AU2004237877A1 - 3' genomic promoter region and polymerase gene mutations responsible for attenuation in viruses of the order designated mononegavirales - Google Patents

Description

S&F Ref: 457034D2

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name and Address of Applicants: Actual Inventor(s): Address for Service: Invention Title: The Government of the United States of America as represented by The Department of Health and Human Services, of Suite 325 6011 Executive Boulevard, Rockville, Maryland, 20852, United States of America Wyeth Holdings Corporation, of Five Giralda Farms, Madison, New Jersey, 07940-0874, United States of America Stephen A. Udem Valerie B. Randolph Brian R. Murphy Joanne M. Tatem Mohinderjit S. Sidhu Spruson Ferguson St Martins Tower Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) 3' genomic promoter region and polymerase gene mutations responsible for attenuation in viruses of the order designated mononegavirales The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c 3' GENOMIC PROMOTER REGION AND POLYMERASE GENE MUTATIONS RESPONSIBLE FOR ATTENUATION IN VIRUSES OF THE ORDER DESIGNATED

MONONEGAVIRALES

00 S 5 Field Of The Invention This invention relates to isolated, Ce recombinantly-generated, attenuated, nonsegmented, negative-sense, single stranded RNA viruses of the Order designated Mononegavirales having at least one attenuating mutation in the 3' genomic promoter region and having at least one attenuating mutation in the RNA polymerase gene. This invention was made with Government support under a grant awarded by the Public Health Service. The Government has certain rights in the invention.

Background Of The Invention Enveloped, negative-sense, single stranded RNA viruses are uniquely organized and expressed. The genomic RNA of negative-sense, single stranded viruses serves two template functions in the context of a nucleocapsid: as a template for the synthesis of messenger RNAs (mRNAs) and as a template for the synthesis of the antigenome strand. Negativesense, single stranded RNA viruses encode and package their own RNA dependent RNA Polymerase. Messenger RNAs are only synthesized once the virus has been uncoated in the infected cell. Viral replication occurs after synthesis of the mRNAs and requires the continuous synthesis of viral proteins. The newly synthesized antigenome strand serves as the template for generating further copies of the strand genomic

RNA.

c 2- 0 The polymerase complex actuates and achieves transcription and replication by engaging the cisacting signals at the 3' end of the genome, in 00 particular, the promoter region. Viral genes are then transcribed from the genome template unidirectionally from its 3' to its 5' end. There is always less mRNA O made from the downstream genes the polymerase C gene relative to their upstream neighbors the nucleoprotein gene Therefore, there is always a gradient of mRNA abundance according to the position of the genes relative to the 3'-end of the genome.

Based on the revised reclassification in 1993 by the International Committee on the Taxonomy of Viruses, an Order, designated Mononegavirales, has been established. This Order contains three families of enveloped viruses with single stranded, nonsegmented RNA genomes of minus polarity (negative-sense). These families are the Paramyxoviridae, Rhabdoviridae and Filoviridae. The family Paramyxoviridae has been further divided into two subfamilies, Paramyxovirinae and Pneumovirinae. The subfamily Paramyxovirinae contains three genera, Paramyxovirus, Rubulavirus and Morbillivirus. The subfamily Pneumovirinae contains the genus Pneumovirus.

The new classification is based upon morphological criteria, the organization of the viral genome, biological activities and the sequence relationships of the proteins. The morphological distinguishing feature among enveloped viruses for the subfamily Paramyxovirinae is the size and shape of the nucleocapsids (diameter 18mm, 1mm in length, pitch of nm), which have a left-handed helical symmetry. The biological criteria are: 1) antigenic cross-reactivity between members of a genus, and 2) the presence of neuraminidase activity in the genera Paramyxovirus, cM 3 0 o Rubulavirus and its absence in genus Moibillivirus. In addition, variations in the coding potential of the P gene are considered, as is the presence of an extra 00 gene (SH) in Rubulaviruses.

S 5 Pneumoviruses can be distinguished from Paramyxovirinae morphologically because they contain Snarrow nucleocapsids. In addition, pneumoviruses have C- major differences in the number of protein-encoding cistrons (10 in pneumoviruses versus 6 in Paramyxovirinae) and an attachment protein that is very different from that of Paramyxovirinae. Although the paramyxoviruses and pneumoviruses have six proteins that appear to correspond in function P, M, G/H/HN, F and only the latter two proteins exhibit significant sequence relatedness between the two subfamilies. Several pneumoviral proteins lack counterparts in most of the paramyxoviruses, namely the nonstructural proteins NS1 and NS2, the small hydrophobic protein SH, and a second protein M2. Some paramyxoviral proteins, namely C and V, lack counterparts in pneumoviruses. However, the basic genomic organization of pneumoviruses and paramyxoviruses is the same. The same is true of rhabdoviruses and filoviruses. Table 1 presents the current taxonomical classification of these viruses, together with examples of each genus.

Table 1 Classification of Nonsegmented, negative-sense, single stranded RNA Viruses of the Order Mononegavirales Family Paramyxoviridae Subfamily Paramyxovirinae Genus Paramyxovirus Sendai virus (mouse parainfluenza virus type 1) 4 0 Human parainfluenza virus (PIV) types 1 and 3 Bovine parainfluenza virus (BPV) type 3 00 Genus Rubulavirus Cc 5 Simian virus 5 (SV) (Canine parainfluenza virus type 2) Mumps virus C1 Newcastle disease virus (NDV) (avian Paramyxovirus 1) Human parainfluenza virus types 2, 4a and 4b Genus Morbillivirus Measles virus (MV) Dolphin Morbillivirus Canine distemper virus (CDV) Peste-des-petits-ruminants virus Phocine distemper virus Rinderpest virus Subfamily Pneumovirinae Genus Pneumovirus Human respiratory syncytial virus (RSV) Bovine respiratory syncytial virus Pneumonia virus of mice Turkey rhinotracheitis virus Family Rhabdoviridae Genus Lyssavirus Rabies virus Genus Vesiculovirus Vesicular stomatitis virus Genus Ephemerovirus Bovine ephemeral fever virus Family Filovirdae Genus Filovirus Marburg virus 5

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For many of these viruses, no vaccines of any kind are available. Thus, there is a need to develop vaccines against such human and animal pathogens. Such 00 vaccines would have to elicit a protective immune response in the recipient. The qualitative and quantitative features of such a favorable response are O extrapolated from those seen in survivors of natural Cl virus infection, who, in general, are protected from reinfection by the same or highly related viruses for some significant duration thereafter.

A variety of approaches can be considered in seeking to develop such vaccines, including the use of: purified individual viral protein vaccines (subunit vaccines); inactivated whole virus preparations; and live, attenuated viruses.

Subunit vaccines have the desirable feature of being pure, definable and relatively easily produced in abundance by various means, including recombinant DNA expression methods. To date, with the notable exception of hepatitis B surface antigen, viral subunit vaccines have generally only elicited short-lived and/or inadequate immunity, particularly in naive recipients.

Formalin inactivated whole virus preparations of polio (IPV) and hepatitis A have proven safe and efficacious. In contrast, immunization with similarly inactivated whole viruses such as respiratory syncytial virus and measles virus vaccines elicited unfavorable immune responses and/or response profiles which predisposed vaccinees to exaggerated or aberrant disease when subsequently confronted with the natural or "wild-type" virus.

Early attempts (1966) to vaccinate young children using a parenterally administered formalininactivated RSV vaccine. Unfortunately, several field c 6 0 Strials of this vaccine revealed serious adverse reactions the development of a severe illness with P unusual features following subsequent natural infection 00 with RSV (Bibliography entries It has been 5 suggested that this formalinized RSV antigen elicited an abnormal or unbalanced immune response profile, Spredisposing the vaccinee to RSV disease c Thereafter, live, attenuated RSV vaccine candidates were generated by cold passage or chemical mutagenesis. These RSV strains were found to have reduced virulence in seropositive adults.

Unfortunately, they proved either over or underattenuated when given to seronegative infants; in some cases, they also were found to lack genetic stability Another vaccination approach using parenteral administration of live virus was ineffective and efforts along this line were discontinued Notably, these live RSV vaccines were never associated with disease enhancement as observed with the formalininactivated RSV vaccine described above. Currently, there are no RSV vaccines approved for administration to humans, although clinical trials are now in progress with cold-passaged, chemically mutagenized strains of RSV designated A2 and B-1.

Appropriately attenuated live derivatives of wild-type viruses offer a distinct advantage as vaccine candidates. As live, replicating agents, they initiate infection in recipients during which viral gene products are expressed, processed and presented in the context of the vaccinee's specific MHC class I and II molecules, eliciting humoral and cell-mediated immune responses, as well as the coordinate cytokine-patterns, which parallel the protective immune profile of survivors of natural infection.

c 7 This favorable immune response pattern is contrasted with the delimited responses elicited by inactivated or subunit vaccines, which typically are 0 largely restricted to the humoral immune surveillance arm. Further, the immune response profile elicited by some formalin inactivated whole virus vaccines, e.g., O measles and respiratory syncytial virus vaccines C- developed in the 1960's, have not only failed to provide sustained protection, but in fact have led to a predisposition to aberrant, exaggerated, and even fatal illness, when the vaccine recipient later confronted the wild-type virus.

While live, attenuated viruses have highly desirable characteristics as vaccine candidates, they have proven to be difficult to develop. The crux of the difficulty lies in the need to isolate a derivative of the wild-type virus which has lost its diseaseproducing potential virulence), while retaining sufficient replication competence to infect the recipient and elicit the desired immune response profile in adequate abundance.

Historically, this delicate balance between virulence and attenuation has been achieved by serial passage of a wild-type viral isolate through different host tissues or cells under varying growth conditions (such as temperature). This process presumably favors the growth of viral variants (mutants), some of which have the favorable characteristic of attenuation.

Occasionally, further attenuation is achieved through chemical mutagenesis as well.

This propagation/passage scheme typically leads to the emergence of virus derivatives which are temperature sensitive, cold-adapted and/or altered in their host range one or all of which are changes O8 c- 8- 2from the wild-type, disease-causing viruses i.e., changes that may be associated with attenuation.

Several live virus vaccines, including those 00 for the prevention of measles and mumps (which are paramyxoviruses), and for protection against polio and rubella (which are positive strand RNA viruses), have O been generated by this approach and provide the c- mainstay of current childhood immunization regimens throughout the world.

Nevertheless, this means for generating attenuated live virus vaccine candidates is lengthy and, at best, unpredictable, relying largely on the selective outgrowth of those randomly occurring genomic mutants with desirable attenuation characteristics.

The resulting viruses may have the desired phenotype in vitro, and even appear to be attenuated in animal models. However, all too often they remain either under- or overattenuated in the human or animal host for whom they are intended as vaccine candidates.

