeprogenyto infectother
transformedcells afterlysing and killingthe host cell,hasbecomea majorareaof therapeuticcancer
research.Therearesomecharacteristics that an idealreplication-competent, oncolyticvirusshould
possess aboveand beyondthoseviruses that function simplyasdeliveryvectors: (i) be easyto engi-
neerand to producein largequantities;(ii) selectivity to neoplastic cellalone; (iii)minimaltoxicity
to normal tissue;(Iv)showproliferationwithin and systematic killingof tumor tissuewhich itself
maybe rapidlypropagating; (v) abilityto disseminate throughout the tumor massand possibly to
sitesof invasion distant from the initial inoculation site; (vi) carrylow or bearable toxicity;(vii)
genomically stable,thus avoidingthe generation of toxic, undesirable mutants that could pose a
danger; (viii) incorporatea "fail-safe" mechanismfor inactivation;(ix) absence of potentialspread
to the generalpopulation; and (x) enduringefficacy despiteprospectof encounteringa mounting
immune response to replicating viruses. Replication-conditional HSV-based vectors have great
potential in the treatment of varioustypesof cancersincludingbrain tumors.64-66.77,214-220
So far, several oncolytic HSV vectors havebeen developed. The first generationof thesevec-
tors contained mutation in a single gene that restricted their replication to dividingcells. Three
such HSV-I mutants were constructed: (i) dbpTk containing a deletion in the tk gene;221 [ii)
hrR3 containing an insertion of the E. coli lac-Zgenein the earlygeneUL39, encoding the large
subunit of the viral RR (ICP6);222.223and (iii) R3616 containing I kb deletionsin both copiesof
the y34.S gene, encodingthe neurovirulence factor ICP34.S.224-226
TK mutants are highlyneuroattenuated and when used in differentmousemodelsof various

nervous system-derived tumor types, showed a slowed tumor growth and prolonged survival.
However,clinicaltrialswerenot pursued because of (i) undesirable levelof toxicityat high titers
and (ii) its TK-negative status made it resistant to traditional anti-herpetic treatments,a major
disadvantage should anyviral toxicityto arisein treated patients.221,227
ICP6 mutants havebeen tested as replicative anti-canceragents, aloneor in combinationwith
acyclovir/gancyclovir, as the mutants retain their sensitivity to such anti-virals. Moreover, the
RR mutants havebeen shown to display an increased sensitivityto gancyclovir, comparedto the
wt virus. Theserecombinantviruses showedenhanced killingof rumor cells in vitro and showed
improved survival of animals. However, likeTK mutants, they can cause fatal encephalitis when
used at sufficient dose and were not thought to provide a sufficient margin of safetyfor testing
in humans.223•228
It hasbeen shownthat deletionof ICP34.5,the neurovirulence factoressential for HSVpatho-
genicity, providesthe greatestdegreeof attenuation of any individual mutation where the virus
can still replicatein actively dividingcells. R3616, the prototype HSV-l deletedin both copiesof
y34.5, had demonstrated attenuated neurovirulence but with maintainedanti-gliomaactivityand
wasfound to produce no encephalitisin a nude mousemodel.22,116.229.230 The useof y34.5mutated
virusesdemonstrated considerable anti-tumor efficacy, combined with a good safetyprofile and
differentversionsofHSV ICP34.5-deleted are currentlyin human clinicaltrials.m
Followingpreclinicaltestingwith the abovementioned oncolyticvectors,secondgeneration
vectors with multigenicmutations werecreated. G207 contains deletion in both y34.5loci and
a lacZ gene insertion in the ICP6 gene.232 These multiple mutations made the reversion to wt
highly unlikelyand conferred several important safety advantages. Moreover, G207 retains its
susceptibilityto standard anti-HSV therapiessuch as acyclovir, sincethe tk geneis intact. After
oncolytic activityand safetyevaluationstudies in the mouse model.P' G207 neurotoxicitywas
further evaluatedin nonhuman primates.233 Thedataobtained in the previousexperimentallowed
to moveinto PhaseI clinicaltrials234 and,presently, enrollmenthasbegunfor sequentialPhases Ib/
II trials employingG207 as an anti-tumor agent for malignant gliomas. Almost simultaneously,
HSVl716, derivedfrom the parent wt strain HSV-117+ in which both the copiesofy34.5 have
been deleted,alsounderwentclinicaltrialsto evaluate its toxicityin patientswith recurrenthuman
glioma,235.236 afterit wasdemonstrated to be avirulentin mice.237.238
Oncolytic herpesvirus have been also studied as an oncolytic anti-tumor therapy against a
varietyof tumors differentthan GBM and anaplastic astrocytoma,suchashuman breastcancerin
a brain metastaticmodel,25 colorectalcancerand liver rnetastases.P? prostate cancer,240 pancreatic
cancer241.242 and head and neck squamous carcinoma.iv In manyof these studies, the efficacy of
G207 has been comparedwith that of the abovementioned NVI020, which hasshownto be too
attenuated to work asa liveviralvaccine.1l7.244 Moreover, NV1020 is currentlybeinginvestigated
in Phase I clinicaltrials for patients with colon cancerthat has metastasized to the liver and has
proven recalcitrant to chemotherapy. This is also the first trial to investigate administration via
intravascular delivery. In fact,oncolyticviruses canbe administeredlocally, bydirect intratumoral
inoculation,or systemically, byintravascular administration. However, the routeofadministration
ofthe virusdid influenceefficacy, aswasobservedin the animal model.
Despite the promising resultsobtained with the engineered HSV-l based oncolyticvectors
describedabove,it is likely that a multimodal approach to eradicatecancer will be more effective
with the final goal to improvesafetyand efficacy of the system. At this regard, oncolytic HSV
vectors havebeen further modifiedto augmenttheir anti-tumor efficacy, by incorporation of ex-
pressioncassettes for the delivery ofvarious transgenes. Moreover, ifthe therapeuticgeneischosen
carefully, this maybe synergistic with the anti-tumor effectof virus replication.
Molecules includinga number ofinterleukins and interferonshavebeen testedwith oncolytic
HSVs. Among these, IL-4,245 IL_12,25.246.247 IL_IO,245 GM_CSF,231.247and B7.1/ 48 which increase
tumor immune recognition.This approach also reducesthe possibilityof toxicityderived from
the systemic administration of the cytokine,
Oncolytic HSVshavebeen tested, which encode differentpro-drug-activatingsystems other

than the endogenousTK activityof the virus. Both the S-fluorocytosine (S-FC) pro-drug/yeast
cytosinedeaminase(CD) genesystem,249 alone or in combination with the TK/gancydovir sys-
tem186 and the cytocrome P-4S0/cyclophosphamide (CPA) system. 250.251 were shown to induce
beneficial effects.
Nevertheless, ICP34.S mutants replicate with considerable reduced efficiency in most tumor
cells. comparedto wt HSV.Toimprovetumor-selective replication, an ICP345 deletedHSV with
enhancedgrowth characteristics wasisolatedafterserialpassages on a tumor cellline.Thismutant
wasalsofound to giveimproved anti-tumoractivityin vivo,without compromisingsafety.252These
improvedcharacteristics werefound to derive fromasecondsitesuppressor mutation in the unique
short region, which determinesthe expression of the US1I geneasan IE rather than a L geneand
the deletion ofUS12, encodingthe ICP47 protein, contributing the improvementof anti-tumor
immune response.v' Thisviralisolatewasshown to exhibit enhanced anti-tumor effect. 254
Most of the oncolytic HSVsanalyzedhavebeen based on serially passaged laboratorystrains
of HSV. Thesestrains haveprobablylost some of their aggressive properties. It has in fact been
recentlydemonstrated that an oncolyticHSV,]S1IICP34.S-II CP47-I GM-CSF, derivedfrom a
clinicalisolate ofHSV-I.possesses a higherabilityto kill tumor cells in vitroand in vivo. Moreover,
in a modelof mouselymphoma,micecuredwith this viruswereprotected againstfurther tumor
challenge.!"
In earlyclinicaltrials,however, treatment with the current generationof oncolyticviruses did
not significantly affecttumor growth.234.235 Thissuboptimal resultmayreflectviralgenedeletions,
which can reduce the replicative potential of viruses in tumor cells. For example, deletion of the
y345 genesignificantly reducedviralgrowthevenin rapidlydividingcells.223A varietyof strategies
are beingpursued to enhance the potency of oncolyticviruses. Overall.the resultssofar obtained
demonstratethat incorporatingsuicide and/or cytokinetransgenes in theviralgenomecanincrease
anti-tumor efficacy, especially if usedin combinationwith preexisting anti-cancertreatmentssuch
aschemotherapyor radiotherapy.66.255 Another strategyis to clonetherapeuticgenesinto the viral
genome to arm the virus with additional cytotoxic mechanisms that augment the direct lytic
functionsof the virus.Particularly attractivein this context arecytotoxicmechanisms with potent
bystander effectcapable of eliminatingtumor cells that the virus cannot reach. To this purpose.
it has been recentlydemonstrated that incorporation of cell membranefusion capabilityinto an
oncolyticHSV cansignificantlyincrease the anti-tumor potencyof the virus.256.257Theseoncolytic
HSVswereconstructed bythree differentmethods: (i) screeningfor the syncytialphenotypeafter
random mutation of a well-established oncolyticHSV (to obtain Fu-I'O): (ii) insertionof the gene
encodingthe hyperfusogenic membraneglycoproteinofgibbonapeleukemia virus(GALV.fus)into
the genome of an oncolytic HSV (to generateSynco-Z): and (iii) incorporation of both of these
two membrane fusion mechanisms into a single oncolytic HSV (to generate Synco-2D).These
vectorshavebeen tested for their anti-tumor activityagainstliver, breast, ovarianand metastatic
prostate cancers showinga significant increase in viral oncolysis; this may lead to an enhanced
clinicalperformance, especially in the late stagecancerpatients.
Conclusion
Thedifferentkindsof vectorsthat derivefrom HSV-I wereconceived to takeadvantage ofthe
biological properties of this virus.Therefore, recombinant HSV-I vectors.either attenuated or
defective, attempt to exploitdifferentadaptationsofHSV-1 to the nervesystem, such aslatency,
the presenceof powerful neurospecific promoters, or the occurrenceof viral genes controlling
neuroinvasiveness or neurovirulence. Sofar. promisingresultshavebeenobtained in treatment of
several modelsofPNS and CNS diseases,154.258.259 in treatment of pain17.161 and usingsuchvectors
as tools for investigation of behavioral traits. like learning and memory'>'and for neuroscience
research in general.146Although thesevectorshavebeen used mainlyfor gene transfer to neurons
or glialcells. theyholda bigpotentialasvectorvaccines," both againstinfectious disease and cancer.
In fact. they can efficiently deliver genes to other cell types,includingepithelial cells, fibroblasts,
myoblasts, myotubes, embryonic and adult cardiomyocytes and celllines derived from gliomas,
hepatocellular carcinomas, osteosarcomas, epidermoid carcinomas and many other human and
murine malignancies. In no case, the vectorgenomes integrateinto host chromosomes, therefore
precludingthe riskofinsertional mutagenesis. Theother type ofvector, namdytheamplicon vectors,
attempts to exploitthe capacity of the virus capsid to accommodate more than 150 kb of foreign
DNA. HSV amplicons possess the uniquefeature to possibly ddiver entiregenomic lociincluding
allupstream regulatory dementsand downstream intronsand to converttheminto humanartificial
chromosomes. One of the majorareas of interestin amplicon devdopmentregards thepossibility to
producestilllargeramountsof purified vectors than thosegenerated bycurrentprocedures. Tothis
purpose,different suggestions regardthe improvement of the structureof theamplicon plasmids, of
the helpervirus systems and of the transcomplementing celllineswhere the amplicon vectorstocks
arebeingproduced.Asasecondpoint,thereis the possibilityofcontrollingtransgenic expression for
therapeutical applications and to avoid transgenic silencing. Thiscan be achieved since helper-free
amplicons do not express proteinsenhancingexpression, like ICP4,27and22,orproteinsprotecting
fromsilencing, likeICPO. Asa consequence, transgene expression depends on celltype, multiplicity
ofinfection andcellcycle.It ispossible thatplacingtransgenic expression underthecontrolofgenuine
cellular regulatory sequences will resolve, at leastin part, this difficulty.142.260.261
Much moreworkremains to be carriedout, especially ifweintend to prolongtransgene expres-
sionand to improve celltargeting. However, althoughshort-termtransgene expression represents a
greatlimitationfor the useofvectors in the genetherapyof diseases, this is not necessarily the case
when considering their usefor geneexpression that are associated with certainbehaviors that are
often transient.
Another goalto increase the efficacy of the HSV vectors and to decrease the undesired effeCts
such as infection of healthy cells is to target infection to specific tissues or organsor to restrict
transgene expression to predefined subsets of cells, Geneticmodifications to the genomeofHSV-l
vectors havebeengenerated to preferentially targetviralinfection and/or replication to tumor cells
versus normalcells.'?' Targeting viralinfection to particularcells can beobtainedbymodifying the
firststepsof the virus lifecycle, i.e.,adsorptionand penetration. Efforts for engineering the HSV-l
envelope to obtain targetedinfectionare currently in progress.16,262-264 AlteringHSV-l host range
hasproveda formidable taskbecause H5Y-I infection is a complex process involving the actionof
several glycoproteins in cellattachment, entryand cell-to-cell spread.
Asa final consideration, althoughthe vectorology areaof research is stillin continuousdevelop-
ment,certainly, moreworkshouldbedoneinorderto betterunderstand thevector/hostinteractions.
Anyway,it canbeinferred, fromwhatit isknownon HSV-l immunebiology, that allthe threetypes
ofHSV vectors, includingamplicons, will inducean anti-viral cellular response, at leastin somecell
types and will stimulate both theinnateandadaptive branches oftheimmuneresponse in theinfected
organism. These responses caneventually resultin the elimination of the vectoror in the silencing
of the therapeutic transgenes. Finally, it can be predicted that the large size insert capacity of the
amplicongenome, that allowthesevectors to express several viralor cellular proteinswell-known
to down-regulate or to inhibit the anti-viral and immuneresponses, will be a majoradvantage of
amplicons overother vectors to fightagainst the silencing cellular forces.
Acknowledgements
This work was supported by MIUR-FIRB-2001 (RBNEOI27YS-002), by grants from
the Istituto Superiore di Sanita (ISS), the Italian Concerted Action on HIV-AIDS Vaccine
Development (ICAV), the Italian Ministry for the University and Scientific Research (FISR),
the Italian National Institute of Health (ProgramStemCells,CS 126.1),aswellasbythe French
societies Association Francaise contre lesMyopathies (AFM) and Association pour la Recherche
sur le Cancer (ARC) and from grants form European Commission (THOVLEN project and
HEVARproject, FP6).
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CHAPTER 11
Virus-Like Particles as aVaccine

Delivery System:
Myths and Facts
Polly Roy" andRob Noad
Abstract
accines against viral diseasehave trad itionally relied on attenuated virus strains or inactiva-
V tion of infectious virus. Subunit vaccinesbased on viral proteins expressedin heterologous

systemshave been effectivefor some pathogens, but have ofien suffered from poor immu-
nogenicity due to incorrect protein folding or modification. In this chapter we focus on a specific
classofviral subunit vaccine that mimics the overall structure ofvirus particles and thus preserves
the native antigenic conformation ofthe immunogenic proteins. Thesevirus-likeparticles (VLPs)
have been produced for a wide range of taxonomically and structurally distinct viruses, and have
unique advantages in terms of safety and immunogenicity over previous approaches. With new
VLP vaccines for papillomavirus beginning to reach the market placewe argue that this technology
has now 'come-of-age' and must be considered a viable vaccine strategy.
Introduction
There are many infectiousviruses that remain major threats to public health (seeTable 1).Where
an effective vaccine exists,vaccination is usually the most cost-effective long-term protection against
diseaseand spread for most viruses.The principle ofvaccination is to generate sufficient immunity
to protect from infectious disease. Thus the vaccine stimulates the body's natural defensesagainst
disease through use of a benign 'decoy' that mimics the virulent pathogen. The more similar a
vaccine is to the natural disease, the better the immune response to the pathogen on subsequent
exposure. In general, resistance to virus infection depends on the development ofan immune re-
sponse to antigens present on the surface ofvirions or virus-infected cells. Therefore identification
ofprotective antigens is the first step in the development ofeffectiveviral vaccines.
Currently many successfulviral vaccines have been developed and are in use. These vaccines
are predominantly based on live attenuated or inactivated viruses. The live attenuated vaccines
such as measles, mumps, rubella, oral polio, smallpox, varicella and yellow fever are a weakened
form ofthe "wild" viruses. These attenuated virus vaccines rely on limited replication ofthe virus
in the host following vaccination. Immune responses induced are similar to those from natural
infections and often these vaccines are effective after a single dose. However, such vaccines may
cause severe reactions in some patients, which are ofien the result ofthe limited replication ofthe
att enuated virus following vaccination. In contrast to attenuated live virus vaccines, inactivated
(or killed) vaccines can not replicate, as their genetic material or overall structure are purposefully
destroyed. These vaccines are safer than live vaccines but generally not as effective,requiring 3-5
·Corresponding Author: Polly Roy-London School of Hygiene and Tropical Medicine,

Keppel St., London, WCl E 7HT, U.K. Email: polly.roy@lshtm.ac.uk
Pharmaceutical Biotechnology, edited by Carlos A. Guzman and Giora Z. Feuerstein.
©2009 Landes Bioscience and Springer Science+Business Media.
Table 1. Viruses thataremajor health threats
Virus Disease
HIV AIDS
RSV Respiratory Infection
Hepatitis B Liver Cancer
Hepatitis C Cirrhosis/Cancer
Epstein Barr Virus lymphomas, Nasopharyngeal carcinoma
HPV Cervical Cancer
Measles Pneumonia (infants)
Influenza Pneumonia
Abbreviations: HIV human immunodefi ciency virus, RSV rous sarcoma virus, HPV human
pappilomavirus.
dosesasantibodytiter falls overtime.Theylackthe self-boostingqualities ofliveattenuatedvaccines

but are saferin the sensethat the inherent dangersassociated with virus replicationare avoided.
Thesevaccines are madeaswhole cellvaccines (suchasInfluenza,polio,rabiesand hepatitisA) or
asfractionalor subunit vaccines such ashepatitis B. Subunit vaccines are basedon the delivery of
only a limited number of viral proteins,often the major protein in the capsidor envelope that is
sufficient to conferprotectiveimmunity. Thesevaccines arean incrementalstep saferthan inacti-
vated vaccines becausesubunit vaccines can be prepared independent to the culture of replicating
virus. Indeed, any remaining possibilityof incomplete inactivation or batch to batch variation
in the safetyof the vaccineis eliminated. However, subunit vaccines havetraditionally suffered
from one important drawback; often singleproteins when expressed and purified in the absence
ofother viral componentsare less immunogenicthan those that areincorporated into infectious
virus.Thisisprobablybecause a proportion of this protein ispresentin a misfoldedconformation
relative to the nativeprotein. Thus, more doseswith higher amounts of antigen are required to
achievethe samelevel ofprotection.
A majoradvance in subunit immunogenproduction hasbeen assembly ofproteinsasvirus-like
particles (VLPs) usingprotein expression technologyin yeast, insect or mammalian cells. VLPs
are a highly effective type of subunit vaccines that mimic the overallstructure of virus particles
without any requirement that they contain infectiousgeneticmaterial. Indeed, manyVLPslack
the DNA or RNA genome of the virus altogether, but havethe authentic conformation of viral
capsidproteins seenwith attenuated virusvaccines, without anyof the risksassociated with virus
replication or inactivation.
VLP preparations are all based on the observation that expression of the capsid proteins of
manyviruses leadsto the spontaneousassembly ofparticlesthat arestructurallysimilarto authentic
virus.':' In practicalterms,the fact that VLPsmimic the structure of virusparticlesusually means
that VLPsshouldelicitstronghumoralresponse and that lowerdoses of antigenrelative to subunit
vaccines aresufficient to elicitsimilarprotectiveresponse. In addition to their abilityto stimulate
B cell mediated immune responses, VLPshavealso been demonstrated to be highly effective at
stimulatingCD4 proliferative and =(CTL) responses.l? Thisfeatureof VLPvaccines is likely to
be a major contribution to their effectiveness in the field. It is also becomingincreasingly clear
that preciseprime-booststrategies canbe important to how effective vaccinationisasa strategyto
control disease.Therefore, the addition of VLPto the 'arsenal' of vaccine strategies for anydisease
extends the type ofprime-boostregimethat can beemployed.
To date, VLPs have been produced for many differentviruses that infect humans and other
animals (seeTable2 and review)," One of the most strikingfeatures of this group is that it is ex-
tremely diverse in terms of the structure of the individualviruses. It includesviruses that havea
singlecapsidprotein, multiplecapsidproteinsand thosewith and without lipid envelopes. Clearly
Virus-Like Particles asa VaccineDelivery System 147
Table 2 Baculovirus derived VLPs that havebeen tested as vaccines
Proteins
VLP Family Expressed Vaccine Tested In VLP Refs.
Papillomavirus, Papillomaviridae Humans (licensed) 10,11 ,15-20,73

Norwalk and Calciviridae Mice, cats, humans 26,27,74-78
Norwalk-like (Phase I)
viruses, Feline
calicivirus
Hepat itis E virus Hepeviridae M ice, cynomologous 28-30
monkeys
Porcine parvovirus, Parvoviridae Pigs, dogs, mink 21-23,79,80,81
mink enteritis
parvovirus, Canine
parvovirus, B19,
adeno-associated
virus
Chicken anemia Circoviridae 1, 2 (chicken Chickens 82-85
virus, Porcine anaemia virus)
circovirus
5V40, jC viru s, Polyomaviridae M ice, rabbits (in vitro) 32,86 ,87
murine
polyomavirus
Polio virus Picornaviridae 1 (polyprotein) 88
Bluetongue virus, Reoviridae 4 (bluetongue) Sheep (bluetongue) 4,35,42-49,89
Rotavirus 2-3 (rota) Mice, pigs (rota)
Hepatitis C Viru s Flaviviridae 3 M ice, baboons 7,51,53
HIV, SIV, FIV, Retroviridae 2 Mice, guinea pigs 2,3,50,90-96
Visna virus, FeLV,
BLV, Rous
Sarcoma virus
Newcastle Disease Paramyxoviridae Chickens 97
V irus
SARS Coronav irus Coronaviridae 3 Mice (in vitro) 54
Hantaan virus Bunyaviridae 3 Mice 98
Influenza A virus Orthomyxoviridae 2-4 Mice 52,60,61
Infectious Bursal Birnaviridae 1 Chickens 34,52,99,100
Disease virus
Abbreviations: BTV Bluetongue virus, HIV Human immunodeficiency virus, SIV simian immuno-
deficiency virus, FIV feline immunodeficiency virus, FeLV feline leukem ia virus, SV40 simian virus
40, rota rotaviru s.
not allofthe VLPs that are generated to date are appropriate vaccine targets, some VLPs have been
generated to facilitate in fundamental understanding of virus assembly process, morphogenesis
or architecture ofviruses. However, an important point remains that the structure of the target
virion is not limiting to the successofVLP production. Although various expression systems have
been employed for VLP production, this chapter will mainly focus on insect cell culture produced
VLPs that are being developed as candidate vaccines. The rationale behind this is that among all
expression systems, insect cells, together with baculovirus expressing system, appear to be one of
the most promising for VLP technology for development ofviral vaccines (Fig. 1).
148 Pharmaceutical Biot~chno/Qgy
Figure 1. Key stages of intracellular assembly of VLPs using the baculovirus system. a)
Baculovirus acts as a vehicle to efficiently deliver DNA, encoding recombinant proteins,
to the nucleus of insect cells. b) Viral DNA is uncoated and replicates in the nucleus. c)
Recombinant protein expression is driven by strong very-late viral promoters. d) Viral mRNA
is used for the synthesis of recombinant proteins. e) VLPs are assembled by the interaction
of proteins w ithin the cytoplasm.
Insect Cells and Baculovirus Expression System as Preferred System

for VLP Production
As stated above, a varietyof protein expression systems are available to express recombinant
proteins and particles. Howevercertain criteriafor generationofVLPs as prophylacticvaccines,
particularly forhumanviralinfection,mustbe considered. In orderforaVLPto bea realisticvaccine
candidate, it needs to be produced in a safeexpression systemthat is easyto scaleup to large-scale
production.Table2 shows baculovirus expressed/insect cellproducedVLPsthat havebeendemon-
strated to be highlyimmunogenicand potential vaccine candidates.Thisinsectcell-based protein
production systemhas manyadvantages for VLP production. Firstly, extremely largeamounts of
correctly folded recombinant proteins can be produced in high-density cell-culture conditions
in eukaryotic cells. Secondly, baculovirus expression systems havebeen developedfor expression
of multiple foreign proteins simultaneously from a single recombinant virus facilitating capsid
assembly in each infectedcell. Thirdly. asthe insectcellsthat are usedfor vaccine production can
be cultured without the need for manunalian cellderivedsupplements. the risksof coculture of
opportun istic pathogens is minimized. Fourthly. the baculovirus used for recombinant protein
expression has a narrow host range that includesonly a fewspecies ofLepidopteraand therefore
represents no threatto vaccinated individuals. Finally the baculovirus system is amenable to scale-up
for largescalevaccine production,"
Virus-LikeParticles asa Vaccine Delivery System 149
VLPsProduced forStructurally Simple Non-Enveloped Viruses

For a number ofnonenveloped virusesviral capsidsare formed by only one or two major pro-
teins and thus are relatively easyto manipulate for generation of VLPsbyheterologousexpression
systems. Examplesofthese are the VLPsformed by the expressionofthe major capsidprotein of
Papillomaviruses, Parvoviruses, Calciviruses, Circovirses, Polyomaviruses and Hepatitis E virus
(Table 2). All of these viruses are nonenveloped and have a single, virallyencoded protein that
forms the major structural component of the virion. Papillomavirus VLPs are among the most
completelystudied ofthis collection of VLPs and are at the most advancedstagewith respectto
production ofa usefulvaccine. VLP ofPapillomavirusesare formed from the over expressionof
the major capsidprotein Ll. lO•12These particlesare highly immunogenic and are ableto stimulate
both humoral and cellmediated immune responses.P'" Human Papillomavirus (HPV) isthe lead-
ing causeofcervicalcancer.Globally, approximately70% ofall cervicalcancer casesareassociated
with two serotypesofHPY, HPV-16 and HPV-lS. VLPsproduced in insect cellshavebeen used
successfully for Phase I and II human clinicaltrials in largenumbers and were shown to be highly
efficacious. 1S•19 Moreover, GlaxoSmithKline's cervicalcancervaccinecandidate (Cervarix:"') target-
ing HPV 161 IS iscurrentlyundergoingPhaseIII clinicaltrialsinvolvingmore than 30,000women
worldwide. In this Phase III randomized, double-blinded trial conducted in multiple centres in
Denmark, Estonia. Finland, Greece. the Netherlands and the Russian Federation. All vaccinees
receivedthe HPV VLPs(HPV-16I1S AS04) asfollows: 15S 10-14yearsold healthy girls and 45S
15-25 yearsold young women receivedthe candidate VLP vaccineaccording to a 0,1 ,6 month
schedule and anti-HPV antibody titers wereassessed. At month seven 100 per cent seropositivity
wasachievedin both groups for HPV 16 and IS although average antibody titers for both HPV
typeswereat leasttwo-foldhigher in 10-14 year-oldgirls.Thevaccinewastolerated byallpatients
and no vaccinerelated seriousadverseeffectswere detected. Further, the follow-upsmdy clearly
demonstrated the sustained efficacy of HPV-16I1S VLPs up to 4.5 years.19,20 In conclusion, the
bivalent HPV vaccine is highly immunogenic and safe and induces a high degree ofprotection
against HPV-16 and HPV-lS infection and associatedcervicallesions.
Thesestudiesarenot onlyan important demonstrationofthe effectiveness ofHPVVLP vaccine,
and that multi-serotype VLPs are effective, but also highlight the fact that insect cell produced
VLPsare a realisticalternativeashuman vaccines againstviraldisease. It should alsobe mentioned
at this point that a tetravalent (HPV-61III 1611S)VLP vaccine, Guardasil'" (Merk),produced in
yeastcellswasapproved by FDA in]une 2006 for use in women aged 9-26.
VLP vaccinesfor variousdiseases causedby parvovirusinfections are alsoat an advancedstage
although as yet none have undergone such large scaletrials as those reported for HPV. Synthesis
ofmajor structural proteins VP2 ofcanine parvovirus (CPV) and porcine parvovirus (PPV) led
to assembly ofVLPs in insect cells. 21.22 Vaccinationtrials of CPV VLPs in dogs and PPV VLPS
in pigswere highly encouraging.i':" In one efficacy assaydogs that receivedaslittle as or 10 ~or
25 ~ ofCPV VLP werecompletelyprotected from virus infection when challengedwith virulent
virus. Furthermorea singlesubcutaneousdose 00 ~same CPV VLP with 50 ~ ISCOM adjuvant
wasableto protect mink againstchallengewith the anti-genically similarvirus,mink enteritisvirus
(MEV),2l Similarlyit has been reported recently that a singleimmunization with 0.7 ~ ofPPV
(porcine starin) VLPs yielded complete protection in targeted animals against infectious PPV
strains.P Indeed microgram doses ofVLPs in gilts were not only highly immunogenic. but were
alsoveryefficientin preventing trans-plancentalvirus transmission and significantly reduced the
number of reproductive failures. In addition, the feasibilityof safelarge-scale production of the
porcine parvovirusVLPvaccine hasbeen establishedcomplyingwith the EuropeanPharmacopoeia
requirements,"
Calicivirusstudies have relied heavilyon the production of proteins in heterologous systems
mainly due to the fact that it is not yet possible to grow the virus in cell culture. Thus, VLP to
Norwalk-like viruses have been extremely useful as sources of diagnostic antigen to monitor
diseaseoutbreaks. Norwalk virus VLP have also been shown to be effective at stimulating IgG,
IgA and humoral responsesin mice.24•2s Preliminary PhaseI trials in humans to test the safetyand
150 Pharmaceutical Bjot~chnology
immunogenicity of insect cell expressed Norwalk virus VLPs has confirmed that they are both
safeand effectively stimulate IgG and IgA responses.26.27
VLPsfor Hepatitis E havebeen assembled usinga truncated form of the viruscapsidprotein."
In immunization studies in mice these VLPswereable to induce systemic and mucosalimmune
responses following oral adminisrrarion.P" Furthermore, oral administration of the Hepatitis
E VLPs to cynomologousmonkeys induced IgM, IgA and IgG responses and was sufficient to
protect against infection and disease on challengewith virus," Thus there is clear potential for
the application of theseVLPsas a vaccinefor hepatitis E.
VLPpreparationsto Circoviruses and Polyomavirusareat a less advanced stage.VLP formation
hasbeen reported for Circovirusbut asyetno seriousattempt hasbeenmadeat vaccine production.
Vaccination of rabbitswith VLPsfor human JC virusin the presenceof adjuvantallowedproduc-
tion ofa hyperimmuneserumthat effectively neutralizedinfectiousviruspreparadons." However,
in the absenceofadjuvant there was no response. This pattern of responseis unusualfor VLPsin
general,which often stimulatestrong immune responses evenin the absenceofadjuvant.Indeed,
VLPs of murine polyomavirus were able to stimulate a strong immune responsein the absence
of adjuvant when administered as a single 610 ng dose.32 Intriguingly, these particles appear to
be particularlystablewith no alteration ofparticle morphology or reduction in immunogenicity
even after 9 weeksstorageat room ternperature.P
VLPs ofStructurally ComplexViral Capsids with Multiple ProteinLayers

Viral particles that contain multiple interacting capsid proteins present more of a technical
challengethan those that are formed by one or two major capsidproteins. Particularly, it is far
more difficultif the assembling proteins of capsidsare encoded by multiple discrete mRNAs, but
not processedfrom a singlepolyprotein as in the caseof picornaviruses. This is due the fact that
for efficientassembly of a VLP the interacting capsidproteins must be expressed in the vicinity to
each other, in other words in the samecell Assembly of VLPsbyprocessingof polyproteins have
been achievedboth for poliovirus" and for InfectiousBursaldiseasevirus" usingthe baculovirus
expressionsystem. More complex assembly of multilayered, multiprotein VLPs have also been
efficiently produced for the members of the Reoviridae. Theseviruseshave capsidsmade up of
concentric layers of different capsidproteins. Co-expression in insect cellsof2-4 ofthese capsid
proteins, depending on the virus and the particle made, has allowedthe production ofVLP that
are empty of the segmented dsRNA viral genome, but are otherwise indistinguishable from
authentic viral particles.4•35The first member of the Reouiridae for which VLPs were described
is Bluetongue virus (BTV), an insect transmitted animal virus. This remains the systemin this
familyfor which the largestvarietyofdifferentVLPsand recombinant singleantigen subunit im-
munogens made by baculovirus expression systems has been tested. In addition, the requirement
for efficientco-expression of viralcapsidprotein in the sameinsect cellin this systemhas resulted
in the development ofbaculovirus multigeneexpression vectors.36.37We will focuson this system
in somedetail asit highlightsboth the effectiveness ofVLP vaccines and someof the technological
advances that havebeen made for the production ofVLP with complexarchitecture.
Bluetongue disease affects mainly sheep and cattle and is classified as an emergingdiseasein
Europe." The disease is causedby bluetongue virus,BTY,which has a multi-layered icosahedral
structure formed by nonequimolar amounts of sevenviral proteins (VP1-VP7). Three of these
structural proteins (VP1,VP4, VP6) are dispensable for the formation of VLPsas they playonly
an enzymaticrole in the virus transcription rnachinery.P The remainingfour structural proteins
(VP2, VP5, VP3 and VP7) are organisedin two capsids. The inner capsidactsasa scaffold for the
assembly of outer capsidthat is responsible for cellentry and hence contains the major candidate
for virus neutralisation."
Expression of all four major structural proteins of BTV was achieved by construcing a
baculovirus that simultaneously expressed all four proteins.YThe advantage of this approach
over co-infection with several baculoviruses each expressing a single protein is that equivalent
conditions are achievedin all infected cells. Thus assembly ofVLP is more efficientasexpression
Virus-Like Particles asa Vaccine Delivery System 151
A
VP2
VP5
I VP7
« VP3
B 600
500
- . · - 10 J,g VlP
?;
.& 400 · · ·. · · 5OI'll VLP
=
~ _100 ~gVLP
i!' 300 - -0- - 200 ~g VLP

