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WO1995032000A1 - Immunogenes de polyproteines de vih - Google Patents

Immunogenes de polyproteines de vih Download PDF

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Publication number
WO1995032000A1
WO1995032000A1 PCT/US1995/006346 US9506346W WO9532000A1 WO 1995032000 A1 WO1995032000 A1 WO 1995032000A1 US 9506346 W US9506346 W US 9506346W WO 9532000 A1 WO9532000 A1 WO 9532000A1
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WIPO (PCT)
Prior art keywords
hiv
polyprotein
immunogen
full length
gpl60
Prior art date
Application number
PCT/US1995/006346
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English (en)
Inventor
Gale E. Smith
Craig S. Hackett
John D. Knell
James Debartolomeis
Franklin Volvovitz
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Microgenesys, Inc.
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Application filed by Microgenesys, Inc. filed Critical Microgenesys, Inc.
Priority to AU25178/95A priority Critical patent/AU2517895A/en
Publication of WO1995032000A1 publication Critical patent/WO1995032000A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to HIV polyprotein immunogens that find application as diagnostic reagents in the detection of HIV infection, as prophylactic or interventive vaccines against HIV infection, and as therapeutic agents in the treatment of HIV-I infection.
  • the present invention relates to the HIV polyprotein immunogens, gpl60/p24 and gpl60/p55.
  • the Human Immunodeficiency Virus and particularly Type-1 (HIV-1) , is a retrovirus which causes a systemic infection with a major pathology in the immune system and is the etiological agent responsible for Acquired Immune Deficiency Syndrome (AIDS) (Barre-Sinoussi et al . , 1983, Science, 220.868- 871; Popovic et al . , 1984, Science 224;497-500) .
  • AIDS Acquired Immune Deficiency Syndrome
  • HIV M Lymphadenopathy-Associated Virus
  • HIV has a genome totaling about 9 kilobases (kb) , with a nucleotide sequence containing seven major open reading frames corresponding to the gag, pol, env, vif, tat, rev, and nef genes (U.S. Patent No. 5,130,247).
  • the genes gag, pol, and env code respectively for core subunits, viral enzymes such as reverse transcriptase or protease, and outer surface (envelope) subunits.
  • the gag gene codes for a primary translation product of 55kD which is posttranslationally processed to produce at least three structural proteins: p24, the major internal core protein, and pl5 and pl7, which are produced in lesser amounts (Marcus-Sekura et al . , 1990, Aids Research and Human Retroviruses 6(3. :317-327) . Marcus-Sekura et al . report that early development of p24 antigenemia may be associated with increased risk of developing AIDS.
  • the gag protein is capable of directing the assembly of virion particles independent of other retroviral elements and plays an important role early in the infection of a cell (Luban et al . , 1993, Cell 73:1067-1078..
  • the env protein is initially synthesized as a 160,000 molecular weight glycoprotein (gpl60) that is then cleaved into a 120,000 molecular weight external glycoprotein (gpl20) and a 41,000 molecular weight transmembrane glycoprotein (gp41) .
  • gpl60 160,000 molecular weight glycoprotein
  • gpl20 120,000 molecular weight external glycoprotein
  • gp41 molecular weight transmembrane glycoprotein
  • envelope proteins are the major target antigens for antibodies in AIDS patients (Barin et al., 1985, Science
  • gpl20 A recombinant form of gpl20 and the whole HIV envelope gene product have been expressed in mammalian cells (Lasky et al . , 1986, Science 233:209- 221) and insect systems (Cochran et al . , 1987, Vaccines, pub. Cold Spring Harbor Laboratory Press pg. 384-388) .
  • V3 region is located in HIV gpl20 approximately between amino acids 296 and 331 and has been suggested as the major neutralizing site of HIV.
  • the V3 region has been reported to be strain specific (Broliden et al, 1992, PNAS USA, vol. 89, pg. 461-465 and Robert-Guroff et al., 1994, J. Virology, vol. 68, pg. 3459-3466).
  • T-cell immunity rather than serum immunoglobulin is essential in the recovery from a variety of viral infections.
  • HIV appears to elicit high levels of antibody and only weak T-cell responses, favoring development of the carrier state and ultimately disease.
  • the weak T-cell responses and low frequency of neutralizing antibodies to HIV may have several possible explanations, including "molecular mimicry" i.e., structural relatedness between antigenic determinants of the HIV envelope and those of the class II major histocompatibility complex (MHCII) that normally interact with the CD4 receptor.
  • MHCII major histocompatibility complex
  • the immune system may suppress both T-cell and B-cell responses directed against MHCII determinants, resulting in what appears to be poor T-cell immunogenicity of HIV and gpl60 in humans.
  • T4 lymphocytes T4 lymphocytes
  • CD4 T4 lymphocytes
  • the CD4 molecule is also responsible for cell fusion or syncytia formation mediated by the virus, whereby HIV-infected cells fuse with uninfected cells expressing CD4 (Zhou et al., 1989, Biochemical et Biophysica Acta 989:301-313). HIV infection may be conferred by both free virus and infected cells.
  • An effective AIDS vaccine might be expected to elicit antibodies which would bind to the envelope of HIV and prevent it from infecting T cell or other susceptible cells.
  • An effective AIDS vaccine also may be expected to elicit T-cell immunity against HIV infected cells.
  • Protein immunogens intended for use as vaccines are generally given to healthy individuals before they are exposed to a disease organism with the intention of blocking infection in the event of exposure to the disease organism or lessen the severity of clinical symptoms following infection.
  • an HIV protein immunogen as a post- HIV exposure immunotherapeutic in the treatment of AIDS (Salk, 1987, Nature, 327:473-476. reviewed by Redfield et al . , 1992, AIDS Res, and Hu. Retroviruses 8.:1051-1058) and in interventive therapy, i.e., treatment of HIV-infected pregnant women with the aim of preventing transmission of HIV to the newborn (Schultz et al .
  • an HIV protein immunogen may be administered as a pre- HIV exposure immune prophylactic to block HIV infection
  • an HIV protein immunogen may be administered post-HIV infection to alter the course of clinical disease and/or lessen the severity of clinical symptoms, following infection.
  • An HIV immunogen may also be administered to HIV infected pregnant women to alter their course of-HIV infection in an effort to prevent transmission to the newborn.
  • Efforts to develop an AIDS vaccine have focused on recombinant forms of the HIV env glycoprotein gpl20 and gpl60 (Stanhope et al . , 1993, The Journal of Infectious Disease 168:92-100; Daher et al .