Even as to current vaccines in use, there is still a need for more efficacious vaccines. For example, the current measles vaccines provide reasonably good protection. However, recent measles epidemics suggest deficiencies in the efficacy of current vaccines. Despite maternal immunization, high rates of acute measles infection have occurred in children under age one, reflecting the vaccines' inability to induce anti-measles antibody levels comparable to those developed following wild-type measles infection As a result, vaccineimmunized mothers are less able to provide their infants with sufficient transplacentally-derived passive antibodies to protect the newborns beyond the first few months of life.

I

V 9 Acute measles infections in previously immunized adolescents and young adults point to an additional problem. These secondary vaccine failures 00 indicate limitations in the current vaccines' ability S 5 to induce and maintain antiviral protection that is both abundant and long-lived (11,12,13). Recently, yet another potential problem was revealed. The CI hemagglutinin protein of wild-type measles isolated over the past 15 years has shown a progressively increasing distance from the vaccine strains (14).

This "antigenic drift" raises legitimate concerns that the vaccine strains may not contain the ideal antigenic repertoire needed to provide optimal protection. Thus, there is a need for improved vaccines.

Rational vaccine design would be assisted by a better understanding of these viruses, in particular, by the identification of the virally encoded determinants of virulence as well as those genomic changes which are responsible for attenuation.

Summary Of The Invention Accordingly, it is an object of this invention to identify those regions of the genome of the RNA viruses of the Order Mononegavirales where mutations result in attenuation of those viruses.

It is a further object of this invention to produce recombinantly-generated viruses which incorporate such attenuating mutations in their genomes.

It is still a further object of this invention to formulate vaccines containing such attenuated viruses.

These and other objects of the invention as discussed below are achieved by the generation and 0 10 isolation of recombinantly-generated, attenuated, nonsegmented, negative-sense, single stranded RNA viruses of the Order Mononegavirales having at least 00 one attenuating mutation in the 3' genomic promoter region and having at least one attenuating mutation in the RNA polymerase gene.

SIn the case of measles virus, at least one C attenuating mutation in the 3' genomic promoter region is selected from the group consisting of nucleotide 26 (A nucleotide 42 (A T or A C) and nucleotide 96 (G where these nucleotides, as well as others delineated in this application (unless stated otherwise), are presented in positive strand, antigenomic, that is, message (coding) sense, and at least one attenuating mutation in the RNA polymerase gene is selected from the group consisting of nucleotide changes which produce changes in an amino acid selected from the group consisting of residues 331 (isoleucine threonine), 1409 (alanine threonine), 1624 (threonine alanine), 1649 (arginine methionine), 1717 (aspartic acid alanine), 1936 (histidine tyrosine), 2074 (glutamine arginine) and 2114 (arginine lysine).

In the case of human parainfluenza virus type 3, at least one attenuating mutation in the 3' genomic promoter region is selected from the group consisting of nucleotide 23 (T nucleotide 24 (C -4 T), nucleotide 28 (G T) and nucleotide 45 (T and at least one attenuating mutation in the RNA polymerase gene is selected from the group consisting of nucleotide changes which produce changes in an amino acid selected from the group consisting of residues 942 (tyrosine histidine), 992 (leucine C 11 Sphenylalanine), 1292 (leucine phenylalanine), and 1558 (threonine isoleucine).

In the case of human respiratory syncytial 00 virus subgroup B, at least one attenuating mutation in m 5 the 3' genomic promoter region is selected from the Sgroup consisting of nucleotide 4 (C G) and the Sinsertion of an additional A in the stretch of A's at c nucleotides 6-11, and at least one attenuating mutation in the RNA polymerase gene is selected from the group consisting of nucleotide changes which produce changes in an amino acid selected from the group consisting of residues 353 (arginine lysine), 451 (lysine arginine), 1229 (aspartic acid asparagine), 2029 (threonine isoleucine) and 2050 (asparagine aspartic acid).

In another embodiment of this invention, attenuated virus is used to prepare vaccines which elicit a protective immune response against the wildtype form of the virus.

In yet another embodiment of this invention, an isolated, positive strand, antigenomic message sense nucleic acid molecule (or an isolated, negative strand genomic sense nucleic acid molecule) having the complete viral nucleotide sequence (whether of wildtype virus or virus attenuated by non-recombinant means) is manipulated by introducing one or more of the attenuating mutations described in this application to generate an isolated, recombinantly-generated attenuated virus. This virus is then used to prepare vaccines which elicit a protective immune response against the wild-type form of the virus.

In still another embodiment of this invention, such a complete wild-type or vaccine viral nucleotide sequence is used: to design PCR primers for use in a PCR assay to detect the presence of the CN 12- 0 corresponding virus in a sample; or to design and select peptides for use in an ELISA to detect the presence of the corresponding virus in a sample.

00 p 5 Brief Description Of The Figures O Figure 1 depicts the passage history of the CI Edmonston measles virus The abbreviations have the following meanings: HK human kidney; HA human amnion; CE(am) chick embryo; CEF chick embryo fibroblast; DK dog kidney; WI-38 human diploid cells; SK sheep kidney; plaque cloning. The number following each abbreviation represents the number of passages.

Figure 2 depicts a map of the measles virus genome showing putative cis-acting regulatory elements at and near the genome and antigenome termini. Top a schematic map of the measles virus genome, beginning at the 3' end with 52 nucleotides of leader sequence (1) and ending at the 5' terminus with 37 nucleotides of trailer,sequence Gene boundaries are denoted by vertical bars; below each gene is the number of cistronic nucleotides. Bottom an expanded schematic view of the 3' extended genomic promoter regions of genome and antigenome, showing the position and sequence of the two highly conserved domains, A and B.

The intervening intergenic trinucleotide is denoted as well. Nascent 5' RNAs encompassing the A' to B' regions are presumed to contain the regulatory sequence at which the N protein encapsidation initiates.

Figure 3 depicts a genetic map of the RSV subgroup B wild-type strains designated 2B and 18537 (top portion), the intergenic sequences of those strains (middle portion) and the 68 nucleotide overlap between the M2 and L genes (bottom portion). The RSV c 13 0 2B stain has six fewer nucleotides in the G gene, encoding two fewer amino acid residues in the G protein, as compared to the 18537 strain. The 2B 00 strain has 145 nucleotides in the 5' trailer region, as compared to 149 nucleotides in the 18537 strain. The 2B strain has one more nucleotide in each of the NS-1, O NS-2 and N genes, and one fewer nucleotide in each of C the M and F genes, as compared to the 18537 strain.

Detailed Description Of The Invention Transcription and replication of negativesense, single stranded RNA viral genomes are achieved through the enzymatic activity of a multimeric protein acting on the ribonucleoprotein core (nucleocapsid).

Naked genomic RNA cannot serve as a template. Instead, these genomic sequences are recognized only when they are entirely encapsidated by the N protein into the nucleocapsid structure. It is only in that context that the genomic and antigenomic terminal promoter sequences are recognized to initiate the transcriptional or replication pathways.

All paramyxoviruses require the two viral proteins, L and P, for these polymerase pathways to proceed. The pneumoviruses, including RSV, also require the transcription elongation factor, M2, for the transcriptional pathway to proceed efficiently.

Additional cofactors may also play a role, including perhaps the virus-encoded NS1 and NS2 proteins, as well as perhaps host-cell encoded proteins.

However, considerable evidence indicates that it is the L protein which performs most, if not all, the enzymatic processes associated with transcription and replication, including initiation, and termination of ribonucleotide polymerization, capping and 0 0 c 14 0 polyadenylation of mRNA transcripts, methylation and perhaps specific phosphorylation of P proteins. The L protein's central role in genomic transcription and 00 replication is supported by its large size, sensitivity S 5 to mutations, and its catalytic level of abundance in the transcriptionally active viral complex (16).

8 These considerations led to the proposal that C- L proteins consist of a linear array of domains whose concatenated structure integrates discrete functions Indeed, three such delimited, discrete elements Within the negative-sense virus L protein have been identified based on their relatedness to defined functional domains of other well-characterized proteins. These include: a putative RNA template recognition and/or phosphodiester bond formation domain; an RNA binding element; and an ATP binding domain. All prior studies of L proteins of nonsegmented negative-sense, single stranded RNA viruses have revealed these putative functional elements (17).

Without being bound by the following, it is reasonable to presume that these non-protein coding, promoter and other cis-acting genomic regulatory domains are important determinants of the efficiency with which transcription and replication by measles virus (MV) and other viruses of the Order Mononegavirales are actualized, in association with the L protein, and that they may therefore be virulence determinants for these viruses as well.

In summary, the invention is believed to encompass a coordinate set of changes between the cisacting regulatory signal genomic promoter region) and the polymerase gene which results in attenuation of the virus while retaining sufficient ability of the virus to replicate. Attenuation is C' optimized by rational mutations of the 3' genomic promoter region and the polymerase gene, which provide the desired balance of replication efficiency: so that 00 the virus vaccine is no longer able to produce disease, yet retains its capacity to infect the vaccinee's cells, to express sufficiently abundant gene products 0 to elicit the full spectrum and profile of desirable C- immune responses, and to reproduce and disseminate sufficiently to maximize the abundance of the immune response elicited.

Without being bound by the following, attenuating mutations in the extended promoter (3' genomic promoter region) and in the polymerase gene are believed to affect the display of cis-acting signals and the conformation of the polymerase complex engaging these signals. For example, when encapsidated, the promoter RNA is coiled in a helical array. Changes in promoter sequence may affect the relative positions at which the conserved signals are displayed relative to one another. Specifically, the measles wild-type 3' genomic promoter region has a pyrimidine (uracil) at positions 26 and 42 (the antigenomic message sense sequences have the purine adenine). The vaccine strains have purines at those positions (the antigenomic message sense sequences have the corresponding pyrimidines; see Table 3 in Example 1 below). The larger purines may change the distance and/or angular display between the conserved domains of the promoter in measles, positions 1-11 and 87- 98), resulting in an altered spatial presentation of the cis-acting signals to the polymerase.

Animal studies have demonstrated a decrease in viral replication sufficient to avoid illness but adequate to elicit the desired immune response. This likely represents a decrease in transcription, a c- 16- Sdecrease in gene expression of virally encoded proteins, a decrease in antisense templates and, therefore, the production of fewer new genomes. The 00 resulting attenuated viruses are significantly less 'c 5 virulent than the wild-type.

c- The attenuating mutations described herein may be introduced into viral strains by two methods: CI Conventional means such as chemical mutagenesis during virus growth in cell cultures to which a chemical mutagen has been added, selection of virus that has been subjected to passage at suboptimal temperature in order to select temperature sensitive and/or cold adapted mutations, identification of mutant virus that produce small plaques in cell culture, and passage through heterologous hosts to select for host range mutations. These viruses are then screened for attenuation of their biological activity in an animal model. Attenuated viruses are subjected to nucleotide sequencing of their 3' genomic promoter region and polymerase genes to locate the sites of attenuating mutations. Once this has been done, method is then carried out.