!
Loo
z
r¥ne
100
0
0 20 60 80 100 120
c
Inocu lum CRI V1r. em 1a
(day. post c ha''-nge)
10,.gVLP 00 none
50 ,ogVLP 00 none
l00~gVLP 00 none
2OOI'llVLP 00 none
SalIne 6.5 0 • .,.4·14
Figure 2. Summary of pro duc tio n and tes ting of VLPs for Blue to ngue virus. A) Left, car toon
showi ng the multi-laye red structure of BTV VLPs. Right, e lectron micrograph of negatively
stained BTV VLPs. B) Summary of neutra lizing a ntibody respo nse to VLP vaccination in
Me rino sheep. She ep were vaccinated with two doses of VLPs with dose ranging from 10 Ilg
to 20 0 ug as indicated. Neuralising antibody titre was followed for 117 days, at whic h point
the sheep we re c hallenged w ith virulen t BTV. C) Ta ble showing clinical reac tion index (CRI)
and length of Virae mia in sheep vaccinated with va rious doses of VLP an d control. No signs
of blueton gue disease or virae mia we re detected in any of the VLP vacci nated a nimals .
is controlled at the level of the cell. rather than the level of the culture as is the case with mixed
infections. BTV VLPs (Fig. 2) are structurally indistinguishable from virus particles bu t lack the
segmented. double-stranded (ds) RNA virus genome normally present in infectious virus."
Antibodies raised to purified BTV VLPs gave high levels of neutralizing antibodies against
the homologous BTV serorype.i In subsequent clinical trials 1 year-old Merino sheep were vac-
cinated with various amounts (10-200 fLg) ofVLPs for BTV serotype 10. All vaccinated animals
developed demonstrable neutralizing antibodies 39•40 and when challenged with virulent virus after
four months of vaccination were completely protected from disease. In contrast, unvaccinated
control animals developed typical BT clinical symptoms. Even at doses as low as 10 fLg VLP was
sufficient to protect animals from any signs ofdisease. Further efficacy tests were performed where
VLPs from two different serotypes were combined to vaccina te the same animal. In these animals
VLPs vaccination provided complete protection against the rwo vaccine serorypes and also partial
protection fromchallenge with relatednonvaccine serotypes.The protectiveefficacy ofvaccination

in thesetrialsextendedovera long(14 month) period.P'Ihis observationraises the possibilitythat
a broad spectrumvaccine againstall 24 BTV serotypes is a possibilitybycombiningVLPsfrom a
relatively smallnumber of serotypes.
The BTV system also demonstratesthe efficiency ofVLP vaccines relative to immunization
with subunit vaccines based on dissociated antigens or unassembled recombinant antigens. In
addition the assembled VLPsthe two componentsofthe BTV outer capsid, VP2 and VP5, were
alsopreparedand testedin vaccination studies.While 100 ~ VP2,the majorserotypedetermining
antigen,wasonlypartiallyprotectiveforashort duration (75days) againstvirulentviruschallenge,
50 ~ ofVP2 combined with 25 ~ VP5 was protective." In contrast, 10 ~ VLPs (containing
only 1-2 ~ VP2) affordeda better level of protection for a much longer duration." Thesestud-
ies demonstrate that assembly of antigensinto VLPsresultsin a more effective immunogen than
deliveryof separately isolatedproteins.
In addition to BTV, VLP have also been produced for rotavirus, another member of the
Reoviridae. Intriguingly, VLPsformedfrom the twoinner structuralproteinsaloneof the rotavirus
capsid have been shown to be effective immunogens in animal modelsY-48Indeed in mice even
intrarectal immunisationwhich inducesa localmucosalresponse issufficient for protection from
rotavitus infection .v The data from these immunogenicityexperiments are encouragingand it is
possible rotavirusVLP mayprovidea viablealternative to the livevitus vaccine for rotavitus.
VLPs from Viruses with LipidEnvelopes

Manypathogenicviruses suchasInfluenza, HIV and HepatitisC aresurroundedbyan envelope,
a membranethat consists of a lipid bilayerderivedfrom the host cell,insertedwith vitusglycopro-
tein spikes. Theseproteins are the targetsofneutralizingantibodiesand areessential components
ofvaccine. Due to the inherent properties of lipid envelope, assembly ofVLPs in insect cellsfor
thesevitusesis a different type oftechnicalchallenge to those produced for vituseswith multiple
capsids. Nevertheless, efficient formation ofVLPs of a number of enveloped viruses in insectcells
hasbeen reported.Forexample, VLPsof HepatitisC virus,several retroviruses, SARSCoronavirus
and influenza A havedemonstratedcorrectassemblyof the the lipidenvelope with theglycoproteins
inserted.so-ss Indeed,for retroviruses, it hasbeenpossible to producehybridVLPsthat contain the
gagcapsidprotein fromone virus(SIV) and the envelopeprotein from another (HIV)S6 in insect
cells. Although none of the retrovirus derivedVLPsare yet at the stagethat they are beingusedin
clinicalvaccinetrials,initial experiments in anitnalmodelsare promising.s7.s8
VLPs for SARS Coronavitus as a basisfor vaccinationwere produced rapidlyfollowing the
SARSoutbreak in 2002-2003.S4.SSHoweverthe control ofSARS Coronavirusbyepidemiological
measures, continued lackof re-emergence of the virus,and difficulties workingdirectlywith the
virus haveseverely limited the developmentof SARSVLPs as vaccine. Despite this, anti-serum
raisedin mice againstinsect cellderivedSARSVLPswereableto neutralizea retroviruspseudo-
typed with the SARSS protein (Fig.3).
The Hepatitis C VLPs(Fig. 1)havebeen tested in miceand baboonsand shown to be effective
at stimulatingboth cellularand humoralimmune responses.?·S3oS9 In one experiment, 6-8 weekold
female BALBlc micewere immunizedintramuscularly three times,at three week intervalswith
20 ~ insect cellderivedH CV VLP, produced byco-expressing HCV coreE1-E2. Because of the
lackofasuitableanimalmodelfor H CV infectionsa recombinantvaccinia vitusexpressing HCV
structural proteins (vvHCV.S)wasusedasa modelsystem. Vaccinated micewerechallenged three
daysafterthe finalimmunizationwithvvHCV.S and then five dayslaterthe ovaries ofinfectedmice
wereharvested and the vaccinia virustitre determined. Fiveout ofseven vaccinatedanimalshad
no detectablevacciniavirus in the ovaries at this point. The remainingtwo anitnalshad five logs
lowervacciniatitres compared to control mice'? In addition, this study wasable to demonstrate
that the VLPsefficacy wasbasedlargely on its stimulation of CD4+ and CD8+ T-cell responses.
A further study in baboons has demonstrated that the VLPsare welltolerated and can stimulate
broad and long-lastingHCV targeted immune responses.P
Virus-Like Particles asa Vaccine DeliverySystem 153
B
600 ,
500
Mouse serumaamples
SARSVLP V8cD1e
Figure 3. Summary of production and testing of VlPs to SARS coronavirus. A) left, cartoon
and right, electron micrograph of VlPs produced by co-expression of E, M and S proteins of
SARS coronavirus . These VlPs were used to raise anti-sera in mice and the ability of these
anti-sera to protect against infection with a SARS 5 protein pseudotyped lentivirus were as-
sessed. B) le90 neutralising antibody dilution for SARS S pseudotyped lentiv irus, using sera
from 3 mice immun ized with SARS VlP, rotavirus VlP and serum obtained from a SARS
convalescent patient.
To date, the most structurally complicated enveloped virus particle that has been used to
generate VLP is influenza.VLPs for InfluenzaA H9N2 and H3N2 have been produced by other
groups.PThese studies have shown that expression of the major structural protein MI alone is
sufficient result in the budding ofvirus-likevesicles from insect cells.? Also, co-expressionofMl
with M2, HA and NA leads to the assembly of influenzaVLP and MI-HA and MI-HA-NA VLPs
confer protection from lethal challengewith the same type influenza A in mice.60•61 VLP produc-
tion was also successfully achieved by co-expressing HA, NA, MI and M2 from influenza virus
A/Udorn/72 (H3N2) using a single recombinant baculovirus.P To date none ofthese influenza
VLP havebeen tested in humans. However the potential that HA and NA could be incorporated
directly into these VLP from circulating influenza strains without passage in tissue culture has
particular advantage for the control of rapidly changing influenzaA virus.
Future andAlternative Directions

In addition to the use ofVLPs as direct immunogens, the efficiency with which they stimu-
late cellular and humoral responses has made them prime candidates as carrier moleculesfor the
deliveryof epieopes, DNA and smallmolecules targetingother diseases. This has been facilitated
by the excellent structural information that is often available for virus particlesallowingrational
designof vaccines where epitopes are exposedon the surfaceof the VLP. Manyof the VLPsthat
have been developedas vaccines in their own right have also been tested as delivery systems for
other molecules. It is not possible here to providea full accountof this approach, as the literature
on deliveryand display usingVLPsis at leastaslargeas that on VLP production for direct immu-
nization (for reviewseeref 62). Howeverit is necessary at least to introduce this important area
ofVLP-based vaccine development.The use ofVLPs as carrier molecules for epitopes for other
diseases isnot limited to thoseVLPsthat areformed from the capsids of economically significant
viruses. The reasonthat manyVLPsmakeexcellent carriermolecules for the delivery of epitopes
in vaccines is most likely because the particulate VLP structure is readily taken up into antigen
presenting cells and thus is able to prime long lasting CTL responses in addition to antibody
responses.6.63.64 Certainly accumulatedevidence on VLP vaccines suggests that they are efficient
at stimulating both cellularand humoral immune responses. H 64-66 Notable work has been done
in this areawith both the hepatitis B core particles, human papillomavirus VLPsand parvovirus
VLPsdisplayingT-cellspecific epltopesfromanother protein on their capsid.5.64-66.67 Thesestudies
demonstrate that like bacterialepitope displaysystems VLPs are efficient stimulators of MH C
class I and class II responses.v ThusVLPshavegreatpotential asepitope displaysystems for other
diseases. Theonly majordrawbackfor this approachisthat the requirementof the capsidprotein
to assemble often constrains the sizeof the foreign sequence that can be tethered to the VLP.
One approach that maybe of use to overcome this constraint would be to link foreign protein
sequences to capsidproteins in such a way that they extend the N or C termini of the protein
and extendeither insideor outside to particle/" Ofcourse,this isonly suitablewhereone or both
termini ofthe protein are exposedon the insideor outside faceof the capsid. So far, there are no
VLP that we are awareof that havefullyexploitedthe potential of this approach but it has been
successfully employedfor other protein-basedparticulate structures that are similar to VLPs in
their stimulation of B-cell and T-cell responses and requirement for complexprotein-protein
interactions for particleassembly/"?'
Perspectives: Myths andFacts

Despite the accumulatedevidence of the potential ofVLPs as potent immunogensfor many
viralsystems that wehavediscussed, thereremains someresistance to the VLPapproachasageneral
vaccinationstrategyfor diseases causedby viruses. In part this is due to some high profiledisap-
pointing resultsfor VLP vaccines in the earlystages of development, for example an ineffective
earlyvaccine for HIV basedon TyVLPS.72 Thisexampleraises a point of caution for VLPvaccine
designers. In general. VLPs stimulate efficient cellular and humoral immune responses but, as
with anyvaccine, they relyon the long term host response to be effective. VLPsdesignedto work
in immunocompromisedindividuals need to overcome the samechallenges to efficient immune
responseas any other vaccine approach. The notion that VLPsare ineffective vaccines is clearly a
myth that isexplodedby the imminent release of two new VLP-based HPV vaccines. Indeed, the
accumulateddata from the fieldsuggests that VLPsare more effective than manyother types of
subunit vaccines, becausethey are more conformationally authentic and are safer than manylive
viruspreparationsbecause they are usually freeof viralgeneticmaterial.VLPproduction doesnot
appear to be limited to anyone type of virus or virusfamily, nor is it significantly limited by the
complexityofthe virus particle."
The use of insect cells as a protein expression system offers excitingopportunities for the
synthesis of conformationally authentic VLPs that are formed from the intracellularassembly
ofmultiple proteins expressed in the samecell. The advantage of this system overothers used for
protein expression is its capacityfor industrial scale synthesis of largeand multiple proteins and
the fact that insectcells are the natural replicationreservoirfor manypathogenicviruses. Thusthe
basiccellularmachinerythat normallyprocesses the infectiousform of the virusispresent within
the expression system and available to produce authentic VLPs.
Virus-Like Particles asa Vaccine DeliverySystem 155
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CHAPTER 13
Immune Modulators with Defined

Molecular Targets:
Cornerstone to Optimize Rational Vaccine Design
Thomas Ebensen and Carlos A. Guzman"
Abstract
accination remains th e most valuabl e to ol for preventing infectious diseases. However,
V th e performance ofmany existing vaccines should be improved and there are diseases for
wh ich vaccines are still not available. The use ofwell-defined antigens for the generation of
subunit vaccines has led to products with an improved safety profile. However, purified antigens are
usually po orly immunogenic, making essential th e use ofadjuvants. Despite the fact that adjuvants
have been used to increase th e immunogenicity of vaccines for more than 70 years, only a hand-
ful has been licensed for human use (e.g., aluminium salts, the micro-fluidized squalene-in-water
emul sion MF59 and monophosphoryllipid A). Thus, the development of new adjuvants which
are able to promote broad and sustained imm un e responses at systemic and muco sal levels still
remains as a major challenge in vaccinology. Recent advance s in our understanding ofthe immune
system have facilitated the identification of new biological targets for screening programs aimed
at the discovery ofno vel immune stimulators. This resulted in the identification ofnew candidate
adjuvants, wh ich made po ssible the modulation of the immune responses elicited according to
specific need s. A number of promising adjuvants which are currently under preclinical or clinical
development will be described in this chapter.
Introduction
A key requirement for the immune system is the discr iminat ion between self and nonself,
particularly in the context ofmicrobial agents able to cause disease. The recognition ofpathogenic
micro-organisms is in pan performed by th e presence of pattern recognition receptors (PRR),
such as th e Toll-like receptors (TLR), on cells from the innate immune system. Thi s system has
also evolved other mean s to ident ify potentially dangerous entities, such as the CD ld-mediated
recognition of cerarnide s: th e complement system ; specialized receptors enabling natural killer
cells to sense "nonself", "missing-self" and "induced-self "; and the Nod proteins by wh ich unique
microbial motifs are detected (e.g., peptidoglycan), thereby initiating pro-inflammatory signaling
cascades.u ·s On the other hand, the recognition ofdangerous enti ties by the innate immune system
is a prerequisite for th e stimulation of pathogen- specific adapt ive immune responses.F It is no w
known that adaptive immunity dependencyon th e innate immune system results from the need for
antigen pro cessing and presentation. These fun ction s are d isplayed by professional antigen present-
ing cells (APC), such as dendritic cells (D C) .N The initial uptake and phagocyto sis ofmicrobes by
APC is facilitated by recognition ofpathogen-associated molecular patterns (PA M P) by PRR.This
·Corresponding Author: Carlos A. Guzman-s-Department ofVaccinology and Applied

Micob iology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 0 -38124
Braunschwe ig, Germany. Email: carlos.guzmanwhehnholtz-hzt.de
PharmaceuticalBiotechnology, edited by Carlos A. Guzman and Giora Z . Feuerstein .
leadsto the activationof DC maturation, aprocessentailingup-regulationof majorhistocompat-

ibility complex (MHC ; class I and class II) and costimulatory(e.g., CD40, CD80 and CD86)
molecules, together with the production of differentcytokines,which in turn resultsin optimal
antigenprocessingand presentationto naiveCD4+ and CD8+ T-cells. A wellorchestratedinnate
and adaptiveimmune response usually leadsto pathogen eradicationand long-lasting immunity
against the specific agent (Fig. 1). In contrast, failureto efficiently discriminatebetween selfand
nonself can lead to uncheckedspreadof the pathogen or autoimmunity.
Although highly effective vaccines are currentlyavailable for a number of infectiousdiseases,
vaccineformulations can still be improved. The use of well-defined antigensfor the formulation
has led to lessreactogenicsubunit vaccines, which unfortunatelyareless immunogenicthan their
liveor wholecellinactivatedcounterparts.Thishascreateda majorneedfor efficient adjuvants. On
the other hand, conventionalvaccines are usually administeredbythe systemic route.Thismainly
promotes the elicitationof systemic immunity.However, most pathogensenter the body through
mucosal surfaces. Therefore, systemic vaccines are suboptimal, since they fail to induce a local
mucosalresponse able to blockthe earlystages of infection.This roadblockcan be eliminated by
administeringantigensbythe mucosalroute. Furthermore, mucosal vaccination offers several ad-
ditional benefits, suchaseasyadministrationlogistics and high acceptance bythe public,asa result
of the lack of pain.?However, mucosalvaccines haveto overcome formidablebarriers. Antigens
administered by this route can be clearedbynon specific mechanisms (e.g., ciliaractivity, mucous
entrapment, peristaltisrn), are degraded by enzymes and may be affectedby extremepH , before
havingthe chanceof reachingAPC. One of the strategies to overcome the poor immunogenicity
Innate immunity Adaptive immunity
.
..........• cellular _'~\.
,1 \ .
vv
'.
I .
I
I
I
I
I
I
'.. I
, '---I
I
. ..
• Iflb/ll I
''o..~cs."
.. "'" .. .. .. I
I
.. , I
., I
I
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,I
hurnoral r "
._.- . No effecto r (..-v:t6on

-_•• Stront..ffKtDr function
Figure 1. The success or failure of an antigen-specific immune response depends on the

inte rface between innate and adaptive immun ity. For a successful removal of a pathogen it
is essential that microbes are recognized by components of the innate immune system. The
systems for microbial sensing are complementary and they are involved in the development
of the ensuing adaptive immune response. Adaptive responses may tend toward tolerance or
productive act ivation of T helper Type 1 (Th1) or Type 2 (Th2) cells. Different responses are
required for the elimination of different microbes (e.g., clearance of intracellular pathogens
usually requires robust Th1 and CTL responses).
Immune Modulators withDefinedMolecular Targets 173
is the co-administration of antigenswith mucosal adjuvants." Thesecompounds generatea local

microenvironment conductive towards antigen processingand presentation, thereby promoting
the elicitation of robust responses at both local and systemic levels. I I
Thechemicalnature of the adjuvantsand their mode ofaction areextremelyvariable. Potential
mechanismsbywhich theyexerttheir biological activityarethe "depot"effect,antigentargetingto
APC, improvementof antigenprocessing and presentation and immuneactivationor modulation
through the up-regulatedexpression ofcellularmediators.However, it is also important to consider
that their biologicalactivities maynot onlyfavorthe elicitationof adaptiveresponses, but also the
appearanceof side effects. A strong stimulation may result in local or general inflammationand
tissuedestruction,with the resultingdistressfor the vaccinees. On the other hand, the stimulation
ofvigorousresponses isinsufficient to protect againsta specific pathogen. It isessentialto promote
an adequate type of response. Otherwise, we mayeven lead to immune pathological reactionsor
a more severe course of infection. Thus, the identification of adjuvantsable to promote predict-
able responses is a major issuein vaccinology. The lack of adjuvantswhich are able to stimulate
cell-mediatedresponses representsalso a bottleneck. Only a few moleculeshavebeen identified
exhibitingthisproperty,beingmostof them unacceptable forhumanusedue to their reactogenicity.
Therefore,the choiceofa specific adjuvant for a formulation reflects a compromisebetween the
required immune modulatory effectand an acceptable(i.e., low) levelof side effects.
Dozens of adjuvants have demonstrated their efficacy in preclinical and clinical studies.
Nevertheless,only a few, suchaluminum-basedsalts,12,13the squalene-in-wateremulsionMFS914•15
and monophosphoryllipid A (MPL)I6-19 have been approved for human use (Table 1). In fact,
aluminum saltsare at the moment the most widelyused adjuvantsfor both human and veterinary
Table 1. Adjuvants included in human and veterinary vaccines
Adjuvant Administration Route Side Effects
Aluminium based salts Systemic Mild systemic reactions

(93%),27 local side effects
(e.g., suprapubic pain and
vesical tenesmus)20,28
Squalene oil-in-water emulsion Systemic Local side effects29,30
Monophosphoryl lipid A (MPL) Systemic and mucosal Pyrogenic at high doses"
Endogenous human cytokinesor Systemic May cause adverse effects
hormones, (e.g., INFs, IU2 , GMCSF)
Bacterial toxoids (e.g., derivatives of Systemic and mucosal Mild side effects, may result
cholera toxin and E. coli heat labile toxin) in retrograde homing to
neural tissues when
administered by nasal
route32,33
Muramyldipeptide (MDP), lipid A Systemic and mucosal Currently under study
derivatives
Lipopeptides, MALP-2 Systemic and mucosal
Saponins (e.g., QS-21 ) Systemic and mucosal May cause allergic
reactions when given at
high dosages
Immunostimulating complex (ISCOMs) Systemic and mucosal Currently under study
CpG-ODN Systemic and mucosal Conflicting data on effect
following long term
administration, may cause
mild damage."
vaccines. Although severe side effects are rare, alum has the potential to cause sterile abscesses,
eosinophilia and myofasciris.fThere are also concern regardingthe potential role of aluminum
in neurodegenerative diseases."Alum inducesstrongantibody and Th2 responses. Therefore, ad-
juvantsableto promote Th1 and CTL responses arestill needed. Interestingly, the useof alum in
combination with ILI2 can redirectresponses fromTh2 towardsaThl-dominant response." On
the other hand, MFS9 promotes both humoral and cellularimmune responses. 14.1S.22.23Squalene
or squalaneemulsionsareefficient adjuvants whichcan bestabilizedbymicro-fluidization, sothat
the emulsionscan be frozenor kept for years at room temperature, allowingalsotheir sterilization
by terminal filrradon." Antigensare added after emulsification, so that conformationalepitopes
are not lost by denaturation, as well as to facilitate manufacturing. 24.25 Clinical trials of several
MFS9-adjuvantedvaccines, which were performed in different age groups (from newborns to
elderly), havedemonstrated their safetyand immunogenicity. However, PhaseIVsmdiesareman-
datory,sincethe useof oil-based adjuvants maybe associated with a higher riskfor autoimmunity
in experimentalmodels." Thisseems to be relatedwith the hydrocarbon's abilityto induce ILI2,
IL6 and TNFa. Whether this is of relevance for human vaccinationis matter a discussion, since
immunotoxicity depends on many factors, such as the species, genetic makeup, route, dose and
duration ofthe administration. In fact, up to now,clinicalstudiesdo not seemto support a high
risk for autoimmune disease.
Saponinsare a chemically heterogeneous group of sterolglycosides and triterpene glycosides,
which are common constituents of plants. They are known to cause substantialenhancement of
immuneresponses sincethe 1920s. Naturallyoccurringsaponinsfrom QUillaja saponaria stimulate
humoralresponses againstT-dependenr andT-independentantigens and CTL responses." Despite
their usein animalvaccines, the developmentof saponin-based formulations for humanshasbeen
impeded by their complexityand concernsabout toxicity.3S,36 On the other hand, ~ilA, which
resultsfrom partial purificationfrom crudefood-grade extracts, iscontained in severalveterinary
vaccines. Further purificationprovided concentrated saponin fractions, such as QS-21, which is
currently under clinicalinvestigation in humans.37.39 Interestingly, purifiedsaponinsseems to be
alsoeffective asadjuvantswhen delivered byoral route."
Saponinshavebeen combined with cholesterol and other lipidsto generateimmunosdmular-
ingcomplexes (ISCOMs), which areopen cage-like structureswith build-inadjuvantactivitythat
promote antibody,T helper and CTL responses. ISCOMs seemto enhance antigen targetingto
APC , as well as their subsequent uptake, processing and presentation. The use ofISCOMs also
result in the production of pro-inflammatorycytokines, such as Ill, IL6 and ILl2. 40 Liposomes
representa relateddelivery system, which alsohasbuild-inadjuvantproperties.Theyarevesicular
structureslimited bya bilayermembranecomposedof phospholipidsand cholesterol.Liposomes
can carry both membrane associated antigens, as well as water solublemolecules. Their physical
properties are highly variable, depending on the composition and the manufacturingmethod.
This allows the optimization of the designfor specific tasks (e.g., targeting, co-incorporation of
adjuvants). Liposomes havea longhistoryasvehicles forantigendelivery.41.42 Recently, the so-called
virosomes havebeen developed, byincorporatingthe hemagglutininfrom the influenzavirusinto
llposomes.vr" Thisglycoprotein guidesvirosomes to APC and promote their fusionwith the en-
dosomalmembraneat lowpH.Thisin tum leadsto the cytoplasmic release of the antigens, thereby
providingoptimal processing and presentation in the context of MH C class I molecules.
Thereare other candidateadjuvants in advance stages of preclinicaland clinicaldevelopment,
such as a new generation of water-in-oil emulsions [e.g., Montanide, CSA 720), which were
demonstrated ableto triggermoreefficient humoralresponses than alumin several animalspecies
and humans.47-49A smallimmunomodulatory peptide, CEL-l 000, alsopromoted the elicitation
of protective Thl responses against infectiousagents and rumors in experimental animal mod-
els.SO.51 Nevertheless, despitethe availability of a number of molecules with adjuvantproperties,to
optimize rational vaccinedesigna broaderpalette of adjuvants ableto promote differenttypesof
immune responsewould be necessary. In this context, the availability ofimmune-modulators with
well-definedmoleculartargetswould representa clearasset. This approachwould not only allow
Immune Modulators with Defined Molecular Targets 175
fine-tuningand customizingresponses accordingto the specific needs, but would also facilitate
the prediction ofpotential sideeffects.
Immune Modulators with Defined Molecular Targets

The advent of whole-genome sequencingof bacterialpathogenshas revolutionizedthe field
ofvaccinology. This approach not only provides the full arrayof potential candidateantigensfor
vaccine design, but hasalsorevealed new PAMP,whichmight exertimmunomodulatoryactivities
byactingon PRR.52 Adjuvantexhibitingthis property delivera dangersignalto the host immune
system through the activation of PRR, thereby mimickinginfectiousagents. This results in the
expression ofsolublemediators(e.g., cyrokines,chemokines) and APC activation. Thestimulation
of the innateimmunesystem in turn determinesand shapesadaptive responses.Theexploitationof
this knowledge for the identification of newmoleculartargetsisfacilitated byrecentdevelopments
on mass mutagenesis, high throughput screening, gene expression profiling and combinatotial
chemistry," However, the intrinsichuman geneticvariationis a majorfactor which might leadto
differential responses afterstimulationwith PRR agonists.Therefore, immunogenetics isa critical
buildingblock of discovery programs.54
TLRAgonists
The mammalianTLR family consists of at least 13 members(10TLRs (TLRI-1O) in human
and 12 TLRs (TLRI-9 and TLRII-13) in mice) havebeen found and eachTLRis involved in
recognizingavarietyof microorganism-derivedmolecularstructures(Table2).55.56 TLR comprise
a extracellular domain with dozensofleucine-richrepeatmotifs,a trans-membranedomain (with
the exceptionofTLR3) and a cytoplasmic Toll/ILlR (TIR) domain similar to that of the III
receptors (ILlRs).57The TLR1, TLR2, TLR4, TLRS, TLR6 and possibly TLRII are expressed
on the cellularsurface, whereas TLR3, TLR7, TLR8 and TLR9 are believedto reside insidethe
cells (e.g., endoplasmicreticulum and/or endosomes). Each TLR is expressed in a varietyof im-
mune cells, includingmacrophages, DC and Bvcells, aswellasin other celltypes(e.g.,endothelial
cells, epithelial cells). TLR can recognize molecular patterns conserved among but unique to
microbes, which usually do not existin the host. Pathogen recognition by TLR provokes a rapid
activationof the innate immune system by inducing production of pro-inflammatorycytokines
and up-regulationof costimulatorymolecules. The activatedinnate immunesystem subsequently
leadsto effective adaptiveimmunity. Dependingon the TLR triggered, specific signalingpathways
are activated (Fig. 2). DifferentTLR can exert distinct, but to some extent alsooverlapping sets
of biological effects." StimulationofTLR2 and TLR4 failed to increase CTL responses, whereas
ligandsofTLR3, TLRS and TLR7 exhibitedmoderateactivity. In contrast,stimulationofTLR9
dramatically increases CTL responses.f
Agonists able to either control the over-expression ofTh2 cytokines or skew the ThI :Th2
balancetowards a Thl profilewould be of clinicalrelevance for the control of allergic disease/"
In fact, different TLR ligands were demonstrated to be able to inhibit Th2 cell activation and
IgE-dependent release ofTh2 cytokines in animal experimentalmodels of allergy.61.6Z TLR are
alsoimportant for the stimulationofTh2-typeresponses, sincethey augmentthe overall maturity
ofDC.63.64 Someof the initial studies performed with native ligandsgave conflictingresultsdue
to contamination with other moieties. However, the availability of well-defined syntheticderiva-
tives made possible to dissect the signaltransduction events triggered by the specific activation
of a TLR . Thus, the exploitationof well-defined TLR agonistsfor the establishmentof immune
prophylacticor therapeutic interventions seems to be an extremely promising field. However,
safetyaspects need to be carefully addressed during preclinical and clinicaldevelopment, since
TLR seem to be involved in the pathogenesis of human diseases (e.g., autoimmunity,allergies,
asthma and cardiovascular diseases).
In thiscontext, bacteriallipoproteinsand their syntheticanalogues arestrongimmunemodula-
tors during infection, signalingthrough TLR2/TLRI heterodimers.Their synthetic derivatives,
suchasPam3CyS-SK" havebeenexploitedasadjuvants and modulatorsofT-cellresponses. Previous
Table 2. Toll-like receptors (TLRs) and theirligands59
Interaction
With Other Ligand Ligand
TLR TLR (Exogenous) (Endogenous) Source
TLRl TLR 2 Peptidoglycan, triacyl HSP70 Gram-positive bacteria,

Iipopeptides, diacyl Measles virus
TLR2 TLRl lipopeptides, Iipoteichoic
and TLR6 acid, glycolipids
TLR3 dsRNA, siRNA mRNA West Nile virus, mouse CMV,

Schistosoma mansoni,
synthetic RNA molecules
TLR4 Lipopolysaccharide, HSP70, Gram-negative bacteria,

taxol, RSV fusion protein, ~ -defensin 2, plants, respiratory syncytial
mouse mammar y tumor fibr inogen, virus, mouse mammary
virus envelope protein fibronectin, tumor virus, Bacillus
phosphorylcholine, glycan hyaluronic acid anthracis, helminths
TLRS Flagellin Flagellated bacteria
TLR6 TLR2 Lipopeptides Bacteria