  • Macaques were protected against simian immunodeficiency virus (SIV) infection by vaccination with a combination of recombinant vaccinia virus expressing SIV env followed by boosting with purified SIV envelope gpl60 produced by recombinant baculovirus-infected cell (Hu et al . ,
  • Diagnostic tests developed to detect HIV infection include enzyme-linked immunosorbent assay (ELISA) , immunofluorescent assay (IFA) , radioimmune precipitation (RIP) assay, Western blot, antigen identification, methods that rely on gene probes (Levinson et al., 1988, Crit. Rev. Clin. Lab. Sci. 26(4) :277-302) , skin test that measures immune response (EP 370,573), and methods that use a test strip (EP 288093).
  • ELISA enzyme-linked immunosorbent assay
  • IFA immunofluorescent assay
  • RIP radioimmune precipitation
  • fusion proteins comprised of fragments of the envelope and/or gag HIV proteins have been reported by Nakata et al . (European Patent No. 572737, published 12/08/93), Shafferman (European Patent No. 509006, published 10/21/92), Himmler et al . (European Patent No. 570357, published 11/18/93), Kagaku Oyobi Kessei (Japanese Patent Number 1179687, published 7/17/89), Broker et al . (European Patent No. 305777, published 3/8/89) and Burke et al . (United States Patent No. 5,130,248).
  • the baculovirus system has been demonstrated to be of general utility in producing HIV-1 proteins and other proteins.
  • the baculovirus Autographa California nuclear polyhedrosis virus (AcNPV) has been used as a vector for the expression of the full length gpl60 and various portions of the HIV-1 envelope gene in infected Spodoptera frugiperda (fall armyworm) cells (Sf9 cells) (Hu et al . , 1987, J. Virology 61:3617) .
  • the present invention relates to HIV polyprotein immunogens that find application as diagnostic reagents in the detection of HIV infection, as prophylactic or interventive vaccines against HIV infection, and as therapeutic agents in the treatment of HIV-I infection.
  • An HIV polyprotein immunogen is defined herein as one comprising at least one isolated epitope from HIV gpl20 and at least one isolated epitope from an HIV gag protein. . _ . ..
  • HIV polyprotein immunogens such as gpl60/p24 and gpl60/p55
  • diagnostic reagents in the detection of HIV infection
  • prophylactic or interventive vaccines against HIV and as therapeutic agents in the treatment of HIV-I infection.
  • the present invention is based, in part, upon the unexpected finding that after immunization with a HIV polyprotein immunogen such as, gpl60/p55, the immunogenicity of the gag protein(s) is greatly enhanced as compared to administration of the gag gene product(s) alone or mixed with envelope protein.
  • the present invention is also based upon the unexpected finding that the presence of the gag gene in the polyprotein immunogen does not interfere with immune responses to gpl60.
  • the present invention is also based, in part, upon the unexpected finding that good T-cell responses against both gpl60 and p24 were observed after immunization with the HIV polyprotein immunogens, gpl60-p55 and gpl60-p24, as measured by delayed type hypersensitivity reaction (DTH) .
  • DTH delayed type hypersensitivity reaction
  • HIV infected chimps boosted with the HIV protein gpl60 showed a rise in antibody to the HIV gag protein, p24, as well as to gpl60. This boosting of the anti-p24 response may be due to improved presentation and/or processing of HIV antigens.
  • mice injected with a polyprotein HIV immunogen comprising gpl60/p24 showed increased immunogenicity as measured by ELISA over injection of either HIV protein alone or in combination.
  • the polyprotein immunogens of the present invention may provide the immune system with more HIV epitopes to attack, thereby eliciting an increased production of antibodies and T-cell responses to HIV.
  • a particularly preferred polyprotein immunogen of the present invention is one comprising 5 HIV proteins, gpl20, gp4l, pl7, p2 , and pl5 (herein referred to as "gpl60/p55") , wherein gpl20, pl7, and p24 are isolated full length proteins, and gp41 and pl5 are truncated proteins.
  • gp41 amino acids encoding a region homologous to amino acids encoding human HLA antigens are removed and pl5 is truncated to enhance recombinant expression.
  • Another preferred polyprotein immunogen of the present invention is one comprising 3 HIV proteins, gpl20, gp41, and p24 (herein referred to as
  • HIV polyprotein immunogen of the present invention is one comprising the HIV polyprotein immunogens gpl60/p24 or gpl60/p55, and a full length HIV polymerase gene product.
  • the HIV polyprotein immunogen is further comprised of one or multiple modified variable region(s) within the gpl20 wherein the variable region is replaced by one or more repeats of a neutralizing epitope.
  • an HIV polyprotein immunogen is comprised of a modified variable region 3 (V3 loop) , wherein the amino acids adjacent to the tip of the V3 loop are replaced by one or multiple repeats of the amino acid sequence Glu Leu Asp Lys Trp Ala (herein referred to as "ELDKWA”) , a neutralizing epitope from a conserved region of gp41.
  • V3 loop modified variable region 3
  • ELDKWA amino acid sequence Glu Leu Asp Lys Trp Ala
  • an HIV polyprotein immunogen is further comprised of a second non-HIV protein.
  • the HIV polyprotein immunogen is further comprised of the Hepatitis B surface antigen (HBsAG) .
  • HBsAG Hepatitis B surface antigen
  • the present invention also relates to the use of HIV polyprotein immunogens as diagnostic reagents for the detection of HIV.
  • a diagnostic reagent comprising a polyprotein immunogen of the present invention, comprising both envelope and gag epitopes, would have the advantage of being capable of detecting antibodies to both HIV envelope and gag proteins.
  • An HIV polyprotein immunogen of the present invention may be used as a diagnostic reagent in assays that detect HIV infection such as, for example. Western blots, ELISAs, dot blots or slot blots, skin tests or on test strips, immunofluorescent assays (IFA) , and radioimmune precipitation (RIP) .
  • anti-polyprotein immunogen antibodies may find application as diagnostic reagents for the detection of HIV antigens in infected persons and blood products.
  • the present invention further relates to novel DNA constructs encoding the HIV polyprotein immunogens, host cells expressing the HIV polyprotein immunogens and methods for purifying the HIV polyprotein immunogens.
  • the HIV polyprotein immunogen of the present invention can be economically prepared using genetic engineering techniques.
  • the present invention also encompasses formulations of HIV polyprotein immunogens suitable as prophylactic or interventive vaccines against HIV infection or as therapeutic agents useful in the treatment of HIV infection, whether or not in combination with soluble CD4, other antivirals, immunomodulators, antibiotics, cytokines or other vaccines suitable in the treatment of HIV infection.