A preferred means of introducing attenuating mutations comprises making predetermined mutations using site-directed mutagenesis. These mutations are identified either by method or by reference to closely-related viruses whose attenuating mutations are already known. One or more mutations are introduced into each of the 3' genomic promoter region and the polymerase gene. Cumulative effects of different combinations of coding and non-coding changes can also be assessed.

The mutations to the 3' genomic promoter region and polymerase gene are introduced by standard recombinant DNA methods into a DNA copy of the viral c171- CMI 17 Sgenome. This may be a wild-type or a modified viral genome background (such as viruses modified by method l thereby generating a new virus. Infectious 00 clones or particles containing these attenuating S 5 mutations are generated using the cDNA "rescue" system, which has been applied to a variety of viruses, 8 including Sendai virus measles virus (19); CI respiratory syncytial virus rabies (21); vesicular stomatitis virus (VSV) and rinderpest virus these references are hereby incorporated by reference. See, for measles virus rescue, published International patent application WO 97/06270, designating the United States for PIV-3 rescue, U.S. provisional patent application 60/047575 for RSV rescue, published International patent application WO 97/12032, designating the United States these applications are hereby incorporated by reference.

Briefly, all Mononegavirales rescue systems can be summarized as follows: Each requires a cloned DNA equivalent of the entire viral genome placed between a suitable DNA-dependent RNA polymerase promoter the T7 RNA polymerase promoter) and a self-cleaving ribozyme sequence the hepatitis delta ribozyme) which is inserted into a propagatable bacterial plasmid. This transcription vector provides the readily manipulable DNA template from which the RNA polymerase T7 RNA polymerase) can faithfully transcribe a single-stranded RNA copy of the viral antigenome (or genome) with the precise, or nearly precise, 5' and 3' termini. The orientation of the viral genomic DNA copy and the flanking promoter and ribozyme sequences determine whether antigenome or genome RNA equivalents are transcribed. Also required for rescue of new virus progeny are the virus-specific trans-acting proteins needed to encapsidate the naked, c- -18- 0 single-stranded viral antigenome or genome RNA transcripts into functional nucleocapsid templates: the viral nucleocapsid (N or NP) protein, the O polymerase-associated phosphoprotein and the 5 polymerase protein. These proteins comprise the active viral RNA-dependent RNA polymerase which must O engage this nucleocapsid template to achieve C transcription and replication.

The trans-acting proteins required for measles virus rescue are the encapsidating protein N, and the polymerase complex proteins, P and L. For PIV- 3, the encapsidating protein is designated NP, and the polymerase complex proteins are also referred to as P and L. For RSV, the virus-specific trans-acting proteins include N, P and L, plus an additional protein, M2, the RSV-encoded transcription elongation factor.

Typically, these viral trans-acting proteins are generated from one or more plasmid expression vectors encoding the required proteins, although some or all of the required trans-acting proteins may be produced within mammalian cells engineered to contain and express these virus-specific genes and gene products as stable transformants.

The typical (although not necessarily exclusive) circumstances for rescue include an appropriate mammallian cell milieu in which T7 polymerase is present to drive transcription of the antigenomic (or genomic) single-stranded RNA from the viral genomic cDNA-containing transcription vector.

Either cotranscriptionally or shortly thereafter, this viral antigenome (or genome) RNA transcript is encapsidated into functional templates by the nucleocapsid protein and engaged by the required polymerase components produced concurrently from coc- 19 0 Stransfected expression plasmids encoding the required virus-specific trans-acting proteins. These events and processes lead to the prerequisite transcription of OO viral mRNAs, the replication and amplification of new 5 genomes and, thereby, the production of novel viral C progeny, rescue.

0 For the rescue of rabies, VSV and Sendai, T7 C- polymerase is provided by recombinant vaccinia virus VTF7-3. This system, however, requires that the rescued virus be separated from the vaccinia virus by physical or biochemical means or by repeated passaging in cells or tissues that are not a good host for poxvirus. For MV cDNA rescue, this requirement is avoided by creating a cell line that expresses T7 polymerase, as well as viral N and P proteins. Rescue is achieved by transfecting the genome expression vector and the L gene expression vector into the helper cell line. Advantages of the host-range mutant of the vaccinia virus, MVA-T7, which expresses the T7 RNA polymerase, but does not replicate in mammalian cells, are exploited to rescue RSV, Rinderpest virus and MV.

After simultaneous expression of the necessary encapsidating proteins, synthetic full length antigenomic viral RNA are encapsidated, replicated and transcribed by viral polymerase proteins and replicated genomes are packaged into infectious virions. In addition to such antigenomes, genome analogs have now been successfully rescued for Sendai and PIV-3 (25,27).

The rescue system thus provides a composition which comprises a transcription vector comprising an isolated nucleic acid molecule encoding a genome or antigenome of a nonsegmented, negative-sense, single stranded RNA virus of the Order Mononegavirales having at least one attenuating mutation in the 3' genomic promoter region and having at least one attenuating 0 CA 20 a mutation in the RNA polymerase gene, together with at least one expression vector which comprises at least one isolated nucleic acid molecule encoding the trans- OO acting proteins necessary for encapsidation, 5 transcription and replication N, P and L for 0 measles virus; NP, P and L for PIV-3; N, P, L and M2 0 for RSV). Host cells are then transformed or C- transfected with the at least two expression vectors just described. The host cells are cultured under conditions which permit the co-expression of these vectors so as to produce the infectious attenuated virus.

The rescued infectious virus is then tested for its desired phenotype (temperature sensitivity, cold adaptation, plaque morphology, and transcription and replication attenuation), first by in vitro means.

The mutations at the cis-acting 3' genomic promoter region are also tested using the minireplicon system where the required trans-acting encapsidation and polymerase activities are provided by wild-type or vaccine helper viruses, or by plasmids expressing the N, P and different L genes harboring gene-specific attenuating mutations (19,28).

If the attenuated phenotype of the rescued virus is present, challenge experiments are conducted with an appropriate animal model. Non-human primates provide the preferred animal model for the pathogenesis of human disease. These primates are first immunized with the attenuated, recombinantly-generated virus, then challenged with the wild-type form of the virus.

Monkeys are infected by various routes, including but not limited to intranasal, intratracheal or subcutaneous routes of inoculation (29).

Experimentally infected rhesus and cynomolgus macaques have also served as animal models for studies of 21 0 <d vaccine-induced protection against measles Protection is measured by such criteria as disease signs and symptoms, survival, virus shedding and 00 antibody titers. If the desired criteria are met, the attenuated, recombinantly-generated virus is considered a viable vaccine candidate for testing in humans. The "rescued" virus is considered to be "recombinantly- CI generated", as are the progeny and later generations of the virus, which also incorporate the attenuating mutations.

Even if a "rescued virus is underattenuated or overattenuated relative to optimum levels for vaccine use, this is information which is valuable for developing such optimum strains.

Optimally, a codon containing an attenuating point mutation may be stabilized by introducing a second or a second plus a third mutation in the codon without changing the amino acid encoded by the codon bearing only the attenuating point mutation.

Infectious virus clones containing the attenuating and stabilizing mutations are also generated using the cDNA "rescue" system described above.

Measles virus serves as a useful model for this invention, because sequence data are now available as described herein for the disease-causing wild-type virus and for the disease-preventing vaccines which have a demonstrated history of efficacy.

Measles virus was first isolated in tissue culture in 1954 (31) from an infected patient named David Edmonston. This Edmonston strain of measles became the progenitor for many live-attenuated measles vaccines including Moraten, which is the current vaccine in the United States (Attenuvaxy

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Merck Sharp Dohme, West Point, PA) and was licensed in 1968 and has proven to be efficacious.

0 N- 22 Aggressive immunization programs instituted in the mid to late 1960s resulted in the precipitous drop in reported measles cases from near 700,000 in 0 1965 to 1500 in 1983. In parallel, other vaccine S 5 strains were also developed from the Edmonston strain C (see Fig. Schwarz (Institut Merieux, Lyon, France), O Zagreb (Zagreb, Yugoslavia) and AIK-C (Japan). These C- other vaccines have also proven to be efficacious and have been used extensively. An early, reactogenic, underattenuated vaccine strain (Rubeovax: Merck Sharp Dohme) produced measles-like illness in children and its use thus was discontinued. It, however, was further attenuated successfully to produce the Moraten vaccine strain (see Fig. 1) Live measles virus vaccine provides a success story of the development of an efficacious vaccine and provides a model for understanding the molecular mechanisms of viral vaccine attenuation among nonsegmented, negative-sense, single stranded RNA viruses.

Because of its significance as a major cause of human morbidity and mortality, measles virus (MV) has been quite extensively studied. MV is a large, relatively spherical, enveloped particle composed of two compartments, a lipoprotein membrane and a ribonucleoprotein particle core, each having distinct biological functions The virion envelope is a host cell-derived plasma membrane modified by three virus-specified proteins: The hemagglutinin (H; approximately 80 kilodaltons and fusion (F 1 2 approximately 60 kD) glycoproteins project on the virion surface and confer host cell attachment and entry capacities to the viral particle (16).

Antibodies to H and/or F are considered protective since they neutralize the virus' ability to initiate infection (34,35,36). The matrix approximately 37 c- 23- 0 kD) protein is the amphipathic protein lining the membrane's inner surface, which is thought to orchestrate virion morphogenesis and thus consummate 0 virus reproduction The virion core contains the 15,894 nucleotide long genomic RNA upon which template activity is conferred by its intimate association with 0 approximately 2600 molecules of the approximately 60 kD C- nucleocapsid protein (38,39,40). Loosely associated with this approximately one micron long helical ribonucleoprotein particle are enzymatic levels of the viral RNA dependent RNA polymerase (L; approximately 240 kD) which in concert with the polymerase cofactor approximately 70 kD), and perhaps yet other virus-specified as well as host-encoded proteins, transcribes and replicates the MV genome sequences (41).

To date, the entire nucleotide sequences (only for the Edmonston B laboratory strain and the AIK-C vaccine strain), coding potential, and organization of the MV genome have been reported (33).

The six virion structural proteins are encoded by six contiguous, non-overlapping genes which are arrayed as follows: Two additional MV gene products of as yet uncertain function have also been identified. These two nonstructural proteins, known as C (approximately 20 kD) and V (approximately 45 kD), are both encoded by the P gene, the former by a second reading frame within the P mRNA; the latter by a cotranscriptionally edited P gene-derived mRNA which encodes a hybrid protein having the amino terminal sequences of P and a new zinc finger-like cysteine-rich carboxy terminal domain (16).

In addition to the sequences encoding the virus-specified proteins, the MV genome contains distinctive non-protein coding domains resembling those c 24 directing the transcriptional and replicative pathways of related viruses (16,42). These regulatory signals lie at the 3' and 5' ends of the MV genome and in short internal regions spanning each intercistronic boundary.