(e.g., MALP-2)
TLR7 ssRNA Ul snRNP synthetic compounds

Imidazoquinoline, (spliceosomal viru ses (e.g. influenza virus
Resiquimod, Imiqu imod complex) HIV-l)
autoantigens
TLR8
TLR9 CpG DNA, hemozo in Chromatin Bacteria, synthetic ODN,

complex DNA viruses, Plasmodium
falciparum
TLRll Profilin-like molecule Toxoplasma gondii,

uropathogenic bacteria
studies suggested that Pam.Cys-Sk, is able to ameliorate established allergic airway inflamma-
tion by promoting Thl response rather than by affecting regulatory Tvcells." On the other hand.
its co-administration with anti-CD3 antibodies resulted in modulated immune responses. by
inducing the proliferation ofhoth regulatory and effector T-cells in the absence ofAPe. Thus. it
seems that Pam3Cys-S~ improves adaptive responses by expansion ofeffectors and mitigation of
suppressive activity ofTregs.66.67
Immune Modulators withDefinedMolecular Targets 177
inhibitory effect stimulatory effect
Figure 2. The Thl/Th2 network. IL4 and IFNy do not promote a direct inhibition of Thl or
Th2 cells differentiation, but rather block the differentiation of these subsets from naive
precursors.
Natural and synthetic derivatives of diacylated lipopeptides from Mycoplasma spp. and
Francisella tularensis, such as the macrophage-activating lipopeptide of2 kD (MALP-2). induce
the maturation and activation ofAPC with release of soluble mediators able to act on bystander
cells via activation of the TLR2/TLR6 heterodimer.68•69 Their co-administration with antigens
by either systemic or mucosal route results in the elicitation of strong humoral and cellular
responses. 68•70 Additional studies demonstrated that MALP-2 promotes aT-cell-independent
activation and maturation of Bvcells, increasing also the frequency ofIg-secreting cells. Activated
B-cells exhibited increased expression of both activation markers and ligands which are critical
for cross-talk with T-cells (e.g.• CD19. CD25. CD80. CD86. MHC I, MHC II and CD40).
Immunization ofmice lacking T-cells also showed that MALP-2-mediated stimulation ofTLR2/
TLR6 was unable to circumvent the need ofT-cell help for efficient antigen-specific B-cell acti-
vation. On the other hand. immunization of mice lacking B-cells demonstrated that B-cells are
critical for MALP-2-dependent improvement ofT-cell responses.T " Thus. B-cell stimulation by
PRR seems to be a basic mechanism which can be exploited to improve the immunogenicity of
vaccine formulations.
The lipopolysaccharide (LPS). a component ofthe outer membrane ofGram-negative bacteria.
stimulates adaptive responses. Since the 1970s it was known that the adjuvant effect depends on
the integrity of the lps locus.7; .""7 Then. positional cloning showed that the ips locus was identical
to tlr410cus.-~ However. the toxicity of the LPS prevented its clinical use. making necessary the
development ofnon toxic derivatives. Among them. MPL, a derivative oflipid A from Salmonella
minnesota. was proven to be safe and effective as a vaccine adjuvant for humans. " A new class of
fully synthetic lipid A mimetics (e.g.• aminoalkyl glucosaminide 4-phosphates, RC-529) have
been engineered to target human TLR4 and are showing promise results as vaccine adjuvants and
therapeutics against a wide range of infectious agents." Thus. it seems that the potent immune
modulatory properties ofTLR4 agonists allow their exploitation as both vaccine adjuvants and
stand-alone therapeutics.
Synthetic double stranded polyribonucleotides act at the levelofTLR3, inducing the production
IFN and other cytokines.This activity can beexploited to increase the effect ofanti-viral drugs. For
example, the anti-retroviral effect ofzidovudine is enhanced by combination with polyribonucle-
orides." Poly I:C was one ofthe first therapeutic agents used to treat HIV and leukemia patients,
but it was abandoned due to toxicity," Synthetic polyribonucleotides also induce the activation
and maturation ofDC. In vitro results demonstrated that a novel non toxic analogue ofpoly I:C
(Ampligen) effectively induce in vitro maturation ofmonocyte derived human DC with sustained
production ofILl2. DC primed with tumor lysates and matured with synthetic dsRNA may offer
a valid alternative for optimizing Th 1 anti-tumor responses in cancer patients."
The binding of bacterial flagellin to TLR5 results in DC stimulation and promotion ofThI
responses. 1.84.85 This depends, at least in part, in the up-regulated expression ofILl2 resulting from
phosphorylation ofp38 and c-jun N-terminal kinase 1/2. Monomeric flagellin acts as local and sys-
temic pro-inflammatory factor via activation ofthe TLR 5-NF-kappaB axisand IL8 production.58.86
Co-administration of tetanus toxoid (TT) with flagellin to mice by intranasal route resulted in
significantly enhanced TT-specific humoral responses in the mucosal and systemic compartments.
Vaccinated mice were completely protected after challenge with a 200 -fold minimum lethal dose
oftetanus toxin. FlaB colocalized with CD Llc as patches in putative DC and a clear increment in
the number ofTLR5-expressing cells was also observed in cervical lymph nodes. 87•88
Imiquirnod, a synthetic imidazoquinoline which was approved in 1997 for the topical treatment
of genital warts , exhibits anti-viral and anti-tumor activities . Imiquimod binds TLR7 and TLR8
on DC leading to the production of pro-inflammatory cytokines, such as IFNa, which in turn
stimulates both the innate immune system and the cellular arm of the acquired immune system
(e.g.• improvement of CTL responses). 89.90 This suggests that imiquimod mimics natural ligands
ofTLR7 and TLR8 (e.g., ssRNA from viruses). However. recent experimental and clinical data
indicate that imiquimod also exerts direct pro-apoptotic activity on tumor cells." Imiquimod is
insoluble in water but in most clinical studies its incorporation into an oil-into-water cream emul-
sion (1-5%) was well-tolerated with mild-to-moderate drug-related side effects, such as itching,
burning sensation, pain , erythema. erosion and oedema." An analogue, the so called resiquimod
(R848), is able to promote similar 'Ih l -biased responses . but at 10-fold lower dosages than imi -
quimod." Initial clinical studies suggested that resiquimod reduces the recurrence rate ofgenital
herpes, however. Phase III trials were suspended due to lack ofefficacy.89
In the course ofnatural microbial infections. TLR9 recognizes double stranded DNA which,
in contrast to mammalian DNA. is characterized by the abundance of unrnethylated deoxycyti-
dyl-deoxyguanosine dinucleotides (CpG). The stimulatory activity of microbial DNA can be
mimicked by synthetic oligodeoxynudeotides (ODN) containing such motifs (CpG-ODN).94
The TLR9-mediated stimulation ofthe vertebrate innate immune system and subsequently. ofthe
adaptive immune system, allows the use ofTLR9 agonists as highly effective vaccine adjuvants
for infectious diseases, as well as stand-alone or part of combination therapies against cancer.95.96
TLR9 shows a restricted cellular and sub-cellular pattern ofexpression, which changes also between
animal species. CpG-ODN acts on human B-cells and plasmacytoid DC increasing the produc-
tion ofpro-inflammatory cytokines and promotingAPC maturation and Th 1 responses. F:" These
biological activities enable CpG-ODN to act as adjuvants when co-administered with a given
antigen by systemic or mucosal route, thereby leading to improved antigen-specific responses.
These effects are optimized by maintaining close physical contact between the CpG-DNA and
the immunogen. Ongoing clinical studies also indicate that CpG -ODN are well-tolerated in hu-
mans. 98.99 During infection. recognition of CpG DNA of intracellular pathogens skews immune
response towards a Th 1 dominant pattern. This can be also desirable in therapeutic interventions
aimed at the prevention ofallergic responses.
ImmuneModulators withDefinedMolecular Targets 179
Bacterial Toxins and Their Derivatives

Cholera toxin (CT) and its close relative the heat-labile enterotoxinof Escherichia coli (HLT)
are AlB moiery toxins.They are composed by an A subunit, which is responsible for the enzy-
matic activityand a pentamer ofB subunits, which mediatesthe binding of the holotoxin to the
receptorspresent in the membraneof the target cells. CT and LT-Ibind to the ganglioside GMt,
LT-IIabinds with high affinityto the ganglioside GD lb and with low avidityto the gangliosides
GD la and GM 1and LT-IIb binds with high affinityto GD la. lOO,101 Thesetwo toxins are powerful
adjuvants when co-administeredwith antigens by either systemic or mucosalroure.!" The mo-
lecularmechanisms by which they are ableto stimulatethe innate and adaptiveimmunesystems
are not fullyelucidated. However, direct effects on T-ceilsand APC have been observed, which
provideinsightsinto how thesemolecules mayexerttheir activity. In vitro studiesperformedwith
primaryB-ceils and macrophages showedan increasedphosphorylationofdifferentsignaling mol-
ecules, includingErk1l2 and p38.103In this context, the B subunit of CT inducessignalingevents
resultingin cellularactivation,expression of surface molecules and production of cyrokines, by
promoting a transactivation of two transcriptionalelements, the cyclic AMP-responsive element
and NF-KB.103 Theobserved GMI-dependent nucleartranslocation ofNF -KB in DC demonstrares
the criticalrole playedby this receptor in the stimulation of signaltransduction cascades.P' On
the other hand, there is a paucity of information regardingthe structurally related membersof
the serogroup II HLT, namelyLT-IIaand LT-IIb, which havedifferent binding specificities for
ganglioside receptors.
The use of CT and LT in humans was initially hampered by their high toxicity. However,
site-directed mutagenesis has permitted the generation ofLT and CT derivatives with reduced
toxicity, which retain their adjuvanticiry,'?' Different mutants, such as LTK63 (LT with a ser-
ine-to-lysine mutation at position63 of the A subunit),havebeenexploitedassystemic or mucosal
adjuvants.106 A preferentialinduction ofTh2-type responses wasobservedwhen these molecules
wereincorporated in the vaccine formulations. Interestingly, their usealsopreventedor alleviated
autoimmune diseases, thereby demonstrating their wide-ranging effects on the immune system.
The observation that this improvement is associated with the generation of regulatory T-celIs,
which in turn inhibit pathogenicTh1 responses, explains in part the two apparentlycontradictory
outcomesafterexposure to CT-likeenterotoxins. Nevertheless,further investigations are required
to unravelthe mechanisms leadingto either adjuvanticity or toleranceinduction to fullyexploit
their potential in the context of vaccine designand immunorherapies.!"
CDld Agonists
NKT-cellsrepresenta unique subsetofimmune regulatoryT-cells, whichareableto recognize
glycolipids presentedbythe MH C class I-likemoleculeCD 1d. Because of their biological proper-
ties, NKT-cells are attractivetargetsfor the developmentof immunotherapies. The prototypical
NKT-celiligand agalactosylceramide (aGalCer) , originallyisolatedfrom a marine sponge,has
potent immunemodulatory activities. aGalCer isa ligandof invariantVa14+ NKT-celIs, which
is presented by CD Id on APC. Administration of aGalCer to mice resultsin a rapid activation
of NKT-cells, which is characterized by cytokine secretion, surface receptor down-regulation,
expansionand secondaryactivation of a varietyof bystander cells from the innate and adaptive
immune systems. Differentstudieshavedemonstrated the efficacy of aGalCer againstmetastatic
tumors,infectionsand autoimmunediseaseS.108. l ll Studiesperformedwith structuralanalogues of
aGalCer alsoshowedthat ~-anomeric GalCer can induce CD Id-dependenr biological activities
in mice,albeit at lowerpotency than n-anomericGalCer.1l 2 The response ofNKT-celis to distinct
GalCer analogues seems to differboth at quantitativeand qualitative levels.Thesefindings indicate
that specific glycolipids could be exploitedto fine-tuneNKT-cells responses, therebyshapingthe
elicited immune responses in vivo. This approach will certainlyfacilitate the developmentof ef-
fective and safeNKT-cell-based immunotherapies.iv'P!"
Different studies have also demonstrated that aGalCer could act as an effective adjuvant
when co-administered with antigens by either systemic or mucosal route. When aGalCer was
administered with the model antigen ovalbumin by intranasal route to CS7BL/6 and BALBlc
mice. strong ovalbumin-specific humoral and cellular immune responses were stimulated at
systemic and mucosallevd, characterized by a mixed ThllTh2 cytokine profile. Vaccinated mice
also showed complete protection against challenge with the ovalbumin-expressing tumor cell
line EG7. Furthermore, intranasal vaccination with the hemagglutinin from the influenza virus
A/PR/S/34 co-administered with aGalCer also conferred significant protection against viral
infection. Interestingly. when aGalCer was given with a replication-deficient adenovirus to mice.
significantly enhanced immune responses were detected The adjuvant effect induced by intrana-
sal co-administration with aGalCer was blocked in CD Id-I- mice. indicating that the immune
responses were exclusivdy dependent on the CD Id molecule present on APe. Experiments
performed using CFSE-Iabded aT-I cells which were adoptivdy transferred into syngenic mice
showed that naive T-cells were activated and stimulated to differentiate into functional effector
T -cells.1l6The efficacy ofaGalCer as adjuvant was also demonstrated in the context ofother vac-
cine rdevant antigens (e.g.• malaria, influenza . hemagglutinating virus ofJapan}.l13·117.1l8 Despite
the excellent results obtained using aGalCer, there are major drawbacks preventing the efficient
transfer into the clinic. such as its poor solubility. To provide soluble formulations. non organic
solvents or detergents are needed. which represent a safety concern and might affect the immu-
nological properties of some antigens. However. a new pegylated derivate. aGalCerMPEG, has
been recently described. which is water-soluble and retains both the specificity for the CD Id
receptor and the immune stimulatory properties when tested in vitro or in vivo, even at 33-fold
lower concentrations ofthe active moiery.!"
Cytokines
The process by which mammals meet the dual need of an immediate response to danger and
initiation oflong-term protection is regulated by pro-inflammatory cytokines, which are primar-
ily produced by cells from the innate immune system. Therefore, cytokines have been widdy
exploited as natural adjuvants for the design ofimmunotherapeutic or prophylactic interventions.
Many cytokines were demonstrated to be able to enhance protection against infectious agents and
tumors. both in preclinical and clinical studies. One of the most exploited ones is ILl2, which
plays a key role linking the innate and acquired immune systems. ILI2 is critical for activation of
NK cells. promotion ofCTL development, T-cell independent induction ofIFNy, activation of
differentiated CD4+ and CDS+ T-cells, development ofTh 1 responses. provision ofcompiemen-
cary immune-regulatory signals to IL2 and defense against intracellular pathogens.P? Therefore.
also other members ofthe ILl2l1L23/1L27 family, whi ch share ligand and receptor subunits and
play overlapping roles in innate and adaptive responses are also considered as prime candidares,"!
The use ofIL2 also gave promising results. The mechanism ofanti-tumor activity ofthis cytokine
is related to its ability to expand and activate the NK and CTL subsets.!" There is also increasing
evidence that local or systemic effector cell dysfunction, which is characteristic of patients with
advanced cancer, can be reverted by IL2. Another important cytokine, Il.l S, is produced by
several leukocytes in response to infections. IL IS exhibits many homologies to IL2 and. like IL2 ,
stimulates NK cells.!" Chemokine-antigen fusions also resulted in enhanced immunogenicity.
The rational use of combinations between eytokines and chemokines could promote the target-
ing ofantigens to APC, their subsequent maturation. the attraction ofcritical bystander cells, the
steering of cellular immune responses toward a Th 1 response pattern, the improvement of CTL
responses and the enhanced production ofsystemic IgG and secretory !gA. Several review articles
have provided additional information about the most commonly used eytokines and chemokines
for immunotherapics.P'P?
Cell Wall Components

Freund's complete adjuvant (FCA) has been the adjuvant of choice for animal experimenta-
tion for decades. A water-in-oil emulsion is generated. in which water droplets containing antigen
are emulsified in mineral oil containing killed mycobacteria or their cell walls. However. its high
Immune Modulators withDefinedMolecularTargets 181
toxicity precludes its use in humans. Nevertheless, subsequent studies allowed the isolation ofthe
active components and a better understanding of the underlying mechanisms of adjuvanticity.
This resulted in the identification of molecules exhibiting similar immune modulatory proper-
ties and an adequate safety profile. N-acetylmuramyl-L-alanyl-D-isoglutarnine (I.e., muramyl
dipeptide; MDP) was the first. l3l This is a synthetic derivative of a component present in the
bacterial peptidoglycan. The activity ofMDP can be attributed to its ability to cause the release
of multiple cytokines, Animal studies established that MDP exhibits a broad array ofimmuno-
logical effects, including: (i) enhancement or suppression of antibody levels dependent on the
time of administration relative to antigen; (ii) improvement of cell-mediated immunity; (iii)
enhancement of nonspecific immunity to bacteria, viruses, fungi and parasites; (iv) stimulation
of natural resistance to tumors; (v) promotion of eytokine release and (vi) pyrogenicity. MDP
is recognized by NOD2, but not by TLR2 or heterodimers formed by TLR2 with TLRI or
TLR6.132 In contrast to intact and diacylated MDP, derivatives with a single octanoyl or stearoyl
fatty acid chain were found to activate TLR2 and TLR4 (see above) and exert their activities
through the MyD88-dependent pathway on APe. 133 Studies performed using a Mycobacterium
tuberculosis animal experimental model also showed that NOD2 and TLR are two non redundant
recognition mechanisms which synergize for the induction of pro-inflammatory cytokines.!"
Interestingly, MDP also exerts pronounced neuropharmacological activities, probably through
the interaction with 5-hydroxytryptamine receptors. However, some ofthese biological activities
are undesirable for clinical use.m Thus, structurally well-defined synthetic derivatives from the
MDP were generated,which exhibit improved pharmacological properties.73.136.137.139 Among these
non toxic derivatives can be mentioned the adamantylamide dipeptide (AdDP), MDP-Lys (LI8)
and murabutide. The AdDP is a synthetic compound in which the dipeptide was combined with
the anti-viral compound amantadine. AdDP exerts its adjuvant properties when administered by
either systemic or mucosal route , leading to the elicitation ofstrong humoral and cellular responses
at both systemic and mucosal Ievels.r'"!"
Co-Stimulatory Molecules
Processed antigenic peptides are presented by APC to T -cells in the context ofMHC mol-
ecules, which are the only physiological ligands for the T-cell receptor (TCR). However, several
additional receptors form pan ofthe immunological synapsis. In fact, ligation ofthe TCR to the
MHC Il-peptide complexes (signal 1) leads to various T-cell effector functions, depending on the
specific nature of the costimulatory receptors (signal 2). Thus, surface molecules such as CD40
and CD 1S4 are central to the cross-talk between T-cells and antigen-presenting cells. The study
of the immune respon ses against intracellular pathogens showed the relevance ofCD40-CD 154
interactions in the regulation ofILl2 and IFNy secrerion .!" Furthermore, anti-CD40 antibod-
ies can be effective adjuvants when administered either separately (high dosages) or conjugated
(low dosages) to the antigen. In the former case, it is likely that side effects such as splenomegaly
would occur, whereas when conjugates are used, side effects may be avoided.145.146 GM-CSF has
been shown to be synergistic with IL 12 or CD 154 for induction ofCTL responses and the triple
combination of GM-CSF, ILI2 and TNFa appears to induce the most effective protection in
some experimental models. The combination of costimulatory molecule s with vaccine antigens
has also resulted in a synergistic effect.147Another potential target molecule is CD134, a member
of the TNFR superfamily. CD 134 is transiently expressed on T-cells following ligation of the
TCR. The CD 134 ligand, known as OX40L, is a TNF family member expressed on APe. The
OX40/0X40L pathway offers opportunities for both enhancing responses through use ofagonists
(adjuvants) and abrogating unwanted responses, such as those generated in autoimmune diseases,
through the use of blocking reagents and antagonists.!" Another widely studied costimulatory
pathway is the CD28-CD80/CD86. CD28 is constitutively expressed on T-cells and can be
seen as analogous in funct ion to CD40 on B-cells. The ligands for CD28 are CD80 and CD86
(also known as B7-1 and B7-2). They are members of the immunoglobulin superfamily, which
182 PharmaceuticalBiotechno/qgy
are transientlyexpressed on activated APe. Forcedexpression ofB7 on tumor cells, whichdo not
normallyexpress it, resultedin enhancedT-cellresponses.149-152
Bis-(3',5')-Cyclic Dimeric Guanosine Monophosphate (cdiGMP)

In many bacteria bis-(3',S')-cyc1ic dimeric guanosine monophosphate (cdiGMP) signaling
determinesthe timingand amplitudeof complex biological processes, such asbio6lmformation,
virulenceor photosynthesis. Besides its role as an intracellular and intercellularsignaling mol-
ecule in prokaryotes, c-di-GMP also affects eukaryotes. 153•IS4This interestingmolecule inhibits
cancer cells in vitro, by affecting basal and growth factor-induced cell proliferation. ISS Recent
studieshavealsodemonstratedthat cdiGMP exhibitspotent adjuvantproperties. Subcutaneous
co-administrationofantigenswith cdiGMP promotesthe elicitationofvigorous antigen-specific
humoraland cellular immuneresponses,whichwerecharacterized byabalanced Thl11h2 response
partern.l" Similarresultswereobtained when cdiGMP wasusedasmucosaladjuvant. However,
the 19GI /lgG2a ratioofantigen-specific antibodies andthe profiles ofsecretedeytokines suggested
that a mixedThl11h2 response patrern is promoted when cdiGMP is administeredby mucosal
roure.!" IntraperitonealinjectionofcdiGMP in miceresultedin the localreetUitment and activa-
tion ofmonocytesand granulocytes. Human immatureDC cultured in the presence of cdiGMP
showed increased expression of the costimulatory molecules CD80 and CD86, the maturation
markerCD83 and MHC class II molecules.Il.Iz, IFNy, 118,MCP-I, IFN-y-inducible protein
10 and RANTES werealsoproduced bycdiGMP-treatedDC, whichexhibitedenhancedT-cell
stimulatoryactivity.1S8 It seems that the activation of the p38 MAPK in human DC and an ERK
phosphorylation in human macrophages may be involved in cdiGMP signaling. However, the
underlyingsignaling eventsneed to be further eluddared.l'"
Conclusion
The advent of subunit vaccines rendered obviousthe pressing need for developing efficient
and safeadjuvants. This is particularlytrue when mucosalvaccination strategies are cominginto
consideration, sinceonly a few moietieshavebeen identifiedso far exhibitingthis property. In
particular,thereisa high demandfor adjuvants ableto stimulatecellular immunity, whichisessen-
tialfor combatingintracellularpathogensand tumors. In this context,it wouldbe helpfulto have
well-characterized moieties, which are ableto exert their biological activitythrough stimulation
ofdefinedcellulartargetsand/or signaling cascades. Thisisnowpossible due to the identification
ofagonistsof PRR presentof APC, whichare thought to providea link betweenthe innate and
adaptiveimmune system. An in depth understandingof the underlying mechanisms of action of
thesemoieties, togetherwith the availability of syntheticderivatives with a well-defined structure
will certainlyreduce the likelihoodof undesiredside effects. It would be alsofeasible to choose
the most appropriateentity in order to stimulatepredictableimmuneresponses, accordingto the
specific clinicalneeds. Another aspectwhich requires attention is the minimizationof effector
functionsin untargetedcells.Tothis end,effortsarebeinginvested to achieve the specific targeting
ofadjuvants and/or formulations.Thetargetingencompasses both organand cell-specific delivery
[e.g., local lymph nodes,solid tumors, APe). This is expected to facilitate optimal stimulation
with negligible side-effects. Thus, basic research in immunologywill continue being the main
drivingforcefor innovationin the vaccinology field. Increase predictabilityfor candidatestested
in preclinicaland clinicalstudieswill facilitate the cost-efficient development ofefficient vaccines
with an optimal safetyprofile.
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144. Subauste CS. CD154 and rype-I eytokine response: from hyper IgM syndrome to human immunode-
ficiency virus Infecdon.] Infect Dis 2002; 185 Suppl. I:S83-9.
145. Barr TA. Carlring ], Heath AW. Costimulatory agonisrs as immunological adjuvants. Vaccine 2006;
24( 17):3399-407.
146. Cairing]. Barr T, Heath AW. Adjuvanticity of anti-cD4Oin vaccine developrnenr, Curr Opin Mol Ther
2005; 7(1):73-7.
147. Ahlers ]D, Bdyakov 1M, Berzofsky]A. Cytokine , chemokine and costimulatory molecule modulation
to enhance efficacyof HIV vaccines. Curr Mol Med 2003; 3(3):285-301.
148. Sugamura K, Ishii N. Weinberg AD. Therapeutic targeting of the effector T-cdl costimulatory molecule
OX4O. Nat Rev ImmunoI2004; 4(6) :420-31.
149. Dohring C. Angman L, Spagnoli G er al. T-hdper- and accessory-cell-independent cytotoxic responses
to human tumor cells eransfecred with a B7 rerroviral vector, Int ] Cancer 1994; 57(5):754-9.
150. Chen L. McGowan P, Ashe S et al. B7-1/CD80-rransduced tumor cells dicit better systemic im-
munity than wild-type tumor cells admixed with Corynebacterium parvum. Cancer Res 1994 ;
54(20):5420-3.
151. Chen L. McGowan P, Ashe S et aI. Tumor immunogenicity determines the effeer ofB7 costimulation
on Tvcell-medlaced tumor immunity. ] Exp Med 1994; 179(2):523-32.
152. Kalus RM. Kantor ]A. Gritz L et al. The use of combination vaccinia vaccines and dual-gene vac-
cinia vaccines to enhance antigen-specific Tscell immunity via Tscell costimulation. Vaccine 1999;
17(7-8):893-903.
153. Romling U, Amikam D. Cyclic di-GMP as a second messenger. Curr Opin Microbiol 2006;
9(2):218-28.
154. Romling U. Gomdsky M. Galperin MY. C-di-GMP: the dawning of a novd bacterial signalling system.
Mol Microbiol 2005; 57(3):629-39.
155. Karaolis DK. Cheng K, Lipsky M et aI. 3', 5'-Cyclic diguanylic acid (c-di-GMP ) inhib its basal and
growth factor-stimulated human colon cancer cell proliferation. Blochern Biophys Res Commun 2005;
329(1 ):40-5.
156. Ebensen T, Schulze K. Riese P et aI. The bacterial second messenger cyclic diGMP exhibits potent
adjuvant properties. Vaccine 2007; 25(8):1464-9.
157. Ebensen T, Schulze K. Riese P et aI. The bacterial second messengercdiGMP exhibits promising activity
as mucosal adjuvant. Clin Vaccine Immuno12007; 14(8):952-8.
158. Karaolis DK, Means TK, Yang D er al. Bacterial c-di-GMP is an immunostimulatory molecule. ]
Immuno12007; 178(4):2171-81.
CHAPTER 14
Innovative Approaches to Develop

Prophylactic and Therapeutic Vaccines
against HIV/AIDS
Aurelio Cafaro,' Iole Macchia,' Maria Teresa Maggiorella, Fausto Titti
and Barbara Ensoli"
Abstract
T
he acquired immunodeficiency syndrome (AIDS) emerged in the human population in
the summerof 1981.Accordingto the latest United Nations estimates,worldwideover33
millionpeopleareinfectedwithhuman immunodeficiency virus (HIV) and the prevalence
rates continue to rise globally. To control the alarmingspread of HIV, an urgent need exists for
developinga safeand effective vaccine that prevents individualsfrom becoming infected or pro-
gressing to disease. To be effective, an HIV/AIDS vaccineshould induce broad and long-lasting
humoraland cellularimmuneresponses, at both mucosal and systemic level. However, the nature of
protectiveimmuneresponses remains largely elusive and this represents one of the majorroadblocks
preventingthe developmentofan effective vaccine. Here wesummarize our presentunderstanding
of the factorsresponsible for resistance to infection or control of progressionto disease in human
and monkey that may be relevant to vaccinedevelopment and brieflyreviewrecent approaches
which are currently being tested in clinicaltrials. Finally, the rationale and the current status of
novel strategiesbased on nonstructural HIV-1 proteins, such as Tat, Nef and Rev, used alone or
in combination with modified structural HIV-1 Envproteins are discussed.
Introduction
Epidemiology: Main GlobalandRegional Trends
Human immunodeficiencyvirus/acquiredimmunodeficiencysyndrome(HIV/AIDS) remains
one ofthe most serious threatsnot onlyto globalhealth,but also to globaldevelopment.According
to the 2007 AIDS epidemicupdate byWorld Health Organization (WH 0) and theJoint United
Nations Programme on HIV/ AIDS (UNAIDS), approximately 33.2 million people wereliving
with HIV in 2007 1 of whom 2.5 million were due to new infection among adults and children
(Fig. 1).Today, Sub-SaharanAfricacontinues to pay the highest toll to the globalepidemic,with
68% of new infections,76% of the estimated 2.1 million deaths in 2007 and 90% of the 2.5 mil-
lion children livingwith HIV worldwide.Despite major methodological improvements, which
have cut by 16%the former estimates, the statisticsstill indicate that the number of people living
with AIDS increasedin 2007, partly due to expanded access to therapy that reduced the number
ofdeaths. This new analysis also showsthat the pandemic actuallypeaked in the late 1990s and
"Ihese authors have contributed equally to this chapter.
'Corresponding Author: Barbara Ensoli-Director, National AIDS Center, Istituto Superiore di
Sanlta, V.le Regina Elena 299, 00161 Rome, Italy. Email: barbara.ensolletss.lt
Pharmaceutical Biotechnology, edited by CarlosA. Guzman and Giora Z. Feuerstein.
.....
~
" ~.NAI~-
Adults and children estimated to be living with HIV, 2007
~
'~"
Total: 33.2 (30.6 - 36.1) million ~
:to
l::
....
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~
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Figure 1. Adults and children estimated to be living with HIV, 2007. Reproduced by kind permission of UNAIDS (2007) (www.unaids.org).
.s-
~
Innovative Approaches to DevelopProphylactic and Therapeutic Vaccines againstHIV/AIDS 191
the subsequentdeclinemayat leastin part be attributed to the efficacy of prevention campaigns.

While this iscertainlygood news, it hasimportant practicalconsequences on clinicaltrials,which
mayactuallyresult undersizedand lackstatisticalpower.As today, longitudinal prospectivestud-
iesappear the only wayto realistically measurethe incidenceand will haveto be included in the
preparatory studiesfor vaccine testing.'
Rationale and Roadblocks to HIV Vaccine Development

Theincreasing socialand economical, includingtherapy, costsof the pandemicmakethe search
for a preventive and therapeuticHIV/ AIDS vaccine the highestpriorityofthe worldHIV/AIDS
agenda.' Preventive vaccination is aimed at inducingprotectiveimmune responses in individuals
naive to HI¥, whereas therapeutic vaccination is aimed at increasing the potency and breadth
of anti-HIV immune responses in order to avoid or delay anti-retroviraltherapy use in HIV-l
infected individuals,"
Despite advancements in the understanding of HIV biology and pathogenesis' and despite
over20 years of attempts, no vaccine is available to combat the HIV/AIDS epidernic.v' However,
the vast majorityof vaccines evaluatedso far turned out to be safeand immunogenic, with some
of them showingsomelevelof protection in preclinicalmodelsand thereforeadvancedto clinical
testing.8•9
The difficultyin generatingan HIV vaccine capableof eradicatingthe infection depends on
manyfactors. Amongthe mostimportant, the highgeneticvariability of HIV (for a review seerefs.
10,11),the peculiarpropertiesof the envelope (Env)proteins to avoidand evadeimmunerecogni-
tion and neurralizadon," the lack of identifiedcorrelates of immune protection, the complexity
of implementingpreclinicalanimalmodelsl •13•14and conducting efficacy clinicaltrials,especially
in developing countries. In addition to the elusionof antibody (Ab) neutralization,HIV is able
to elude cellularimmune responses bymultiplemechanisms, includingdown-regulation of major
histocompatibilitycomplex(MHC) class I molecules,"emergence of mutants escaping cytotoxic
T-Iymphocytes (CTLs)16 and dysregulation of cyrokineproduction.17·19
Correlates ofProtection
Lessonsfrom the NaturalHistory ofHIV-l and SIVInfection
Thecomprehensionof mechanisms of natural resistance to HIV/AIDS mayhaveimplications
for the identification ofnovelanti-viral strategies and in particularforthe development ofinnovative
diagnostics, therapeuticsand vaccines. It is now clearthat both host and viralfactors contribute
to the outcome of the infection and mayexplainthe higher or lowerindividualsusceptibility to
HIV /AIDS (Table 1). In particular,two phenomena ofnatural resistance to HIV-l infection or
progression to disease havebeen described: individuals that remain uninfected despiteexposure
to the virus [multiplyexposeduninfected (MEU) individuals, also termed exposed-uninfecred
(EU), or highly exposed persistently seronegative (HEPS) individualsj'? and individuals that
becomeinfectedbut do not progress to AIDS [long-termnonprogressors (LTNPs)]. Both MEU
and LTNPs offer valuable clues to elucidate immune mechanisms involved in the resistance or
control of infection, respectively and might thus providea unique resource to identify correlates
ofprotectiveimmunity to HIV.
MEU include HIV-discordant couples having unprotected sex,21.22 sex workers23.25 and
health care workers.26.27 Homozygosis for a mutation in CCRS gene (the 32 bp deletion, i.e.,
CCRS-Delta32allele) ispresently considered the mostrelevantgeneticprotectivefactor 8.3Q (Table
1). Further,in a cohort ofMEU Kenyan sexworkersHIV-l specific CTLs werefound both in the
blood and in the vaginal mucosa." Interestingly, theseCTLs, whichhadsimilarspecificities, recog-
nizedepitopesdistinctfrom thoserecognised byHIV infectedindividualsf and maycontribute to
protection frominfectionwith both cytolytic and noncytotoxicanti-viral effectorrnechanisms.F"
In the Nairobi cohort, HIV-l specific CD4+ T-cells produce interleukin-Z (IL-2) rather than
interferon-y (IFN-y)and display limited activationand celldeath ascomparedto HIV-l infected
....
~
Table 1. Major host factors associated to resistance or control of HIV infection and relevant to vaccine development
Level Resistance Control Reference
Genetic Polymorphism
CCR5 CCR5 a32 homozygosis CCR5 a32 heterozygosis 28,60,61
CCR2 CCR2-641 473
CCR5 & CCL3L1 Polymorphisms of these genes affect the capacity 79
to mount immune responses (DTH)
MHC A2/6802 supertype B57, B5801, B27 and additional polymorphisms in 62,80,412
B, C and outside Band C loci
KIR Activating (KIR3DS1) and inh ibiting (KIR3DL1) 66,474
NK cell receptors binding HLA-B molecules with
isoleucine at position 80 (HLA-Bw4)
Innate Immune Response
Natural antibodies Against CCR5 Against CCR5 475,476
a and II defensins Increased in the genital mucosae 36
CCL5 (RANTES) Increased in the genital mucosae 477
NK Increased production of IFN-y Conserved lytic activity despite altered phenotype 35,478
Adaptative Immune Response
Antibodies Neutralizing IgA in the genital mucosae 37,479 ~
I:.
CD4 ' T-cells Prevalent IL-2 production, limited activation and cell death Polyfunctional, high per cell cytokine production 480 ~~
as compared to infected individuals l:
CD8 ' T-cells Polyfunctional, found also in the genital mucosae, with Polyfunctional, mostly against Gag, highly cyto- 34,63,71
specificities different from those found in infected individuals toxic
l
b:I
l;"
~
CCR5: chemokine (C-C motif) receptor 5; CCR5 a32: a 32-base pair deletion mutant of the CCR5 gene; CCR2: chemokine (C-C motif) receptor 2; CCL3L1: C-C S.
chemokine ligand 3-like 1 (MIP-1a isoform), a high affinity ligand for CCR5; MHC: Major Histocompatibility Complex; KIR: Killer cell Immunoglobulin Receptor; :s
.
CCL5: Chemokine (C-C motif) ligand 5; NK: Natural Killer cells. s-
~
InnovativeApproaches to Develop Prophylactic and Therapeutic Vaccines againstHIV/AIDS 193
individuals'? (Table1).1n addition,an increased production ofIFN-y bynaturalkiller(NK) cells