  • the present invention also relates to the use of the HIV polyprotein immunogens in novel immunization schedules.
  • both the initial immunization and subsequent boosting immunizations may comprise administration of an HIV polyprotein immunogen of the present invention, or one of the immunizations may comprise administration of nucleic acid encoding a single HIV protein or an HIV polyprotein immunogen through gene therapy techniques, administration of vaccinia virus encoding an HIV polyprotein immunogen, or administration of a single HIV protein.
  • the present invention also relates to pharmaceutical compositions comprising HIV polyprotein immunogens of the present invention, further comprising a pharmaceutically acceptable carrier, which can be used as a prophylactic vaccine or therapeutic agent to induce a cellular or humoral immune response in individuals at risk for or subject to HIV infection.
  • Figures 1A, IB, 1C, ID, and IE illustrate intermediate plasmids and the final plasmid pA8888 in the cloning of the polyprotein immunogen, gpl60/p24. - 11 -
  • Figures 2A, 2B, and 2C describe the cloning strategy for the HIV polyprotein immunogen, gpl60/p55.
  • Figures 3A, 3B, and 3C describe the cloning strategy for the HIV polyprotein immunogen gpl60/p55- protease-reverse transcriptase.
  • Figure 4 describes the cloning strategy for the HIV polyprotein immunogen, gpl60/p55-HBsAg.
  • Figures 5A, 5B, 5C and 5D illustrate Western Blots of HIV antigens.
  • Lanes A, B and C were loaded with 1 microgra of recombinant HIV p24, recombinant HIV MN gpl60 and recombinant HIV gpl60/p55, respectively.
  • the Western blot is incubated with a 1/1000 dilution of rabbit anti-p24.
  • Figure 5B the Western blot is incubated with a 1/1000 dilution of mouse anti-gpl60.
  • the Western blot is incubated with a 1/1000 dilution of rabbit anti-gpl60/p55 and in Figure 5D, the Western blot is incubated with a 1/1000 dilution of pooled human HIV positive sera.
  • Figure 6 is a Coomassie-stained gel of purified gp 160-p55 polyprotein immunogen.
  • Figures 7A, 7B, and 7C illustrate modification of an MN-like gpl60 V3 loop by the insertion of sequences encoding the ELDKWA epitope.
  • Figure 7A illustrates DNA and Protein sequences of the unmodified HIV-I gpl60 V3 loop. The sequences to be replaced are singly underlined and the GPGR sequence at the tip of the loop is doubly underlined.
  • Figure 7B illustrates the V3 loop with a single ELDKWA replacement. The new sequence is underlined with dots and the other features are indicated as in Figure 7A.
  • Figure 7C illustrates the V3 loop with two ELDKWA replacements. Features are as indicated in Figure 7B.
  • Figures 8A-8G are nucleic acid sequence (SEQ ID N0:1) and deduced amino acid sequence (SEQ ID NO:2) of gpl60-NY5 gp55 in MGS12 (plasmid A9760) .
  • Figure 9 illustrates the immunogenicity of recombinant gpl60-p55 in CD1 mice immunized at week 0 and 4 with 5 icrograms rgpl60-p55 + alum.
  • Figure 10 illustrates the levels of IgG anti-p24 in CD1 mice after immunization with recombinant gpl60- p24.
  • Figure 11 illustrates the levels of IgG anti-gp 160 after immunization with recombinant gpl60-p24.
  • the present invention relates to HIV polyprotein immunogens that find application as diagnostic reagents in the detection of HIV infection, prophylactic or interventive vaccines against HIV infection or therapeutic agents useful in the treatment of HIV-I infection.
  • a polyprotein immunogen as used herein is one comprising at least one isolated HIV gpl20 epitope and at least one isolated HIV gag epitope.
  • a preferred polyprotein immunogen is one further comprising at least one isolated epitope from HIV gp41.
  • the amino acid sequence of HIV envelope in virus particles obtained from different patients or even the same patient at different times exhibits a high degree of variability (Stryer's Biochemistry . 1988, 3rd edition Freeman and Co. publishers) .
  • the term HIV includes HIV derived from any HIV viral isolate.
  • HIV retroviral isolates The nucleotide sequence and deduced amino acid sequence of HIV retroviral isolates are disclosed in for example, Wain-Hobson et al . (1985, Cell 40:9- 17; Ratner et al . , 1985, Nature 313:277-284; and Sanchez-Pescador et al . , 1985, Science 22_7:484-492) .
  • an isolated HIV epitope refers to the HIV epitope as being removed from at least one other component with which it is associated in nature.
  • full length envelope protein refers to an isolated envelope protein that has all the amino acids found in the naturally occurring protein, some of which may have homology to human HLA proteins.
  • truncated as used with envelope protein or gag protein refers to an isolated protein that lacks a contiguous group of amino acids, some of which have homology to human HLA proteins.
  • the env gene of the HIV retrovirus, gpl60 encodes the glycoprotein of the viral envelope, which is important in the attachment of the virus to the surface of the host.
  • the sequence of gpl60 predicts 28 potential N-linked glycosylation sites with estimated molecular weight of this gpl60, including sugar residues, being approximately 145,000.
  • the env protein of HIV is initially synthesized as a 160,000 molecular weight glycoprotein (gpl60) that is then cleaved into a 120,000 molecular weight external glycoprotein (gpl20) and a 41,000 molecular weight transmembrane glycoprotein (gp41) which anchors both itself and the gpl20 to the viral particle.
  • gpl60 refers to the HIV envelop protein comprised of gpl20 and gp41.
  • the preferred gpl20 of the present invention has the signal peptide removed.
  • the preferred gp41 of the present invention is a truncated HIV gp4l having amino acids encoding a region homologous to amino acids encoding human HLA antigens removed.
  • the preferred gp41 of the present invention has amino acids 1-722 of the naturally occurring HIV gp41 envelope protein, as is shown in Figures 8A-8G.
  • the gag gene of HIV is made up of pl7, p24, and pl5.
  • the preferred pl5 gag gene of the present invention is truncated and is lacking 63 amino acids (189 nucleic acids) as is shown in Figures 8A-8G.
  • the HIV gag protein p55, containing pl7, p24 and the truncated pl5 is encoded by nucleic acids 1-1308, whereas the naturally occurring full length HIV gag protein, p55 is encoded by nucleic acids, 1-1497.
  • Antibody titers against p2 appear to be a useful indicator regarding the progression to clinical disease in HIV infection. The titer of anti-p24 antibody rises soon after infection and its eventual decline signals the onset of clinical manifestations (ARC then AIDS) .