5 The former encode the putative promoter and/or C- regulatory sequence elements directing genomic O transcription, genome and antigenome encapsidation, and Sreplication. The latter signal transcription termination and polyadenylation of each monocistronic viral mRNA and then reinitiation of transcription of the next gene. In general, the MV polymerase complex appears to respond to these signals much as the RNA-dependent RNA polymerases of other non-segmented negative strand RNA viruses (16,42,43,44).

Transcription initiates at or near the 3' end of the MV genome and then proceeds in a 5' direction producing monocistronic mRNAs (40,42,45). As the polymerase traverses the MV genomic template, it encounters putative stop/start signals which, in 3' to 5' order, are: a semi-conserved transcription termination/polyadenylation signal (A/G U/C UA A/U NN

A

4 where N may be any of the four bases) at which each monocistronic RNA is completed; a non-transcribed intergenic trinucleotide punctuation mark (CUU; except at the H:L boundary where it is CGU); and a semiconserved start signal for transcription initiation of the next gene (AGG A/G NN C/A A A/G G A/U, where N may be any of the four bases) (45,46). Since some polymerase complexes fail to reinitiate, the abundance of each MV mRNA diminishes in parallel with the distance of the encoding gene from the genomic 3' end.

This mRNA gradient directly corresponds to the relative abundance of each virus-specified protein. This indicates that MV protein expression is ultimately controlled at the transcriptional level (44).

0 0 cI 0 SThe 3' and 5' MV genomic termini contain non-protein coding sequences with distinct parallels to P the leader and trailer RNA encoding regions of VSV 00 Nucleotides 1-55 define the region between the genomic 3' terminus and the beginning of the N gene, while 37 additional nucleotides can be found between O the end of the L gene and the 5' terminus of the C genome. However, unlike VSV, or even the paramyxoviruses Sendai and NDV, MV does not transcribe these terminal regions into short, unmodified or sense leader RNAs (47,48,49). Instead, leader readthrough transcripts, including full-length polyadenylated leader:N, leader:N:P, leader:N:P:M, and of course full-length antigenome MV RNAs are transcribed (48,49). Thus, the short leader transcript, the key operational element determining the switch from transcription to replication of the VSV single-stranded, negative polarity genome (50,51,52), seems absent in MV. This leads to consideration and exploration of alternative models for this crucial reproductive event (42).

Measles virus, as well as all other Mononegavirales except the rhabdoviruses, appears to have extended its terminal regulatory domains beyond the confines of leader and trailer encoding sequences For measles, these regions encompass the 107 3' genomic nucleotides (the genomic promoter region", also referred to as the "extended promoter", which comprises 52 nucleotides encoding the leader region, followed by three intergenic nucleotides, and 52 nucleotides encoding the 5' untranslated region of N mRNA) and the 109 5' end nucleotides (69 encoding the 3' untranslated region of L mRNA, the intergenic trinucleotide and 37 nucleotides encoding the trailer).

Within these 3' terminal approximately 100 nucleotides 0 0 c- 26 0 of both the genome and antigenome are two short regions of shared nucleotide sequence: 14 of 16 nucleotides at the absolute 3' ends of the genome and antigenome are 00 identical. Internal to those termini, an additional region of 12 nucleotides of absolute sequence identity have been located. Their position at and near the O sites at which the transcription of the MV genome must C- initiate and replication of the antigenome must begin, suggests that these short unique sequence domains encompass an extended promoter region.

These discrete sequence elements may dictate alternative sites of transcription initiation the internal domain mandating transcription initiation at the N gene start site, and the 3' terminal domain directing antigenome production (42,48,53). In addition to their regulatory role as cis-acting determinants of transcription and replication, these 3' extended genomic and antigenomic promoter regions encode the nascent 5' ends of antigenome and genome RNAs, respectively. Within these nascent RNAs reside as yet unidentified signals for N protein nucleation, another key regulatory element required for nucleocapsid template formation and consequently for amplification of transcription and replication. Figure 2 schematically shows the location and sequence of these highly conserved, putative cis-acting regulatory domains.

Terminal non-protein coding regions similar in location, size and spacing are present in the genomes of other members of the genus Paramyxoviridae, though only 8-11 of their absolute terminal nucleotides are shared by MV (42,54). The genomic terminii of the Morbillivirus canine distemper virus (CDV) displays a greater degree of homology with its MV relative: 73% of the nucleotides of the leader and trailer sequences cM 27 o of these two viruses are identical, including 16 of 18 at the absolute 3' termini and 17 of 18 at their ends No accessory internal CDV genomic domain- 00 sharing homology to that of the MV extended promoter 5 has been found. However, there is a 20 nucleotide long c stretch lying between CDV genomic nucleotides 85 and 0 104 and 15,587 and 15,606 in which 15 of the Ci nucleotides are complementary (Gene Bank accession number AF 14953). This indicates that CDV, like MV contains an additional region within its non-coding 3' genomic and antigenomic ends that may provide important cis-acting promoter and/or regulatory signals Additionally, the precise length of the 3'leader region (55 nucleotides) is identical among several members of the Family Paramyxoviridae (MV, CDV, PIV-3, BPV-3, SV and NDV). Further evidence for the importance of these extended, non-protein coding regions comes from analyses of a large number of distinct copy-back Defective Interfering Viruses (DIs) recently cloned from subacute sclerosing panencephalitis (SSPE) brain tissue. No DI with a stem shorter than the 95 5' terminal genomic nucleotides was found. This indicates that the minimal signals needed for MV DI RNA replication and encapsidation extend well beyond the 37 nucleotide long trailer sequence to encompass the additional internal putative regulatory domain (56).

As exemplified in part by measles virus, this invention is directed to the concept that important virulence/attenuation determinants reside in viral genomic non-protein coding regulatory regions and in the transacting transcription/replication enzyme complex with which these cis-acting elements must interact. The cis-acting domains are found both at the 3' and 5' ends of the MV genome, flanking the six 0 0 c 28 0 contiguous genes encoding viral structural proteins; and within the MV genome as short regions encompassing 0 internal intergenic boundaries. The former encode the 0 putative promoter and/or regulatory sequence elements directing the vital processes of genomic transcription, genome and antigenome encapsidation, and replication.

0 The latter signal transcription termination and C- polyadenylation of each monocistronic viral mRNA and then reinitiation of transcription of the next gene.

The transcription/replication enzyme, RNA dependent RNA polymerase molecule can modulate transcription and/or replicative efficiency, thereby determining the abundance of cytopathic viral gene products and/or virion progeny.

Proof of the concept of this invention for measles virus is obtained by first determining the nucleotide sequences of the non-coding regulatory regions genomic promoter region) and the coding regions of the L gene (with predicted amino acid sequences) of the progenitor Edmonston wild-type MV isolate, together with available measles vaccine strains derived from this isolate (see Figure 1).

Independent other wild-type isolates were examined for comparative purposes as well.

The nucleotide sequences (in positive strand, antigenomic, message sense) of four wild-type and five vaccine measles strains, as well as the deduced amino acid sequences of the RNA polymerase (L protein) of these measles viruses, are set forth as follows with reference to the appropriate SEQ ID NOS. contained herein: 29 0 Virus Nucleotide Sequence L Protein Sequence Wild-Type Edmonston SEQ ID NO:1 SEQ ID NO:2 00 1977 SEQ ID NO:3 SEQ ID NO:4 S 5 1983 SEQ ID NO:5 SEQ ID NO:6 Montefiore SEQ ID NO:7 SEQ ID NO:8 CI Vaccine Rubeovax" SEQ ID NO:9 SEQ ID Moraten SEQ ID NO:11 SEQ ID NO:12 Zagreb SEQ ID NO:13 SEQ ID NO:14 AIK-C SEQ ID NO:15 SEQ ID NO:16 Each measles virus genome listed above is 15,894 nucleotides in length. Translation of the L gene starts with the codon at nucleotides 9234-9236; the translation stop codon is at nucleotides 15783- 15785. The translated L protein is 2,183 amino acids long.

Note that nucleotide 2499 of 1983 wild-type measles virus is indicated as in SEQ ID NO:5. In fact, the base is actually a mixture of and Also note that nucleotide 2143 of Rubeovax T vaccine virus is indicated as in SEQ ID NO:9. In nine clones sequenced, this base was in seven and in two; thus, this base can be or In addition, the Schwarz vaccine virus genome is identical to that of the Moraten vaccine virus genome (SEQ ID NO:11), except that at nucleotides 4917 and 4924, Schwarz has a instead of a Nucleotide differences distinguishing the 3' genomic promoter region and nucleotide and amino acid differences distinguishing the L gene and L protein sequences of the Edmonston wild-type isolate, vaccine strains and other independently isolated wild-type ,1cI 30 Sviruses were then compared and aligned (see Tables in Example 1 below).

As shown in Table 3, there were three 00 mutations from the 3' genomic promoter region (in 5 antigenomic, message sense) of the progenitor wild-type MV isolate and the derivative vaccine strains: At nucleotide position 26, from to at position C- 42, from to or from to and in the case of Zagreb only, at position 96, from to In addition, the other examined wild-type isolates differed from both the progenitor wild-type isolate and the vaccine strains at position 50 by having "A" instead of The predicted amino acid sequences of the L genes of measles vaccine strains (Rubeovax, Moraten, Schwarz, AIK-C and Zagreb) and wild-type isolates (1977, 1983 and Montefiore), differ from the progenitor strain (Edmonston) at 49 positions in the 2183 amino acid long open reading frame (see Tables 4 and 5 in Example 1 below).

These amino acid differences can be divided into four categories: Positions where one vaccine strain differs from the progenitor, as well as from other vaccine and wild-type strains, suggesting a potential attenuation site.

Specific differences between all wildtype and all vaccine sequences; these may also constitute important attenuation sites.

3) Residues where chronologically newer wildtypes differ from older wild-types; which may be attributable to genetic drift.

Positions where one or more vaccine strains and/or wild-type strains have common amino acids and differ from all the other strains; these c- 31changes may represent lineage-specific, potentially attenuating changes within the vaccine strains and relatedness among the wild-type isolates, respectively.

SThere were four category changes where one vaccine differed from the other vaccines, as well C as the wild-type strains. Two of these were in Moraten O and Schwarz (amino acids 331 and 2114) and two were in C- AIK-C (1624 and 2074). These mutations are of special interest because all of these viruses are good vaccines. Thus, these positions are sites for attenuation.

Only one position, 1717, fits into category with all wild-types having aspartic acid and all vaccines having alanine. Interestingly, this position is in one of two areas where the L genes of measles and canine distemper virus (which are otherwise highly homologous) do not show exceptional conservation. This difference makes it more likely that 1717 is a key position for an attenuating mutation in measles.