wasfound in thesesexworkers" which, together with increased levels of chemokines and alpha
and bera-defensins (for a review seeref 36), indicatea substantialrolefor innate immunityin the
observedprotection. Absmayalsohavean important rolesincein MEV HIV-I-specific IgA but
not IgG weredetected in the genitalmucosae, which displayed neutralizingacdviry" (Table1).
Takentogether,thesedata suggest that localand to alesser extentsystemic, innateand adaptive
immune responses maydevelop upon sexual intercourses with HIV-I infected partners, which
protectfrombecomingovertly infected.1ntriguingly, quittingsexworkresultedin diminished CTL
frequency and increased riskof infection, suggestingthat long-termmemorywasnot inducedand
persistentantigenicstimulationwasrequiredto maintain protectiveCTL responses'" or IgA.37.38
This short-term immunity may also be consistentwith allogeneic responses triggeredby sexual
intercourses with multiplepartners,whichhavebeenshown to confertransientimmunity,39.40 an
observationthat has led to the proposalof alloimmunization for HIV-I vaccine design."
LTNPs are HIV-I infected people that remain clinically healthy for over 10 years with low
(50-2,000 RNAcopies/mlplasma,alsodefinedas"controllers") to undetectable «50 RNAcopies/
ml plasma, alsodefinedas "elitecontrollers") viralload and a minimallossofCD4+ T-cells in the
absence of anyanti-retroviral therapy.42-45 Nonprogression appears to depend on multiplefactors.
SomeLTNPshavemutationsinviralgenes that influence replication and/or immunecontrol,such
asthose codingfor the regulatoryand accessory proteins Nef Rev, Tat,Vif Vprand VpU.46-52The
LTNPsharboringviruses withmutationsin nefareofspecial interestbecause ofthe reportedefficacy
of vaccination of macaques with an attenuated simianimmunodeficiency virus (SIV) carryinga
deletionin the nefgene(SIVililef).53-55However, follow-up ofthe well-characterized SidneyBlood
BankCohort revealed progression in 3 out ofthe 8 individuals infectedwith the verysameHIV.56
Ofnote, progression wasobserved alsoin newbornsand a fewadult macaques infectedwith the
SIVAnef57Thus, HIV-I (and SIV) carryinga mutation in nef(andlong-terminal repeat)are not
safeand, more importantly, the immune responses induced by these attenuated viruses are not
effective at efficiently controllingthe infectionovertime.Further attenuationsof the SIVililef to
ameliorate their safetyprofilereducedthe degreeofprotection from superinfection." indicating
that much work needs to be done to implementthis kind of vaccine.59
Asmanyas25-30%LTNPsmayhave specific genetic polymorphisms in the HIV-I coreceptors
CCR5 and CCR2, as wellas in the geneencodingthe CCR5 ligandmacrophage inflammatory
protein l -c, which areassociated with an impairmentofviralattachment and thus infectiviry60·61
(Table1).
Certain MHC haplotypes (B57, BS801 and to a lesser extent B27) are over-represented in
LTNPS62 and, togetherwith a lowertotal magnitudeand breadth of HI V-specific CTL responses
preferentially targeting Gag, significantly correlate with nonprogression.f Of interest, human
leukocyteantigen (HLA)-B restrictedCTLs to HIV are oflow avidity, express low levels of the
exhaustion markerPD-I and accounts for the vastmajorityofpolyfunctionalCTLs, confirming
the relevance of polyfunctionality (especially IFN-y and IL-2, see below) and shedding some
light on the protectiveroleplayedbyHLA-Balleles.r'The highestfrequency of theselowavidity,
polyfunctionalCTLs wasobserved in B57+ individuals, whichisover-represented in LTNPs.This
is somewhatparadoxical in viewof the known capability of HIV to downregulate through Nef
the expression of MH C-I A and B molecules hamperingthereforetarget recognitionespecially
bylowavidityCTLs.65However, sinceB57isalsoa ligandfor the inhibitory NK receptortermed
KIR3DL1,whichhasrecently beenshownto stronglyassociate with nonprogression/fit has been
proposed that the HIV-drivenMH C downregulation mayrelieve this inhibition, triggering NK
cell activity against the virus.? It is conceivable that, under these opposing pressures, HIV be
forcedto limit the degreeofMHC downregulation to avoidNK surveillance, thus explaining the
persistence oflow aviditypolyfunctional CTLs, whichhowever keepin checkthe virus.It will be
ofinrerest to determinewhethertheseCTLs areover-represented in LTNPsinfectedwithviruses
carryingmutationsin Nefthat hamperMHC downregulation. Takentogether, thesefindings and
hypothesessuggestthat NK cells actively participatewith CTLs to the containment ofinfection

and ways to stimulatethis arm ofthe innate immunity should be consideredin vaccine design.
Concerning the enhanced and qualitatively different polyfunctional activity [simultaneous
production of two or more cytokines(TNF-a, IL-2, IFN-y,MIP 1-~) in conjunction with mark-
ers of degranulation,such as CD107a expression] of HI V-specific CD8+T-cells from LTNPs,68
the capability of these T-cells to coproduce IL-2 appears the most important feature, possibly
related to its ability to sustain proliferation, expanding the number of effector cells.69.70 Along
the sameline,virus controlhas been associated with high frequencies ofHI V-specific CTLs with
a "nonexhausted" phenotype (HLA-DRHigh, CD38 Low) , which is consistent with retention of
polyfuncrionaliry and a low degreeof immunoactivationand a potent exvivocytotoxicactivity
againstautologousinfectedCD4+T-cells.71Theimportanceof polyfunctionalityis also suggested
by in vitro experiments showing that fully competent autologous monocyte-derived dendritic
cells(MDDCs) pulsedwith Gagwerecapableto triggerthe expansionof CD8+T-cellsin LTNPs
but non in progressors, whoseCD8+T-cellswereunableto produce IL-2.72 Thesedata should be
carefully consideredwhendesigningtherapeuticvaccine trials. In addition to the cytolyticactivity,
HIV-specific CD8+T-cells havebeenreported to secreteunknown anti-viral solublefactor(s)(for
a reviewsee ref 73) that has been associated with nonprogression.o"
CD4+ T-cellsare pivotal for induction and maintenanceof effective T- and B-cellresponses
(for a reviewsee ref 76). Of interest, CD4+T-cells from individuals that control infection are
polyfunctional and secretehigher amount of cytokines (IFN-y,TNF-a and IL-2) as compared
to monofunctional cells.TIhese findings highlight the importanceof multi-parametric analysis of
HIV-specific immuneresponses in order to correctlyidentifythe functionalpropertiesrelevantto
protection or control ofinfection. However, whether theseimmunological features playa keyrole
in controllingthe infectionor merelyreflecta preservedimmunesystem remainsto be elucidated.
To gainmore insightsinto host geneticfactorsimpactingcontrol ofinfection, Consortia arebeing
formed to allowanalyses oflarge cohorts of patients.78-80 Resultsfrom these studiesindicate that
control ofinfection is stronglyinfluencedby host geneticfactors,of which only someare related
to adaptiveimmunity,underscoringthe presentneed to searchand identifynovelimmunological
mechanisms (Table 1).
Despiteabroaderneutralizing antibodies(NAbs)response wasreportedin LlNPs ascompared
to patients with progressive disease,81.82 no apparent rolefor NAbs in suppression of HI V replica-
tion could be demonstrated in elite controllers as well as in patients respondingto HAART.83
Nevertheless, plasmafrom LTNPs has been recentlyscreenedwith random peptide phagelibrar-
ies in order to identify mimotopes capable of inducing relevant NAbs against conformational
epiropes."
Another valuable modelto investigate correlates of protection isrepresentedbySIV infection
in the natural host in which the virus typically doesnot induce AIDS despitechronic high levels
of virus replication. A better understanding of the mechanisms underlying the lack of disease
progressionin Africangreenmonkeys(AGM)85 and sootymangabey monkeys (SM) mayprovide
cluesto the pathogenesis ofimmunodeficiencyin HIV-infectedhumans.86-88 Recentfindingssup-
port the ideathat in the natural host a strongand rapid immunosuppressive response mediated by
regulatoryT-cells(Tregs)abrogates immune hyperactivation, resultingin a more benign disease
outcome.89•90 Sincecontrol of infection appearsto occur earlyafter infection, natural immunity
is also believed to be key, either by combating directly the virus, or by instructing an effective
adaptiveimmune response." Strikingly, protection from progression mayoccur in these animals
despite severe lossof CD4+T-cells both in the blood and in the mucosae, indicating that CD4+
T-cell depletion per se is not sufficient to drive progression and that these species haveevolved
mechanisms to compensatefor the T-helper loss.92-94 Lowlevels ofT-cellactivationand apopto-
sis,preservedlymph nodes architecture and no sign of immunodeficiency werethe hallmarksof
nonprogression in thesemonkeys. Takentogether,the findings in human and nonhuman primates
suggestthat bluntingofinitial inflammatoryresponse and immunehyperactivation correlatewith
maintenance of polyfunctionalT-cellsand lackof phenotypes associated to progression (lossof
polyfunctionality and especially ofIL-2 production, upregularion of CD38, PD-l, CTLA-4,

downregulation of CD 107) and should therefore be the goals of vaccines aimed at containing
the virus. As a corollary, immunizationstrategies resultingin sustainedvaccine antigen load and
immune hyperactivation may be detrimental. Finally, some concerns about the criteria used to
defineprotection as control of infection. In fact, the abovestudiesin the natural hosts as wellas
those in vaccinatedanimals9s.96question the concept that plasmaviremialevels and CD4+T-cell
countsarealways reliable indicatorsofvaccine efficacy. Extendedfollow-up of "bonafide" protected
animalsis therefore recommended.
What we Have Learnedfrom Vaccination Studies

Although the correlates of protectiveimmunityare still elusive,97.9s thereissubstantialevidence
that preventive and therapeutic vaccine approaches elicit both humoral (broadly NAbs)99 and
cell-mediated (T-helper and -cytotoxic) responses'P' (Table 2). NAbs are believed not to playa
pivotal rolein earlyHIV-l clearance in the natural infection,sincethey appearwhen substantial
destruction of the gut associated lymphoidtissue(GALT) hasalreadyoccurredand the virushas
colonizedvirtuallyeverytissue.IOI.IOZConversely, it is conceivable that the induction ofa robust
Ab response by vaccinating prior to exposure to the virusmayprotect from infection.
In order to prevent infection by a diverse range of HIV-l isolates, a vaccine must elicit Abs
that are broadlyneutralizing. The HIV Env glycoproteins gp120and gp41 mediate binding and
entry into target cellsand are the main viral targets for NAbs. However, the domains of gp120
and gp41 which are criticalto cellattachment and entry are hidden from the attackingAbs, thus
enablingHIV to evade neutralization.l'"
Nevertheless, a fewmonoclonalAbs(mAbs)with broad neutralizingactivityhavebeenidenti-
fiedin HIV-l positive patient seraand wellcharacterized (2FS,4EIO,2G12and bI2) .1Q4.106 Passive
transferof these mAbsprotected neonatal macaques from subsequentchallenge with SIVI07 and
delayviral rebound after anti-viral treatment interruption in acutelyand chronicallyinfectedpa-
tients.IOSHowever, polyspecific reactivity with autoantigens, reported for 2FS and 4El 0, the two
NAbs targeting the membrane proximalexternalregion of gp41, suggests that their production
might be regulatedbytolerance mechanisms.l'" Thisraises doubts about the feasibility and safety
of inducing in healthyindividuals Abswith these specificities.I10
Although NAbsdo not appearto contribute to the controlofviremia in acuteHIV-l infection,
Abs to the Envcan be detected at the timeof reduction ofplasmaviremiaand other effectorfunc-
tionsofAbsmayplaya rolein viralclearance.'!' In fact,significantreduction ofset-pointviralloads
and preservation of centralmemoryCD4' T-Iymphocyte countswereobservedin rhesus macaques
challenged with a pathogenicSIV (SIVmacZ39) and passively immunized7 dayslater with SIVmacZ39
specific NAbs, despite the rapid disappearance of the NAbs from the plasma."! Further, recent
data indicate that HIV-l infectedprimarycells and in particular macrophages and dendritic cells
(DCs), are more susceptible than celllines to Ab effectorfunctions, including neurralizanon,'!'
suggesting that the effectiveness of the Ab responses elicited so far by vaccination might have
been underestimated. Moreover, there is evidence that immunization mayinduce memoryB-cell
responses that can last for several years and that can be boosted upon recallvaccination.l 'Y"
Finally, antibody-dependent cell-mediated cytotoxicity (ADCC) has recently beenreconsidered
as an important Ab effectorfunction that maycontribute to HIV control (for a review see ref
116). ADCC is an immune mechanism in which Abs bind target cells, makingthem vulnerable
to the attack by immune cells carryingreceptorsfor the Fe portion of the Ab (FcR)such as NK
cells, yb T-cells, neutrophils and macrophages. Although ADCC against HIV and SIV has been
reported since many years,I17·123 only recentlyit was shown to correlatewith protection against
SIVin a prime/boost AIDS vaccine approachin rhesus macaques.P" More recently,the protection
affordedbypassive immunizationwith the broadlyneutralizingmAb b12 againstan intravaginal
challenge with the RS-tropic SHIVSFl6ZP3 wasstronglydiminished in monkeysimmunizedwith a
b12variantdevoidofFeR bindingactivity,IZSunderscoringthe relevance ofadditionalAb effector
functions even in the context of neutralization.This is of importance for several reasons: (i) Abs
....
Table 2. Key scientific issues to successfully develop an fnv-based vaccine according to a Working Group convened by the Global HIV ~
Vaccine fnterprise t
Structure-Assisted Immunogen Design - To improve understanding of the structural basis of antibody binding to the HIV-1 Env glycoprotein
- To stabilize gp120 into more immunogenic forms or to scaffold conserved neutralization epitopes
into foreign prote ins
- To identify epitopes capable of inducing neutralizing antibodies or antibodies contributing to
neutralization (additive or synergistic effect)
To Assessthe Role of Fc Receptors and Complement - To standardize assays that measure these anti-viral activities
- To assess their biologic relevance in passive protection experiments in animal models using
antibodies that exhibit the different effector functions in vitro
Assay Standardization and Validation - To standardize and compare neutralizing antibody assays in order to identify the most reliable
assayor combination of assays to measure neutralization
- To use more than one assay to assure that all neutralizing antibodies are detected
- To prioritize standardization of the PBMC assaybecause it is the only one partially validated in
passiveantibody experiments in animal models
- To validate neutralization assays based on new technologies in passive antibody experiments in
animal models
- To generate new SHIVs from nonclade B viruses to address the above issues
Immunoregulation of B-cell Responses - Better understanding of B-cell responses regulation (and dysregulation) is needed in order to
ident ify the best way to induce long-lasting neutralizing antibodies
~
- To identify genes that are associated with the wide variation in neutralizing antibody responses in
HIV-1-infected individuals and in vaccine recipients ~
~
l::
- To study in nonhuman primate models the potential functional contributions of B-cells to HIV :l'.
infections £
....
b:I
~.
- To establish a research consortium to study fundamental B-cell biology as it relates to HIV-1
~
vaccines So
;s
tpLoS Medicine, www.plosmedicine.org December 2007, Volume 4, Issue 12 e368 1867-1871.

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Innovative Approaches to Develop Prophylactic and Therapeutic Vaccines againstHIV/AIDS 197
do not need to be neutralizing. (ii) effectorcells belongto nativeimmunityand arethereforeready

to go, (iii) ADCC does not attack the virus. but clears infected cellsdisplaying viral antigens. a
mechanismrerninescentofT-cell immunity. However. unlikeCTLs, ADCC doesnot distinguish
infectedcellsfrom uninfectedoneson whichviralparticlesor antigensareabsorbed(for example,
solublegp120on membraneCD4). Despitethis limitation,it isconceivable that a preventive vac-
cine inducinghigh titers of AbsmediatingADCC should be effective at curbingviral replication
at the verybeginning, tipping the balancein the virus-host dynamics in favorof the host.
Although these recent advancements havespurred new interest in the feasibility of inducing
protectingAb responses. manyvaccine approaches, such asthosebasedon DNA and viralvectors,
haverecentlyfocused on the induction of cellularimmune responses.6.126-128The rationaleis that a
cellularimmune response against HIV, although unable to providesterilizingimmunity,should
hopefullyenablevaccines to control virusreplicationfollowing infection, contain viralload,slow
down progression toward disease and reducethe probabilityof secondaryinfections.!"
Long-lived memoryvirus-specific T-cellresponses havebeenshownto be criticalto the control
ofviralreplication in manychronicinfections includingcytomegalovirus, Epstein-Barr virus. human
papillomavirus,hepatitis C virusand alsoHIV (for a reviewsee ref.29). Despite a largebody of
evidence suggesting that CTLs,13O.131 T-helpercells I3O.131and Tregs" playan important rolein con-
trollinginfection.direct proofis stilllacking. FUrther. in contrast to Ab responses, T-cellresponses
are technicallymore difficultto investigate. In fact,in most vaccine studiesIFN-yELISpotis used
to evaluateCTLs because of its ease. robustness, reproducibilityand sensitivity.132.134 However,
whilethis assay issuitable to measure vaccine immunogeniciry, IFN-yproduction byCTLs alone
did not correlatewith control of infection135.136 (for a reviewsee ref 137). Similarly, multimer
staining technology has provided powerfully insights into dynamics of epitope specific CTLs
and their pressure to selectescape viralvariants. However. this technique requiresknowledge of
MHC haplorypes and at present it can be applied to a limited number of specificities, makingit
unsuitableto monitoring of the globalT-cellresponse.
More recent advancements in the measurementof T-cell responses demonstrated that poly-
functional CD4+ and CD8+T-cell responses are a much better correlateof control of infection
or survival. Prolonged(4 years) control of SHIV-89.6P infection in macaques primed with DNA
and boosted with modified vaccinia virus Ankara (MVA), both encoding Gag, Pol and Env of
SHIV-89.6P, correlated with low breadth and frequency of polyfunctional (IFN-y and lL-2)
CD4+and CD8+ T-cells.138 The low breadth and frequencyof these responses might reflect the
limited antigenicstimulationdue to the strongsuppression ofvirusreplication. However. it might
also be due to containment of the detrimental immunoactivationobserved in pathogenic HIV
and SIVinfections.sincenaturallySlY-infectedsootyrnangabeys, whichhavea high viralloadbut
limited immune activation,developasimilarpattern of CD8· T-cellresponses.P? In a SIVmodel,
the prolonged survival of rhesus macaques vaccinatedwith plasmid DNA followed by boosting
with replicationdefective adenoviral vectorsencodingSIVGag. Poland Envand challenged with
S1Vmac2s1 correlatedwith the magnitudeand preservation of Gag-specific polyfunctional central
memory CD4+ and CD8+ Tcells, while set-point viral load was not predictive.96.98 In a similar
study, preservation of polyfunctional CD4+T-cells during the first two weeks of infection wasa
strong predictor of prolonged survival and it wasassociated to the rapid appearance of Abs neu-
tralizingthe challenge virus.which mayhavecontributed to the significant reduction of set point
viralloadobservedin vaccinated animals. l 40 Intriguingly, this prolongedsurvival occurreddespite
vaccinatedanimalsunderwent a substantial, although not massive, CD4+T-celldepletion in the
gut, suggesting that CD4loss in the gut isnot invariably associated to progression.asindicatedby
the studiesin naturalhosts.92•94 Takentogetherthesedata suggest that preservation ofvirus-specific
polyfunctionalT-cells is an important predictor of control or prolongedsurvival.The differences
observedin these studiesmaybe due to the differentvaccinestrategies. which mayhavedifferent
correlates of protection. aswellas to the virustype and dose chosenfor the challenge (for a review
see ref. 14). The relevance of the challenge virusis suggested by the monovalentAdS-gag vaccine
developed by Merck (MRKAds), which was shown to elicit a potent CTL response in rhesus
monkeysand protect from disease progression upon intravenous challenge with the pathogenic
SHIV-89.6P.141.142 but to a muchlesser extentagainstan intrareetalchallenge with SIVmac239.143
Notably. thelimitedprotection upon SlVmac239 occurredin the presence ofT-cellresponses that
correlatedwith protection in the former study.144 Thus. the recent failure (no reduced transmis-
sionor viralloadin vaccinees without pre-existing immunityto the vector)of the PhaseII clinical
trial based on the MRKAd5 trivalentvaccine would suggest SIV as a more rigorous challenge
virus and better predictor of vaccine efficacy in human.145.146 An additional factor complicating
the identification of correlates of protections is the possibility that T-cell responses effective at
controllingprogression in LTNPs,when induced byvaccination prior to infection mayactually
favourthe selectionofescape mutantsand accelerate progression.!" Suchan acceleration mayalso
occurbecause ofthe expansion of HI V-specific CD4+T-cellsinducedbyvaccination. whichactu-
allyincreases the number of susceptible target cells to the incomingvirus.148Thus.despitemajor
advancements in the field. solidcorrelates of protection arestill lacking. The recent introduction
in the HIV vaccine field of midsize Phaselib test ofconcept (TOe) trialsbasically acknowledges
thisweakness and isaimedat obtainingvaluable informationaboutvaccine efficacy beforemoving
to much larger. longerand costly PhaseIII efficacy trials.
General Strategies Adopted to Induce Protective Immunity

VaccinesAimedatInducingNeutralizingAbs: Vaccines Based on HIV-l Env
Protein
Former subunit vaccine candidatesweremainlyfocused on the useof the structural protein
Envas the immunogenaimedat blockingvirusadsorption to the target cells byinducingbroadly
NAbs.99•149 However. vaccination with the monomeric HIV Env subunit (gpI20). elicited Abs
that wereableto neutralizelab-adaptedbut not primaryvirusisolates1so and homologous but not
heterologousviruses in preclinical challenge models.151.152 Thus.despitenumerousattempts.these
approaches havefailedso far in elicitingdurable.cross-clade NAbs needed to achieve sterilising
immunity.as recentlysoberly confirmedby the failure of the first two PhaseIII clinical trialsof
gp120 envelope subunits.AIDSVAX BIB and AIDSVAX B/E byVaxGen. tested in over 5.000
at-riskvolunteersin the United Statesand in Thailand.respectivdy.IS3.IS6The reasonfor this fail-
ure waslikely relatedto the complex structure of Env and its high variability.ls7.ls9 Further.heavy
glycosylation ofthe gp120 molecule creates a glycan shield.protecting the protein from incom-
ing NAbs. a phenomenon unknown at the time of the first Env immunizations.P" In fact. Env
hides its Ab bindingsitesunder the protein loopsand the heavily glycosylated siteson its surface.
hamperingrecognition of relevant. mostlyconformational. epitopesby NAbs.161.163Despite the
tremendouseffortand the soberingresults. HIV-l Envremains akeytargetfornewvaccine strate-
gies(Table2). In the recentyears muchefforthasbeendevoted to the constructionof oligomeric
[erimeric) formsofEnvwhichclosely mimicthe structureofthe nativeprotein presenton the viral
envelopel64.165 and have beenshownto besuperiorat inducingAbs directedtowards conformational
epitopesl66,167and capable of neutralizingboth T-cell-line adapted (X4) and selected(R5 and X4)
primary isolates of HIV-I,168.169as comparedto the monomericgp120.170 However. Env trimers
are extremely unstableand several approaches havebeen undertaken to stabilize them. In one ap-
proach.the gp120-gp41 cleavage sitewasdisruptedbymutagenesis. generating an uncleaved form
ofgp140 (gpI4OUNc) .17I.176 Someof these uncleaved formsof the Envproteins weremoderately
superior to monomericgp120 for induction of NAbs in smallanimalmodels!67.177.18o
In another approach. an intermolecular disulfide bond betweengp120and gp41 (SOSgp140)
wasintroduced. with an expectation to induce both neutralizingand fusion-blocking Abs.I8I •183
Primingwith DNA encodinga membrane-bound form of the SOS gp140 protein followed by
repeated immunizationwith the solubletrimers resultedin high titer Absthat neutralizedneu-
tralizationsensitive labstrainsand. to amuchlesser extent. primaryheterologous HIV-l strains.184
Sincethe gp41-gp41interactions in 50S gp140weretoo weakto maintaintheproteinin atrimeric
configuratlon.F' a single residuechange. 1559P. within gp41 was iruroduced .F' This variant of
InnouatiueApproathes toDtvtlopProphylactic and Thuaptutic VaccinesagainstH1V/A1DS 199
SOS gpl4O, designated SOSIP gpl4O, appear to be fully cleaved, to be predominantly trimeric
and to have favourable antigenic properdes .l'" In a recent study, comparison ofthe immunogenic-
ity of SOSIP gp140 trimers with uncleaved gp140 trirners and monomeric gp120 using a DNA
prime-protein boost immunization regimen in rabbits, indicated that SOSIP gp 140 trimers were
superior to gp140 uNc and gp 120 proteins at inducing NAbs.170.184 and SOSIP gp 140 trimers with
Env from other clades have been generated, which have comparable or better stability and capability
to induce NAbs as compared to a prototypic strain OR-FL, subtype B).I64,185.186
In a third approach, heterologous trirnerization domains at the terminus ofthe gp41-ectodo-
main187,188were introduced to stabilize the molecule and 30 aminoacids in the second hypervari-
able region (V2) were deleted to expose neutralizing epitopes shielded by V2 .189,190 This particular
variant, termed AV2 Env, has been developed and tested in preclinical models including rabbits
and monkeys and is currently being evaluated in a gag + env DNA/PLG prime-AV2 Env protein
boost preventive Phase I triaI.168.169,177,I89-191
Since critical neutralizing epitopes are displayed very transiently upon binding to CD4, an-
other strategy developed by Merck and by the University of Maryland!" is based on covalently
linked monomeric gp120 or oligomeric gp140 to soluble CD4 or to synthetic mimetics of the
CD4 receptor in order to induce the conformational changes that take place upon binding of
the virus to CD4 prior to virus entry, thus revealing critical neutralizing epitopes, such as those
involved in coreceptor binding. This approach was tested in macaques and shown to elicit broadly
cross-reactive NAbs.193Finally, broadly NAbs are presently being exploited to select peptides from
phage-display libraries that mimics the Env neutralizing epitopes (mimoropes) with the goal of
identifying immunogens capable to elicit broadly NAbs (for a review see ref. lOS).
Vaccines Aimedat Inducing Cellular Immunity: Vaccines Based on Gag, Pol

or Nonstructural HIV-l Proteins: Rev, Tat andNef
Due to the obstacles encountered in the preparation of an anti-Env vaccine providing sterilis-
ing immunity and the protective role ofcellular immunity (see correlates ofimmunity section), a
second generation ofvaccines based on the structural protein Gag has been developed, with the
concept of inducing strong and broad T-helper and CTL responses, which, would contain virus
replication, thereby protecting from disease progression and reducing virus transmission to healthy
individuals. In fact, Gag protein seems to be the most potent of all HIV-l antigens in eliciting
CTLs, it is more conserved in its immunodominant epitopes than Env and, mo st importantly, the
breadth ofCTLs to Gag but not to other HIV proteins appears to correlate with nonprogression
in a large cohort stu dy conducted in South Africa on untreated individuals." Ongoing trials with
the Gag antigen show promising induction of cellular immunity in primates and humans (for a
review see ref. 194).
A Gag-Pol DNA vaccine has been recently tested and resulted safe and well tolerated. However,
no HIV-specific Ab responses and only low-magnitude HIV-specific T-cell respon ses were de-
tected.!" Further, evidence from preclinical testingofa Gag/Env DNA vaccine in monkeys indicate
that initial control (Le., undetectable plasma viremia level) against challenge with the pathogenic
virus SHIV89.6P was overcome over time by the appearance ofescape mutants despite apparently
preserved anti-viral humoral and cellular responses. 196.1 97 Alternative strategies have been recentl y
considered, based on the new concept of "reverse vaccinology" with the aim of blocking virus
replication and disease onset by targeting nonstructural HIV regulatory genes such as Tat , Rev
and Nef, which are essential for replication and infectivity: 53
In particular, these proteins share desirable features for the generation of a promising vaccine
since they exert key functions in the early virus life cycle, contribute importantly to infectivity
and pathogenicity, induce a broad immunity and they are highly conserved in their immunogenic
domains across HIV-l clades I98,199 (Table 3). In fact , these proteins are produced very early after
infection, Tat and Nef even before HIV integration in quiescent T-cells in which they promote
cellular activation and viral replicanon.P' Emerging data indicate that, despite their small size,
regulatory and accessory proteins are targeted by cellular immune responses very early in the course
""~
Table 3. Rationale to use Rev, Tat, and Nef for an HIVIAIDS vaccine
Evidence Description Reference
Pathogenetic Rev, Tat and Nef are expressed very early and strongly dysregulate the immune system contributing 15,200,205,241,251
importantly to the establishment of infection and to disease progression
Epidemiological In asymptomatic individuals responses to these nonstructural proteins significantly correlated with non- 74,201,225 ,229,481
progression to disease
Immunological Rev, Tat and Nef are conserved in their functional and immunogenic regions (both B- and T-cell 198,212,215,216,482
epitopes). Further, Tat and Nef display immunomodulatory effects on APCs exert ing adjuvant effects and
determining the type of immune response elicited
Preclinical Tat, Rev and Nef, either alone or in combination, have demonstrated in preclinical models to be safe and For a review, see refs. 240,483
to elicit broad and specific immune responses and, more importantly, to control viral replication and to
block disease progression
Clinical Tat, Rev and Nef either alone or in combination have demonstrated in Phase I trials to be safe and to For a review, see ref. 240
~
elicit broad and specific immune responses
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Innovative Approaches to Develop Prophylacticand Therapeutic Vaccinesagainst HIVIAIDS 201
of natural HIV-l infection and contribute importantly to the total Hlv-J -specific CD8+T-cell
responses. sincemultiple CTL epitopeshavebeen identifiedin functionallyimportant regionsof
these proteins.20I'204 Furthermore, they haveimmuno(dys)regulatory effects aimed at facilitating
target cellrecruitment and activation, further promoting HIV replicationand spreading. 20 5-207Of
note. Tat and Nef arealsofound extracellularly and in thisform they exerteffects on differentcell
types.includingchemotacticactivityfor HIV target cells. 208
In particular.extracellular Tat can enter both infected and uninfecredcells, whereit promotes
HIV replicationor modulates the expression of cellulargenes, respectively (for a review see ref.
209). Among others. extracellular Tat upregulates the expression of chemokine receptors and
HIV coreceptors. CCR5 and CXCR4.21O,211 Extracellular Tat has also important effects on im-
munoregulatoryfunctions (for a review seeref.209) (Table3). In particular. bioactive solubleTat
selectively bindsand entersboth immatureand matureDCs (iDCs and mDCs. respectively).drives
iDCs maturation and activationtoward aT-helper 1 [Th-I) inducing phenorype.i" gainsaccess
to the MH C class I pathwayof presentation,213.214 and modulatesthe proteasomecatalyticsubunit
composition. modifying the hierarchyof the CTL epieopes presented in favor of subdominant
and crypticepitopes,215.216This latter activitymight be of relevance sinceone wayused by HIV-l
to escape CTL recognition is to mutate residues in the epitope that prevent or impair processing
and presentation.217.218Accordingly.in the majorityof the multiplyexposeduninfectedsexworkers
of the Nairobi cohort, CTLs recognize epitopes chat are either subdominant or not recognized
in infected women." It remains to be seenwhether Tat contributes to it in the courseof natural
infection.whether targetingTat impactson this typeof immuneevasion,or whether this property
of Tat maybe exploitedto inducebroaderT-cellresponses by includingit in HIV /AIDS vaccines
targeting other antigens. Indeed. preliminarydata indicate that in mice co-immunizationwith
Gag and Tat induces CTL responsesagainst 11 different T-cell epitopes, as compared to mice
vaccinated with Gag alone, which only responded to 6 epitopes."? Both cellularand humoral
Tat-specific immunity may contribute to the control of infection and/or disease progression.
Because HIV-infected cells express Tat veryearlyafter infection, vaccine-induced anti-TatCTLs
mayeliminateinfected cells and block HIV infection at an earlystage.220In fact. rapid induction
of anti-TatCTLs hasbeenreported in naiverhesusmacaques acutelyinfectedwith the pathogenic
SIVmac239 molecularclone,leadingto the selectionofapparentlyless aggressive virusvariants.P!
Notably. anti-TatCTLs werefound moreeffective thananti-GagCTLs at suppressing virus repli-
cation in Mamu-A*OI rhesus macaques.i" Ofoutmost importance. these data havebeen recently
confirmedin patientsenrolledprior to seroconversion and in which a strong temporal correlation
betweenanti-TatCTLs appearance (asearlyas8 dayspostinfecrion),viralload decline and CD4+
T-cells recovery wasfound."
Consistent with this hypothesis. the presenceof Tat-specific CTL responses correlates with
nonprogression to AIDS both in SIV-infected monkeys and in HIV-positive individuals.223•226
Furthermore.anti-TatAbscansequester the extracellular protein. thus preventingthe extracellular
Tat-drivenenhancementofinfection and immunedysregulation associated with them. Strikingly,
anti-TatAbs,whicharefoundonlyin a minority(15-20%) of HIV-I infectedindividuals. arealmost
exclusively present during the asymptomatic phase of infection and correlatewith nonprogres-
sion to AIDS.227.231 Whether this indicatesthat neutralizationof extracellular Tat mayimpact on
disease progression or ismerelya reflection of an underlyingeffective and broad immuneresponse
iscurrentlyunder investigation. Tat is alsohighlyconservedin its immunodominant domains. as
suggested bythe observationthat serafrom Ugandanand South Africanindividuals infectedwith
nonclade B HIV-l strains cross-react with the Tat protein of an HIV-l cladeBsrrain.!"
Vaccines basedon HIV-l Tat (both proteinand DNA) haveprovento besafeand immunogenic
in miceand protectivein monkeys.232'236However. theseresultshavenot been confirmedbyother
investigators utilizingdifferentTat formulationsand vaccination strategies.237•239 Whether these
apparentlyconflictingresults are due to the nature of the vaccine antigen (DNA, nativeversus
inactivated Tat protein. vectored antigen), the monkey species. the route of the administration.
the antigen dose and schedule of immunization, the adjuvant used, or the virus challengedose,
still remains to be elucidated.24O
Rev is also absolutely required for HIV replication since it facilitates the nuclear export of
intron containing viral mRNAs allowingthe transition from the early to the late phase of gene
expressionand proviruseslackingRev do not produce virions.i" While it is presently unknown
whether Rev is releasedextracellularly and exertseffectson neighbouring cells, Rev, like Tat and
NeE, is often targeted by CTLs in HIV-positive individuals'?' and is broadly conserved among
different HIV-l cladesin its functionally constrained and immunodominant domains at the N
rermlnus.t? However, spontaneously occurring mutations in Rev reduce HIV-l structural gene
expression to levels undetectable byCTLs and mayrepresenta mechanism to escapeimmune rec-
ognition. 205Ofimportance, HIV-l Tat and Revare the dominant viral proteins produced before
Nefdown-regulatesMHC classI moleculeson the cellsurface,hampering recognition ofinfected
cellsby CTLs65 (Table 3). Vaccine basedon Revand Tat hasproven protective in monkeys.s" Rev
alone or in associationwith Nef and Tat has been used for therapeutic vaccinationand resulted to
be safeand immunogenic in HIV-l infected individuals.244-246
HIV-l Nef protein is a myristoylated,membrane-associated cytoplasmicprotein abundantly
expressedin the earlyphaseofHIV-I replicationand releasedin the extracellularmilieu.247.248 Nef
protein serves multiplefunctionsand islikelyto contribute to viralpathogenesis bydownregulating
CD4 and MHC classI on the surfaceofinfected cells249(Table 3). Among its biologicalactivities,
Nef mediates receptor down-regulation, T-cell activation and cytoskeleton rearrangement.250.251
The Nef protein alsoentersB-cells and suppresses immunoglobulinclass-switch contributing to the
evasionofprotective T-cell-dependent IgG and IgA responses.P' In addition, Nefinterferes with
the ability ofCTLs to kill infected T-cellsbydecreasingthe surfaceexpressionofMHC classI on
HIV-l-infected Tcells" and favoursspreadingofHIV to T-cellsby increasingthe expressionof
DC-SIGN on DCs, which traps infectiousHIV parricles'" and bypromoting DC marurarion.i"
Further,differentlyfrom Tat, Nefinduces FasLupregulation and apoptosisin bystandercellsboth
in vitro and in vivo,255 whereasit selectivelysparesinfected cells,256 contributing to immuneevasion
and pathogenesis. Finally,infectionofMDDCs with awild type but not with a nefdefective HIV-l
induces the releaseofsolublefactors recruiting and activatinglymphocytes, which consequently
become targets for productive HIV lnfecrion.P'Taken together these data strongly support the
view ofNef as an important vaccinecandidate alone or in associationwith other HIV antigens
(for a reviewseeref 240). Immunization with plasmidDNA or a MVAvector expressingNefwas
demonstrated to be safeand immunogenic in preclinical and clinicalstudies.258.260
VaccinesCombining StructuralandNonstructural HIV--l Gene Products