  • the polymerase gene codes for a precursor of three different enzymes; protease, reverse transcriptase and integrase. The polymerase genes are described in Marcus-Sekura et al . , 1990, (Aids).
  • the polymerase coding region is the only one in which significant homology has been found with other retroviral proteins sequences, i.e., there are conserved regions of homology in the protease and reverse transcriptase sequences among retroviruses (Wain-Hobson et al . , 1985, Cell 40:9-17).
  • V3 loop has been shown to be relatively strain-specific.
  • an HIV polyprotein immunogen contains the V3 loop from one HIV isolate removed and switched with another strain- specific V3 loop allowing targeting of the vaccine to a predominant HIV strain in a given population.
  • a variable region is replaced by one or more repeats of a neutralizing epitope.
  • the neutralizing epitope is the amino acid sequence ELDKWA which is located on the highly conserved ectodo ain of gp41.
  • a V3 loop mutant was constructed having 30 bp upstream of the amino acids GPGR at the tip of the V3 loop (Figure 7A) replaced by the sequence ELDKWA and the two flanking amino acids ( Figure 7B) .
  • a construct was produced having the 30 bp upstream and downstream of the V3 loop replaced by tandem ELDKWA repeats ( Figure 7C) .
  • HIV polyprotein immunogen of the present invention may comprise at least one isolated epitope from HIV gpl20 and at least one isolated epitope from an HIV gag protein.
  • a preferred HIV polyprotein immunogen of the present invention is one comprising at least one isolated epitope from HIV gpl20, at least one isolated epitope from HIV gp41 and at least one isolated epitope from an HIV gag protein.
  • a particularly preferred HIV polyprotein immunogen of the present invention is one comprising an isolated full length HIV gpl20, and an isolated full length HIV gag protein.
  • Another particularly preferred HIV polyprotein immunogen of the present invention is one comprising an isolated full length HIV gpl20, a truncated HIV gp41 and an isolated full length gag protein.
  • the gpl20 comprises a modified variable region wherein the variable region is replaced by one or more repeats of a neutralizing epitope.
  • the variable region is replaced by one repeat of the neutralizing epitope.
  • the variable region is replaced by multiple repeats of the neutralizing epitope.
  • the variable region is the V3 loop and the neutralizing epitope is Glu Leu Asp Lys Trp Ala from gp41.
  • Another preferred HIV polyprotein of the present invention is one comprising an isolated full length HIV gpl20, a truncated HIV gp41, an isolated full length gag protein and an isolated full length polymerase protein product.
  • a particularly preferred HIV polyprotein immunogen of the present invention is one comprising 5 HIV proteins, gpl20, gp41, pl7, p24, and pl5, wherein gpl20, pl7, and p24 are isolated full length proteins and gp41 and pl5 are truncated proteins (herein referred to as "gpl60-p55") .
  • gp41 amino acids encoding a region homologous to amino acids encoding HLA antigens are removed.
  • HIV pl5 is truncated, thereby increasing stability and expression of pl5.
  • Another particularly preferred HIV polyprotein immunogen of the present invention is one comprising 3 HIV proteins, gpl20, gp41, and p24 (herein referred to as "gpl60/p24”) , wherein gpl20 and p24 are isolated full length proteins, and wherein the gp41 is a truncated protein having amino acids encoding regions homologous to amino acids encoding human HLA antigens removed.
  • HIV variants Over 100 HIV variants have been identified through molecular cloning and restriction analysis, including the isolates RF, ARV, LAV, WMJ-1, and MN (U.S. Patent No. 5,142,025).
  • the subject invention is illustrated by the polynucleotide and deduced amino acid sequence of an MN-like isolate as shown in Figures 8A-8G, the appropriate amino acid regions from any HIV isolate can be used in preparation of the polyprotein immunogens of the present invention.
  • Appropriate amino acid regions of individual isolates of HIV can be determined by any alignment algorithm known to those of skill in the art and include, for example, the alignment method of Feng and Doolittle, Meth. Enzvmology ______:375-387, 1990;
  • polyprotein immunogens of the present invention may be produced in expression systems as discussed infra, or the polyprotein immunogens may be expressed individually then micro-encapsulated in bio- resorbable materials in various combinations.
  • the particles may form naturally as virosomes or hetero-multimers containing various combinations of polyprotein immunogens.
  • An expression vector encoding the HIV polyprotein immunogen may contain the nucleic acid encoding the individual HIV proteins of a polyprotein immunogen in tandem and in frame, with no intervening nucleic acid, such that the individual HIV proteins are produced as fusion proteins or alternatively, in tandem with intervening nucleic acid, which intervening nucleic acid may encode other proteins or, for example, enzymatic cleavage sites.
  • the nucleic acid encoding the individual proteins may be in any order, i.e., nucleic acid encoding gpl20 may be 5' or 3' to the nucleic acid encoding the HIV gag protein(s) .
  • transformation encompasses any introduction of an expressed heterologous gene in a cell whether by transfection, infection, electroporation, nuclear injection, or by protoplast fusion.
  • the polynucleotide sequence encoding the HIV proteins or their functional equivalents can be used to generate recombinant molecules which direct their expression.
  • DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence, may be used in the practice of the invention as long as they are capable of eliciting an immunogenic response but not necessarily an identical immunogenic response of its counterpart naturally occurring HIV protein.
  • DNA sequences include those which are capable of hybridizing to the individual HIV protein sequence under stringent conditions.
  • stringent conditions refers to those hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50°C.
  • a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml) , 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC and 0.1% SDS.
  • formamide for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 m
  • Altered DNA sequences which may be used in accordance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product.
  • the gene product itself may contain deletions, additions or substitutions of amino acid residues within the HIV protein gene sequence, which result in a silent change thus producing a functionally equivalent HIV protein.
  • Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipatic nature of the residues involved.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.
  • DNA sequences of the invention may be engineered in order to alter the HIV protein coding sequence for a variety of ends including but not limited to altera ⁇ tions which modify processing and expression of the gene product. For example, mutations may be intro ⁇ quiz using techniques which are well known in the art, e.g. site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, phosphorylation, etc. For example, it may be preferable to alter the HIV coding sequence to eliminate certain regions, for example variable regions.
  • the coding sequence of an HIV protein could be synthesized in whole or in part, using chemical methods well known in the art. See, for example, Caruthers et al., 1980, Nuc. Acids Res. Symp. Ser. '.21S-233 ,' Crea et al . , 1980, Nuc. Acids Res. 9(10) :2331; Matteucci et al . ,
  • the protein itself could be produced using chemical methods to synthesize the HIV amino acid sequence in whole or in part.
  • peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high perform ⁇ ance liquid chromatography. (e.g., see Creighton, 1983, Proteins Structures And Molecular Principles. W.H. Freeman and Co., N.Y. pp. 50-60). The composi ⁇ tion of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g. the Edman degradation procedure; see Creighton, 1983, Proteins. Structures and Molecular Principles, W.H. Freeman and Co., N.Y., pp. 34-49).
  • the present invention provides a method for producing a recombinant HIV polyprotein immunogen comprising: (a) culturing a host cell transformed with the recombinant DNA vector containing a polynucleotide encoding the polyprotein immunogen and which expresses the HIV polyprotein immunogen; and
  • the polyprotein immunogens of the present invention may be produced in a variety of expression systems including bacterial systems, yeast systems, insect systems and mammalian systems, each system showing differences in the extent and nature of glycosylation of the HIV proteins.
  • a preferred expression vector/host cell system of the present invention is the baculovirus Autographa California nuclear polyhedrosis virus in Spodoptera frugiperda (fall armyworm) cells described in European Patent No. 265,785.
  • the insect cells contain the post translational modification enzymes to perform glycosylation, fatty acid acetylation, alpha amidation and phosphorylation that occur in mammalian cells so that eukaryotic proteins produced are similar to the authentic protein in their biological activity, structure and antigenicity.
  • the baculovirus/insect expression system utilizes the strong polyhedrin gene promoter to drive the expression of foreign genes. This system enables the production of high levels of protein due to the strength of the viral promoter.
  • the rod shaped virion and large genome can accommodate large fragments of DNA.
  • Many baculoviruses have genomes in the 140Dbp range and one isolated from the corn earworm (Heliothi ⁇ zea) has a genome of 240 Kbp.
  • the host cells which contain the coding sequence and which express the biologically active gene product may be identified by at least four general approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) the presence or absence of "marker" gene functions; (c) assessing the level of transcription as measured by the expres ⁇ sion of polyprotein immunogen mRNA transcripts in the host cell; and (d) detection of the gene product as measured by immunoassay or by its biological activity.
  • the presence of the polyprotein immunogen coding sequence inserted in the expression vector can be detected by DNA-DNA or DNA- RNA hybridization using probes comprising polynucleotide sequences that are homologous to the polyprotein immunogen coding sequence, respectively, or portions or derivatives thereof.
  • the recombinant expres- sion vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g. thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.).
  • certain "marker" gene functions e.g. thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.
  • certain "marker" gene functions e.g. thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.
  • a marker gene can be placed in tandem with an polyprotein immunogen sequence under the control of the same or different promoter used to control the expression of the polyprotein immunogen coding sequence. Expression of the marker
  • transcriptional activity for an polyprotein immunogen coding region can be assessed by hybridization assays.
  • RNA can be isolated and analyzed by Northern blot using a probe homologous to a polyprotein immunogen coding sequence or particular portions thereof.
  • total nucleic acids of the host cell may be extracted and assayed for hybridization to such probes.
  • the expression of a polyprotein immunogen protein product can be assessed immunologically, for example by Western blots, immunoassays such as radioimmuno-precipitation, enzyme-linked immunoassays and the like.
  • Purification of the polyprotein immunogens can be achieved by employing conventional methods in combination.
  • purification of the immunogens can be accomplished by performing an appropriate combination of the following methods: methods to collect the transformed cells, e.g. _ centrifugation, filtration, precipitation, etc. ; preparation of cell extracts by, e .g. homogenization, freeze-thaw procedures, ultrasonic treatment, and pressure-vacuum treatment, etc.; and purification of the polyprotein immunogens through preciptation by means known in the art, i.e., with organic solvents, ultracentrifugation, column chromatography, electrophoresis, etc.
  • antibodies to epitopes of the recombinantly produced polyprotein immunogens include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by an Fab expression library.
  • Neutralizing antibodies, i.e., those which neutralize the biological activity of HIV are especially preferred for therapeutics.
  • various host animals may be immunized by injection with a polyprotein immunogen including but not limited to rabbits, mice, rats, etc.
  • a polyprotein immunogen including but not limited to rabbits, mice, rats, etc.
  • Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete) , mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, immunoactive peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynehacterium parvum.
  • BCG Bacilli Calmette-Guerin
  • Corynehacterium parvum include BCG (bacilli Calmette-Guerin) and Corynehacterium parvum.
  • Monoclonal antibodies to a polyprotein immunogen may be prepared by using any technique which provides for the production of antibody molecules by continuous - 25 -
  • Antibody fragments which contain specific binding sites of a polyprotein HIV immunogen may be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed (Huse et al . , 1989, Science. 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity the polyprotein HIV immunogen of interest. 5.7. Uses of Polyprotein Immunogens
  • HIV has been established as the primary etiologic agent in the pathogenesis of AIDS and related disorders which may be transmitted by infected individuals or exchange of infected blood products. Infection of humans with HIV leads to production of antibodies directed against HIV proteins. During the course of infection, the immune profile changes, with antibodies to HIV envelope and HIV gag proteins appearing at different times during the course of infection. The detection of those anti-HIV antibodies forms the basis for numerous diagnostic assays known to those of skill in the art including, for example, Western blots, dot blots and slot blots, ELISAS
  • the present invention provides polyprotein immunogens that find application as diagnostic compositions in assays designed to detect antibodies to HIV proteins.
  • Diagnostic compositions comprising HIV polyprotein immunogens of the present invention provide the advantage of enhanced sensitivity of detection due to simultaneous detection of antibodies to HIV envelope and gag proteins.
  • Figures 5A, 5B, 5C and 5D illustrate that an HIV polyprotein immunogen of the present invention, gpl60/p55, is able to detect antibodies to both p24 and gpl60 in human HIV positive sera in a Western blot.
  • HIV polyproteins immunogens of the present invention may be administered to healthy individuals and/or animal species in order to prepare polyclonal antibodies and/or hybridoma cell lines or transgenic animals expressing immunoglobulms which may be used as diagnostic reagents. Accordingly, the present invention provides anti-HIV polyprotein immunogen antibodies that find application as diagnostic compositions in assays designed to detect HIV proteins.
  • the present invention provides HIV polyprotein immunogens that find application in preventing and treating HIV infection as well as in preventing transmission from infected mother to newborn.
  • HIV polyprotein immunogens that find application as prophylactic vaccines which may be administered pre- or post-exposure to HIV infected individuals to prevent or alter the course of infection, or during pregnancy to intervene in the transmission of HIV from infected mother to newborn.