There were five positions, 149, 636, 720, 2017 and 2119, where both chronologically newer wildtypes (1983 and Montefiore) differ from older wildtypes (Edmonston and 1977), which therefore fit into category These differences suggest genetic drift rather than denoting sites of attenuating mutations.

Not included in this total are 16 positions where Montefiore (the 1989 isolate) differed from the rest (see Table Thesecould be either genetic drift (category or random change (category The remaining 23 positions are category with one or more of the viruses differing from the consensus.

Three of these positions (1409, 1649, 1936) are potentially attenuating category mutations.

These are changes where two vaccine strains have a common change from the progenitor wild-type strain.

c 32 0 SThese changes may be connected with the vaccine lineage leading to the Rubeovax TM and Moraten vaccines (Figure 1 00 Applicants have found that their AIK-C vaccine strain nucleotide sequence differs from the published sequence (33) at 21 positions, including one O insertion and one deletion. Several of these C- differences result in coding changes including two in the L gene (at amino acids 1477 and 2008).

Thus, the additional changes accrued within the L gene sequence as the measles progenitor strain is progressively attenuated to achieve a replicative capacity optimized for live vaccine purposes appears to be constrained and delimited. Presumably, this limited tolerance in the number and location of L gene changes is imposed not only by the need to preserve the multifunctional capacities of the polymerase, but also by the preexisting 3' promoter changes with which the evolving L protein must interact to achieve transcription and replication. In other words, optimal virus attenuation requires coordinate linked) changes in the polymerase protein and the cis-acting regulatory elements on which it acts.

The 3'-leader displays the least tolerance for change, allowing highly selected changes during the attenuation process at nucleotide position 26 (always the change of from to and at position 42 (the change of from to or from to (in antigenomic, message sense). In the case of Zagreb only, there is a single further change, from to "A" at position 96, which may be important when combined with Zagreb L gene-specific changes. The 3'-leader region seems to have undergone only one instance of genetic drift since 1954, with a change of to "A" at position 50 (see Table 3).

c 33 0 The net change in the 3' genomic promoter region during the attenuation process is the replacement of two pyrimidines by two purines in 00 genomic sense in all MV vaccine strains. The coevolution of the L gene during these attenuation C processes is believed to reflect selection of subtle Schanges favoring reproduction of the viruses in eC different host cells. All the vaccine strains were grown in chick embryo (CE) or chick embryo fibroblast (CEF) cells during their attenuation process (Figure In addition, some vaccine strains have been exposed to unique host cells; Zagreb vaccine was grown in dog kidney cells and human diploid cells, while the AIK-C vaccine was adapted to sheep kidney cells. Moraten and Rubeovax T were exclusively developed in CE and CEF.

Some of the lineage-specific L gene changes (position 1649 in Rubeovax

M

Moraten and Schwarz vaccines and the change at position 1717 in all vaccines) represent a subset of adaptations of the L gene to the 3'-leader to modulate the transcription/replication processes for vaccine attenuation. Additionally, individual vaccine-specific changes (category may provide additional fine tune modulation of virus replication/transcription for each vaccine strain.

Based on Table 3 and the foregoing discussion, the key attenuating mutations for the MV 3' genomic promoter region are nucleotide 26 (A T), nucleotide 42 (A or A and nucleotide 96 (G A) (in antigenomic, message sense).

Based on Table 4 and the foregoing discussion, the key attenuating sites for the L protein are as follows: amino acid residues 331 (isoleucine threonine), 1409 (alanine threonine), 1624 N 34 0 <d (threonine -+alanine), 1649 (arginine methionine), 1717 (aspartic acid ->alanine), 1936 (histidine tyrosine), 2074 (glutamine ->arginine) and 2114 00 (arginine ->lysine). It is understood that the m 5 nucleotide changes responsible for these amino acid Schanges are not limited to those set forth in Table 4 Q of Example 1 below; all changes in nucleotides which result in codons which are translated into these amino acids are within the scope of this invention.

Human parainfluenza virus type 3 (HPIV-3) is another nonsegmented, negative-sense, single stranded enveloped RNA virus. HPIV-3 belongs to the Family Paramyxoviridae (see Table The genome of HPIV-3 is 15,462 nucleotides long and encodes six non-overlapping protein-encoding genes Five of the genes encode a single virion structural protein each, which are designated NP (corresponding to the N protein of MV), M, F, HN (hemagglutinin-neuraminidase) and L. The sixth mRNA encodes the P protein, and by an overlapping 5' proximal open reading frame (ORF) encodes the C protein, and by the RNA editing mechanism, also encodes the D protein.

Like MV, HPIV-3 consists of a 3'-nonprotein coding leader region of 55 nucleotides, but unlike measles (where it is 37 nucleotides), it has a 44 nucleotide long 5'-trailer region. The polymerase transcribes the genome in a linear, sequential, startstop manner which is guided by transcription signals in the RNA template.

Attempts to develop a live attenuated HPIV-3 vaccine by passaging the wild-type virus JS strain through cell culture at sub-optimal temperature has produced promising results Several "cold passage" (cp) mutants were isolated for evaluation from different passage levels of the JS strain. One such N 35 0 mutant resulted from 45 serial passages and was designated This virus exhibited three interesting 00 properties: cold adaptation the ability to replicate efficiently at the suboptimal temperature of 0 C; temperature sensitivity inability to replicate in vitro at temperatures greater than or Cl equal to 39°C; and small plaque morphology. This mutant appeared to be a promising vaccine candidate because: its ca, ts and small plaque phenotype is stable after passage in cell culture; its replication is restricted in both the upper and lower respiratory tract of hamsters; and it induced significant protection in hamsters against subsequent challenge with wild-type HPIV-3 (58,59).

Evaluation of this strain in the rhesus monkey showed the attenuation mutations in cp45 to be a combination of ts and non-ts mutations Subsequent evaluation in chimpanzees indicated that cp45 appeared to be satisfactorily attenuated while still able to induce a high level of protection against wild-type virus challenge Later preliminary clinical evaluation of cp45 in seronegative human infants and small children suggested that this candidate vaccine strain is suitably infectious and attenuated, as well as being moderately immunogenic (61).

The cp45 strain has been grown in both fetal rhesus lung (FRhL) and Vero cells as follows: The PIV- 3 cp45 virus grown in FRhL cells was prepared by inoculating confluent FRhL cell monolayers in tissue culture flasks at an MOI 0.1-1.0. The infected cell cultures were fed with EMEM medium and incubated at 32 0 C. About seven days later, when maximal cytopathic effects (synctyia) were observed, the virus was cM 36 0 harvested by subjecting the cultures to one freeze-thaw cycle, pooling the fluids and then storing the virus at -70 OC.

OO The PIV-3 cp45 virus grown in Vero cells was prepared by inoculating with virus a bioreactor culture of confluent monolayers of Vero cells on microcarrier beads which was continuously stirred. The infected CI bioreactor culture was maintained at 30 0 C. The virus was harvested 4-5 days later when syncytial CPE was observed. The culture fluid containing the virus was stored at -70 C.

The nucleotide sequences (in positive strand, antigenomic, message sense) of the HPIV-3 JS wild-type strain (89) and the cp45 vaccine strain grown in FRhL and Vero cells, as well as the deduced amino acid sequences of the RNA polymerase (L protein) of these HPIV-3 viruses, are set forth as follows with reference to the appropriate SEQ ID NOS. contained herein: Virus Nucleotide Sequence L Protein Sequence Wild-Type JS SEQ ID NO:17 SEQ ID NO:18 Vaccine FRhL cp45 SEQ ID NO:19 SEQ ID Vero cp45 SEQ ID NO:21 SEQ ID NO:22 Each PIV-3 virus genome listed above is 15,462 nucleotides in length. Translation of the L gene starts with the codon at nucleotides 8646-8648; the translation stop codon is at nucleotides 15345- 15347. The translated L protein is 2,233 amino acids long.

As detailed in Example 2 and Table 6 therein below, based upon the differences between the wild-type 0 0 C 37 0 JS strain and the FRhL-grown cp 45 mutant vaccine strain, the key attenuating mutations for the HPIV-3 3' genomic promoter region are nucleotide 23 (T 00 nucleotide 24 (C nucleotide 28 (G and l^- CM 5 nucleotide 45 (T (in antigenomic, message sense).

SAs also detailed in Example 2 and Table 6 therein Sbelow, key attenuating sites for the L protein of HPIV- 3 include the following: amino acid residues 942 (tyrosine ->histidine), 992 (leucine phenylalanine) and 1558 (threonine ->isoleucine).

In addition, the Vero-grown cp45 mutant vaccine strain contains an additional mutation resulting from a coding change in the L gene at amino acid residue 1292 (leucine phenylalanine).

It is understood that the nucleotide changes responsible for these amino acid changes are not limited to those set forth in Example 2 below; all changes in nucleotides which result in codons which are translated into these amino acids are within the scope of this invention.

Human respiratory syncytial virus (RSV) is yet another nonsegmented, negative-sense, single stranded enveloped RNA virus. RSV belongs to the Subfamily Pneumovirinae and the genus Pneumovirus (see Table 1).

Two major subgroups of human RSV, designated A and B, have been identified based on reactivities of the F and G surface glycoproteins with monoclonal antibodies More recently, the A and B lineages of RSV strains have been confirmed by sequence analysis (63,64). Bovine, ovine, and caprine strains of this virus have also been isolated. The host specificity of the virus is most clearly associated with the G attachment protein, which is highly divergent between 38 the human and the bovine/ovine strains (65,66), and may be influenced, at least in part, by receptor binding.

0 RSV is the primary cause of serious viral 00 pneumonia and bronchiolitis in infants and young S 5 children. Serious disease, lower respiratory tract disease (LRD), is most prevalent in infants less Sthan six months of age. It most commonly occurs in the c nonimmune infant's first exposure to RSV. RSV additionally is associated with asthma and hyperreactive airways and it is a significant cause of mortality in "high.risk" children with bronchopulmonary dysplasia and congenital heart disease (CHD). It is also one of the common viral respiratory infections predisposing to otitis media in children. In adults, RSV generally presents as uncomplicated upper respiratory illness; however, in the elderly it rivals influenza as a predisposing factor in the development of serious LRD, particularly bacterial bronchitis and pneumonia. Disease is always confined to the respiratory tract, except in the severely immunocompromised, where dissemination to other organs can occur. Virus is spread to others by fomites contaminated with virus-containing respiratory secretions, and infection initiates through the nasal, oral, or conjunctival mucosa.

RSV disease is seasonal and virus is usually isolated only in the winter months, from November to April in northern latitudes. The virus is ubiquitous, and over 90% of children have been infected at least once by 2 years of age. Multiple strains cocirculate. There is no direct evidence of antigenic drift (such as that seen with influenza A viruses), but sequence studies demonstrating accumulation of amino acid changes in the hypervariable regions of the G r CI 39 0 protein and SH proteins suggest that immune pressure may drive virus evolution.