Experimental evidenceon the role played by regulatory and strucrural HIV gene products in
HIV infection and pathogenesis represents the rationale to develop new vaccination strategies
based on the combination of the two classes ofproteins (Table 4). Thesestrategies range from a
"minimalistic" approachin whichonly two antigens,one regulatory(Tat or Nef) and one structural
(1:1V2-Env) HIV protein are combined, to a "maximalistic" one, which isaimed at imitating a live
attenuated vaccineand therefore combinesmanyHIV structural and nonstructural genes.Several
of these new generation vaccines are currently under evaluation in preclinical and clinical trials
in the context of the European AIDS Vaccine Integrated Project (AVIP) (http://www.avip-eu.
org).261.266 Such combined vaccines should be able to generate immune responsesto both viral
products, which are expressed early (regulatory proteins) or late [serucruralproteins) during the
viral life cycle, thus maximizing immune targeting ofviral infection. The criteria for an advance-
ment of any of these combined vaccines towards Phase II/III trials in Developing Countries are
safety and the demonstration of stronger and broader immune responses against each antigen,
compared to those elicited upon immunization with each antigen separately.
In particular, the combination ofthe regulatory HIV-l protein Tat with the structural protein
1:1V2-Env, represents one of the most recently developed HIV vaccine candldaees.>' Preclinical
studies in mice and monkeyshaveshown that the Tat/1:1V2-Envvaccineissafe, immunogenic and
~
IS
.~
~.
Table 4. Combined vaccine candidates to be tested in Phase I trials within the AVIPConsortium : previous work and development stage ~
-s
of single components <l
S.
~
Clinical ~
Single Mice Monkey GMP Approval for Trials (Performed
Combined Vaccine Cand idates Component s Immunogenicity Mice Efficacy' Efficacy Development Human Use or Ongoing» r
-sS"'"
Tat + AV2Env' (clade B) Tat + + + + Completed (P + T)
AV2Env (clade B) + + + Pending To be started (P) -s'<l""
~
Nef + AV2Env (clade B) MVA'-Nef + + + + + Completed (T) i:l
~
AV2Env (clade B) + + + Pending To be started (P) 1:;-
Completed (T) IS
Nef, Rev, Tat, Gag, RT, Env Nef, Rev, Tat + + + + + 'l:>.."
Gag, RT, Env + + + + To be started
~
Multi HIV B-c1ade Ags and epitopes' Multigene· + + + + Completed (P + T) i:!
Multi HIV A-clade Ags and epitopes" Multigene + + + Pending To be started
l...
1:;-
Multi HIV C-c1ade Ags and epitopes" Multigene + + + Pending To be started ~
;::
Multi HIV FGH-c1ade Ags and epitopes' Multigene + + + Pending To be started ii-
~
~
'Mice efficacy is evaluated as the capability of HLA-A2 transgeni c C57BI/6 mice to elimi nate the engraftment of HIV-l /M uLV-infected syngeneic splenocyte s ~-
injected int raperltona llv.v" 2P: Preven tive ; T: Therapeutic; 3L\V 2Env: Env deleted in V2 (see text for further deta il s); 4M VA : Modified Vaccinia An kara ~
vi rus; 5M ulti HIV, also terme d MultiHIV DNA vacci ne is a pl asmid exp ressing an antige nic fusio n protein compo sed of the regul ator y HIV-l proteins ~
Rev, N ef and Tat, Gag p17/p24 and a stretch of 11 cytotox ic T-Iymphocyte (CTL) epitope clusters fro m Pol and Env, wh ich w as cloned into a nov el
DNA vector named the Ge ne Transport Unit (GTU). Four differe nt pl asmids expressing the same im munogens but originating from subty pes A , B, C
~
t;
v,
consensus, or FG H ancestral seq uences, are currently und er evaluat ion; 6M ultigene is a coc ktail of seven plasmids enc odi ng cl ade B Nef, Rev, Tat,
RT, clade A and B Gag and clade A, B and C Env prot eins.
N
e
protects monkeys againstan intrarectalchallenge with SHIVSFl62p4 (ref 267 and EnsoliB,unpub-
lished data) or an intravenous challenge with SHIV89.6P.268 Basedon thesepromisingresults, a
PhaseI clinicaltrialwill start in 2008.Theminimumcriteriumof success forEnv-specific responses
will be the induction of NAbs against the vaccine strain [Le••homologous neutralization)in at
least 50%ofthe trial participants.
The other minimalistic approach developed within the AVIP consortium is based on the
combination ofNefand 1:1V2-Eny26'l (Table4). Thisvaccine iscomposedof the nefgeneinserted
into the MVA(seebelow)in combinationwith the same1:1V2-Env protein mentionedbefore. As
for the Tat/1:1V2-Envapproach.the Nef/1:1V2-Envvaccine will be administeredin HIV-negative
volunteers. seekingto inducemostlyanti-Nefcellular immunityand anti-Envhumoralimmunity.
in particular NAbs,to prevent (or reduce)virusentry and to controlvirusreplication. As for the
Tat/1:1V2 Envvaccine. criteriafor advancement of the Nef/1:1V2 Envvaccine beyond PhaseI will
be provensafetyand broaderand morepotent immune responses againstthe componentsof the
combinedvaccine. ascomparedto thoseobtaineduponvaccination with thesingle antigens. Phase
I trialswith Nef or 1:1V2-Env alone havebeen completed189•2S9 preclinical testingin macaques of
the Nef/1:1V2-Env combinedvaccine will be carriedout in 2008 and preventive PhaseI studies
will followshortly.
The multigene strategy represents another attractivevaccine approach based on the design
ofa cocktailof geneticimmunogens (DNA constructs)encodingseveral viralcomponentsfrom
varioussubtypesofHIV-I. includingstructuraland regulatoryproteinsaswellasviralenzymes. 266
Twomultigeneapproaches arebeingevaluated in the AVIP Consortium.one containingacocktail
ofsevenplasmidsencodingNef Rev. Tat. Gag. RT and Envantigens. termed HIV multigene;266
the second one, basedon the genescodingfor Rev, Tat. Net Gag. p17, p24 full length antigens.
alsoincludesover20 T-cellepitopesfrom Pol.Protease and Envantigens and is thereforetermed
Multi-HIVantigens/epitopes.265 Aprophylactic Phase I trialwith the HIV multigenewasrecently
conducted in Sweden and shown to be safeand highlyimmunogenic. More than 90% of the 38
volunteers mounted T-cell responses (proliferative and IFN-y ELISpot responses) against the
vaccine antigens upon administration with a needle-free device (Biojector) of DNA encoding
Env(cladeA, B. C), Rev(B),Gag(p17/p24, A and B),RT (mutated,B)followed bya boost with
MVAexpressing Env, Gagand Polof CRFOIA_E.269 Basedon theseencouraging results showing
that thisadministration strategy and the useofGM-CSF asadjuvant increases the immunogenicity
of a DNA prime followed byMVAboostingin human, a newpreventive PhaseIIII studystarted
in 2006 in Tanzania.F? As part of the AVIPprogram, a therapeuticPhaseI-II trial has recently
started in UK and Sweden with the aim of comparingin individuals infectedwith HIV cladeB
the immunogenicity ofthe multigenevaccine basedon cladeBantigens with that of the cladeA-C
multigenevaccine. Ofnote, both vaccines includea newlydeveloped plasmidencodingTar/Nef
as a singlefusionprotein.266The Multi-HIV antigens/epitopes approachexploits a noveldelivery
systemtermed genetransport unit (GTU), a proprietary technologyof FIT BIOTECH, which
increases the immunogenicity of DNA vaccines therebyavoiding the need for an heterologous
boosting.265 PhaseI studiescarriedout in Finland haveproven that GTU-MultiHIV (B clade) is
safeand immunogenic in healthyand HIV-I infectedindividuals and it iscurrently beingtestedin a
therapeutic Phase lIa clinical trialin SouthAfrica. 265 Asecondgeneration vector,called Auxo-GTU,
wasmorerecently preparedand aconstructsexpressing multipleantigens andepitopesfromseveral
clades (A, B,C, FGH) wasmadeand it isgoingto be evaluated in a PhaseIIII trial in 2008.
To address the issue of viraldiversity, a novelstrategyto broaden cross-clade T-cellresponses
has been recently proposed. Accordingto this strategy, polyvalent vaccines can be made that
comprise a mosaic of several naturallyoccurringsequences computationally optimizedto include
the maximumnumberofpotentialT-cellepitopesfrom relatively conserved and immunologically
relevantHIV-I prorelns.f" In vivotesting will verifythe validityof this approach.
Innovative Approaches to DevelopProphylactic and Therapeutic Vaccines againstHIVIAIDS 205
Key Issues Relevant to HIV Vaccine Development: How to Get the

Right Responses in the Right Places
Mucosal Vaccines
Sincemost ofthe HIY-I infectionsarecausedbymucosaltransmission both horizontally(sexual
intercourse) and vertically(child deliveryand breast-feeding), an AIDS vaccine must primarily
elicit a robust immune responseat the mucosal surfaces.'!" Most ofthe AIDS vaccinecandidates
that are currently in clinicaltrials around the world are deliveredbyintramuscular or intradermal
injection. Theseroutes of administration induce Ab and cellularimmune responsesin peripheral
lymphoid tissues (systemic immunity), although evidence in human of mucosal responsesupon
intramuscularvaccinationwith a recombinantcanarypoxHIY-I vaccine havealsobeen reported.272
Furthermore, studies ofSlY infection in macaquesindicated that, regardless ofthe route ofinfec-
tion, the gastrointestinal and vaginal mucosae represent the major site of virus replication and
amplificationand the initial sitesofCD4+T-celldepletion.273-275 In particular a rapid depletion of
CD4+ T-cellshas been observed in the vaginaof SlY-infected macaques, particularly among the
CCRS+CD4+ subset that is the preferential target for elimination by SlY infection.276
Severalstudies indicated that secretory IgAinhibit virus assembly and intracellularrelease and
playan important role in inhibiting HIY transmissionvia the mucosalroute, in multiply exposed
females.277.278 Further, multiplerectalexposures to lowdosesofSlY induced MHC class l-restricted
cytotoxic responses that protected against a mucosal challenge with an heterologous virus,"?
Similarly, studies in the macaque model provided insights on the protective role ofhigh-avidity
mucosal CTL responsesgenerated upon lntrarectal vaccination.28o.281 Taken together these data
suggestan important protective role for IgA and CTLs at the portal of virus entry. In this regard,
mucosalimmunisation appearsto be moreeffective than systemicvaccinationat elicitinghumoral
(IgAand IgG) and cellular(CDS+CTLs) immune responses.282.283However,not all the mucosal
routes of vaccineadministration are equallygood at inducing immune responsesat the different
mucosalsurfaces in the body. For instance, oral vaccines areeffective at preventing infections that
primarilytarget intestinal tissues, but arenot veryefficientat inducing IgAin the vagina,one ofthe
main ports ofentry ofHIY-I.284 A fewstudies havesuggestedthat localadministration ofprotein
vaccines to the mucosaofthe genitourinary tract induces a weak to modest localimmunity at the
site ofimmunization.283,285 In contrast, intranasal (IN) immunization has been shown to induce
local immunity not only in the nasal-associated lymphoid tissue and lung, but also in the female
genital tract in rodents,286.287human 288 and nonhuman prlmaees.P" Of note. it has been recently
reported in the mouse that the nasalassociatedlymphoid tissueis also an inductive site.290 Taken
together, these data makethis type ofimmunization, which ismore practical than vaginalvaccina-
tion, appealing to AIDS vaccineresearchers. In this regard. immunization ofHIY-l seronegative
women with an Env subunit vaccineadministered either intranasally or intravaginally, together
with a mucosal adjuvant shown to be effective in mice, failed to induce detectable 19A or IgG.291
These negativeresults should not stop the attemps to use the IN route. In fact, variousprotein-,
DNA-and RNA-basedimmunopotentiating adjuvants/deliverysystems aswellasoflivebacterial
and viral vectors, which may differ in their ability to induce a specifictype of immune response
(e.g.•CTLs versusantibody responses) at the desiredsite,292 are available for IN immunisation and
should be evaluated. However,sincethe noseisawell-knownport ofentry for neurotropic viruses,
there are safety issues that will need to be fully addressed before testing IN vaccines in clinical
trials. Ofnote, tonsillar immunization with an attenuated SlY (SlY~Nef) induced systemicim-
mune responsesand conferred protection upon intrarectal challengeof rhesus macaqueswith a
pathogenic Sly.293It will be ofinterest to determine whether safervaccineapproaches willafford
similarprotection . Recent reports havesuggestedthat combinations of mucosaland systemicim-
munizations mayenhance both mucosaland systemicimmune responses.294'297
Mucosal tissuesare rich in antigen presenting cells(APCs), specialisedimmune cellsthat are
involvedin the induction and regulation ofanti-viralimmunity. DCs represent the most potent
APCs for naiveTvlymphocytes and they are among the first cellsin the body to come in contact
with HIV.298 Thesecellsare criticalin the earlyphasesofinfection.workingassentinels, alerting

the immunesystem and controllingitsearlydecisions. Furthermore,theyexertacrucialrolein the
induction and regulationofadaptiveimmune responses.i" Therefore,it iscriticalto designvaccine
formulations capable ofproperlytargetingand stimulatingDCs to inducestrongimmuneresponses
at mucosalsites.It islikely that the stimulireceived byDCs in the peripheralcompartmentsaffect
their ability to activateT-cellsand/or B-cells as wellas the type ofT-cell response elicited.P? Of
interest. in the mouse,a singlelntracolorecral administrationofa replicationdefective adenoviral
vectorexpressing OVAor Herpessimplex virus(HSV)-2 glycoprotein Bantigentargetedmucosal
DCs. which migratedto the draininglyrnph nodesand induced adaptiveimmuneresponses at the
rectalandvaginallevelthat protected the animals againstachallenge (byeitherroute)withvaccinia
expressing OVAor a lethaldoseofHSV-2, respectively.'?' Similarprotection wasnot affordedby
IN. intravaginal. or subcutevaccination. suggesting that adenoviralvectors.whichnaturallytarget
DCs. maybe particularlysuitableto induce protectivehumoral and cellularimmuneresponses at
sitesthat representthe majorportalsofentryofHIV. It remains to beseenwhetherthesepromising
results are confirmedin human. In fact. intramuscularimmunization with replication-defective
AdSvectorsexpressing HIV-l gag.pol, or nef failedto reducetransmission or lowerviralload in
high risk individualsthat becameinfected.l 46 In this regard. manipulation of antigen presenting
cellsto elicit virus-specific cellularresponses is a promisingtool to control persistantviralinfec-
tions.302-309 In fact.studiesin monkey'"? and human'!' indicatethat inactivatedwholevirus-pulsed
DC vaccines maybe an effective strategyfor treatingpeople with chronic HIV-l infection.
Delivery Systems
Subunit (proteins or peprides)vaccines aregenerally verysafe. with well-defined components.
However. these antigens are often poorly immunogenic and adjuvants are required to induce
a measurable and supposedlyadequate immunity. Thus. a vast array of delivery systems (e.g.•
micro/nanoparticles, emulsions. ISCOMS. liposornes, virosomes and virus-like particles). im-
munomodulators (cytokines, chemokinesor costimulatorymolecules) and, as mentioned above.
evenautologousDCs pulsedwith viralantigenshavebeen proposedand are presentlybeingused
to increasethe efficiency of vaccines against HIV/AIDS (Table S). Furthermore.several highly
attenuated replicatingand nonreplicatingvectors have been or are being tested in a number of
preclinicaland clinicaltrials[ref 240 and Table 6)
Thesestrategies. reviewed elsewhere.P" haveproven effective in controllingviremiaand pro-
gression to AIDS in nonhuman primates.but observations in earlyphaseclinicaltrialsin humans
have not been promising. In fact. some of the trials had to be stopped at variousstages due to
adverse reactionsto the delivering vector312or the inabilityof the expressed immunogen to cover
genetically diverse isolates prevalent in the geographical areas. 313 Nevertheless. the outcome of
several ongoing clinicaltrialsis expectedto delivergood newsabout safevaccine delivery vectors
and. ifpossible, an effective vaccine againstaparticularstrain ofHIV-1 9 (Table6). Herewebriefly
reviewthe different approaches utilized to delivervaccines that are currentlybeing evaluatedin
clinicaltrials (TableS).
PlasmidDNA
Genetic vaccines (naked-DNA vaccines). employDNA plasmidsas"Trojanhorse"vectorsto
delivergenesthat codefor HIV epiropes (fora review seeref 314).Theseexpression vectorsremain
in their episomalform into the host cellwherethey produce peptidesthat induce cellularimmu-
nity. Compared to viraland bacterialvectors.DNA plasmidsfocusthe immuneresponse on more
narrowlyon HIV insert sequences. do not induce (and are not affectedbypre-existing) immunity
to the vector.are cheapand haveseveral regulatory. safety. handling advaneages.l" Immunization
with DNA plasmidscontaining HIV inserts has been demonstrated to elicit substantialcellular
responsein miceand nonhuman primates.315.316 but not in humans,"?
Therefore. many strategies have been undertaken to enhance the immunogenicityof genetic
vaccines. which includedelivery systems. modifications of thevaccine construct,fonnulation with
immunostimulatory rnolecules.l" In particular promoter modification and inclusion of genes
;s
Table 5. Major HIV-vaccine approaches .is'
~
~.
Types of HIV Vaccine Advantages Disadvantages Type of Response Elicited
~
-s
Whole HIV Viruses <!
- Killed/Inactivated Viruses Simple to prepare; they might present HIV Litt le efficacy in nonhuman primates; Few NAbs, no CTL response s,
'"
~
surface proteins in a relatively native conforma- safety concerns (inactivation efficiency) ~
tion depending o n the inactiv ation pro cedure; no
mutation or reversion ~
~
- Live Attenuated Viruses Mimic natural infecti on; high levels of protect ion Safety conce rns: mutation; potential Long-lasting cell- media ted and ~
in animal mod els reversion to virulenc e hum oral immunity -s<!
Recombinant Viral Proteins Safe; simple and inexpensive to prep are; defin ed Immunogenic response is restricted to Target humoral immunity, no CTL
..s-
t"""
(Subunit Vaccines) composition (mostly structural protein s) selec ted ant igens; responses are not respon se ~.
durable; no protection in two effica cy
trial s; adjuva nt required
lt;J
Peptides These vaccines use small pieces of HIV protein s Poorly immunogenic in human trials; Poorly im munogenic in human
trials; adjuvant requ ired
-s~
as an immunogen; safe, inexpensive, potentiall y stability issue ~
~
useful for broad antigenic diversity ,,"
Naked DNA Safe; stable; no cold chain required; inexpensive; Poorl y imm unogenic in humans; co n- Cellular immune respon ses; ~
potential to encod e multiple anti gens; immu - cerns abo ut DNA integration into hum an heterologous pr ime-b oost strate- sR~
nogenic in animals; prolon ged immunity; mor e cells gies needed to induce humor al ~
effective in heterol ogou s prim e-boost strategies respo nses in primates '~" .
Viral Vectors The vaccine is a w eakened vi rus, unrelated Complica ted to prepare; viral escape Dependi ng on the vector, sys-
to HIV, into wh ich HIV genes are inserted; mutants; potenti al immunodysregulator y temic and mu cosal humoral and ~
high-level produ ct ion of protein antigens directl y effect of the vector proteins ; pre-existin g cell-mediated immune responses ~
with in cells of the immunized host, potenti al imm unity t;
c..,
adjuvant effects of the viral delivery system itself;
delivery of antigen dir ectly to co mpo nents of the
immune system, such as antigen-presenting cells
N
<::>
continued on next page '-1
N
Table 5. Continued ~
Types of HIV Vaccine Advantages Disadvantages Type of Response Elicited
• Poxviruses Highly immunogenic. they grow to high tite rs, Safety concerns Mucosal and systemic antibody
are very stable when lyophilized and are capable and T-cell responses
of accepting large transgene sequences; potential
to be administered by different routes
- M VA Safe because they do not replicate in mammalian Limited immunogenicity and durability Both CTl and Ab responses
- NYVAC cells; possibility of introducing large amounts of of immune responses in humans at both systemic and mucosal
DNA; effective in nonhuman primate models sites, depending on the route of
immunization
- Canarypox (CPV) ex. Safe because they do not replicate in mammalian Limited immunogenicity T-cell responses
ALVAC cells; no concerns about pre-existing immunity
- Fowlpox (FPV) against these vectors in humans
- Adenoviruses (Ad) Safe (replication incompetent Ad), stable; highly Responses limited by pre-existing Robust mucosal and systemic
immunogenic; wide tropism; high effici ency of immunity (especially to AdS) humoral and cellular immune
cellular uptake; can be administered at mucosal responses, especially when
sites replication-competent Ad vectors
are used
- Adeno-Associated Viruses They establish a persistent infect ion in the host Poorly immunogenic in human; in mice Humoral and cellular immune
(AAV) they induce T-cells with an altered responses
phenotype and functionally impaired ~
- Alphaviruses: They can infect a large number of animal cell Anti-vector immune response Humoral and cellular immune '"
- Sindbis (SIN), types and transiently express high amounts of vi - responses
~
R
l>
- Venezuelan equine ral and heterologous protein s; safe because their
encephalitis (VEE), replication occurs exclusively within the host-cell l
b:l
- Semliki Forest (SFV) cytoplasm; lack of preexisting immun ity; ability c;"
~
virus to induce apoptosis of transduced cells, favoring s..
lS
DC cross-priming
'"
lS
Table 5. Continued .l:lSi"
~.
Types of HIV Vaccine Advantages Disadvantages Type of Response Elicited ~
~
-s
- Herperviruses (HSV) Dur able immunity; abi lity to activate the inn ate Safety con cerns in immunocompro- Humoral and cell ular immune
<l
immune system; ability to acco m mo date large mi sed individuals; pre-existing immunity respon ses
inserts of DNA
S.
~
~
- Rhabdoviruses (VSV) Rapid growth; relatively safe; mu cosal efficiency Potent ial safety concerns in immuno- HIV speci fic CTL respon se b
comprom ised ~
- Polioviruses They grow to high titers; easy to prepare and Pre-ex isting immun ity Mu co sal humoral and cell ular -ss-
to deliver orall y; stable in the intestinal tract immune responses
wh ere they infect the mucosal M -cell s respon - -s"'<l"
~
sible for antigenic presentation; immunogenic in ...i:""
~
....
nonhum an prim ates
l::.
Bacterial Vectors Simple and inexpensive to prep are; probabl y lS
Stability issue Humoral and cellular respon ses; l:l..
safe; they can be administered at mu cosal site mu cosal immunity
~
- Mycobacterium (BeG) Safe in human s; easily administ ered and Safety issues especially in children and Mucosal and systemic humoral ~
affordable; able to indu ce long-lasting immunity; immunocompromised hosts and cellular iimune responses l~.
muco sal delivery of HIV antigen s; ability to
accommodate large inserts; adjuvant activity ~
~
- Listeria Sti mulates innate and adaptive im mune Safety issues especi ally in ch ildren and CD4' and C DB" T-cell respon ses S·
~
responses immuno-comprom ised hosts ~
I:i
- Salmonella Safe; inexpensive; offers the benefi t of oral Safety issues especially in children and Mu cosal and system ic hum oral ~.
delivery; muco sal targeting immuno-compromised hosts; poor and cellular immune respon ses
immunogenic ity in hum ans ~
VLPs Safe; mimi c the virus particl e, d isplaying HIV Di ffi cult to prep are Good cellular and humoral ~
prote ins in a relatively native co nfor matio n imm une responses at system ic b
c..,
and mu cosal level
Microparticles Enhance the bioavalaibility of the antigen, Instability and manufacturing difficulties CTL response
reducing the numb er of doses in the
immunization schedul e; targeting of DCs N
~
encoding cytokines that increasethe expansionof antigen-specific T-cells (IL-2 and IL-IS), or
attract and inducethe maturationofAPCs (GM-CSF and B-chemokines) havebeenevaluated (for
a reviewseerefs.318. 319). Furthermore,novelformulationsare beingpursued to increase the in
vivoexpression ofDNA vaccines and to protect them from rapiddegradation,involvingadjuvants
or carrierssuch as Iiposomes, bacterialendotoxin, macroglobulins, CpG oligodeoxinucleotides,
peptides and polymers (described below).320-322 Nevettheless, DNA vaccines recently tested in
clinical trials displayed limited immunogenicity195.323-325 and most DNA vaccines are presently
deliveredas a prime in heterologousprime-boostsstrategies (seebelow).
Bacterial Vectors
Among bacterialvectors.liveattenuated recombinant~cobacterium spp. and entericbacteria
such as Salmonella spp. are microorganisms that can be administered at a mucosalsurfaceand
should be ableto specifically induce mucosalcellularand humoralimmuneresponses/" Bacterial
DNA vaccine delivery demonstrated in vivo efficacy in several experimental animal models of
infectious diseases.l" Attenuated strainsof Salmonella spp.havebeen developedaspotential vec-
tors for stimulating immune responses in the gastrointestinal mucosa .l" An advantage of these
vectors is the possibilityto exploit the Type III secretionsystem. a multicomponent systemthat
allowdeliveryofantigensdirectlyinto the cytoplasm, favouring MH C class I antigen-processing
and presentation.F'
A Salmonella in which the SIV gagtransgenehad been fused to a Type III-secretedbacterial
protein wasusedin a Salmonella-prime/MYA-boostregimento stimulateSIV Gag-specific CTL
responses in the gastrointestinaltract of rhesusmacaques.P" Although low levels of CTLs were
detected afterthe priming,upon MYA boostingstrongCTL responses weredetected in the blood
and in the colonic mucosa. However. no protection against intrarectal challenge with SIVmac239
was observed.P" In a PhaseI dose escalation trial oral delivery of Salmonella expressing HIV Gag
resulted safeand induced strong immune responses to Salmonella antigens, but modest immune
responses to Gag.m.332
Mycobacterium booisBacillus Calmette-Gulrin (BCG) is another promisingvector. BCG has
a longrecord of safetyin humansand isableto inducelong-lasting imrnuniry.P! However, despite
extensive testingin smallanimals (miceand guineapigs),evidence in nonhuman primatesofprom-
isingimmunogenicity334.335 and efficacy againstan homologouschallenge afterasingleinoculation
of rBCG expressing the HIV MN V31oop,336 recommendations byWHO and UNAIDS to further
explorethe useofrBCG asa potential vectoredvaccine for HIY,337no clinicaltrial has started yet
with this vaccine approach. While pre-existing immunity to BCG doesnot seemto be a problem,
use of this vector in developing countrieswhere Mycobacterium tubercolosis (and tubercolosis) is
highly prevalent and even BCG vaccination may be fatal in immunodeficient children.r" raise
some concernson the feasibility of largescalevaccination with this platform.
Another interestingvectorisrepresented byListeria monocytogenes (Lm). afacultative intracel-
lular bacterium that enters the cellby phagocytosis and colonizes the cytosolof the host cell.339
Several properties makeLm an attractiveHIV vaccine vector. First.this bacteriumis a good agent
to stimulate innate as well as adaptiveimmune responses sinceit specifically infectsand induces
maturation of DCs. Second. foreign antigensencoded by Lm are efficiently processedand pre-
sented by both MHC class I and MHC class II molecules. thus activatingboth CD8- and CD4+
antigen-specific T_cells.340-342 Third,Listeria-derived vaccine vectors maybegiven orally.343 In animal
models,oral or parenteral immunization with Lm engineeredto express a number of HIV/SIV
antigensinduced strongcell-mediated immune responses, but demonstratedlittle efficacy against
a SIVmac239 challenge in macaques vaccinatedagainst Envand Gagin DNA prime-rLm boost
regimen.339.344.345 However, as Lm can causeseriousinfectionsin neonates,pregnant women and
immunocompromised hosts, different attenuation strategies are being undertaken to overcome
safetyissues associated with the useof liveLm asvaccine vector in humans. In particular,a liveat-
tenuatedLm 346and a killedbut metabolically active Lm 347 havebeenrecently developed and shown
preserved immunogenicity and efficacy in tumor mouse models.346-349 In nonhuman primates,
Innovative Approaches to DevelopProphylacticand Therapeutic VaccinesagainstHIVIAIDS 211
vaccination with an attenuated strain termed Lmdd expressing HIV-l gag induced Gag-specific
'f-cell responses upon oral administration, whereas combined oral/intramuscular administration
induced strong Gag-specific systemic and mucosal Ab responses. This difference was also evident
for anti-vector Ab, indicating that the route of administration strongly influences the type of re-
sponse elicited. Ofnote, a very late boost failed to induce a robust increase ofanti-Gag Ab titers,
suggesting that anti-vector Ab may severely limit the vaccine immunogeniciry.P" In this regard , an
heterologous DNA prime-oral Lm boost strategy appears a more promising approach, in that it
induced in rhesus macaques mucosal SIV-Gag-specific CD8' T-cells expressing the a4~7 integrin
gut-homing reccptor.l"
Viral Vectors
Safety concerns about live attenuated viruses and inactivated vaccines352J53have led scientists
to look for better and safer ways ofmaking an AIDS vaccine. Most of the more promising AIDS
vaccine candidates currently beingdeveloped and tested use viral vectors, viruses that are not harm-
ful and act as the delivery system to carry HIV antigens to the immune system. 354The potential
advantage ofthe viral vector strategy is to mimic as closely as possible the efficacyoflive-attenuated
vaccines, while at the same time offering much greater safety. Since several of such vectors are
replication competent, the emergence ofviral escape mutants may represent a concern. Further,
although used as vectors, these are actually viruses that have developed multiple and sophisticated
ways to modulate and evade the defense system,355 which may affect the immunogenicity of the
transgene, both qualitatively and quantitatively. Also, some of the proteins of the vector may be
highly immunogenic, thus hampering the immunogenicity ofthe transgene. Thus , a better knowl-
edge of the se aspects is critical when designing vaccination approaches based on viral vectors. A
number of different viruses have been developed as vectors for vaccines. The different vectors all
have their own advantages and disadvantages. Among viral vectors, poxviruses and adenoviruses
have received the most attention in the design of HIV vaccine.
Poxvirus Vectors
Several poxviruses , relatives ofvaccinia (the smallpox vaccine) , are attractive vectors since thei r
large genome allows for the inclusion of multiple heterologous genes, including those encoding
antigens, costimulatory molecules and cytokines, Moreover, poxvirus vectors may be used for mu-
cosal immunization.F" Attenuated vaccinia strains such as MVA and NYVAC (derived from the
Copenhagen strain by further deletion of 18 open reading frames encoding molecules implicated
in pathogenicity and host-range regulatory functions) are the most frequently used poxvirus vec-
to rs. These vectors have been shown to be safe in immunocompromised macaques and in human
Phase IIII clinical trialS.357.36O Pre-existing immunity to vaccinia is ofa limited concern since its use
for smallpox vaccination has ended more than twenty years ago. Most of the recombinant HIV
vaccine usingpoxviruses are effective in nonhuman primate models ,361.364 however, they have much
less immunogenicity and less durable immune responses in humans.
Vaccination with rMVA alone has failed to show sufficient immunogenicity in preventive
and therapeutic Phase I clinical trials 365.366 and because of the inherent high immunogenicity of
the vecto r MVA is currently used as boost for DNA vaccines (see below) . However, therapeutic
vaccination with MVA-nef was safe and induced novel immune respon ses in the majority of the
14 volunteers.259.367
Other poxvirus vectors presently being tested include canarypox'" and fowlpox (for a review
see ref 369). Despite promising results in monkeys.V? Canarypox vectors expressing different HIV
genes have shown limited immunogenicity in humans even at high doses, which were associated to
high reactogenicity.371.m Results from a Phase II trial recently cond ucted using the recombinant
cana rypox ALVAC vCP 14S2A administered alone or together with rgp 120 failed to demonstrate
sufficient imm un ogenicity to grant advancement to Phase III trials." ! Based on composite data
from Phase I and Phase II trial s, Aventi s and the Thai Ministry of Health, together with the US
National Institutes ofHealth (NIH) and the US Military HIV Research Program, have launched a
tv
~
Table 6. Preventive and therapeutic ongoing HIV-vaccine clinical trials
Vaccines Based on HIV Structural Gene Products
Phase (1,11,11I)
Vaccine Type Trial N° Organizer, Producer Vaccine Product (Clade) Start Date
DNA HVTN 070 Univ. Pennsylvania DNA gag, pol , env (B):I: Il-12 or Il-15 DNA I (preventive) Sep-07
Env DNA St. jude, NIAID DNA env (A, B, C, D, E) I (preventive) May-OS
Protein C86P1 SGUl, Richmond Pharma- Prime: HIV-gp140-lT-K63 I (prevent ive) Sep-06
cology, Novartis Vaccines Boost: HIV-gp140-MF59
HVRF-380 -131004 Moscow Institute Env, Gag (B) I (preventive) Mar-06
Immunology
Vectored Antigen
• Poxviruses HPTN 027 NIAID, Sanofi AlVAC-HIV vCP1521 env (B, E) I (preventive) Oct-06
RV138IVR811 USMHRP A lVAC- HIV vCP205 env gag, pol (B) I (preventive) Mar-06
HIVIS 02 Karolinska Institute, SMI, MVA env (E), gag (A), pol (E) I (preventive) jan -06
USMHRP
RV 158/WR 1143 USMHRP,WRAIR MVA gp160, gag, pol (A, E) I (preventive) jul-05
- Adenoviruses VRC012 NIAID-VRC Ad35 env (A) I (preventive) May-07
AdS env (A) ~
HVTN 054 NIH-VRC Ad gag, pol (B), env (A, B, C) I (preventive) jul-05
Heterologous Prime Boost HVTN 072 NlAID Prime: DNA env (A) I ( preventive) May -07
~
~
;;:
Boost: AdS or Ad35 env (A) ~,
HVTN 049 NIAID, Chiron Prime: DNNPlG microparticles gag, env (B) I (preventive) jan-OS Ii:
....
b;j
Boost: oligomeric V2-deleted gp140 (B)-MF59 c;'
~
continued on next page S.
:s
s"-
~
S'
;:s
Table 6. Continued l;;'"
~.
Vaccines Based on HIV Structural Gene Products ~
-s
<l
Phase (1,11,11I) S.
~
Vaccine Type Trial N° O rganizer, Producer Vaccine Product (Clade) Start Date
...
H V TN 042/AN RS NIAID, ANRS Prime: AlVAC-H IV vCP1452 env, gag, po l + CTl 1/11 (preventive) Apr -04 ~
~
VAC019 epi top es fro m nef/pol (B) ~
Boost: L1PO-5 or AlVAC (CTl epi topes) fro m Gag,
Pol, Env (B) ~'"'"
RV144 DoD,Thailand Pr ime: A lVAC env (B, E) III O ct-03 ~
j;;"-
MOPH,N IA ID Boost: Gag (B), Pol (B), Env (B, E) pro teins ~
::;.
TAVEG,Sanofi,VaxGen l:.
;:s
l:l...
POl A I47490 University of M aryland Prime: Salmonella Typhi env (B) I (preventive) Jun-03 t;:l
Boost: Env Protei n(B) '"
-s~
NOl AI05394 N IAI D Prime: D NA env, gag (A, B, C, E) I (preventive) M ay-03 ~
:::.....
Boost: Env, Gag prote in (A, B, C, E)
~
R
Vaccines based on combined HIV structural and nonstructural gene products ;;;'
~
~l:i
DNA N/A Guangxi CDC M ulticlade D NA plasm ids (B, C) I (preventive) M ar-OS ~.
H IVISOl Karolinska Institute, SM I, D NA env (A, B, C), gag (A, B), RT (B), rev (B) I (preventive) Feb-OS
Vecura
~
~
040254; 04- 1-0254 NIA ID M ulticlade DNA plasmids: gag, pol, nef (B), env (A, B,C) I (preventive) Aug-04 ::.::
b
c..,
Protein 108706 GlaxoSmithK line Gag, Pol, Nef 1/11 (preventive) Feb -07
HV TN 064 DA IDS, Protein ep itopes Env, Gag, Pol, Vpu (B) andlor DNA I (preventive) Jan-06
Pharmexa-Epimmune gag, po l, vpr, nef (A, B, C, D, F, G)