  • immunization with gpl60/p55 or gpl60/p24 was shown to increase immunogenicity to gag proteins as well as induce a good T-cell response, in vivo.
  • HIV polyprotein immunogens that find application as therapeutic agents in the treatment of AIDS which may be administered post-exposure to alter the course of infection, for example to increase cellular and humoral responses, or to lessen the severity of disease.
  • HIV polyprotein immunogens it may be sufficient for the immunogen to match antigenically the infecting type of HIV in the patient to be treated (Salk, 1987, Nature 327:473- 476) .
  • the HIV polyprotein immunogen of the present invention may be used in a treatment regime alone or in combination with soluble CD4 (Lasky et al . , 1987 , Cell 50:975-985 and EP 538371) other anti-virals, immunomodulators, antibiotics, cytokines or other vaccines suitable in the treatment of HIV infection.
  • a polyprotein immunogen of the present invention may find application as the active ingredient in a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier, which can be used as a prophylactic vaccine or therapeutic agent to induce a cellular or humoral immune response in individuals at risk for or subject to HIV infection.
  • Useful animal models for vaccine development include, for example, chimpanzees, which are susceptible to infection with
  • the HIV polyprotein immunogens of the present invention may be administered at times of pre-exposure and/or post exposure to HIV.
  • the HIV polyprotein immunogens may be administered as part of an immunization/boost schedule wherein both the initial immunization and subsequent boosting immunizations may comprise administration of an HIV polyprotein immunogen, or one of the immunizations may comprise administration of nucleic acid encoding a single HIV protein or an HIV polyprotein immunogen through gene therapy techniques, administration of vaccinia virus encoding an HIV polyprotein immunogen, or administration of an individual HIV protein.
  • the HIV polyprotein immunogens of the present invention may be introduced into a host through recombinant live virus vectors, such as for example, the vaccinia virus vector (Hu et al . , 1986, Nature 310:537-540; Zarling et al . , 1986, Nature 323:344-346; and Chakrabarti et al . , 1986, Nature 320:535-537) and recombinant adenovirus vectors (Hung et al . , 1988, UCLA Svmp. Mol. Cell. Biol.. n.s.
  • live virus vectors such as for example, the vaccinia virus vector (Hu et al . , 1986, Nature 310:537-540; Zarling et al . , 1986, Nature 323:344-346; and Chakrabarti et al . , 1986, Nature 320:535-537) and recombinant adenovirus vectors (Hung
  • This invention also encompasses the introduction of nucleic acid into a cell or tissue of a host animal either in vitro , in vivo or ex vivo .
  • Methods for introducing nucleic acid into such cells or tissues include methods for in vitro introduction of nucleic acid such as the insertion of naked RNA or DNA (such as by the injection of the nucleic acid into a tissue) , the introduction into a tissue or a cell modified ex vivo to transcribe and express the HIV polyprotein immunogen, the provision of nucleic acid in liposomes or other carrier, the use of a vector such as a virus, retrovirus, phage, plasmid, etc., or techniques such as electrophoration.
  • Other known methods for the introduction of nucleic acid into cells are suitable for the practice of this invention.
  • the route of administration of host cells or plasmids comprised of nucleic acid encoding HIV polyprotein immunogens of the present invention includes, for example, viral infection with a suitable virus, retrovirus phage, direct injection into a target cell or tissue of the host cell in a suitable carrier as described in Wang et al . , 1987, (PNAS USA 84 . ⁇ 7851-7855; Kaneda et al. (1989, Science 243:375- 378) and Ono et al . (1990, Neurosci. Lett.
  • Formulation and methods and routes of administration of the HIV polyprotein immunogens of the present invention will vary depending on the intended application, i.e., whether used as a prophylactic vaccine or therapeutic agent, and the desired immune response i.e., maximizing the T-cell response or the B-cell response.
  • the polyprotein immunogen is formulated in a manner such that it is small and soluble.
  • the polyprotein immunogen is formulated in a manner such that it is large and insoluble and may include an adjuvant, such as, for example, Freund's adjuvant or alum, and may be administered, for example, intramuscularly.
  • an adjuvant such as, for example, Freund's adjuvant or alum
  • an HIV polyprotein immunogen of the present invention may be administered more frequently than for vaccination purposes.
  • the HIV polyprotein immunogens of the present invention may be administered alone or in co-therapy with other drugs useful in the prevention or treatment of HIV infection.
  • United States Patent No. 5,130,247 describes examples of antiviral, immunomodulators, antibiotics and vaccines that could be used in co- therapy with the polyprotein immunogens of the present invention.
  • Dosage amount of a polyprotein immunogen and the interval between immunizations and boost may be adjusted individually. The dose for adults of one administration of polyprotein immunogen will generally be in the range of 0.001 to 1000 micrograms.
  • Vaccines formulations may contain an additional component, called an adjuvant.
  • Adjuvants are materials which help stimulate the immune response.
  • Adjuvants in common use in human vaccines are gels of aluminum salts (aluminum phosphate or aluminum hydroxide) , and are usually referred to as alum adjuvants (Bomford, 1985, Animal Cell Biotech. 2_:235- 250, Academic Press Inc. London) .
  • a preferred adjuvant of the present invention is alum. 6.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) were propagated in Spodoptera frugiperda cells at 28°C using PNMFH insect medium (GIBCO BRL/Life Technologies, Gaithersburg, MD) supplemented with 5% fetal calf serum (FCS) . Procedures for growing AcNPV derivatives, determining viral titer and analyzing infected cell DNA followed standard methods.
  • a standard plaque assay procedure was used to obtain viral plaques from dilutions of the media harvested from the co-transfection. Individual plaques were examined under a microscope and scored for the presence or absence of polyhedra.
  • DNA from large-scale (500ml) plasmid preparations was purified by CsCl gradient by procedures deemed to be routine to those skilled in the art. Miniprep DNA was prepared by the boiling method.
  • PCR was performed with, per 200 ⁇ l, l ⁇ g of 5' and 3' oligonucleotide primers, 50mM deoxyribonucleotide triphosphate (dNTP) , lOOng template DNA and 1 unit of Taq DNA Polymerase (Perkin- Elmer, Norwalk, CT) in 1 x Taq Polymerase buffer.
  • dNTP deoxyribonucleotide triphosphate
  • lOOng template DNA 1 unit of Taq DNA Polymerase (Perkin- Elmer, Norwalk, CT) in 1 x Taq Polymerase buffer.