In mouse and cotton rat models, both the F 00 and G proteins of RSV elicit neutralizing antibodies 5 and immunization with these proteins alone provides longterm protection against reinfection (67,68).

In humans, complete immunity to RSV does not CI develop and reinfections occur throughout life (69,70); however, there is evidence that immune factors will protect against severe disease. A decrease in severity of disease is associated with two or more prior infections and there is evidence that children infected with one of the two major RSV subgroups may be somewhat protected against reinfection with the homologous subgroup observations which suggest that a live attenuated virus vaccine may provide protection sufficient to prevent serious morbidity and mortality.

Infection with RSV elicits both antibody and cell mediated immunity. Serum neutralizing antibody to the F and G proteins has been associated, in some studies, with protection from LRD, although reduction in upper respiratory disease (URD) has not been demonstrated.

High levels of serum antibody in infants is associated with protection against LRD, and adminstration of intravenous immunoglobulin with high RSV neutralizing antibody titers has been shown to protect against severe disease in high risk children (70,72,73). The role of local immunity, and nasal antibody in particular, is being investigated.

The RSV virion consists of a ribonucleoprotein core contained within a lipoprotein envelope. The virions of pneumoviruses are similar in size and shape to those of all other paramyxoviruses.

When visualized by negative staining and electron microscopy, virions are irregular in shape and range in CI 40 0 diameter from 150-300 nm The nucleocapsid of this virus is a symmetrical helix similar to that of other paramyxoviruses, except that the helical diameter 00 is 12-15 nm rather than 18nm. The envelope consists of l^- C 5 a lipid bilayer that is derived from the host membrane and contains virally coded transmembrane surface glycoproteins. The viral glycoproteins mediate c-I attachment and penetration and are organized separately into virion spikes. All members of paramyxovirus subfamily have hemagglutinating activity, but this function is not a defining feature for pneumoviruses, being absent in RSV but present in PVM Neuraminidase activity is present in members of the genera Paramyxovirus, Rubulavirus, and is absent in Morbillivirus and Pneumovirus of mice (PVM) RSV possesses two subgroups, designated A and B. The wild-type RSV (strain 2B) genome is a single strand of negative-sense RNA of 15,218 nucleotides (SEQ ID NO:23) that are transcribed into ten major subgenomic mRNAs. Each of the ten mRNAs encodes a major polypeptide chain: Three are transmembrane surface proteins F and SH); three are the proteins associated with genomic RNA to form the viral nucleocapsid P and two are nonstructural proteins (NS1 and NS2) which accumulate in the infected cells but are also present in the virion in trace amounts and may play a role in regulating transcription and replication; one is the nonglycosylated virion matrix protein and the last is M2, another nonglycosylated protein recently shown to be an RSVspecified transcription elongation factor (see Figure These ten viral proteins account for nearly all of the viral coding capacity.

The viral genome is encapsidated with the major nucleocapsid protein and is associated with 41 0 0 CM 41 0 the phosphoprotein and the large polymerase protein. These three proteins have been shown to be necessary and sufficient for directing RNA replication 00 of cDNA encoded RSV minigenomes Further studies have shown that for transcription to proceed with full processing, the M2 protein (ORF 1) is required (74).

When the M2 protein is missing, truncated transcripts C- predominate, and rescue of the full length genome does not occur (74).

Both the M (matrix protein) and the M2 proteins are internal virion-associated proteins that are not present in the nucleocapsid structure. By analogy with other nonsegmented negative-stranded RNA viruses, the M protein is thought to render the nucleocapsid transcriptionally inactive before packaging and to mediate its association with the viral envelope. The NS1 and NS2 proteins have only been detected in very small amounts in purified virions, and at this time are considered non-structural. Their functions are uncertain, though they may be regulators of transcription and replication. Three transmembrane surface glycoproteins are present in virions: G, F, and SH. G and F (fusion) are envelope glycoproteins that are known to mediate attachment and penetration of the virus into the host cell. In addition, these glycoproteins represent major independent immunogens The function of the SH protein is unknown, although a recent report has implicated its involvement in the fusion function of the virus (78).

The genomes of two wild-type RSV subgroup B strains (2B and 18537) have now been sequenced in their entirety (see SEQ ID NOS:23 and 25, discussed'below).

Genomic RNA is neither capped nor polyadenylated (79).

In both the virion and intracellularly, genomic RNA is tightly associated with the N protein.

c 42 0 The 3' end of the genomic RNA consists of a 44-nucleotide extragenic leader region that is presumed 0 to contain the major viral promoter (Fig. The 3' 0 genomic promoter region is followed by ten viral genes in the order 3 '-NS1-NS2-N-P-M-SH-G-F-M2-L-5' (Fig. 3).

The L gene is followed by a 145-149 nucleotide extragenic trailer region (see Figure Each gene C begins with a conserved nine-nucleotide gene start signal 3'-GGGGCAAAU (except for the ten-nucleotide gene start signal of the L gene, which is 3'-GGGACAAAAU; differences underlined). For each gene, transcription begins at the first nucleotide of the signal. Each gene terminates with a semi-conserved 12-14 nucleotide gene end G U/G U/A ANNN U/A A 3 (where N can be any of the four bases) that directs transcription termination and polyadenylation (Fig. The first nine genes are non-overlapping and are separated by intergenic regions that range in size from 3 to 56 nucleotides for RSV B strains (Fig. The intergenic regions do not contain any conserved motifs or any obvious features of secondary structure and have been shown to have no influence on the preceding and succeeding gene expression in a minreplicon system (Fig. The last two RSV genes overlap by 68 nucleotides (Fig. The gene-start signal of the L gene is located inside of, rather than after, the M2 gene. This 68 nucleotide overlap sequence encodes the last 68 nucleotides of the M2 mRNA (exclusive of the Poly-A tail), as well as the first 68 nucleotides of the L mRNA.

Ten different species of subgenomic polyadenylated mRNAs and a number of polycistronic polyadenylated read-through transcripts are the products of genomic transcription (74).

Transcriptional mapping studies using UV light mediated C 43

O

genomic inactivation showed that RSV genes are transcribed in their 3' to 5' order from a single promoter near the 3' end Thus, RSV synthesis 00 appears to follow the single entry, sequential transcription model proposed for all Mononegavirales (16,81). According to this model, the polymerase (L) O contacts genomic RNA in the nucleocapsid form at the 3' C< genomic promoter region and begins transcription at the first nucleotide. RSV mRNAs are co-linear copies of the genes, with no evidence of mRNA editing or splicing.

Sequence analysis of intracellular RSV mRNAs showed that synthesis of each transcript begins at the first nucleotide of the gene start signal The end of the mRNAs are capped with the structure m7G(5')ppp(5')Gp (where the underlined G is the first template nucleotide of the mRNA) and the mRNAs are polyadenylated at their 3' ends Both of these modifications are thought to be made cotranscriptionally by the viral polymerase. Three regions of the RSV 3' genomic promoter have been found to be important as cis acting elements These regions are the first ten nucleotides (presumably acting as a promoter), nucleotides 21-25, and the gene start signal located at nucleotides 45-53 Unlike other Paramyxovirinae, such as measles, Sendai and PIV- 3, the remainder of the leader and non-coding region of NS1 gene of RSV was found to be highly tolerant of insertions, deletions and substitutions (83).

Additionally, by saturation mutagenesis (wherein each base is replaced independently by each of the other three bases and compared for translation and replication efficiencies) within the first 12 nucleotides of the 3' genomic promoter region, a Utract located at nucleotides 6-10 was shown to be S- 44 0 highly inhibitory to substitutions In contrast, the first five nucleotides were relatively tolerant of a number of substitutions and two of them at position 00 four were up-regulatory mutations, resulting in a four- 5 to 20-fold increase in RSV-CAT RNA replication and transcription. Using a bi-cistronic minireplicon system, gene-start and gene-end motifs were shown to be C signals for mRNA synthesis and appear to be self contained and largely independent of the nature of adjoining sequence (84).

The L gene start signal lies 68 nucleotides upstream of the M2 gene-end signal, resulting in gene overlap (Fig. 3) The presence of the M2 gene-end signal within the L gene results in a high frequency of premature termination of L gene transcripts. Full length L mRNA is much less abundant and is made when the polymerase fails to recognize the M2 gene-end motif. This results in much lower transcription of L mRNA. The gene overlap seems incompatible with a model of linear sequential transcription. It is not known whether the polymerase that exits the M2 gene jumps backward to the L gene-start signal or whether there is a second, internal promoter for L gene transcription It is also possible that the L gene is accessible by a small fraction of polymerases that fail to start transcription at the M2 gene-start signal and slide down the M2 gene to the L gene-start signal.

The relative abundance of each RSV mRNA decreases with the distance of its gene from the promoter, presumably due to polymerase fall-off during sequential transcription Gene overlap is a second mechanism that reduces the synthesis of full length L mRNA. Also, certain mRNAs have features that might reduce the efficiency of translation. The initiation codon for SH mRNA is in a suboptimal Kozak C 0 Ssequence context, while the G ORF begins at the second methionyl codon in the mRNA.

RSV RNA replication is thought (74) to follow the model proposed from studies with vesicular S 5 stomatitis virus and Sendai virus (16,81). This C- involves a switch from the stop-start mode of mRNA O synthesis to an antiterminator read-through mode. This results in synthesis of positive sense replicationintermediate (RI) RNA that is an exact complementary copy of genomic RNA. This serves in turn as the template for the synthesis of progeny genomes. The mechanism involved in the switch to the antiterminator mode is proposed to involve cotranscriptional encapsidation of the nascent RNA by N protein (16,81).

RNA replication in RSV like other nonsegmented negative-strand RNA viruses is dependent on ongoing protein synthesis Predicted RI RNA has been detected for the standard virus as well as RSV-CAT minigenome (74,85). RI RNA was 10-20 fold less abundant intracellularly than was the progeny genome both for the standard and the minigenome system. The nucleotide sequences (in positive strand, antigenomic, message sense) of various wild-type, vaccine and revertant RSV strains, as well as the deduced amino acid sequences of the RNA polymerase (L protein) of these RSV viruses, are set forth as follows with reference to the appropriate SEQ ID NOS. contained herein: 46 Virus Wild-Type 2B 18537 Vaccine 2B33F 2B20L Revertant 2B33F TS(+) 2B20L TS(+) Nucleotide Sequence SEQ ID NO:23 SEQ ID NO:25 SEQ ID NO:27 SEQ ID NO:29 L Protein Sequence SEQ ID NO:24 SEQ ID NO:26 SEQ ID NO:28 SEQ ID SEQ ID NO:31 SEQ ID NO:33 SEQ ID NO:32 SEQ ID NO:34 Each RSV virus genome that is 2,166 amino acids long.

other nucleotide information is encodes an L protein Genome length and as follows: Virus Wild-Type 2B 18537 Vaccine 2B33F 2B20L Revertant 2B33F TS(+) 2B20L TS(+) Genome Length 15218 15229 15219 15219 15219 15219 L Start Codon 8502-8504 8509-8511 8503-8505 8503-8505 8503-8505 8503-8505 L Stop Codon 15000-15002 15007-15009 15001-15003 15001-15003 15001-15003 15001-15003 As detailed in Example 3 (especially Tables 7 and 8) below, the key attenuating mutations for the RSV subgroup B 3' genomic promoter region are nucleotide 4 (C and the insertion of an additional A in the stretch of A's at nucleotides 6-11 (in antigenomic 0 0 c- 47 0 message sense). As also detailed in Example 3 below, the key potentially attenuating sites for the L protein of RSV are as follows: amino acid residues 353 00 (arginine lysine), 451 (lysine arginine), 1229 n 5 (aspartic acid asparagine), 2029 (threonine isoleucine) and 2050 (asparagine aspartic acid). It is understood that the nucleotide changes responsible c-I for these amino acid changes are not limited to those set forth in Example 3 below; all changes in nucleotides which result in codons which are translated into these amino acids are within the scope of this invention.