w
N
~
Table 6. Continued
Vaccines Based on Combined HIV Structural and Nonstructural Gene Products
Phase (1,11,111),
Vectored Antigen
- Poxviruses IAVI C003 ADARC, IAVI, Rockefeller MVA env/gag-pol, nef-tat (C) I (preventive) Nov-06
HIV-POL-OOl Bavarian Nordic MVA HIV polytope vaccine I (preventive) Oct-06
IAVI DOOl IAVI, Therion TBC-M4 MVA env, gag, tat, rev,nef aRT (C) I (preventive) Dec-OS
IAVI C002 IAVI-ADARC MVA env/gag-pol, nef-tat (C) I (preventive) Jan-OS
- Adenovirus (Ad) AINS04-AS218 NlAID, Merck, HVTN AdS gag, pol, nef (B) II (therapeutic) Sep-OS
HVTN OS7 NIAID Ad gag, pol (B), env (A, B, C) I (preventive) Nov-04
- Adeno-associated IAVI A002 IAVI, Targeted Genetics AAV gag, PR, aRT (C) II (preventive) Nov -OS
viruses (AAV)
Heterologous Prime Boost HIV NAT 064 The National Centre in Prime: DNA gag, pol, tat/rev, env (A, E) I (preventive) May-07
HIV Epidemiology and Boost: rFPV gag, pol, tat/rev, env (A, E)
Clinical Research, The
University of South Wales
~
l>
HVTN 067 NIAID, DNA vaccine and MVA (alone or in I (preventive) Apr-07
Pharmexa-Epimmune prime-boost regimen) env, gag, pol, vpu (B); ~
gag, pol, vpr, nef (A, B, C, D, E, G) ~
;:
HVTN 069 NIAID Prime: DNA gag, pol, nef (B), env (A, B, C); I (preventive) Nov-06
Boost: Ad gag, pol (B), env (A, B, C) l
b:l
l; "
~
continued on next page s,
;s
'"~
~
::t
.;;;-
~
Table 6. Continued ~.
~
-s
Vaccines Based on Combined HIV Structural and Nonstructural Gene Products ~
So
~
Phase (1,11,111), ~
Vaccin e Type Trial N° Organizer, Produc er Vaccine Product (Clade) Start D ate ~
~
V RC-Ol l N IAI D, VRC Prime: D NA gag, po l, nef (B), env (A, B, C) or I (preve ntive) May -06 ~
Ad gag,po l (B),env (A, B, C)
Boost: Ad gag, po l (B) env(A, B, C) ~ ""
~
HVTN 065 NJAID, Geovax Prime: D NA gag, pro, RT, env, tat, rev, vpu (B); I (preventive) Ap r-06 t-
~
Boost: M VA gag, po l, env (B) ~.
HV TN 06 8 DAIDS, N IAID, VRC Pri me: Non repl icating Ad gag-po l (B), env (A, I (preventive) M ar-06
B, C) or DNA gag, pol, nef (B), env (A, B, C);
'~"
Boost: Ad gag-pol (B), env (A, B, C)
Si
EuroVacc 02 EuroVacc Foundatio n Prime: DNA env, gag, po l, nef (C) I (prevent ive) Feb-06
"i:l
Boost: N YVAC env, gag, pol , nef (C) 1::t.....
IAVI VOO l IAVI, NIAJD Prime: D NA gag, po l, nef (B), env (A, B, C); I (preve ntive) Nov-05
~
Boost: Ad gag, pol (B), env (A, B, C) R
HTV N 063 NIAI D, W yeth Prime: D NA gag (B) ::l: IL-15 I (preventive) Sep-05 ii '
~
Boost: D NA gag (B)::l: IL-15 or DN A gag (B) ::l: ~
l:i
IL-12 ~.
HTVN 060 NIAI D, Wyet h Prime: DNA gag (B) ::l: IL-12 I (preventive) Aug-05
Boost: D NA or peptide gag (B) or gag, env, nef ~
(B) + GM-CSF ~
H IV IS 03 M UCHS, Karolinska Prime: DN A env (A, B, C), gag (A, B), RT (B), 1/11 (preventive) De c-06 b
e;"
Institute, SM I, Vecura, rev (B);

USMH RP Boost: M VA env (E), gag (A), po l (E)
N
...
""
~
0,
Table 6. Continued
Vaccines Based on Combined HIV Structural and Nonstructural Gene Products
Phase (1,11,11I),
RV 172/WR 1218 USMHRP, NIAID Prime: DNA gag, pol, nef (B), env (A, B, C); 1/11 (preventive) May-06
Boost: Ad gag, pol (B); env (A, B, C)
HVTN 204 NIAID, Vical, GenVac Prime: DNA gag, pol, nef (B), env (A, B, C); II (preventive) Sep-OS
Boost: Ad gag, pol (B), env (A, B, C)
HVTN 055 NIAID, Therion Prime: MVA env, gag (B) + tat, rev, nef, pol (B); I (preventive) Sep-04
Boost: FPV env, gag (B) + tat, rev, nef, pol (B)
Whole HIV DCV-02 Hospital Clinic Barcelona DCs pulsed with inactivated autologous HIV I/II(therapeutic) Nov-06
5P01A157127-2 NIAID HIV immunogen (whole killed gp120-depleted 1/11 (therapeutic) Oct-05
HIV inactivated)
2000456-01 H Ottawa Health Research Institute Remune (vaccine) and AlVAC (vaccine) 1/11 (therapeutic) Sep-05
ADARC: Aaron Diamond AIDS Research Center; ANRS: Agence Nationale de Recherches sur Ie SIDA (France); DAIDS: Division of AIDS; DoD: US Department
of Defense; EuroVacc: European Vaccine Effort Against HIV/AIDS; Guangxi CDC: Guangxi Centre for Disease Control and Prevention; HPTN: HIV Prevention ~
Trials Network; HVTN: HIV Vaccine Trials Network; IAVI: International AIDS Vaccine Initiative; MoPH: Ministry of Public Health; MUCHS: Muhimbill University
College of Health Science; NIAID: US National Institute Allergy and Infectious Diseases; NIH: US National Institutes of Health; SGUL: St George's, University ~
of london; SMI: Swedish Institute for Infectious Disease Control; St. Jude: St. Jude Children's Research Hospital; TAVEG: Thai AIDS Vaccine Evaluation Group; ~
l:
USMHRP: US Military HIV Research Program; VRC: Vaccine Research Center; WRAIR: Walter Reed Army Institute of Research; MVA: Modified Vaccinia An- ::-.
E:
kara; FPV; fowl poxvirus; vCP: viral Canarypox; LT·K63: nontoxic mutant of Escherichia coli heat labile enterotoxin (IT); PLG: Polylactide- coglycolide; GM-CFS: b:I
1;;'
Granulocyte-macrophage colony stimulating factor.
-
~
s,
:s
.
~
Innovative Approaches toDevelop Prophylactic and Therapeutic Vaccines agaimt HIV/AIDS 217
PhaseIII trialwith a HIV-l Env-based vaccine in which a canarypox prime isfollowed byboosting
with the VaxGen gpl20 protein (Table6).
Similarly. despitepromisingresultsin the monkey model.l" a PhaseI therapeutic trial with a
fowlpox vector expressing Gag and Pol failed to displayany immunogenicityof the transgenes,
althoughit inducedanti-vector Abs.37SImmunogenicity wasdemonstratedin a morecomplex thera-
peutic trial in which immunizationwith fowlpox expressing several HIV antigenswascombined
with HIV lipopeptides(syntheticfragments of HI V proteins associated with lipidsthat facilitate
the induction of a cellularimmuneresponse) and followed byadministration ofIL- 2. confirming
the intrinsicweakimmunogenicityof this vector.376Fowlpox vectoredvaccines performedpoorly
alsoin heterologousDNA prime-fowlpox boost approachesboth in monkeys and PhaseI preven-
tiveor therapeutic trials.375.377.378 Subsequentstudies in the monkey model suggest that the poor
immunogenicityobservedin human as compared to monkeysmight be due to the vaccine dose
used. which might be insufficient to triggeradequate responses.t" However. higher production
costs,multiple inoculations and reactogenicity are seriousobstacles to scalingup vaccine dosing
and might hamper further developmentof vaccines of this type.
Adeno andAdeno-Associated Vectors

Adenoviralvectorshavea broad host rangeand infect both proliferatingand quiescentcells.
The tropism of adenoviruses for mucosalepithelium makesthem extremely attractiveas vectors
for HIV vaccinedevelopment since they can been delivered orally or intranasallyand induce
mucosalimmune responses.P? Recombinantadenovirus vectorscan accommodatelargerinserts,
mediate transient but high levels of protein expression and can be easily produced at high titers.
Furthermore. adenoviruses targets DCs in which they up-regulatecostimulatorymolecules and
MH C class II expression and induceproduction ofTh-l and pro-inflammatory cytokines.381.382 Of
note. Adenovirus-based vaccine candidateshaveproduced the most impressive cellularimmune
responses seenso far. 383Bothreplication-competent and-incompetent vectors arebeingdeveloped
asvaccine againstHIV. However,whilereplication-competent adenovirus vectorsinducestronger
and more persistenthumoral and cellularimmuneresponses comparedto the nonreplicatingvec-
tors. there are safetyconcernsabout their use in clinicaltrials.383.38s
The replication-incompetent recombinant adenovirus Type S (rAdS) is a modified form of
AdS, the virus that causes some formsof the common cold. It is replication-defective to enhance
safetyand represents one of the most promisingviralvectors for HIV vaccines. However. prior
exposure to AdS mayboost anti Ad-Santibody response blunting the expression ofthe transgene
and the percentageof volunteers respondingto the vaccine. This anti-vectorimmunity mayrep-
resent a major problem in the developing world, where the prevalence of prior exposure to AdS
isgreatest.386Thishasprompted the developmentby Merck. Crucelland Transgene, in collabora-
tion with IAVI. of candidatevaccines basedon less prevalenthuman Adenovirus serotypes (Ad6.
Ad3S. Adl l, or Ad 24) to replace the AdS vector in fusion HIV trials.387.39o Another approach
to circumventpre-existing immunity to AdS has been to modify the vector by substituting key
neutralizing epitopes on the surface of viral capsidproteins with those from the less prevalent
serotypeAd48. Suchchimericvector is calledAdSHVR48.391
Finally. pre-existing immunity to adenovirus maybe overcome by heterologousprime-boost
strategies, including DNA priming followed by adenovirus vector boosting.96.392 or the use of
different adenovirus serotypes, including the above mentioned AdSHVR48. for priming and
boosting.393•394
Upon extensive testingin nonhuman primatesMerckfoundout that intramuscular vaccination
with SIV Gag delivered by AdS (El- deleted) was superior to DNA or MVAat inducing CTL
responses and at protecting against disease followingpathogenic intravenous challengedwith
SHIV_89.6P.141.142but not against an intrarectal challenge with SIVmac239.143 Noteworthy.the
limited protection upon SIVmac239 occurredin the presenceofT-cell responses that correlated
with protection in the formerstudy.144 Co-immunizationwith AdScarryingGagand Envwasless
effective than Gagaloneat controllinginfectionin rhesusmacaques challenged intravenously with
218 PharmaceuticalBioftchnology
SHIV-89.6p238confirmingthe detrimental roleofCTL responses to Envobservedin the natural

infection.63 In order to increasethe breadth of response against HIV-l and to improve the vac-
cine efficacy, replicationdefective AdSvectorscarryingpol and nef wereconstructed and a Phase
lIb trial started in 2004 in which 3,000 high riskindividuals wereimmunizedintramuscularly 3
timeswith replicationdefective AdSvectorsexpressinggag, pol and nef(AdSMRKAdStrivalent).
This trivalentvaccinewasgenerally safeand welltolerated at all dosesstudied and immunogenic
eliciting responses against the 3 antigens included in the vaccine. However, preliminary data
indicate that vaccinationdid not protect from infection or loweredviral loads. Further, there
wasan apparent higher susceptibility to infection in vaccinees with pre-existing immunity to the
vector.146.395 Reducedexpression and immunogenicityofthe transgenes, assuggested bythe much
lowerproportion of vaccinees respondingto allthe three HIV antigens(Gag,Poland Nef) in the
group with pre-existing immunityto AdSascomparedto vaccinees with no pre-existingimmunity,
immune activation generated by the response to the vector and/or an increaseof HIV-specific
target T-cells induced by the vaccine!" are some of the hypotheses that have been proposed to
explain these findings. Thus,although verypreliminary, overallpreclinicaland clinicaldata may
suggestthat SIV is a more rigorouschallenge virus and a better predictor of vaccine efficacy in
human and that boostingof pre-existing immunityto the vector mayactuallyenhancethe suscep-
tibility to infection ascomparedto placeboor vaccinees with no pre-existing immunity.Another
nonreplicativeadenoviral vector (deleted of the genes coding for El and E3 proteins) has been
developedby NIH Vaccine Research Center (VRe), together with a DNA-basedvaccine. These
are multicomponent vaccines, which express the Env glycoprotein from clades A, B and C and
the Gag, Pol and Nef proteins from cladeB and aredesignedfor usein a DNA prime-AdS boost
regimenstrategy.396 Despitedifferences in the vaccine design, initiation ofPhaseII trialshas been
postponed to late 2008,when a better understandingofthe reasons of the Merck's vaccinefailure
will clarifywhether vaccination with the VRC candidatewould posethe same risks.
Replication-competent adenoviral vectorshavealsobeen developedasvehicles for AIDS vac-
cines.397Studiesin both chimpanzeeand rhesusmacaquemodelshavedemonstratedthat priming
with replicatingAd recombinantsencoding HIV or SIV genesfollowed by boosting with viral
protein subunits elicitspotent humoral,cellularand mucosalimmuneresponses.385.398-405 Ofnote,
vaccinationofRhesus macaques with Advectorsexpressing HIV-l Tat and Envconferreda strong
protection against a challenge with the pathogenicSHIV 89.6P, which wassuperiorto that pro-
vided by a largervaccine formulation includingSIV Gag and Nef in addition to HIV-l Tat and
Env.268 This underscores the importance ofproperly selecting the antigens to combine together
and providesone of the strongestevidence in favorof the Tat + Envvaccine.
Other viral vectors used as AIDS vaccines in clinicaltrials includeAdeno-associated viruses
(AAV) which are not adenoviruses but are often found in adenovirus infections.406•407These vec-
tors arecurrentlyusedin PhaseI and PhaseII clinicaltrials," However, the weakimmunogenicity
recorded in a multicentric Phase I studr has led to halting the initiation of Phase II trials in
India and spurred a debate on the ethics of conducting the ongoing Phase II trials in Africa in
the faceof such disappointingPhaseI results. Theslightlybetter immunogenicityrecordedat the
highest dose tested in the PhaseI trialsmaysuggestthat a doseincreasecould solve this problem.
However, recent data indicate that, in mice,vaccination with high dosesofAAV expressing Gag
inducedGag-specific effector CD8+CTLs that wereweakproducers ofIFN-y,expressedexhaustion
markersand failedto becomememorycells.Transitionto the memoryphenotype and restoration
of full functionalitywasachievedupon adoptivetransfer, suggesting that chronicexposure to the
trangene might havebeen the cause of the CTL dysfunceion.f"
Other Viral vectors

Other viral vectors have been tested as vaccine vectors for HIV-l and have shown various
degrees of success (for a reviewsee ref. 410). Among them, the alphaviruses include weakened
formsof three viruses named Venezuelan Equine Encephalitis (VEE), Sindbis(SIN) and Semliki
ForestVirus(SFV).The firstalphavirus vectorcandidate,AlphaVax's VEE,isdesignedasa replicon
Innovative Approaches to DevelopProphylactic and Therapeutic Vaccines against HIV/AIDS 219
particles containing self-replicatingRNA encoding the VEE replicase proteins and expressing a
gene of interest in placeof the viralstructural protein genes. An appealingfeatureof alphaviruses
is their known ability to induce apoptosis of transduced cells, favouring DC cross-priming."!
Thesereplicon particleshaveshown protection againstother virusesand haveelicitedsignificant
cell-mediatedand antibody immune responses with SIV antigens,perhaps due to the propensity
of the vector to target antigen-presentingcells.412 The VEE vector is currently being tested in
clinicaltrials.4I3
Viruses belonging to the rhabdovirus family and in particular the vesicularstomatitis virus
(VSV) are also being used. These vectors offer the advantage to be highly flexible, easy to ma-
nipulate and ableto express largeand multipleforeigngenes.414Intramuscularvaccinationof mice
with a single-cycle vector expressing HIV Env elicited strong Env-specific humoral and cellular
responses."? Furthermore, immunization of macaques with recombinant VSVs (rVSVs) express-
ing SIV Gagand HIV Envhas been reported to protect from pathogenic SHIV89.6P.416.417 These
promising resultshaveled to the developmentofrVSVfor use in humans.t" However, sincethe
prototypic rVSVvector was found to be insufficiently attenuated for clinical evaluation, novel
highlyattenuated vectorshavebeendesigned, whicharelessneurovirulentand moreimmunogenic
than the prototypic rVSVvector," ?
Other potentially powerful vaccinedeliverysystems are represented by Polioviruses.f" Both
replication-competent and replication-deficient recombinants have been shown to be immu-
nogenic in nonhuman primates when used through various routes of immunization, including
mucosaldelivery.t" However, restrictionsto the useof thesevectors include the stabilityand size
of heterologous gene inserts422and the presenceof high levels of pre-existingimmunity to polio
vectors in the generalpopulation.
Replication-competent and replication-defective herpesviruses (HSVs), including HSV-I,
represent suitable vaccinevectors against AIDS. Important advantages include broad host cell
range,high infectivityand easyof production of high-titer stocksof viruses, long-termexpression
of foreign antigens and stimulation of both humoral and cellular arms of the immune system.
Vaccination with replication-competent or replication-defective HSVsvaccinevectors express-
ing SIV Env and Nef, protected macaques against a challengewith SIVmac239.423 However, the
overall toxicityand the pre-existingimmunity against the vector may represent a safetyissuefor
their use in humans and current strategies focus on the development of replication-incompetent
virusesused in prime-boost regimenwith DNA. 424
New Particulate Delivery Systems

Microparticles havebeeneffectively usedformanyyearsasparticulatedelivery systems fordrugs,
therapeuticproteins and varioustypesofvaccines includingrecombinantproteins, plasmidDNA,
peptides and other vaccine components (e.g., immune potentiators).42S·426 Among antigen-loaded
microspheres, injectable, biodegradable polymericparticlespreparedwith poly(d.l-lactide-co-gly-
colide)(PLG) or poly(d.l-lactide) (PL) polymersrepresentasuccessful method for in vivodelivery
of peptide, protein or DNA antigens." ?Both particleshavebeen shown to be effective, especially
for oral delivery.t" Antigen instabilityand manufacturingdifficulties havebeen overcomeby the
recent findings that adsorption rather than microencapsulation of the antigen onto PLGA is
easier, cheaperand ensuresbetter antigen stability.429 In comparisonto standard aluminum-based
adjuvants,these microspheres have many desirable features, including the ability to enhance the
bioavalaibility of the antigen,allowingpulsatingantigen release and to reducethe number of doses
in the immunization schedule,mimickingthe conventionalprime-boost regimen.Furthermore,
for adjuvantingvaccines against intracellularpathogens and cancer, selective targeting of PLGA
microparticlesto DCs has been achievedand induction of CTLs has been attained in both small
animalsand nonhuman primares.v"In particular PLGs havebeen demonstrated to enhance the
immunogenicityof DNA vaccines to HIV-Gagand HIV-Envin rhesusmacaques.t" PLG particles
are currently beingevaluatedin a gag + env DNA/PLG prime-AV2 Envprotein boost preventive
PhaseI trial.189
Two novel classes ofbiocompatible core-shell anionic microspheres havebeen used as an ef-
ficientdelivery system for vaccinationwith the Tat protein.432Thesernicrospheres, synthesized by
dispersion polymerization, arecharacterized byan increased shelf-life and the capability of reversibly
adsorbingnativeproteins at their surface. In particular.thesemicroparticles consistofnegatively
charged microspheres, made ofeither poly(styrene) or poly(methylmethacrylate) and in which
hemisuccinatedpoly(vinylalcohol)or EudragitLl OO/S Swereused,respectively, asstericstabiliz-
ers.432Thesemicrospheres preventedTat from oxidation, maintainingthe nativeand biologically
active conformation required for vaccine efficacy and efficiently delivered Tat intracellularly. In
the mousemodel, delivery of Tat by these microspheres wassafeand immunogenic.433•434
VLPs
Virus-like particles(VLPs) are self-assembling. nonreplicating,nonpathogenic particlesthat
aresimilarin sizeand conformationto intact virions.43s.436 VLPsoffera number ofadvantages over
conventional protein immunogens and havebeen therefore consideredas an ideal HIV vaccine
candidare.i" In fact,theseparticlescanbe easily producedin largeamount in heterologousexpres-
sionsystems (baculovirus, vaccinia virus)and easily purified. In addition,sinceVLPslackregulatory
proteinsaswellasinfectious geneticmaterial, theyareboth replication- and infection-incompetent,
makingVLPssaferthan live-attenuatedviruses. Further,VLPsexpress viralproteinsin their native
conformation and generally induce more effective humoral and cellularimmune response than
their solublecounterparts. in both the systemic and mucosalimmune compartments.437-439
However. due to their nonreplicatingproperties,VLPsareless effective at inducingcellularim-
mune responses ascomparedto live-attenuatedviruses or replicatingviralvectorvaccines. For this
reason,novelapproaches arebeingdeveloped in order to increase their immunogenicity, including
DC targeting.440 The mucosaladministration ofVLP vaccines has also emergedas a promising
strategy to elicit mucosaland systemic anti-HIV humoral and cellularimmune responses.r"
To date. numerous typesofVLPs havebeen produced utilisingthe abilityofcapsidand enve-
lope proteins to self-assemble into highlyorganisedparticulatestructures. In particular.the Gag
protein is required for their assembly. budding and release from host cell. VLPs,basedon HIV-I
pSSgag, presentingthe entiregpI20 moleculefrom an UgandancladeA HIV-I isolate, havebeen
shownto inducestrongsystemic and mucosal humoraland cellular immuneresponses in mice.442,443
More recently, IN administration in a mousemodelof theseVLPstogether with the EurocineL3
mucosal adjuvant (a monoglycerides/fatty acid lipid suspensionsr'" in a heterologous (DNA +
VLPs)prime-booststrategyinduced higher titersof NAbs and strongeranti-EnvT-cellresponses
as compared to vaccination with adjuvanted VLPs only.44s Further, a combined multiepitope
VLP-based HIV vaccine (Combi HIVvac) carryingboth B- and T-cell epitopes (from HIV-I
Env,Gag. Poland Nefproteins) resultedsafeand highlyimmunogenicin mice.446 Ofinterest,vac-
cination ofrhesusmacaques with pSSgagVLPsin the absence of adjuvantinduced broad.durable
anti-GagCTLs.447However, therapeuticvaccination with HIV-I p l?Ip24:Tyvirus-like particles.
which contain part of the HIV-I lIl B Gagsequenceand areproduced byexpressing a TYA:p17/p24
fusion genein yeast448did not appear to slowerHIV-I disease progression.t?or to impact CD4+
T-celldecline in patients with advancedHIV infection.4so
Prime Boost Strategies

Manyofthe vaccinestudiescombinevariousapproaches in a prime-boostfashion to optimize
the immune responses elicited. A heterologousprime boost strategyis the administration ofone
type of vaccine(the primer is usually DNA) followed by the administration of another form of
the vaccine(the booster is usually recombinant proteins or attenuated viralvectors).The goal of
this approach is to complement the priming by a differentstimulation of the immune systemto
enhance the body's overallimmune response to HIV, a result that may not be achieved with a
singletype ofvaccine. For example. whileDNA or microparticles areoptimal for inducingT-cell
responses. they arepoor inducersof Ab, which.however, are readily induced upon boostingwith
protein or a recombinant vector. Another advantage of this strategy is that it circumvents the
InnovativeApproachts toDeoelop Prophylactic and Th"aptutic VaccinesagainstHIV/AIDS 221
relatively common and detrimental immunodominanceof the vector that mayresultin a reduced
immunogenicityof the transgeneand the impossibility to use the samevector twice because im-
munity to the vectorstronglyreduces or preventsthe transgeneexpression. Toovercome thislatter
problem sequential immunizationswith different viral vectors have been used as an alternative
prime-boost approach,as reported in nonhuman primate modelsagainst SHIV451 and SIV143.l44
challenges. DNA prime-viral vectorboost approaches mayalsobe exploitedto target mucosalsite
either because of the intrinsic tropism of the vector or because they can be applied mucosally. A
varietyof protocols usingalternative viralvectorsfor both priming and boosting havealsobeen
reported, both alone and in combinationwith DNA and havebeen successful at limitingdisease
progression, but not at offeringprotection againstinfection. Forexample, DNA primingfollowed
bya recombinantMYAexpressing multipleHIV proteinsdid not preventbut effectively controlled
infection upon challenge with pathogenic SHIV89.6P in rhesus macaques.361.452 Based on the
promising resultsin monkeys, GEOVAXis currentlytesting in 4 different PhaseI trials a DNA
prime-MYA boost approachin which primingwith DNA encodingTat, Rev, Vpuand Gagis fol-
lowedbyboostingwith MYAexpressingEnv, Gag,Protease and RT.Preliminary dataindicategood
safetyand CTL responses in over 50%of the vaccineesv! and a PhaseII trial isplanned for 2008.
Similarly, McMichaeland coworkers at the Oxford Universityhaveshown in PhaseI studiesthat
DNA prime-MVA boost HIV vaccines are well-tolerated and immunogenic, but the percentage
of volunteersrespondingto the vector and the durability of CD8· cell-mediated responses have
not matchedsofar the responses observedwith the rAd5vector.132.366.4S4 However, the lackofsolid
correlates of protection and the largebody of evidence showingthat natural control of infection
is not necessarily associated with strong immune responses should not impede advancement of
these type of vaccines to Phase II trials.
International Networking to Ease and Accelerate HIV/AIDS Vaccine

Development
The firstPhaseI trial of an HIV vaccine wasconducted in the USA in 1987. Sincethen, more
than SO candidatevaccines havebeen tested in about 100 PhaselIII clinicaltrials,involving more
than 35,000 healthyhuman volunteers. Two PhaseIII trialshavebeen completedand a third one
is in progress. The vast majority of these vaccine candidates,including those tested in PhaseIII
trials, werebased on structural HIV-1 proteins and primarilyaimed at inducing NAbs. Most of
the effortsto developand evaluate HIV vaccines is borne by the NIH, CDC and WRAIR in the
USA and by ANRS in France, with strong help from the International AIDS Vaccine Initiative
(lAVI) in New York (http:/ / www.iavi.org). the European Union (EU). initiatives in WHO
(http://www.who.int/en)andUNAIDS (http://www.unaids.it) and the recent commitment of
the Bill and Melinda Gates Foundation for a Global Enterprise (http://www.gatesfoundation.
org/GlobalHealth/Pri_Diseases/HIVAIDS).
The HIVVaccineTrialNetwork (HVTN) established byNIAID in 2000,with 25 clinical sites
in four continents, represents a major resource for clinicalHIV vaccine research (http://www3.
niaid.nih.gov/about/organization/daids),The EU has alsoestablisheda comprehensive program
aimed at strengtheningintegration of science amongcountriesof the EU and promoting, among
the others, vaccine development against poverty diseases (i.e., HIV/AIDS, TB, Malaria). The
AIDS Vaccine IntegratedProject(AVIP) (http:/ /www.avip-eu.org),Mucosal Vaccines for Poverty
Related Diseases (MUVAPRED) (http:/ / www.mucosalimmunity.org/ muvapred/index.asp)
and the European Vaccine effort against HIV/ AIDS (EUROVAC) (http:/ /www.eurovac.net).
are among the most important projects recentlycofunded by the EU. In addition, the European
and DevelopingCountries Clinical Trials Partnership (EDCTP) has been created with the aim
of helping developing countries to build up their capacity in testing the efficacy of new drugs,
microbicides and vaccines.
Conclusion
Several advancements have been made over the past few years to improvevaccine strategies
aimed at inducing protection against HIV. Ideally, the aim of an effective vaccine would be to
produce sterilizingimmunity in allrecipients. However, alsoa vaccine ableto control rather than
prevent the infection might haveimportant benefits, reducing HIV levels in the body,delaying
progression to AIDS and initiation of anti-retroviral therapy and reducing the chance of HIV
transmission.
The current knowledge suggests that an effective HIV candidateshould induce both humoral
and cellularimmune responses, to ensuredurableimmunological memoryand to boost both the
adaptative and innate immune system. The latter one is particularlyimportant at mucosalsites
ofHI V transmission.v' One of the majorimpedimentsto the developmentofan HIV-I vaccine
is the lack of knowledgeof the immune correlates ofprotection. Although studiesof MEV and
LTNPs continue to providevaluable informationon mechanisms of naturalprotection, whichcan
then be appliedto vaccine design, it shouldbe kept in mind that immuneresponses in LTNPs may
representa correlateof preservationof immunecompetencein a host containinginfection rather
than the actualfactorscontrollingthe virus.Natural resistance to infectionhasbeen attributed to
a combinationofgenetic,innate and acquiredimmunesystem-mediated mechanisms."Therefore,
a novelapproach for treatment and/or preventionof HIV infection might be representedby the
manipulation of these restriction factorsin order to improveand broaden their activities.456The
earlycontainment of HI V-I and SIV replicationin acutelyinfected individuals and monkeys is
temporally associated with the emergence of a virus-specific CTL response and high levels of
circulatingCTLs areassociated with good clinicalstatus in chronicallyinfected individuals457.458
and acutelyinfected monkeys,"? Importantly, experimentalin vivodepletion ofCD8- T-cellsin
monkeys, abrogatedcontrolofSIV replication duringprimaryinfectionand the animalsdied after
a rapidlyprogressive disease course.460462 While this has generally been interpreted as the defini-
tive proofof the keyroleof CD8- T-cellsin the containment ofinfection, it should be reminded
that alsoNK cellsexpress the CD8 moleculeand their experimentaldepletion maycontribute to
the lossof virus control.
Interestingly, lossof control of infection has been reported also upon B-celldepletion during
primarySIVmac infectionof rhesusmonkeys,460·463.464 suggestingthat eitherAbsareindeed crucial
for containing the virusevenat the verybeginningof the infectionor, more in general, that severe
disturbance of a component of the immune system disrupts the proper function of system as a
whole, underscoringthe integrated nature of the defensesystem360•463.465-469 and the contribution
ofmultiple arms to an effective control of infection (for a reviewseeref 470).
Because of safetyconcerns, traditional immunization approaches, including those based on
liveattenuated and inactivatedviruses, havebeen almostabandoned.Vaccine candidatesbasedon
purifiedor syntheticproteinsaremainlydeveloped to induceNAbs,whereas recentadvances in mo-
lecularbiologyand geneticengineeringhaveled to the development ofa newgeneration ofvaccines,
whichincludes DNA- and microorganism-vectoredvaccines,whichareprimarily aimedat inducing
T-cellresponses. In this regard,vaccinia viruses, canarypox constructs,replication-competent and
replication-defective adenovirus vectorsare the main livevectors currentlybeing evaluated. The
success of these vectors is believed to depend also on their capabilityto trigger innate immune
responses, which would induce proper adaptive immunity. Although replication-competent
adenoviruses have the advantage of persistentlyinfecting the host and stimulating the immune
system,383 safetyissues need to be fullyaddressed before their advancement to clinicaltrials. The
recent failure of the Merck trial clearly indicates that even replication-defective adenoviralvec-
tors maybe harmful in the presence of pre-existing immunity to the vector.Thus,DNA vaccines
with increasedimmunogenicityand microbialvectors that circumvent pre-existing immunity to
the vector are needed. In this regard,optimization and further explorationof new adjuvants for
DNA and protein antigensare currentlybeingheavily pursued.f" Vl.Ps havealsobeen employed
asmulti-epitopevaccine sincetheyofferthe advantages of [i) mimickingthe virionwithout having
the safetyconcernsof live-attenuatedviruses, (ii) inducing both mucosaland systemic immune
responses , (iii) activating both endogenous and exogenous antigen presentation pathways (MHC
class I and II, respectively) and (iv) maintaining the antigens in their native conformation.
Effective vaccination may ultimately require two or more vaccines used in conjunction (het-
erologous prime-boost strategies), an approach to vaccine development that differs from tradi-
tional vaccine design and is presently the preferred strategy for many vaccine candidates against
HIV/ AIDS (Table 6). In this regard, there is a general agreement that when exploited combined,
the vaccine components used for the prime and the boost are expected to stimulate a broader and
more diversified immune response than using any ofthem repeatedly. In addition, the single use
ofa viral or bacterial vector will avoid the interference, on a second administration, ofpre-existing
immunity to the vector.
Effective anti-HIV/AIDS vaccines may require targeting of several HIV-l antigens. Among
these multi-component vaccines novel minimalistic vaccination strategies, combining structural
(~V2-Env) and nonstruetural (Tat or Nef) proteins have been rationally designed to induce NAbs
and T-cell responses against keyearly and late HIV antigens. In particular, preclinical testing ofthe
Tat/ ~V2-Env combination in macaques has shown efficacy (ref. 26S and Ensoli B, in preparation)
and clinical trials with this vaccine candidate will start in 200S .
However, HIV vaccine development still faces significant challenges. The availability ofan ef-
fective HIV vaccine requires scientific and public-health efforts and the establishment ofConsortia
such as the "European Consortia for HIV vaccine development" (includingAVIP, MUVAPRED,
VIAV) , the "N eutralizing Antibody Consortium"; the "HIV Global Enterprise", an international
Consortium ofnongovernamental and governamental organizarions.f? Clinical trials must also be
performed with appropriate ethical rules, especially in developing countries, avoiding duplication
of efforts, using standardized genetic inserts as immunogens and implementing immunological
assaysfor preclinical and clinical testing to compare candidate vaccines. This is important because
the laboratory assaysused to assess immune responses may not be comparable, severelyhampering
decisions about which candidates to pursue for further testing. In addition, new knowledge about
the immune response to HIV is raising concerns that current assays overlook important aspects
of those imm une respon ses.
Open questions remain to be answered, such as how to induce high titers of NAbs; whether
any of the vaccines being currently developed will elicit cellular immune responses that will cor-
relate with protection from infection or disease progression; the type (poly- or mono-functional)
of CD4' and CDS' T-cell responses elicited by the vaccines currently being developed; the mag-
nitude, breath and durability of the vaccine-induced CD4' and CDS' T-cell responses; the best
combination ofvaceines that in the prime-boost immunization strategies will stimulate an immune
response similar to that thought to confer protection from disease progression.
The challenges the scientific community still faces are formidable. However, looking back ,
enormous progresses have been made in each aspect of vaccine development, from basic science
to clinical testing, that let us be optimistic about the eventual generation of effective preventive
and therapeutic vaccines against HIV/ AIDS. While vaccines able to slow disease progression
and decrease transmission rate should be at reach in the medium term , recent advancements in
the generation ofEnv-based immunogens, their association to key regulatory or accessory HIV-l
proteins and present reconsideration of the several Ab effector functions, make us hoping that
even a sterilizing vaccine may be not too far distant.
Acknowledgements
The research activities described in this publication were funded by the EC Commission un-
der the VI Framework Programme of Research and Technological Development (2002-2006),
Project no . LSHP-CT-2004-S034S7 , AIDS Vaccine Integrated Project ("AVIP") and Project no .
LSHP-CT-2003-S03240, Mucosal Vaccines for Poverty Related Diseases ("MUVAPRED").
The authors wish to thank Mrs. P. Sergiampietri for the editorial assistance and Mr. Leonardo
Sernicola for preparing the tables.
Please note that Aurelio Cafaro and Iole Macchia have equall y contributed to this chapter.
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CHAPTER 15
New Strategies to Overcome