  • PCR was performed in a Perkin-Elmer DNA Thermal Cycler; amplification reactions consisted of a 3-minute denaturation at 94 ⁇ C, followed by 30 cycles [94°C, 1 minute; 55°C, 2 minutes; 72°C, 3 minutes], and a final 72°C incubation for 7 minutes. An aliquot of each PCR product was analyzed on a 0.8% agarose gel.
  • the PCR reaction mixture was analyzed on a 0.8% low-melting temperature agarose gel and purified with glass powder (Geneclean Kit: Bio 101, La Jolla, Ca) , using 1.5 ml Nal solution and 10 ⁇ l Glassmilk (BiolOl) binding for 15-20 minutes.
  • the purified PCR fragment was then cloned into the PCR cloning vector Novagen (TA vector Kit, Madison, WI)
  • gpl60/p55 Proteins The cloning strategy for gpl60/p55 involves the steps shown in Figures 2A-2C: a. cloning the gpl60 coding sequence into pUC 19, b. cloning the p55 coding sequence into Novablue TA cloning PCR vector, c. ligating the p55 sequence into the 3' end of the gpl60, and d. cloning the gpl60/p55 fusion protein into the baculovirus expression vector
  • H9 cells (ATCC accession number HTB 176) are infected with an MN-like HIV.
  • the infected H9 cells are then treated with 0.5M EDTA, 100 ⁇ g/ml Protease K, and 0.5% Na Sarcosyl.
  • the cells are collected through centrifugation and phenol extracted.
  • the gpl60 was isolated by performing a polymerase chain reaction (PCR) in combination with two extension primers:
  • the resultant 2.26 Kb PCR product encoding truncated gpl60 was treated with EcoRI and ligated into pUC 18 treated with restriction endonucleases EcoRI and Hindlll.
  • the plasmid pA8268 containing truncated MN- like gpl60 in pUC 18 was digested with restriction endonuclease EcoRI, the EcoRI fragment was isolated and cloned into pUC 19 that was digested with restriction endonuclease EcoRI.
  • the resulting construct pA8714 contains the Mn a -160 gene in the opposite orientation to the /3-galactosidase gene of pUC 19.
  • step b p55 contained in a baculovirus transfer vector, P4087, was isolated by performance of PCR with extension primers:
  • the 5' primer contains a BamHI restriction site and the 3' primer contains a NotI, Kpnl, and BamHI restriction site.
  • This PCR step resulted in a 1.35 Kb PCR product which was ligated into the PCR cloning vector, Novablue TA to form the resultant plasmid A9428.
  • the p55 protein was then isolated from the plasmid A9428 with BamHI restriction endonuclease and ligated into the BamHI site of vector A8714 which had been digested with BamHI and treated with calf- intestinal alkaline phosphatase.
  • step d the fusion protein gpl60-p55 was cloned into a baculovirus expression vector (BEV) MGS12-AIDS, designated A7306.
  • BEV baculovirus expression vector
  • Plasmid A9533 was digested with Kpnl which isolates the gpl60-p55 fusion protein.
  • the BEV, A7306 was digested with Kpnl, treated with calf-alkaline phosphatase and purified on gel electrophoresis.
  • the fusion protein gpl60/p55 containing Kpnl restriction sites was ligated into the BEV plasmid A7306 that was digested with Kpnl.
  • the resultant plasmid was A9760.
  • the cloning strategy for gpl60/p24 fusion proteins involves 4 major steps as shown in Figs. 1A-
  • step a H9 cells (ATCC accession number HTB 176) are infected with an MN-like HIV. The infected H9 cells are then treated with 0.5M EDTA, 100 ⁇ g/ml Protease K, and 0.5% Na Sarcosyl. The cells are collected through centrifugation and phenol extracted.
  • the gpl60 was isolated by performing a polymerase chain reaction (PCR) in combination with two extension primers:
  • the resultant 2.26 Kb PCR product encoding truncated gpl60 was treated with EcoRI and ligated into pUC 18 treated with restriction endonuclease EcoRI.
  • the plasmid pA8268 containing truncated gpl60 in pUC 18 was digested with restriction endonuclease EcoRI, the EcoRI fragment was isolated and cloned into pUC 19 that was digested with restriction endonuclease EcoRI.
  • p24 contained in the baculovirus transfer vector p6617 was isolated by performance of PCR with extension primers:
  • Both primers contain BamHI restriction sites. This step resulted in a 700 bp PCR product which was ligated into the BamHI site of pUC18 forming the plasmid pA8328.
  • step c the p24 protein was then isolated from the plasmid A 8328 with BamHI/Bglll restriction endonuclease and gel purified.
  • the plasmid A 8714 containing the truncated MN gpl60 was treated with BamHI and gel purified.
  • the gel purified, BamHI/Bglll digested HIV protein p24 was ligated into the BamHI site of vector A8714 containing the gpl60 to form the plasmid A8780 which contains the nucleic acid encoding p24 3' and tandem to the nucleic acid encoding the HIV protein gpl60.
  • step d the fusion protein gpl60-p24 was cloned into a baculovirus expression vector (BEV) MGS12-AIDS, designated A7306.
  • BEV baculovirus expression vector
  • Plasmid A8780 was digested with Kpnl/BamHI and gel purified which isolates the MN gpl60-p24 fusion protein.
  • the BEV, A7306 was digested with Kpnl/Bglll and gel purified.
  • the fusion protein gpl60/p24 was ligated into the BEV plasmid A7306 forming the plasmid A8888.
  • the gpl60-p55 is supplied by the vector A9760 as described in Example 6.
  • protease and reverse transcriptase nucleic acid was isolated by performing a polymerase chain reaction (PCR) in combination with two extension primers:
  • the resultant 2.1 Kb PCR product is ligated into a vector with the resultant vector designated A9398, as is shown in Figures 3A-3C.
  • A9398 is digested with NotI to release the protease/reverse transcriptase coding regions and ligated into NotI digested, calf-intestinal alkaline phosphatase digested plasmid A9760 from Example 6.
  • the resultant vector comprised of the polyprotein immunogen gpl60-p55-protease-reverse transcriptase is designated A9931.
  • the gpl60-p55 and protease-reverse transcriptase fragments had a tendency to insert in the opposite orientation at very high frequencies. This problem was alleviated by the addition of up to a 40-fold increase of calf- intestinal alkaline phosphatase (CIP) to the vector and adding up to a five molar excess of insert to vector in the ligation reaction. The frequency of clones in the right orientation was still very low. 0
  • gpl60/p24 anti-sera reveals a major band migrating with a molecular weight of approximately 185Kd.
  • Primer B also contains 27 bp of the MN gpl60 sequence immediately upstream from the epitope to be replaced by ELDWKA.