The attenuated viruses of this invention exhibit a substantial reduction of virulence compared to wild-type viruses which infect human and animal hosts. The extent of attenuation is such that symptoms of infection will not arise in most immunized individuals, but the virus will retain sufficient replication competence to be infectious in and elicit the desired immune response profile in the vaccinee.

The attenuated viruses of this invention may be used to formulate a vaccine. To do so, the attenuated virus is adjusted to an appropriate concentration and formulated with any suitable vaccine adjuvant, diluent or carrier. Physiologically acceptable media may be used as carriers. These include, but are not limited to: an appropriate isotonic medium, phosphate buffered saline and the like. Suitable adjuvants include, but are not limited to MPL TM (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamilton, MT) and IL-12 (Genetics Institute, Cambridge, MA).

In one embodiment of this invention, the formulation including the attenuated virus is intended for use as a vaccine. The attenuated virus may be mixed C 48 with cryoprotective additives or stabilizers such as proteins albumin, gelatin), sugars sucrose, lactose, sorbitol), amino acids sodium Sglutamate), saline, or other protective agents. This 5 mixture is maintained in a liquid state, or is then C dessicated or lyophilized for transport and storage and 0 mixed with water immediately prior to administration.

Cy Formulations comprising the attenuated viruses of this invention are useful to immunize a human or animal subject to induce protection against infection by the wild-type counterpart of the attenuated virus. Thus, this invention further provides a method of immunizing a subject to induce protection against infection by an RNA virus of the Order Mononegavirales by administering to the subject an effective immunizing amount of a vaccine formulation incorporating an attenuated version of that virus as described hereinabove.

A sufficient amount of the vaccine in an appropriate number of doses must be administered to the subject to elicit an immune response. Persons skilled in the art will readily be able to determine such amounts and dosages. Administration may be by any conventional effective form, such as intranasally, parenterally, orally, or topically applied to any mucosal surface such as intranasal, oral, eye, vaginal or rectal surface, such as by an aerosol spray. The preferred means of administration is by intranasal administration.

In another embodiment of this invention, an isolated nucleic acid molecule having the complete viral nucleotide sequence of either the wild-type viruses or vaccine viruses described herein is used to generate oligonucleotide probes (from either positive strand antigenomic message sense or negative strand c 49 0 complementary genomic sense) and to express peptides (from positive strand antigenomic message sense only), which are used to detect the presence of those wild- 0 type virus and/or vaccine strains in samples of body fluids and tissues. The nucleotide sequences are used C to design highly specific and sensitive diagnostic 0 tests to detect the presence of the virus in a sample.

C- Polymerase chain reaction (PCR) primers are synthesized with sequences based on the viral wild-type or vaccine sequences described herein. The test sample is subjected to reverse transcription of RNA, followed by PCR amplification of selected cDNA regions corresponding to the nucleotide sequence described herein which have nucleotides which are distinct for a defined strain of virus. Amplified PCR products are identified on gels and their specificity confirmed by hybridization with specific nucleotide probes.

ELISA tests are used to detect the presence of antigens of the wild-type or vaccine viral strains.

Peptides are designed and selected to contain one or more distinct residues based on the wild-type or vaccine sequences described herein. These peptides are then coupled to a hapten keyhole limpet hemocyanin (KLH) and used to immunize animals rabbits) for the production of monospecific polyclonal antibody. A selection of these polyclonal antibodies, or a combination of polyclonal and monoclonal antibodies can then be used in a "capture ELISA" to detect antigens produced by those viruses.

Samples of the Moraten measles virus vaccine strain were deposited by Applicants with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, under the provisions of the Budapest Treaty for the Deposit of Microorganisms for the Purposes of Patent Procedures 50 0 S("Budapest Treaty") and have been assigned ATCC accession number VR2587. Samples of the HPIV-3 virus Vero-grown cp45 vaccine strain were deposited by 00 Applicants with the American Type Culture Collection, S 5 12301 Parklawn Drive, Rockville, Maryland 20852, under the provisions of the Budapest Treaty and Shave been assigned ATCC accession number VR2588.

CI Samples of the 2B wild-type RSV virus were deposited by Applicants with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, under the provisions of the Budapest Treaty and have been assigned ATCC accession number VR2586.

Given these three deposited strains and the sequence information for these and other strains provided herein, one can use site-directed mutagenesis and rescue techniques described above to introduce mutations (or restore a wild-type genotype) of all the strains described herein, as well as taking these strains and making additional mutations from the panel of mutations set forth in Tables 3, 4 and 6-8 below.

In order that this invention may be better understood, the following examples are set forth. The examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention.

Examples Standard molecular biology techniques are utilized according to the protocols described in Sambrook et al. (86).

S-51- Example 1 Measles 00 Moraten MV vaccine virus was grown once, S 5 directly from the Attenuvax TM vaccine vial (Lot #0716B), Sthe Schwarz vaccine virus was grown once (Lot 96G04/M179 G41D), while the Zagreb and Rubeovax m C-I vaccine viruses were each grown twice in the Vero cells before RNAs were made for sequence analysis.

MV

wildtype isolate Montefiore (56) was passed 5-6 times in Vero cells before extraction of RNA materials and similarly, MV wildtype isolates 1977, 1983 (14) were grown 5-7 times before extracting materials for analysis. Edmonston wild-type isolate received from Dr. J. Beeler (CBER) (see Fig. 1) was the original Edmonston isolate already passaged seven times in human kidney cells and three times in Vero cells before receipt and further passaged once in Vero cells before using for sequence analysis.

RNA was prepared by infecting Vero cells at a multiplicity of infection of 0.1 to 1.0 and allowed to reach maximum cytopathology before being harvested. Total RNA from measles virus-infected cells was extracted using TrizolM reagent (Gibco-BRL).

The total RNA isolated from Vero cell passage material was amplified by the Reverse Transcriptase-PCR (Perkin-Elmer/Cetus) procedure using measles (Edmonston B strain specific primer pairs spanning the 3' and 5' promoter regions and the L gene of the viral genome. Table 2 presents these primer sequences. The primers of SEQ ID NOS:35-54, 74, 77 and 78 are in antigenomic message sense. The primers of SEQ ID NOS:55-73, 75, 76 and 79 are in genomic negative-sense.

c-I 52 Table 2 Primers for PCR and Sequencing MV L Genes and Genomic Termini 00 M 5 9 0 4 7

CATATCACTCACTCTGGGATGGAG

9 0 7 0 (SEQ ID 9 3 7 1

TCAGAACATCAAGCACCGCC,

3 9 0 (SEQ ID NO:36) 9 7 4 1

ACAGTCAAGACTGAGATGAG

7 6 0 (SEQ ID NO:37) 1 0 0 0 1

AAGAGTCAGATACATGTGGA

1 0 0 2 0 (SEQ ID NO:38) 1 0 3 5 1

ACATGAATCAGCCTAAAGTC

1 0 3 7 0 (SEQ ID NO:39) 10 6 7 4 CCGAAAGAGTTCCTGCGTTACGACC1 6 9 8 (SEQ ID 1 1 1 3

CAGTCCACACAAGTACCAGG

1 1 1 0 2 (SEQ ID NO:41) 1 4 lGTCAGAAGCTGTGGACCATC 1 4 0 (SEQ ID NO:42) 118 4 AATATTGCTACAACAATGGC11860 (SEQ ID NO:43) 2

,,,ACTCTTCATTCCTAGACTGG

1 2 2 (SEQ ID NO :44) 1 2 5 4 2

GTCCAATTATGACTATGAAC

1 2 5 6 1 (SEQ ID 1 2 9

AGAACAGACATGAAGCTTGC

1 2 9 1 1 (SEQ ID NO :46) 32 32

CCAACAAGGAATGCTTCTAG

13 2 (SEQ ID NO:47) 1 3 5 5 1

ACAGCACTATCTATGATTGACCTGG

3 5 7 5 (SEQ ID NO:48) 1 3 9 3

GCAACATGGTTTACACATGC

1 3 9 4 9 (SEQ ID NO:49) 1 4 2 8 0

AGATTGAGAGTTGATCCAGG

1 4 2 9 9 (SEQ ID 14 6 2

,AGGAGATACTTAAACTAAGC,

4 64 (SEQ ID NO:51) 1 4 9 8 1

TAAGCTTATGCCTTTCAGCG

1 5 0 0 0 (SEQ ID NO:52) lS 33 7 TTAACGGACCTAAGCTGTGC 1 5 3 5 6 (SEQ ID NO:53) 15 67 1

GAAACAGATTATTATGACGG

1 56 90 (SEQ ID NO:54) 9 2 9

CGGGCTATCTAGGTGAACTTCAGG

2 7 (SEQ ID NO: 9 500ATTTGGATATGGAATATGAG 94 1 1 (SEQ ID NO: 56) 9 8 4

ACTCAACTGAACTACCAGTG

9 2 1 (SEQ ID NO:57) l,,lAAGACATCATGTATTTCAG 0 1 62 (SEQ ID NO:58) 1 0 5 4 9

TTATCAACGCACTGCTCATG

1 0 5 3 0 (SEQ ID NO:59) log 1 9

ATTTTCAGCAATCACTTGGCATGCC

0 8 9 5 (SEQ ID 1 1 2

GCCTCTGTGCAAACAAGCTG

1 1 2 6 1 (SEQ ID NO:61) 1 16 3 9TCTCTAGTTACTCTAGCAGC 1 1 9 (SEQ ID NO:62) 12 0 1

AGGTCGTTGTTTGTGAGGAG

1 1 99 (SEQ ID NO:63) 1 2 3 6 1

TCGTCCTCTTCTTTACTGTC

1 2 3 4 2 (SEQ ID NO:64) c-I 53 1 2 6 9

CCGTCCTCGAGCTAGCCTCG

1267 0 (SEQ ID NO: 1 305 2

CTCCTCCAGGCTCACATTGG

13 33 (SEQ ID NO:66) 13 42 GGGTTGGTACATAGCTCTGC130 (SEQ ID NO:67) 00 13 7 CACCCATCTGATATTTCCCTGATGG134 (SEQ ID NO:68) S 5 14 9