the Drawbacks ofCurrently Available
Flu Vaccines
Epifanio Fichera: Diana Felnerova, Robert Mischler, jean-Francois Viret
and Reinhard Glueck
Abstract
accination represents the most efficient tool to control morbidity and mortality resulting
V from influenza infections in humans. The currently licensed influenza vaccines provide
good protection levels in healthy adults . whereas lower protection is generally achieved
in ageing individuals who are at a higher risk ofdeveloping severe clinical man ifestations. Future
improvements in influenza vaccines should address the needs of high risk groups including the
elderly. small children and chronic patients. Recently. due to the increased incidence of avian
influenza pandemic outbreaks. the prevention of a potential human influenza pandemic turned
into another crucial issue in the influenza vaccination field. The development and validation of
manufacturing processes for efficient and safe pandemic vaccines became one of the top priori-
ties of health. regulatory and funding agencies all over the world. In the pandemic cont ext. the
development of novel vaccines administered via the mucosal route may playa significant role by
reducing virus shedding from infected individuals. This chapter provides insights in the limita-
tions of existing manufacturing processes. new approaches to overcome limitation in vaccine
production. mechanisms ofaction ofcurrent vaccines and discuss potential strategies to improve
the imm unogenicity and efficacy ofinfluenza vaccines.
Introduction
Influenza viruses A . B and C are enveloped viruses with a segment ed . negative single-stranded
RNA genome. They are members ofthe Orthomyxoviridaefamily together with the Thogotovirus
and Isavirus. Within each influenzavirus genus no separate species has been recognized. but there
are clusters ofstrains that can genetically reassort with each orher.P Studies on the ecology ofthe
influenza viruses led the hypothesis that all mammalian influenza viruses derive from an avian
influenza reservoir. Thus. after long periods ofevolution. human viruses originate from reassorted
influenza viruses ofavian/mammalian origin. The level ofnonhuman virus adaptation to the hu-
man host has a considerable impact on the severity ofhuman influenza disease. Human influenza
is mainly caused by Influenza virus A and . to a less extent by Influenza virus B.' Based on the an-
tigenicity ofthe surface glycoproteins hemagglutinin (HA) and neuraminidase (N) the influenza
virus A is further classified in different subtypes. Actually. 16 different HA and 9 different N have
been identified. In the past . human influen za virus A infection has been caused by 3 different HA
·Corresponding Author: Epifanio Fichera -Etna Biotech Sr.l., Piazz a Stes icoro 59, 9513 1
Catania, Italy. Email: efichera @etnabiotech.it
Pharmaceutical Biotechnology. edited by Carlos A. Guzman and Giora Z. Feuerstein.
(H I, H2 and H3) and 2 different N (N 1 and N2) . However,new avianrestricted strains (HSN 1,
H7N2, H7N3, H7N7, H9N2 and HI ON7)have recentlybeen reported to infect humans. v'
Although associatedwith a significantmorbidity and mortality on groups at risk, such as the
elderly, small children and patients sufferingof chronic diseases (e.g... chronic heart, lung and
kidney diseases, diabetes or immunosuppressive conditions), annual epidemicsusuallyresulring
from infections with circulating influenza A strains represent reduced threats, as compared to a
pandemic situation. Indeed, pandemic avian/mammalian influenza viruseswhich were able to
cross a speciesbarrier and infect humans as a result ofgenetic re-assortmentsare associatedwith
severecomplications and high mortality rates,"
Human influenza epidemics are caused by circulating viru ses and usually begin in
November-Decemberin the northern hemisphereand in May-June in the southern hemisphere, fa-
voredbylowertemperatureandlowerrelative humidity? in spiteof theavailabilityofprophylacticvac-
cineswith provenepidemiological and clinicalbenefits. One reasonfor this isthe naturalhighgenetic
instabilityofinfluenzaA viruses. Repeatedlyoccurringpoint mutations in antigenically important
regionsofthe genesencodingboth the viralHA and N without affectingreplicationcapacity,leads
to significantalterationsin the immunogenicityofthese surface glycoproteins (so-called antigenic
drift). The new so originatingvirusstrain is able to escapepre-existing host immunity allowingthe
new strain to spread.6 The frequencyofantigenicdrifts represents one of the most crucialhurdlesin
the manufacturingofeffective inrerpandemicvaccines. To overcome this obstacle, accordingto the
current WHO recommendations, all manufacturers worldwide produce yearly trivalent vaccines
containingantigensfrom two differentsubtypesofinfluenzaA strains(currentlyH IN1and H3N2)
and one strain ofinfluenzaType B. The specific strain selectionis basedon surveillance data from
the worldwidenetwork of nationalinfluenzacentersand WHO collaboratingcenters.Because vac-
cineshaveto be manufacturedbefore the actualepidemicstrainsare known, a failure to anticipate
emergenceof a strain relative to the vaccine will result in a substantialreduction or abrogation of
vaccine-mediated protection
Human pandemic influenza, againstwhich various vaccines are stillunder development, results
ofthe combinationofseveral factorsin a stepwise manner;"Historicalinfluenzapandemicsemerged
throughan adaptationprocess ofavian or mammalian influenza viruses to thehumanhost.Thecrucial
stepsin such adapting processare the developmentofhybrid virus. termed reassortants, harboring
geneticinformationfrom both human and animalviruses. Indeed.due to the factthat the segmented
genome of influenza viruses consistsof eight individualRNA segments, human-avian virus RNA
reassortants can be produced by cdIs co-infected with both viruses.t Thereby completely novel
subtypesoflnfluenza A viruscan emergein a processcalledantigenicshift,which ismechanistically
different from the much more frequent genetic drifi:s resultingofpoint mutations.Thus, genome
segmentationis a characteristic featureof influenzaA viruses enablinga rapid and drasticevolution
ofthe virusresultingin the escapeof immunosurveillance in newlyinfectedimmunologically naive
humans.Currently,the establishment of efficient vaccines to preventpandemicinfluenzarepresents
one ofthe most crucialglobalproblemsto be solvedby scientists and the vaccine industry.
Manufacturing ofInfluenza Vaccines

The influenza virus was for the first time isolated in 1933 by Smith. Andrewes and Laidlaw,"
In the 1940s the virus could be propagated in embryonated hen'seggs.The first vaccinesagainst
influenza were formaldehydeinactivated whole virus vaccines. They havebeen developed by the
US armed services and made commercially available in 1945 in USA. However,these pioneering
vaccineswere highly pyrogenic due to the suboptimal virus purification process and exhibited
low efficacy. An improvement in the purification processwasachievedmer introduction ofzonal
ultracentrifugation in the 1960s.9 Nowadays the purification ofvirus from infected chicken em-
bryos usingzonal ultracentrifugation is still the manufacturing processofchoice for all marketed
influenza. Currently, severalmanufacturersprovide worldwideinterpandemic influenzavaccines
mainly in the form ofinactivated split or surfaceantigen (subunit) vaccines (seeTable 1).10 Split
vaccinesare prepared from sucrosedensity gradient purified whole virusesby fragmenting viral
New Strategies to Overcome theDrawbacks o/CurrentlyAvailableFlu Vaccines 245
particlesinto smallerpiecesusingdetergents. Subunit vaccines contain further purifiedantigenic

proteins.Theconventionalproductscompriseboth majorsurface glycoproteins HA and N and are
generally appliedintramuscularly."FluMist,a coldadaptedinfluenzavirusvaccineisadministered
intranasally; it is commercially available in the USAsince2003 for activeimmunizationof people
between2 and 49 years of age.11 Thisvaccine is a liveattenuated wholevirusvaccine characterized
by a satisfying protective efficacy in adult. However the vaccine faces some issues related to the
biosafetyprofileof liveattenuated influenzaviruses.
The manufacturing of current seasonal influenzavaccines is associated with several hurdles
that maylimit vaccine production capacity. Natural isolates of new epidemicinfluenzastrains to
be included in the recommendedvaccine composition haveto be genetically modified to create
high-yielding strainssuitablefor antigen production. This is achievedusingclassical methods of
virusreassortment usingembryonatedchickeneggs asa growsubstrate.Reassortants aregenerated
by co-infection of eggs with the current epidemicstrain and a high yielddonor strain. Next, the
selectedstrain is used as a seedvirus stock to grow and manufacturethe desiredvirus type. Seed
virus strain bearingthe recommendedHA and N are provided to vaccine manufacturers by one
of the Control Agencies, such as the Center for Biologics Evaluation and Research (CBER) in
the US, or the National Institute for Biological Standards and Control (NIBSC) in the UK. In
order to maximize virusyieldfromeggs, reassortants of influenza A strainsareoftenutilized.These
possess the surfaceproteins from the desiredwild-typeHINI and H3N2 strains, whilethe other
viral proteins are from a high-yielding strain, such as A/Puerto Rico/8/34. For logistic reasons
new virusproduction substratesthat would avoidthe dependenceon henns eggswould be a key
improvementof the manufacturingprocessfor influenzavaccines," Indeed,the limited availability
of eggsas a growingsubstratemaysometimes be critical, especially in an avianflu pandemicsitu-
ation. Further disadvantages linked to eggproduction concern the riskof the presence of residual
Table 1. Currently marketedinterpandemic influenza vaccines
Subunit Vaccines Split Vaccines Whole Virus Virosomes

Aggripal Afluria FluMist (live attenu- Inflex al V
Chiron/Novartis, I CSL Limited,AU ated, intranasal) Berna Biotech/CruceJl, CH
Medimmune, USA
Fluad (MF-59 adju- Begrivac Invivac
vanted) Chiron/Novertis.D Solvay, NL
Chiron/Novsrtis, I
Fluvir in Fluarix
Evans/Ch iron/ GSK, UK
No vsrtis, UK
Influvac FluLaval
Solvay, NL 10 Biomedicals, CA
Fluvax
CSL, Au
Fluviral S/F
JD Biomedicals, CA
Fluzone
Sanofi Pasteur, F
TIV
Medimmune, USA
Vaxigrip
Sanofi Pasteur, F
allergenicegg's proteins in the finalproduct: the presenceof microbialcontaminants that might

interrupt the antigen supplyconsideringthe removal ofthimerosalfrom the production process
and finally the eggwastedisposal Currently, 90%of the worldwide influenza vaccine production
capacityis concentratedin the USAand Europe,which in turn represents only 10%ofthe world's
population.Thecurrentglobalmanufacturingcapacity(-300 milliondoses peryearof the seasonal
trivalent vaccine, containing 15 ug/dose of each antigen) would be inadequate to meet global
needs during a pandemic, especially as it is expected that higher antigen dosageand a two dose
regimewould be required for protection against a pandemic strain.' Novel approaches, includ-
ing application routes other than intramuscular, the useof noveladjuvants, alternativecell-based
substrates for antigen production, new vaccine formulation (naked DNA immunization, viral
vector basedvaccines) could pavethe wayto greatervaccineaccessibility with associated public
health benefits.
Strongeffortsto replace embryonatedhen'seggsasthe growingsubstratein the manufacturing
processhave been exploredafter the introduction of reverse geneticsinto the influenzavaccine
field.. The reverse geneticstechnologyisan egg independentmethod allowingto generateinfluenza
vaccinestrainswith selectedgenecombinations, while avoidingthe time-consuming selectionof
appropriate reassortants," Several manufacturers areactively workingtowardsthe establishment of
influenzaantigen production in mammaliancellculturesor evenin plants. Firstexperiences with
the cellculture system werepromising,howeverthe firstcellline (293T) used to express antigens
could not be licenseddue to regulatoryrestrictions.Theimprovementwasachievedbythe imple-
mentation ofMDCK,12Vero13or PER.C6·14 cells into manufacturingprocess. Accordingto public
available information,Vero (Chiron, D ; BaxterAu), MDCK (Solvay, NL) and PER.C6· (Sanofi
Pasteur)cellculturebasedinfluenzavaccines arecurrently underclinical evaluation"(Sanofi Pasteur
pressreleases 26 Sept2006). In 2007 EMEAhasissuedthe marketingauthorizationvalidthrought
the European Union for the OptaAu (NovartisVaccine and Diagnostics GmbH & Co. KG), an
influenzavaccinebasedon purified Ha and N, derivedfrom viruses propagatedin MDCK cells.
Moreover, the need for faster and more flexible production systems leadsto the tentativedesign
ofnovelrecombinantvaccines. NakedplasmidDNA or immunizationwith recombinantvectors
encoding HA wereprovento be efficient in animalmodels," but wereless successful in humans,'?
PurifiedHA and N vaccine antigensproduced with the baculovirus expression system havebeen
shown to be effective in a mouse model," Recent studies haveshown that adenovirus-vectored
influenzavaccines areableto elicitrobusthumanimmuneresponses ifdelivered inrranasally,'? Using
an adenoviralvector system, a new HA strain can be constructedwithin one month. A featurein
the development ofvectoredvaccines seems to be the establishment of molecular strategies enabling
forviraltropismmodulation. However, due to selection pressure, suchkindof modifications might
leadto expandedtropismor abolishmentof the adenoviral nativetropism,which can in turn lead
to unintended spreadto new and undesiredcelltypes,aswellas to horizontal transmission of the
vector. However, all these approaches are still far from commercialization.
Strategies to Improve the Immunogenicity and Efficacy ofCurrent

Influenza Vaccines
The immunogenicityof influenzavaccines is currentlymeasuredby their capacityto induce
functional neutralizing HA-specific antibodies in serum," which have been proved to provide
acceptable protection against disease. However, the levelof vaccine's protective capacitydiffers
depending on age and health status of population groups. In case of a good antigenic match
between vaccineand circulatinginfluenzastrains healthyadults, who usually undergo only mild
symptoms of disease and recoverwithin time period up to two weeks, show70-90%protection
againstproven influenzaillness upon conventional immunization. As stressed above, individuals
at the highest risksof severe seasonal influenzainfectionsare elderly, aswellas adults or children
sufferingof chronichealth conditions,suchascancer, immunosuppression or immunodeficiency,
cardiac and pulmonary disorders,diabetes and other metabolic diseases, or renal disease, who
require regular medical follow-up or hospital care. Only 50-70%individuals belongingto these
New Strategies to Overcome theDrawbacks ofCurrent/yAvailableFlu Vaccines 247
populationgroupsareprotectedbyconventionalvaccines.21 Lowerefficacy ofvaccination in elderly

might be relatedto decreased function of cellularcomponentsofimmune system." Also, the im-
mune system of smallchildren differs from the one of adults who haveusually been facinga long
history of contactswith flu viruses. Therefore, current influenzavaccines generate more efficient
immune responses in healthyadults than in infants. Moreover, due to the fact that vaccination is
recommendedin children,elderlyand immunocompromisedindividuals, the safetyprofileof the
vaccine should be very carefully considered. Liveattenuated influenzavaccine FluMist exhibits
high efficacy in protection againstinfluenzain healthyadults. However, due to the riskof residual
replicationofvirusin the respiratorytract, it is not recommendedto be usedin high riskpopula-
tion groups." Takenall these aspectstogether,the challenges for future interpandemicinfluenza
vaccine manufacturersis clearly concernedwith the improvementof immunizationstrategies for
individuals belongingto high riskpopulation groups.
The most evident immunogenic effect of conventional influenza vaccines is the generation
of systemic antibodies specific to the surfaceglycoproteins of influenzavirus. New vaccination
strategies are focusedon the induction of more balancedand broadened immune responses. The
improvementof the immunogenicityof vaccines can be achievedby: (i) immunopotentiatingthe
immune responses via the addition of an adjuvant; (ii) the induction of a mucosalimmunity in
addition to the systemic immunity; and (iii)broadeningthe immuneresponses byincludingmore
conservedantigenicepitopes, mainlyderivedfrom internal proteins ofinfluenza viruses."
Immunopotentiating ofImmune Responses byAdjuvants

Currently marketed inactivated split- or subunit-vaccines provide superior safety over tra-
ditional whole-virus vaccines. However, the immunogenicity of these vaccines leaves space for
improvement. Immunogenic properties of subunit vaccines can be improvedby the addition of
immunostimulating molecules that targetsignalingpathways of defense immunity. An appropriate
antigen-adjuvant combination could overcome this obstacle.
In caseof a pandemic flu vaccine, the requirement for a higher antigen dosageseems to be an
important issue. It seems that a conventionalvaccineformulated with an adjuvantcan allowfor
antigensparing. Currently,threeadjuvants areapprovedfor humanuse:Aluminiumprecipitate,25.26
liposome (virosome}" and MFS9 water/oil suspension.P'" Virosomalformulationsof influenza
surfaceantigens have been shown to possess significant adjuvanteffectdue to the repetitivear-
rangement of presented surface antigensas wellas the maintenanceof viral HA in its nativeand
biologically active conformation." In search for optimal adjuvant candidates highly attractive
targetsbecomeTLR-ligands that activate appropriateSignaling pathways in a highly natural and
specific way,31'33Some adjuvant candidatescurrently tested to improveefficacy of influenza vac-
cinesare listed in Table 2.
The general safety requirements for any vaccine are extremely high, driven by the fact that
vaccines are generally administrated to healthyindividuals. Particularrisk/benefit ratio consider-
Table 2. Adjuvant vaccine candidates under evaluation for influenza vaccines
Proteosornes" OMP of Neisseria meningitis Human

(TLR-2/1 ligand)
LTK63/Biovector" Escherichia coli LT mutants Human
CTA-l D[)36,37 CT derivative (Al subunit of Mouse model
CT + Ig-binding element of
Staphylococus aureus
ISCOMATRIX38•40 Quil-A-based In vitro human mode l mouse
model
TLR "2/6 agonist Mouse model
248 PharmaceuticalBi()Uchn()[()gy
ationsare required ifimmunocompromisedindividuals areconcerned.Regardingthe selectionof

adjuvantmolecules the following characteristics shouldbe considered: (i) preferentially, adjuvant
molecules should be produced synthetically rather then biologically, (ii) the sizeand structure of
moleculesshould be precisely defined, (iii) adjuvantsignalingand functional pathways must be
deciphered; and (iv) a specific wayofaction will be preferred.
Improvement ofImmune Responses Using Alternative Application Routes

Theintramuscularapplication route isroutinelyusedforvaccines administration. However, the
implementation ofalternativeroutes, e.g., intranasally, could resultin the triggeringofadditional
immune pathways to those activatedby intramuscularimmunization.
Thepathogenscausingrespiratorydiseases invadetheir host viathe nasal/trachealmucosa. On
the other hand, parenteralimmunizationisnot appropriateto inducea localimmuneresponse in
the respiratorytract.42 Therefore, the earlyviralcolonizationof upper respiratory areaduringinfec-
tion and the postinfectionprocess ofvirussheddingcannot bepreventedbyparenteralvaccines.7.43
Conversely,pre-existingmucosalimmunityisableto reduceor preventboth colonization andvirus
shedding.Thus, the intranasalroute appearsmost attractiveto prevent influenzainfection.
For several years, live-attenuatedinfluenzavaccines for nasalapplicationhavebeen used suc-
cessfully in the Russian Federation.ThecurrentliveRussian vaccine isbasedcold-adaptedvariants
of an H2N2 strain which is reassortedwith epidemic HINI and H3N2 strains and combined
with a cold-adaptedreassortantofinfluenza B virus,"
FluMist,is a live-attenuatedintranasalinfluenzavaccine, has been shown to be safeand effec-
tive in healthy adults, but its role in the generalprevention of influenza is yet to be defined.The
vaccineinduces IgA antibodies in mucosalsecretions, IgG antibodies in serum, as well as CTL
cellularresponses, leadingto an increased efficacy and crossprotective potential between several
subtypesofviruses.
As stressedabove, the livevirusvaccine approach ensuresan acceptable immunogenicitybut
includesinherent safetyconcerns(geneticstabilityofthe vaccine strains, potential reassortments
with circulatingviralstrains,sheddingin immunocompromisedvaccinees).ln addition, the intra-
nasal applicationof vaccine is otten associated with mucosaldiscomfort,inflammatoryreactions
causingsore throat, aswellas febrile reactions in vaccines.t'
The efficacy of a proteosomal influenzavaccine containing outer membrane proteins from
Neisseria meningitidis (Flulnsure, GSK) and a LTK63IBiovector adjuvanted influenza vaccine are
currentlytested in clinical srudies.!
Inclusion ofConserved Epitopes Derivedfrom Influenza Proteins

in VaccineFormulations
The inclusion of new conserved immunoepitopes is expectedto strengthencellular responses
againstinfluenza, facilitating the directelimination of infectedcells from organism. Moreover, due
to the conserved character ofepitopes, thegenerated immunocompetent cells mightbecross-reactive
and mayhavea significant impacton cross-protection betweendifferentvirussubtypes. Thiscould
be of help in the control of pandemic influenza outbreaks." Someinternal componentsof influ-
enzavirusmight haveimpacton the generation of immuneresponses againstinfluenza. The outer
envelope of the virusisbuilt up bya phospholipidic membranewith intercalated, outsideprojected
influenza surface proteinsHA and N. Theinnersideof the membraneislinedbythe matrixprotein
(MI).The integralprotein (M2)isformingion channels within the membrane.Theinternalprotein
nucleocapsid is surroundedby the viralenvelope. The nucleocapsid oflnfluenzaA virusconsistsof
8 genomesegments, packaged into the core. Eachsegmentisformedbyhelically organizednucleo-
protein (NP) bindinga negative-strandssRNA molecule. Threepolymerase polypeptidic subunits
(PA,PBI, PB2)areassociated with the NP-RNA structure.NonstructuralproteinsNS-I and NS-2
are located at the inner site of nucleocapsid I. The functionalcharacteristics of individual proteins
aresummarized in Table3.
Currentlyavailable influenzavaccines whichareparenterally administered areknownto stimulate
effectively MHCl! restrictedimmune responses involving activation ofB-cells and CD4 T-cells
New Strategies to OvercometheDrawbacksofCurrently AvailableFlu Vaccines 249
specific for influenza surface glycoproteins, mainlythe genetically highlyvariable HA.Thisprocess

seemsto be sufficient to achieve good levels ofprotection against the homologoussubtypeof in-
fluenzavirusin healthy adults, however, it issuboptimal in high riskpopulations, e.g.; the elderly.
Sincethe surface glycoprotein NA wasshown to undergo genetic mutations to a lesser extent as
comparedto HA,the molecule haveattractedattention and studies performedsofar have confirmed
the potential ofN-based vaccines to induce efficient immune responses and protection against
Table 3. The protein-components of influenza virus and theirproperties
Protein Segment Biological Anti-Viral or

Properties Immunological
Properties
HA 4 - Binding to sialic Induction of B-cell
acid receptors on responses, C04
cell surface T-cell s responses,
antibody production
- Fusion of viru s
w ith endosomal
membrane inside of
endo somes
NA 6 Release of new ly Induction of B-cell
synthetized viruses responses, antibody
from infected cells produ ction?
NP 5 RNA binding, Induction of COB
nuclear/c ytop lasmic T-cell responses
transport of vi ral
RNA
Ml 7 M atrix protein form- Induction of COB
ing capsid T-cell responses
M2 7 Ion channel through Induction of B-cell
memb rane, impor- responses, antibody
tant in the uncoat- product ion
ing of viruses in
endosomes
Target molecule of
anti-vi rals amanta-
dine and rimanta-
dine
PA 3 Transcript ase Unkn own
PBl 2 Transcriptase Induction of COB
T-cell responses
PB2 Transcriptase Induct ion of COB
T-cell responses
NSl B Effect s on cellular Induction of COB
RNA transport, T-cell responses
splicing , translation,
anti- IFN protein
NS2 B Unknow n Unknow n
drifted viruseS.46.47 Enhancingthe NA in HA basedvaccines could be of help to control influenza

via the induction of more balancedand broadened influenzaspecific humoral responses
The stimulation of cytotoxic CDS- T-cells responses specifically targeted to the conserved
antigenicdeterminantsderivedmostlyfrominternalproteinsseems to be apromisingapproachto
improveefficacy of interpandemicvaccines, aswellasto developefficient pandemicinfluenzavac-
cines. Following MH Cl-reserictedepitopepresentation,cytotoxiclymphocytes induceapoptosis
ofinfectedcells.An approachincludingconserved internalCTL epitopeswouldhavean advantage
over conventional HA-based strategies by potentially conferringan heterologous cross-reactive
immunity betweendifferentsubtypes.48.49 Although not sterilizing, cellularimmunitywasshown
to prevent illnessand deaths in animal models.
Due to HA antigenic drifi:, HA-basedstockpiledvaccines mightonlyprovidelimitedprotection
againstan emergingpandemicinfluenza strain.Themonitoringofpandemicstrainsderived fromthe
sameviralsubtypeallows to identifythe mostconserved humanepitopeswithinthe internalproteins
ofinfluenza virus.50Newstrategies aremostlyfocused on the inclusion ofnucleoprotein (NP),or M1,
M2 proteins into vaccines. The efficient induction of heterologous crossprotective responses after
immunization withimmunodominantNP epitopeshavebeendemonstratedin mousemodels.51.52 A
conjugatedM2 peptidebasedvaccines (M2 coupledto KLH or OMP from Neisseria meningitidis)
weretestedin mouse, ferretand rhesus monkeys. Thevaccines havebeenshownto be highlyimmu-
nogenicand to conferprotectionagainstlethalchallengewith H INI andH3N2 strainsinallspecies.
Monkeyantiseratestedfor reactivity with different strainsof humaninfluenza A werecrossreactive,
however, they failedto reactwith M2 peptidesderived from highlypathogenic H5Nl strain." In
contrast, if the M2 peptidewascoupledto a hepatitisB coreparticlecarrier, the conjugate failedto
generate protective responses evenagainstinterpandemic strains.54 Anotherapproachmadeuseofan
adjuvanted plasmidvaccine encodingthe M proteinencodinggeneapplied topicallyon the skin.The
vaccine inducedcytotoxicand humoralresponses and providedcross-reactive protection in mice,"
A new generationofliveattenuated vaccines against influenza is basedon NSI protein mutants.
The nonstructuralprotein 1 has beenshownto inhibit TypeI interferons mediatedresponses. Such
cytokines havea highimpacton the regulation ofpathogeniceffects inducedbyinfluenza infection.
The attenuation and immunogenicity ofNSI truncated mutants wasconfirmed in vivo in mouse
model, in whichwasableto conferprotection."
Thus, a variety of approaches have been tested in animal, mainly mouse models so far. The
proof-of-concept in humansstill has to be established. The general immunoregularory factors that
need to be considered in the development of vaccines targetingcellular immuneresponses are still
unclear. Up to now we havelearned, mostlyfrom mousestudies, about the immune mechanisms
triggered bysuchvaccines. Antigenavailability, antigenprocessing, epitopestabilityand individual
T-cell repertoires seemto be critical elements to be taken in accountduring the vaccine develop-
ment process. In addition,the formulation ofindividual components needsto be optimizedand an
alternative wayof administration of the vaccine shouldbe evaluated aswell.
Conclusion
Influenza vaccines available todayarein usesinceover50years. During this timetheyunderwent
onlysmallimprovements.Theefficacy ofvaccines isacceptable in healthyadultsbut suboptimalin at
riskpopulation groups, in whichthe infectioncanprogress to verysevere, complicated disease and
evento a fatalend. Furthermore, thereiscurrentlyno efficient tool to efficiently fightthe predicted
next influenza pandemy. The avian H9N2, H7N2 or H5Nl influenza strainshave been already
shownto cause human infections, althoughtheseavain strainsdid not acquire the capability ofhu-
man to human transmission yet.It isessential to improve our understanding of disease mechanisms
to facilitate the development of better measures to control influenza. In order to protect humans
againstpandemicinfluenza threats, aswellas to improve existing interpandemic vaccines for high
risk population groups, new vaccine approaches must urgentlybe identified and developed. That
will imply major scientific and industrial investments in order to convert promising improved
candidatevaccines into marketable products.
New Strategies to Overcome theDrawbacks ofCurrently AvailableFlu Vaccines 251
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CHAPTER 4
The Role ofNanobiotechnology

in Drug Discovery
KewalK.Jain*
Abstract
T
he potential applications ofnanotechnology in life sciences, particularly nanobiotechnol-
ogy, include those for drug discovery. This chapter shows how several of the nanotech-
nologies including nanoparticles and various nanodevices such as nanobiosensors and
nanobiochips are being used to improve drug discovery. Nanoscale assaysusing nanolirer volume s
contribute to cost saving. Some nanosubstances such as fullerenes are drug candidates. There are
some safety concerns about the in vivo use ofnanoparticles that are being investigated. However.
future prospects for applications in healthcare ofdrugs discovered through nanotechnology and
their role in the development of personalized medicine appear to be excellent .
Introduction
Current drug discovery process needs improvement in several areas. Although many target s
are being discovered through genomics and proteornics, the efficiency of screening and valida-
tion processes need to be improved. This chapter will show how nanotechnology will playa role
in improving this process. Nanotechnology is the creation and utilization of materials . devices
and systems through the control ofmatter on the nanometer scale. Given the inherent nanoscale
functional compo nents of living cells. it was inevitable that nanotechnology will be applied in life
sciences giving rise to the term nanobtorechnology,' Technical achievements in nanotechnology
are being applied to improve drug discovery. drug delivery and pharmaceutical manufacturing. A
product incorporating the NanoCrystal° technology ofElan Drug Deliver y Inc (King ofPrussia,
PA). a solid-dose formulation ofthe immunosuppressant sirolimus , was approved by the FDA in
2000. Nanotechnologies are already being used in molecular diagnostics.' Analyses of Signaling
pathways by nanobiotechnology techniques may provide new insight into the understanding of
disease processes, developing more efficient biomarkers and understanding mechanisms ofaction
ofdrugs. This will help in designing new approaches to drug discovery. Various nanotechnologies
used for drug discovery are listed in Table 1 according to various stages ofdrug discovery.3 Selected
technologies are described briefly in the following text .
Role ofNanoparticles in Drug Discovery

Older imaging tools such as fluorescent dyes or polymer sphere s are either too unstable or too
big to effectively perform single-molecule tracking. The role of nanoparticles for drug discovery
has been explored but no one type of nanoparticle is suitable for universal application in drug
discovery.
°Kewal K. Jain-Jain PharmaBiolech, Blaesiring 7, CH-4057 Basel, Switzerland.