  • Primer C contains sequences complementary to the tip of the V3 loop (GPGR) and to the downstream V3 epitope.
  • the SOE technique requires two PCR steps.
  • the first step two separate PCR reactions containing MN gpl60 as the template are set up.
  • the first reaction contains primers A and B, the second primers C and D.
  • the results of the two reactions are double stranded DNA fragments AB and CD which contain 24 bp of complementary ELDKWA sequences at their 3' and 5' ends respectively.
  • double stranded fragments AB and CD are combined along with primers A and D.
  • the primary single stranded hybrids are filled in by the taq polymerase during incubation at 72°C and are then amplified using the external primers A and B.
  • the product of this reaction is a 1.8 Kb fragment of gpl60 in which the V3 loop epitope upstream of the GPGR tip has been replaced by the ELDKWA epitope.
  • Lane 6 shows a Coomassie-stained gel.
  • Lane 1 contains a mixture of molecular weight markers, and lanes 2 through 15 contain samples taken during the purification protocol.
  • Insect cells infected with the recombinant baculovirus AcA9760 are washed by disruption in a nonionic detergent at pH 8. The homogenate is centrifuged and the supernatant, containing cytoplasmic proteins and extracted peripheral membrane proteins, is discarded.
  • the gpl60-55 fusion protein is extracted by disruption in buffer containing nonionic detergent and 6M urea at pH 11.
  • the pH of the homogenate is adjusted to 9.0 with zwitterionic buffer, and the homogenate is incubated for 60 min at room temperature with 100 units/mL of a recombinant nuclease to degrade nucleic acids.
  • the homogenate is centrifuged, and the pellet is discarded.
  • the solubilized protein is shown in lane 2.
  • Anion-exchange Chromatography The supernatant is applied to a DEAE anion- exchange column equilibrated with a pH 9 zwitterionic buffer containing nonionic detergent and 4M urea. Lipids and a number of contaminating proteins either flow through the ion-exchanger (lane 3) or are eluted with 100 mM NaCl (lane 5) . A large number of contaminating proteins remain bound to the column and are eluted with 1M NaCl (lane 9) or 0.5M NaOH (lane 10).
  • polyprotein immunogen gpl60-p24
  • gpl60-p24 induced strong anti-p24 responses with a GMT titer of >100,000 by week 6.
  • Figure 11 demonstrates that antibody responses against gpl60 are good (>100,000 by week 6) regardless if gpl60 is presented alone, mixed with p24, or as part of the gpl60-p24 immunogen.
  • mice immunized with the polyprotein immunogens, gpl60- p55 or gpl60-p24 were also tested for a delayed type hypersensitivity reaction (DTH) by foot pad injection with gpl60 or p24. Good T-cell responses against both gpl60 and p24 in mice immunized with the polyprotein immunogens were observed.
  • DTH reaction the mice described in experiments summarized in Figures 9, 10, and 11 and Tables I, II and III, were challenged with either 25 ⁇ g purified gpl60 or p24 in the hind foot pads. The extent of swelling is measured at 48 hours as a measure of T-cell immunity.

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Abstract

Immunogènes de polyprotéines de VIH, utilisés comme réactifs diagnostiques pour la détection d'une infection à VIH, comme vaccins ou prophylactiques contre l'infection à VIH, et comme agents thérapeutiques dans le traitement de l'infection à VIH-I. La présente invention se rapporte spécifiquement aux immunogènes de polyprotéines de VIH gp160/p24 et gp160/p55.
PCT/US1995/006346 1994-05-20 1995-05-19 Immunogenes de polyproteines de vih WO1995032000A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006519585A (ja) * 2002-11-05 2006-08-31 グラクソ　グループ　リミテッド Ｈｉｖワクチン
JP2006520185A (ja) * 2002-11-05 2006-09-07 グラクソ　グループ　リミテッド Ｈｉｖワクチン
US7597908B2 (en) 1997-01-30 2009-10-06 Novartis Vaccines And Diagnostics, Inc. Use of microparticles with adsorbed antigen to stimulate immune responses

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01179687A (ja) * 1987-12-30 1989-07-17 Chemo Sero Therapeut Res Inst Ｈｉｖ融合蛋白質
US4925784A (en) * 1986-04-04 1990-05-15 Hoffmann-La Roche Inc. Expression and purification of an HTLV-III gag/env gene protein
US5134227A (en) * 1986-02-27 1992-07-28 Centocor, Inc. DNA encoding immunoreactive, chimeric HTLV-III GAG proteins

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5134227A (en) * 1986-02-27 1992-07-28 Centocor, Inc. DNA encoding immunoreactive, chimeric HTLV-III GAG proteins
US4925784A (en) * 1986-04-04 1990-05-15 Hoffmann-La Roche Inc. Expression and purification of an HTLV-III gag/env gene protein
JPH01179687A (ja) * 1987-12-30 1989-07-17 Chemo Sero Therapeut Res Inst Ｈｉｖ融合蛋白質

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AIDS RESEARCH AND HUMAN RETROVIRUSES, Volume 9, Number 10, issued October 1993, MORIMOTO et al., "Use of the Recombinant Chimera Proteins, LacZ-Env and Gag-Env, for Immunological Studies of HIV-1 Infection", pages 971-978. *
ANALYTICAL BIOCHEMISTRY, Volume 161, issued March 1987, SHOEMAN et al., "Comparison of Recombinant Human Immunodeficiency Virus Gag Precursor and Gag/Env Fusion Proteins and a Synthetic Env Peptide as Diagnostic Reagents", pages 370-379. *
JOURNAL OF CLINICAL MICROBIOLOGY, Volume 28, Number 9, issued September 1990, SIITARI et al., "New Sensitive and Specific Assay for Human Immunodeficiency Virus Antibodies Using Labeled Recombinant Fusion Protein and Time-Resolved Fluoroimmunoassay", pages 2022-2029. *
VIROLOGY, Volume 180, issued February 1991, ELLINGER et al., "Cleavage and Purification of Prokaryotically Expressed HIV Gag and Env Fusion Proteins for Detection of HIV Antibodies in the ELISA", pages 811-813. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7597908B2 (en) 1997-01-30 2009-10-06 Novartis Vaccines And Diagnostics, Inc. Use of microparticles with adsorbed antigen to stimulate immune responses
JP2006519585A (ja) * 2002-11-05 2006-08-31 グラクソ　グループ　リミテッド Ｈｉｖワクチン
JP2006520185A (ja) * 2002-11-05 2006-09-07 グラクソ　グループ　リミテッド Ｈｉｖワクチン

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