Email : jain@pharmabiotech.ch
PharmaceuticalBiotechnology, edited by Carlos A. Guzman and Giora Z. Feuerstein.
Table 1. Application of nanobiotechnology at various stages of drugdiscovery

Target identification and validation
Nanoproteomics
Single wall carbon tube nanosensors
High-field asymmetr ic waveform ion mobility mass spectrometry
Investigatingbiomolecular interactions with atomic force microscopy
Study of molecular interactions using cantilevers
Study of molecular interactions using nanoprobes
Lead identification
Biosensor
Nanowire devices
Assays based on endocytosis at the nanoscale
Surface plasma resonance
Nanofluidics, nanoarrays and nanobioch ips
Nanoflow liquid chromatography
Lead optimizat ion
Nanoparticles
Quantum dots
Gold nanoparticles
Lipoparticles
Use of small molecules attached to nanoparticles
Fluorescence planar wave guide technology
Nanomaterials as drug cand idates
Dendrimers
Fullerenes
Nanobodies
© Jain PharmaBiotech
Quantum DotsfOr DrugDiscovery

The use ofquantwn dots (QDs) for drug discovery has been explored.' Several QDs are com-
mercially available. Qdot" conjugates (Quantum Dot Corporation, Hayward, CA) can produce
photo resolutions up to eight times more detailed than the older imaging tools. The Qdot" con-
jugates are almost "an order ofmagnitude" brighter than fluorescent dyes and can be observed for
as long as 40 minutes compared to about five seconds for the dyes. Length ofobservation time is
critical to studying cellular processes, which change rapidly over a span of several minutes. Since
cellular receptors are critical targets for new drug candidates, a more detailed understanding of
the behavior of these receptors can open up new treatment options. QDs are ideal for targeting
cancer for both diagnosis and therapy. However, some limitations for QD use in drug discovery
studies have yet to be resolved. i.e., toxicity, size variation, agglomeration, potential multiple drug
attachment to a single QD and blinking.
Gold Nanoparticles
Gold nanopartieles are the most commonly used nanomaterial in diagnostics and have many
other uses as well such as a connecting point to build biosensors for detection of disease DNA.
Instead ofa fluorescent molecule, a gold nanoparticle can beattached to the antibody and other mol-
ecules such as DNA, which can beadded to the nanoparticle to produce bar codes. Because many
copies ofthe antibodies and DNA can be attached to a single nanoparticle, this approach is much
The RoleofNanobiotechnology in DrugDiscovery 39
more sensitive and accurate than the fluorescent-molecule tests used currently. Although they can
be used for drug discovery, they need to be combined with another technology for visualization.
Gold nanoparticles have been used to demonstrate multiphoton absorption induced lumines-
cence (MAIL), in which specific tissues or cells are fluorescently-labeled using special stains that
enable them to be stu died. Gold nanoparticles can emit light so intense that it is readily possible to
observe a single nanoparticle at laser intensities lower than those commonly used for MAIL, i.e.,
sub-lOO-fs pulse s of790-nm lighr.' Moreover, gold nanoparticles do not blink or burn out, even
after hours ofobservation suggesting that metal nanoparticles are a viable alternative to fluorophores
or semiconductor nanoparticles for biological labeling and imaging. Other advantages ofthe tech-
nique are that the gold particles can be prepared easily, have very low toxicity and can readily be
attached to mole cules ofbiological interest. In addition, the laser light used to visualize the particles
is a wavelength that causes only minimal damage to most biological tissues. This technology could
enable tracking ofa single molecule ofa drug in a cell or other biological samples.
SmallMolecules Attached to Nanoparticles

Multivalent attachment of small molecules to nanoparticles can be used to increase specific
binding affinity and to reveal new biological properties ofsuch nanomaterials. Gelatin nanoparticles
have been used for the attachment ofbiotinylated anti-CD3 antibodies by avidin-biotin complex
formation to provide a carrier system for specific drug targeting to Tdyrnphocytes," A union be-
tween small molecule chemistry and nanotechnology, has the potential ofdevelopment ofa wide
range ofnanomaterials for biomedical application? One ofthe applications is in target screening in
high throughput drug discover y.This technique can enable small molecule modification to impart
desirable biological functions for in vivo visualization of targets and for delivery of therapeutics
thus enabling combination ofdiagnostics and therapeutics, particularly in case of cancer.
Role ofNanoproteomics in Drug Discovery

Proteomics is playing an important rofe in the target identification and validation phases
of the drug discover y proces s. Application of nanotechnologies in proteomics has been termed
nanoproteomics, which is an extens ion of the scope of proteomics on nanoscale. Most current
protocols including protein purifi cation/display and automated identification scheme s yield unac-
ceptably low recoveries, thus limiting the overall process with respect to sensitivity and speed and
the requirement ofgreater amounts ofstarting material. Low abundant proteins and proteins that
can only be isolated from limited source material can be subjected to nanoscale protein analysis,
nano-capture of specific proteins and complexes and optimization ofall subsequent sample han-
dling step s leading to mass analysis ofpeptide fragments. Some nanotechnologies are refining the
application of proteomics for drug d iscovery and examples are described here briefly.
A technology called magnetism-based interaction capture (MAGIC) identifies molecular
targets on the basis ofinduced movement ofsuperparamagnetic nanoparticles inside living cells.8
These nanoprobes capture the small molecule's labeled target protein and are translocated in a
direction specified by the magnetic field. Use ofMAGIC in genome-wide expression screening can
identify multiple protein targets ofa drug. MAGIC can also be used to monitor signal-dependent
modification and multiple interactions of proteins.
Single-Walled Carbon Nanotubes

Single-walled carbon nanotubes as a platform have been used for investigating surface-protein
and protein-protein binding and developing highly specific electronic biomolecule detectors.
Nonspecific binding on nanotubes, a phenomenon found with a wide range of proteins, is over-
come by immobilization of pol yeth ylene oxide chains. A general approach is then advanced to
enable the selective recognition and binding of target proteins by conjugation of their specific
receptors to pol yethylene oxide-functionallzed nanotubes. These arrays are attractive because
no labeling is required and all aspects of the assay can be carried out in the solution phase. This
scheme, combined with the sensitivity of nanotube electronic devices, enables higWy specific
electronicsensors for detecting clinically important biomolecules such as antibodiesassociated

with human autoimmunediseases.
Nanoflow Liquid Chromatography

The use of liquid chromatography (LC) in analytical chemistryis well established but the
relatively low sensitivity associated with conventional LC makes it unsuitablefor the analysis of
certain biological samples. Furthermore,standard LC flow rates are frequently not compatible
with the use of specific detectors,such as electrospray ionization mass spectrometers. Therefore,
due to the analytical demandsofbiological samples, miniaturizedLC techniquesweredeveloped
to allow for the analysis of samples with greater sensitivity than that affordedby conventional
LC. In nanoflowLC (nanoLC), chromatographic separations are performedusingflowrates in
the rangeoflow nanoliterper minute, which resultin high analytical sensitivity due to the large
concentration efficiency affordedby this type of chromatography. NanoLC, in combination to
tandem mass spectrometry, wasfirst used to analyze peptidesand as an alternative to other mass
spectrometricmethodsto identifygel-separatedproteins.Gel-free analytical approaches basedon
LC and nanoLC separations havebeendeveloped, whichareallowingproteornics to beperformed
in faster and more comprehensive manner than by usingstrategies basedon the classical 2D gel
electrophoresis approaches. Protein identification using nanoflowliquid chromatography-mass
spectrometry(MS)-MS (LC-MS-MS) provides reliable sequencing information forlowfemtomole
levelofprotein digests. However, this task is more challenging for subfemtomole peptide levels.
An ion mobilitytechnology, high-field asymmetric waveform ion mobilitymassspectrometry
(FAIMS), has been introduced as an online ion selection method compatible with electrospray
ionization (ESI).FAIMSuses ion separationto improve the detectionlimitsofpeptide ionswhen
usedin conjunctionwith electrospray and nanoelectrospray MS.Thisfacilitates the identification
of low-abundance peptide ions often present at ppm levels in complex proteolytic digests and
expandsthe sensitivity and selectivity of nanoLC-MSanalyses in globaland targetedproteomics
approaches.Thisfunctionality will probablyplayan importantrolein drug discoveryandbiomarker
programsfor monitoring disease progression and drug efficacy.
Atomic Force Microscopy for Drug Discovery

Atomic force microscopy (AFM) has become a well-established technique for imaging
single biomolecules under physiological conditions. The exceptionally high spatial resolution
and signal-to-noise ratio ofthe AFM enables the substructureofindividualmolecules to be ob-
served. Usedas a sensor, the AFM tip can alsoprobe the charges of biological surfaces immersed
in a buffersolution. So far,such approaches havesuccessfully characterized protein interactions
but in the future they could be appliedto imagingand detectingmultipleparameters on a Single
moleculesimultaneously. If a ligand is attached to the end of an AFM probe, one can simulate
variousphysiological conditions and look at the strength of the interaction between the ligand
and receptor in a widerangeof circumstances. Byfunctionalizing the tip, it can be usedto probe
biological systems and identifyparticular chemical entitieson the surface of a biological sample.
This opens the door to more effective useof AFM in drug discovery.
Role ofNanoscale Biosensors in Drug Discovery

Biosensors arecurrentlyusedin the areas oftargetidentification, validation,assay development,
leadoptimization and ADMET, but arebest suited for applications relatedto solublemolecules.
Biosensors can overcome manyof these limitations of currentlyused cell-based assays. Theyare
particularlyusefulin the study ofreceptors, in that biosensors do not require the removal of the
receptor from the lipid membraneof the cellas might be necessary with other assay methods.A
primary applicationof current biosensor technologies is the optimizationof limited-scope drug
librariesagainstspecific targets.
The RoleofNanobiotechnology in DrugDiscovery 41
OpticalBiosensors
Optical biosensors capable of exploitingsurface plasmon resonance (SPR), waveguides and
resonant mirrorshavebeen usedwidelyoverthe past decadeto analyze biomolecularinteractions.
Thesesensors allow the determination of the affinityand kineticsof a wide varietyof molecular
interactions in real time, without the need for a molecular tag or label. Conventional SPR is
applied in specialized biosensing instruments. These instruments use expensive sensor chips of
limited reuse capacityand require complexchemistry for ligand or protein immobilization. A
sensitive techniqueisbeingdeveloped for opticaldetection ofgoldnanoparticle-labeled molecules
on protein microarray by applyingthe surface plasmon resonance and specific molecularbinding
usingrollingcircleamplification."
Cantilevers
Cantilevers transform a chemicalreaction into a mechanicalmotion on the nanometer scale
and this motion canbe measureddirectlybydeflecting a light beamfrom the cantilever surface.'? A
state-of-the-art position sensitive detector isemployedasdetectiondevice. Thestaticmode isused
to obtain information regardingthe presenceof certain target molecules in the samplesubstance.
The surfacestress causedby the adsorption of these molecules resultsin minute deflections of the
cantilever. This deflectiondirectlycorrelates with the concentration of the target substance. The
dynamicmode allows quantitative analysis of massloadsin the sub-picogram area. As molecules
areadsorbed,minimalshiftsin the resonance frequency of an oscillating cantilever canbe measured
and associated with reference data of the target substance. Both modescan alsobe operatedsimul-
taneously. Thecontrolled depositionof functional layers isthe keyto convertingnanomechanical
cantilevers into chemicalor biochemical sensors. Inkjet printing is a rapid method to coat canti-
leverarrays efficiently with various sensorlayers. Applicationsrelevantto drug discovery include
label-free biochemicalassays and investigation ofb iomolecularinteractionsaswellasmultiplexed
assays. Byattachingspecific antibodiesto cantilevers the simultaneousimagingof target antigens
and identificationof antigen-antibodyinteractionshavebeen demonstrated.
NanoHuidics, Nanoarrays and Nanobiochips

Nanofluidics impliesextremereduction in quantity of fluidanalytein a microchip compared
with standard methods. The use of the word "nano" in nanoliter (nl) is in a different dimension
than in nanoparticle,which is in nanometer (nrn) scale. From one printing run that consumes
< 1 nanomole of each compound, large combinatorial libraries can be subjected to numerous
separation-freehomogeneousassays at volumes that are a smallfraction of those used in current
high-throughput methods.
Nanoarrays are the next stage in the evolution of miniaturization of microarrays. Whereas
microarrays are prepared by robotic spotting or optical lithography, limiting the smallest sizeto
several microns,nanoarrays requirefurther developments in lithographystrategies suchaselectron
beamlithography, dip-pen nanolithography, scanningprobe lithography, finely focusedion beam
lithography and nano-imprintlithography.Nanoarrays canmeasure interactions betweenindividual
molecules down to resolutions of aslittle asone nanometer and can be usedin bioaffinitytestsfor
proteins, nucleicacidsand receptor-ligandpairs.
N anomaterials as Drug Candidates

Dendrimers
Dendrimers are a novelclass of three-dimensional nanoscale, core-shellstructures that can be
precisely synthesized for a wide rangeof applications. Specialized chemistry techniquesallowfor
precise controloverthe physical and chemical propertiesof the dendrimers.Theyaremostusefulin
drugdelivery but can also be usedfor the development of newpharmaceuticals with novelactivities.
Polyvalent dendrirnersinteract simultaneously with multipledrug targets.Theycanbe developed
into noveltargeted cancertherapeutics. Dendrimerscan be conjugatedto differentbiofunctional
42 PharmaceuticalBiottchno/qgy
moieties such as folic acid using complementary DNA oligonucleotides to produce clustered
molecules, which target cancer cellsthat over-express the high affinityfolate receptor.11
Fullerenes
A keyattribute ofthe fullerenemoleculesistheir numerous points of attachment, allowingfur
precise grafting of active chemicalgroups in three-dimensional orientations. This attribute, the
hallmark of rational drug design,allowsfor positional control in matching fullerenecompounds
to biologicaltargets. In concert with other attributes, namely the sizeof the fullerene molecules,
their redoxpotential and its relativeinertnessin biologicalsystems, it ispossibleto tailor requisite
pharmacokinetic characteristics to fullerene-based compounds and optimize their therapeutic
effect [25].
Fullereneantioxidants bind and inactivatemultiple circulatingintracellularfree radicals, giv-
ing them unusualpower to stop free radicalinjury and to halt the progressionofdiseases caused
by excess free radicalproduction . Fullerenes provide effective defenseagainstall of the principal
damaging forms ofreactiveoxygenspecies. C-60 fullerene has thirty conjugated carbon-carbon
double bonds, all of which can react with a radicalspecies. In addition, the capture ofradicalsby
fullerenesis too fast to measureand is referred to as «diffusion controlled", meaningthe fullerene
forms a bond with a radical every time it encounters one. Numerous studies demonstrate that
fullerene antioxidants work significantly better as therapeutic antioxidants than other natural
and synthetic antioxidants, at least for CNS degenerative diseases. In oxidative injury or disease,
Fullerene antioxidants can enter cells and modulate free radical levels, thereby substantiallyre-
ducing or preventing permanent cellinjury and cell death. A tris-rnalonicacid derivative ofthe
fullerene C-sixty molecule (C3) functionally replaces manganesesuperoxidedismuraseand acts
as a biologically effective superoxidedisrnutasemirnecic."
Nanohodies
Nanobodies (Ablynx , Ghent, Belgium) are the smallest available intact antigen-binding
fragments harboring the full antigen-binding capacity of the naturally occurring heavy-chain
antibodies. Nanobodies havethe potential of a new generation of antibody-based therapeuticsas
well as diagnosticsfor diseases such as cancer," They are extremelystableand bind antigen with
nanomolar affinity. They combine the advantages of conventional antibodies with important
features of small molecule drugs and can address therapeutic targets not easily recognized by
conventional antibodies such as activesites of enzymes.
Nanobiotechnology and Drug Discovery for Personalized Medicine

Personalizedmedicine simplymeans the prescription ofspecific treatments and therapeutics
best suited for an individual It is also referred to as individualizedor individual-based therapy.
Personalizedmedicine isbasedon the ideaofusinga patient'sgenotype as a factor in decidingon
treatment options but other factorsare alsotaken into consideration.Moleculardiagnosticsis an
important component of personalizedmedicineand nanobiotechnologiesare alreadybeingused
in moleculardiagnostics.Although current effortsusingpharmacogenomics and pharmacogenet-
icsinclude matching the existingdrugs to the right patients for optimal efficacy and safetyfuture
personalized medicines could be discovered and designed for specific groups of patients using
pharmacoproteomics. Nanobiotechnologyshowspromiseoffacilitatingdiscoveryofpersonalized
medicinesapart from facilitatingintegration of diagnosticsand therapeurics,"
Conclusion
The examples given in this chapter cover a number of different nanotechnologies, Some of
these are alreadyestablishedin researchthrough other well known technologiessuch as biosen-
sors and biochips. Nanoparticlesare still used extensively for developingdiagnosticsand someof
the assays for drug discovery.
With a largenumber of nanotechnologiesand nanomaterials,no generalizations can bemade
about the overall safety and toxicity. In vitro diagnostic use does not carry any risks to people
The RoleofNanobiouchnologyin DrugDiscovery 43
but there is a concern for in vivouseof nanoparticles, particularlythose that areless than SO nrn,
which can enter the cells. Therearestill manyunansweredquestionsabout their fate in the living
body. Because of the hugediversityof materials used and the wide rangein sizeof nanoparticles,
these effects will vary a lot. It is conceivable that particular sizes of some materials may turn out
to have toxic effects. Further investigations will be needed. The FDA approval is essential for
clinicalapplications of nanotechnologyand substantialregulatoryproblemsmaybe encountered
in the approval of nanotechnology-based products. Pharmaceuticals, biologicals and devices are
all regulateddifferently by the FDA and it is not yet clear how emergingnanotherapeutics will
be evaluated.
Future ofNanotechnology-BasedDrug Discovery

An increasing useof nanobiotechnologyby the pharmaceuticaland biotechnologyindustries
isanticipated.Apart from innovationsbasedon nanoparticles, several other nanotechnologies are
in developmentfor applicationin lifesciences. In the near future. it maybepossible to fullymodel
an individualcell's structureand function bycomputersconnected to nanobiotechnologysystems.
Such a detailed virtual representationof how a cell functions might enablescientists to develop
noveldrugs with unprecedented speed and precisionwithout any experiments in livinganimals.
Nanotechnology will be applied at all stages of drug development-from formulations for
optimaldelivery to diagnosticapplications in clinicaltrials. Nanobiotechnologywould fit in with
the conceptsfor integration of diagnostics and therapeuticsfor the developmentof personalized
medicine.
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3. Jain KK. The role of nanobiotechnology in drug discovery. Drug Discov Today 2005; 10:1435-1442.
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INDEX
A Bacterial vaccine 110

Bacteriophage 30,50,100,159
Acetylcholine receptor 8, 44,46 Bioinformatic 4, 83, 96-98
Adaptive design 11 Biologic 28-30,32,49-51, 56, 58-60,72,
Adjuvant 64,68 ,69,71 ,72 ,149,150,159, 106,196,245
163,166,167 ,1 71-175,177-182,200, Biological screening 22
202,2~207 ,209 ,210,219 ,220,222 ,
Biomarker 1-11,37,40
246-248 Bystander effect 130, 131, 133
Affinity 17,30,31-34,39,41,42, 47,51 ,
55·58,90,91,100,101,179,192
Agonist 4,45 -47,64,68,69,72-74,1 75, c
178,179,181 ,182,247 Calciumchannel 45
Allergy 63,175 ,216 Calicheamicin 32
Ampliconvector 120,121, 125, 128-130, Cancer 10,13,28,30,32,33,38 ,39,41 , 42,
134 44-47,49,59 ,63,67,71 -73, 90, 91,101 ,
Antagonist 9,10,21 , 45-47,64,69, 71-74, 103,108 ,109 ,112 ,118 ,120,129-134,
181 146,149 ,168 ,1 78,180,182,219 ,246
Antibiotic 13-16,18,19,69,90,91, 93,103 , Cell wall component 64,180
105,110-112,125,159 ,162,167 Chemicalscreening 22
Antibody 8,28-34,38-42,49-52,54-60,68, Chlamydia pneumoniaes 66, 84
72, 90-103, 105-110, 112, 123, 126, 129, Combinatorial chemistry 13, 17, 18,23, 175
146, 149, 151-154, 163, 164, 174, 176, Comparativegenomeanalysis 84, 85, 88
181,182,191,192,19 4-196,205 ,208 , Complementarity 33,98,100
217,219,223,246-249 Conopeptide 44-47
Antigen 29,30 ,32,33,41 , 42,49-53,55-60, Conotoxin 44-47
67, 72, 73,81,82,85,87,88 ,90-98,100, Conventionalvaccinology 81-83
101,105-110,112,122,125-127,131 , Correlate 2,41,53,82,83 ,166 ,191,
145,146,149,150,152 ,154,160 ,163 , 193-199,201, 221-223
164,166-168,171- 175,1 77-182,193, Cultivation 20-23
195,19 7,199 ,201 , 202,204-212,21 4, Cytokine 32, 49,63 ,65 ,67,69 ,72, 102,
217-219,222 ,223 ,244-247,250 125,126,130-133,172-175,178-182,
Antigenometechnology 97,98 191-194,206,210,211,21 7,250
Asthma 64,68-71, 73, 175 CytotoxicT lymphocyte(CTL ) 126,146,
Atherosclerosis 63,64 ,68,69,74 154,1 72,174,175,178,180,181,
Autoimmunedisease 40,63 ,68,72-74,91 , 191,193 ,194,197.199,201 -203,205 ,
174,1 79,181 207-210,213 ,21 7-222,248,250
B D
Bacillus antbracis 66, 84-86,97 Delivery 33,37,39,41,43,59,85,118,
Bacterial artificial chromosome (BAC) 23, 120-123,125 ,126,128-132,145,146,
121,123,125,128 152,154,159,167.168 ,174,182,
Bacterial ghost 160, 164 2~207,209-211 ,219 ,220
Bacterial infection 63,69 ,71 ,74,90-93,96, Dendrimer 38,41
103,105 , 110, 112
Bacterial toxin 179
Dendritic cell 56.63.64.67.71.72. 121. Humoral 146.149.152-154,166.167.174,

125-127.131.163,171.194.195 177,178,180-182.189,195.199,201.
DISC 126 2~210.217-219,220,222,250
DNA-basedvaccine 218 Hybridoma 29.30.33,34.50.98 .100,103.
Drift 244.250 105
Drug 1-8,10,11.13,17-19.22-24.28-30. Hypermutation 34,51.54-57.100
32-35.37-44.47.49,51,57-59.64.67, Hypervariable 199
92-95.101.105-111.125.129.130.133.
159.160,163.164.167-169.178.219. I
221
Drugdiscovery 1,2.6.11.13.17-19.22-24. ICP34.5 122,123.126,131-133
29.30.32-35,37-43 Immediate earlygene 130
Immunemodulator 175
Immunity 55,63.68,72,74.82.90,91.110,
E
112,120.125,127,130,145,146,159.
Earlygene 120,130,131 160.163,166,171.172,175,181,182.
Env 125,126,189,191,195-199,202-205, 191.193-195.197-199,201.204-211.
210,212-221,223 217-219.221-223,244,247 .248,250
Enzyme reactor 160,168 Immunization 30.34. 50. 55,81, 82. 85, 91,
Epidemic 105.189,191.244.245.248 92,100.103,110.125,126,149.150.
152.154,163.166,177,195,198,199,
F 201,202,205.206.208-211.217.219,
222,223.245 -248,250
Fab 30.32.34.101,102.108 Immunogen 34.90.126,146.152.178,196.
Flu 243. 245. 247 198.206.207.216
Fullerene 37,38.42 Immunogenicity 30.33.50.57,59.60,83.
Fusionprotein 28-30,32.58.66,69,101. 97,98.126,145.150.152.159.167,
108,126,129,130.131,164.176,203 . 171.172.1 74,177.180,197.199.203,
204 204.206.208-211,217-222.243 .244.
246-248,250
G Immunoglobulin 28.34.51,52,54.55,63.
72.95,98,100.101,181.192,202
Gancyclovir 131-133 Immunology 212
Genetherapy 118,121.123.125.127-130. Inactivated vaccine 146, 159.163
134 Infectedcellprotein (ICP) 122
Gene transfer 24. 120.123. 128.129, 133. Infectious disease 13.17,19,63.64,67.68,
159.162.166.167 69.71,72,81.90-92,99.102,103,105.
110,118,120.133,145,171,172.178,
H 210
Influenza 66,72,82.107.108.146.147.
Hemagglutinin 66.174,180.243 152,153,174,176.180,243-250
Herpes simplex virus 118.119,206 Innate immunity 55.68.74,193,194
High performance liquid chromatography Intranasal 178,180,205,245.248
(HPLC) 22
High-throughputscreening (HTS) 13, 17.
21.30 .41, 82, 83 L
Hit 17,29 Lategene 120
HIV/AlDS 189,191.200,201.206,216. Latency 120. 126,127,129. 133
221.223 Lead 3.4.13.17-19.23.29.30.38.40.44.
Horizontal genetransfer 24. 159. 162. 167 45,49 ,51.57-59.74.83,85.86.103,
Index 255
110,123,126,129,130,133,146.153. Neutralizing 8,29,90.102,105 ,151.152.

160,169,172-175.181.244,246 192-199,217.223,246
Library 17,18,21-24.31,32,34,40,41,52, Nicotinic 44-46
57.96-98,100,105,194,199 NMDA receptor 44.47
Lipopeptide 14-16,66.173,176,177,217 Norepinephrinetransporter (NET) 44- 47
Long-termnonprogressors (LTNPs) 191, Nosocomialpathogen 105. 111
193,194,198.222
Lymphocyte 29,39.52,54.55,146.191, o
195,202.203.205,250
Oncolyticvector 132
Optimization 21,30 ,31.38-40,174.222
M
Macrophage-activating lipopeptideof2 kD p
(MALP-2) 173.176 .177
Metagenomes 23 Pain 44-47,129.172,173,178
MF59 171,174,212.247 Painpathway 44-46
Microbialdiversity 19,20.23 Pandemic 189,191,243-248.250
Microbialextract 21 Pan-genome 85,87.88
Microorganism 13,19-21.23.24,64.69, Passive vaccination 90-92
81-88,102,175.210.222 rca 29-32.101,163
Microparticle 161.209,212 ,219.220 Peptide 32.34.39,40.44-47.69,96,98.
Monoclonalantibody 28.29 ,32-34, SO, 59, 101,127 ,164.174.181.194,199.206,
90-92, 95, 98-103. 105, 108-110,112, 207.210,215.219.250
123 Personalized medicine 2,37,42.43
MonophosphorillipidA(MPL) 173.177 Phagedisplay 30,32.34, SO. 57. 59, 100.
Mucosal 69. 126, 150, 152, 160. 163, 166, 101
167.171-173. 177-182. 189,205 -211. Porphyromonasgingivalis 57,70.84
217-223.243.247,248 Prime-boost 126,146,207,210,214,217.
Mucosal administration 167,220 219-221.223
Mucosal immunity 209,247,248 Probiotic 85.159.160,168,169
Multi-component 223 Protection 71.81 -83.85.88,91, 92, 98.
Muramyldipeptide 173,181 103.105.107.112.126,129.145,
146.149.151-153.163.166.180 .181,
N 191.193-198.205-207,210,217-219,
221-223.243.244,246-250
Nanobiosensor 37
Nanobiotechnology 37,38,42,43
R
Nanofluidics 38, 41
Nanomaterials 38,39.41, 42 Receptor 2,4,8. 10. 17.28,29 .32,34,
Nanoparticle 37-39.41-43,206 38-42.44-47,49.51,52,55,59,60,
Nanoproteomics 38, 39 63-67.70,86.91,94.98,101,102, 105,
Nanotechnology 37.39 ,42.43 107.108,127,129-131,163.171.175,
Nativebiologics 28-30,32 176.179-181.192.193.195.196.199,
Natural product 1, 13, 17-21,23, 24 201, 202, 211, 249.
Nervoussystem 6,9 ,46. 118, 120. 123, 127, Recombinant 29,30.33,34,49.51,57,81 ,
129,132 91.92,95-98.101,105.109,118.123,
Neuraminidase 243 124.126-128.130,132,133.148.150 ,
Neurotensin receptor 44,45.47 152.153.166,167,205.207.210,211.
Neurotrophic factor 128. 129 217.219.220,221,246
Replication competent vector 129, 131
256 PharmauuticalBjot~chnology
Replicationdefective vector 126 Targeting 5,24,28,34,38,39,51,57-59,95,

Reversevaccinology 81-85,87,88,96,199 103,108,130,134,149,154,159,168,
Ribonucleotidereductase 122,127,129, 173,174,180,182,193,195,199,201,
131,132 202,206,209,219,220,223,250
Ribosome display 32 Targetvalidation 1,3,4,6-8, 11
Tat 126,189,193,199-204,218,220,221,
s 223
Thymidinekinase 122
Saponin 173,174 Toll-like receptor (TLR) 63, 64, 66, 67, 70,
Secondarymetabolites 13, 14,21,23 163,171,176
Serumtherapy 99,103,104 TLRagonist 68,74,175
Shift 1,24,41,244 Translational medicine 1-3,5,7-9, 11
Signaling 4,5,10,37,63,64,65,67,69-71, Trial 5,33,43,45,47,50,71-73,92,100,
73,74,171,175,179,182,247,248 127,129,131-133,149-152,174,178,
Single-chain 30, 58 189,191,194,198-200,202-207,
Single nucleotidepolymorphism 5, 63, 64 210-219,221-223
Sodium channel 44-46 Tumor 5,33,50,57,59,65,66,101-103,
Source 2,4, 13, 19,21,23,29,30,34,39,44, 105,108,120,122,123,125,126,
52,58,66,67,100,105,149,169,176 129-134,159,174,176,178-182,210
Specificity 8,10,17,29,44,46,50,55,
57-59,67,100-102,159,179,180,191,
192,195,197
v
Splitvaccine 244, 245 Vaccine 64,68,69,71-73, 81-88,90-92,
Stability 23, 29, 30,44, 56, 57, 59, 60, 86, 96-98,110,112,118,123,125-127,130,
122,126,127,167,199,207,209,219, 132-134,145-152,154,159,160,162,
248,250 163,166,167,169,171-175,177-182,
Streptococcus agalactiae 81, 84 189,191-223,243-250
Streptococcus pneumoniae 19,68, 84, 86, 98 Vector 23,101,118,120-123,125-134,150,
Streptococcus pyogene 84, 86, 103 197,198,202-211,217-223,246
Subtractive genomeanalysis 85 Venom 44,91
Subunit 13-16,32,65,81,82,90-92,95,98, Viralvector 122, 125, 128, 197,205,207,
112,122,128,129,131,145,146,150, 211,217,218,220,221,246
152,154,163,167,171,172,179,180, Virion host shut-off 122, 123, 127
182,198,201,205-207,218,244,245, Virosome 174,206,245,247
247,248 Virus-like particle (Vhs) 145-154,220
Suicidegene 129-131
Synthetic 13,17,18,33,42,47,57,66,67, y
72,98,100,164,175-178,181,182,
199,217,222 Yeast display 32,101
T
Target 1-11,13-17,21, 23, 24, 29, 30, 32,
37-42,44,46,47,49-51,55-59,65,68,
74,82,90-103,105-112,128,129,131,
134,147,152,163,164,166-168,171,
174,175,177,179,181,182,193,195,
198,201,202,205-207,217-219,221,
247,249

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Pharmaceutical Biotechnology

ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY

RecentVolumes in this Series

Springer Science+Business Media, LLC

Copyright ©2oo9 Landes Bioscience and Springer Science+Business Media, LLC

Printedin the USA.

SpringerScience+Business Media, LLC,233 Spring Street,NewYork, NewYork 10013, USA

Pleaseaddress all inquiries to the publishers:

Pharmaceutical Biotechnology, editedbyCarlos A.Guzman andGioraZ. Feuerstein. Landes Bioscience I

Whiletheauthors, editors andpublisher believe thatdrugselection anddosage andthespecifications and

Library of Congress Cataloging-in-Publication Data

Pharmaceutical biotechnology I editedby CarlosAlberto Guzman, GioraZeevFeuerstein.

To Michela Morgana and Alessia Federica

Pharmaceutical Biotechnology is a unique compilation of reviews addressing

human health, suchasAIDS, aswellasforthegeneration ofimproved vaccines against

Car/os A. Guzman, MD, PhD

CARLOS A. GUZMAN, MD, PhD is Head of the Department ofVaccinology

GIORA Z. FEUERSTEIN, MD, MSC, FAHA is Assistant Vice President

Shizuo Akira Cevayir Coban

Diana Felnerova Ken llshii

Iole Macchia GabriellaMolinari

RobertoManservigi Danilo GomesMoriel

Robert Mischler J. LynnRutkowski

Maria Scarselli Jean-Francois Viret

1. TRANSLATIONAL MEDICINE-A PARADIGM SHIFT IN MODERN

2. NATURAL PRODUCTS IN DRUG DISCOVERY: PRESENT STATUS

Metagenomics for Drug Discovery ••...••.•..•......•••..••.••••••••••..•.•..•....••••......•••••••••...........•••.• 23

3. PROTEIN PHARMACEUTICALS: DISCOVERY

4. THE ROLE OF NANOBIOTECHNOLOGY

5. CONOTOXIN VENOM PEPTIDE THERAPEUTICS 44

6. SHARK NOVEL ANTIGEN RECEPTORS-THE NEXT

7. IMMUNE INTERVENTIONS OF HUMAN DISEASES

8. GENOME-BASED VACCINE DEVELOPMENT:

9. THE ANTIGENOME: FROM PROTEIN SUBUNIT VACCINES TO

10. HSV AS A VECTOR IN VACCINE DEVELOPMENT

11. VIRUS-LIKE PARTICLES AS A VACCINE DELIVERY SYSTEM:

12. APPLICATIONS OF BACTERIAL GHOSTS IN BIOMEDICINE...... 159

13. IMMUNE MODULATORS WITH DEFINED MOLECULAR

14. INNOVATIVE APPROACHES TO DEVELOP PROPHYLACTIC

15. NEW STRATEGIES TO OVERCOME THE DRAWBACKS

To my daughters Michela Morgana and Alessia Federica Guzman; thanks

To my family who supported me in my career development; my wife

We would also like to express our deep acknowledgement to all

Translational Medicine-A Paradigm

Drug Targets-Historical Perspectives

Translational Medicine: Definition

Biomarkers that validate the importance of the

Biomarkers that define the chemical-physical

Biomarkers that define consequences of

Biomarkers that correlate with disease Init iation.

Biomarkers that define likelihood of pat ients to

Figure 1. Utilitarian classification of biomarkers types 1-5

driven by an under appreciation ofthe disease processes. Translational medicine focuses

therapeuticbenefitswhilesecuringasufficient therapeuticindexofsafetyand tolerability.

Target Validation 2. Moderate -----.

1. Weak No Validated Bton. , lcr

Target/Compound 2. Moderate -----, V,hurN but nol '!,1m) It