TGGTTGACAGTACAAATCTG

1 48 (SEQ ID NO:69) 144

OCTGAAATGGGAAGATTGTGC

1444 1 (SEQ ID 14 82

AGCAATCTACACTGCCTACC

1 4 80 (SEQ ID NO:71) 1

S

1 8

TCACAGATGATTCAATTATC

15 1 6 (SEQ ID NO:72) 1 5530GATCCTAGATATAAGTTCTC 5 5 1 1 (SEQ ID NO:73) 1 ACCAAACAAAGTTGGGTAGG2 (SEQ ID NO:74) GGGGGATCClDOATCCCTATCCTGCTCTTGTCCC8 (SEQ ID NO: 200

GATTCCTCTGATGGCTCCAC

1 1 (SEQ ID NO: 76) 1 5 7 2 TAACAGTCAAGGAGACCmAG17 (E ID NO:77) GGGAAGCTT,58OAACCCTAATCCTGCCCTAGGTGG152 (SEQ ID NO:78) 1 58 4 ACCAGACAAAGCTGGGAATAGA157 (SEQ ID NO:.79) Overlapping PCR fragments of the complete viral genome were directly sequenced without cloning to achieve the consensus sequence, by the dideoxy terminator cycle sequencing method using both strands (ABI PRISM 377 sequencer and ABI PRISM sequencing Kit).

To determine the sequence at the absolute termini, a ligation procedure described previously was used To test this hypothesis, the nucleotide sequences were determined for the non-protein coding regulatory regions and the L gene of the progenitor Ednionston wild-type MV isolate, for the available vaccine strains derived from this isolate, as well as for other wild-type strains. Nucleotide (in antigenomic, message sense) and amino acid differences were then compared and aligned as set forth in Tables (differences are in italics): 54 Table 3 Differences in MV 3' Genomic Promoter Region Nucleotide Sequence Virus Edmonston w-t Nucleotide number: 26 42 50 96 A A G G Vaccines: Rubeovax

M

Moraten Schwarz Zagreb

AIIC-C

T C T C T C T T T C G G G G G G G A G G A G A G A G Wild-Types: 1977 1983 Montefiore A A A A A A Table 4 Differences in MV L Nucleotides and Amino Acids Between Edmonston Wild-Type and Vaccine Strains 00 M 331 1409 1624 1649 1717 1887 1936 2074 2114 Edmonston w-t ATT GCA ACC AGG GAT AAC CAT CAA AGA c-IMutation ACT ACA GCC ATG GCT GAC TAT CGA AAA Edmonston w-t I A T R D N H Q R Rubeovax' m vac. I A T K A D H Q R Moraten vac. T A T M A D H Q K Schwarz vac. T A T x A D H Q K Zagreb vac. I T T R A N H Q R AIK-C vac. I T A R A N y R R 2004237877 Table Differences in MV L Nucleotides and Amino Acids Between Wild-Type Strains 10 Dec 2004 81 122 Edmonston w-t GCC GAT Mutation ACC AAT Edmonston w-t A D 1977 w-t. A N 1983 w-t T D Montefiore w-t A D 149

GTT

ATT

V

V

I

I

623

AGG

AAG

R

R

K

R

252

ACA

GCA

T

T

T

A

626

AGA

AAA

R

R

R

K

331

ATT

GTT

I

V

I

I

628

GCA

GAA

A

A

A

E

441

AA

AGA

K

K

K

R

632

ATA

GTA

I

447

AAA

AGA

K

K

K

R

636

CAA

CAT

Q

Q

H

H

500

GAT

AAT

D

D

N

D

637

GTA

ATA

V

I

V

V

513

GTG

ATG

V

M

M

M

641

GAC

AAT

D

D

D

N

570

AAA

AAT

K

K

N

K

645

GAT

AAT

D

N

D

D

613

TAC

CAC

y y

H

y 650

ATG

ATA

M

M

M

I

618 Edmonston w-t GTC Mutation GCC Ednionston w-t V 1977 w-t A 1983 w-t V Montefiore w-t V 621

AGT

AAT

S

N

S

S

2004237877 10 Dec 2004 652 Edmonston w-t GCT Mutation ACC Edmonston w-t A 1977 w-t A 1983 w-t A Montefiore w-t T Table 5 (continued) Differences in MV L Nucleotides and Amino Acids Between Wild-Type Strains 720 723 794 914 970 1044 1294 1569 1705 1745 ATC TAT CGG CGG GCC GGA AGC GTT ATC AAT GTC TGC TGG CAG TCA AGA ACC ATT GTC AGT I Y R R A G S V I N I C W Q A G S V I N V C R R S G T I I N V C R R A R S V V S 1860 1865 1936 2007 2013 2017 2030 2096 2119 2165 Edmonston w-t GTA TTC CAT GAC GAT ACT AAT ATA AAG GTC Mutation ATA TAC TAT GGC GGT ATT AGT GTA CGG ATC Edmonston w-t V F H D D T N I K V 1977 w-t V Y H D D T N I K V 1983 w-t V F Y D G I N I R I Montefiore w-t I F H G D I S V R V S- 58 SExample 2 PIV-3 00 A comparison of sequences (in antigenomic c 5 message sense) of the parental wild-type JS strain of PIV-3 virus and the FRhL-grown and Vero-grown forms of the cp45 mutant are set forth in Table 6. Where a codon change does not result in an amino acid change, Table 6 states "none", followed by the name of the unchanged amino acid.

2004237877 10 Dec 2004 Table 6 of Vero- and FRhL-grown cp45 JS Sequence Comparison strains Gene Region Nucleotide is FRhL Vero Codon Change Am~i -no Acid Change Position cp45 cp45 (number in L) 3' leader 23 T C C 24 C T T 28 G T T T A A NP UTR 62 A T T NP coding 397 T C C GTC -+GCC Val -*Ala 1275 T G G TCT-*GCT Ser-*Ala P coding 2080 T C C AAT -*AAC none/Asn M coding 4347 C A A CCC -+ACC Pro Thr F coding 5536 C T T AAC -+AAT none/Asn 6329 A G G ATA-*GTA Ile-+Val 6419 G A A GCA-+ACA Ala-*Thr HN coding 6847 T C C GGT -*GGC none/Gly 7956 T C C GTT-4GCT Val *Ala L coding 9323 T C C TAT -4TAC none/Tyr (226) 9971 A G G GAA- GAG none/Glu. (442) 11469 T C C TAC -*CAC Tyr -*His (942) 11621 G T T TTG -*TTT Leu,- Phe (992) 12521 A A T* TTA -+TTT Leu -4 Phe (1292) 12581 C T T TTC ->TTT none/Phe (1312) 13318 C T. T ACT -4ATT Thr Ile (1558) mutations 20 21 0 6 SSequence analysis of the parental wild-type JS strain of PIV-3 virus and the FRhL-grown cp45 mutant showed that the latter contained 20 nucleotide changes.

00 Four changes were in the noncoding 3'-leader region at S 5 nucleotide positions 23 (T 24 (C 28 (G ST) and 45 (T A) (in antigenomic, message sense).

SWhen considered in the genomic, negative sense, the change at position 28 from the smaller pyrimidine to the larger purine may change the size of the region flanked by the conserved regions of the 3' genomic promoter region, resulting in an altered spatial presentation of the cis-acting signals to the polymerase.

Nine changes were coding changes in the NP, M, F, HN and L genes. The other seven changes were non-coding or silent changes in the NP, P, F, HN and L genes or the NP untranslated region (UTR). The mutant has been demonstrated to have poor transcription activity at non-permissive temperatures due to its ts phenotype This ts phenotype has now been mapped to the viral L gene Because the cp45 virus has been shown to function normally with regard to mutations in the HN and F glycoproteins this supports the implication that mutations in the 3'leader and L gene contributed to the attenuating phenotype of this virus.

Thus, the four 3' leader specific changes in FRhL-grown cp45 and the three coding changes in the L gene at amino acid positions 942 (Tyr 992 (Leu ->Phe) and 1558 (Thr ->Ile) contributed significantly to the attenuation phenotype of the candidate vaccine strain.

Furthermore, the Vero-grown cp45 mutant vaccine strain contains an additional mutation resulting from a coding change in the L gene (marked 61 0 with an asterisk in Table 6) at amino acid residue 1292 (leucine phenylalanine).

The first two amino acid changes in the L

OO

0 protein (at positions 942 and 992) map to one of the r 5 highly conserved areas among all Paramyxovirus L genes.

The fourth amino acid change (at position 1558) maps to 0 the area joining two conserved blocks corresponding to the change at amino acid 1717 in the MV vaccine strains.

The published literature (89) sets forth only 18 changes between the antigenomic message sense sequences of the JS and FRhL-grown cp45 strains.

Sixteen of these changes were found by applicants.

The published literature did not report four changes found by applicants: in the 3' leader at nucleotide 45 (T in the NP UTR at nucleotide 62 (A or the changes in amino acids in the NP protein resulting from the changes at nucleotide 397 (T leading to the amino acid change (Val ->Ala) and nucleotide 1275 (T leading to the amino acid change (Ser -+Ala) (nucleotide changes in antigenomic, message sense). Nor did the published literature report the additional potentially attenuating mutation in the L protein found by applicants in the Vero-grown cp45 strain resulting from the change at nucleotide 12521 (A leading to the change in amino acid 1292 (Leu -+Phe).

62 ^1- 0 Example 3 RSV Subgroup B 00 The temperature-sensitive (ts) phenotype is m 5 strongly associated with attenuation in vivo; in addition, some non-ts mutations may also be attenuating. Identification of ts and non-ts attenuating mutations was achieved by sequence analysis and evaluation of ts, cold-adapted and in vivo growth phenotypes of RSV mutants and revertants.

The genomes of the following five RSV 2B strains have now been completely sequenced: 2B parent, 2B33F, one revertant designated 2B33F 2B20L and one revertant designated 2B20L The 2B33F and 2B20L strains are ts and ca and are described in U.S.

Serial No. 08/059,444 which is hereby incorporated by reference. After identifying regions where mutations in 2B33F and 2B20L are located, nine additional isolates of 2B33F "revertants" obtained following in vitro passaging at 39 0 C and in vivo passaging in African Green Monkeys or chimpanzees, and nine additional isolates of 2B20L "revertants" obtained following in vitro passaging at 39 0 C have been sequenced in those regions. The ts, ca, and attenuation phenotypes of many of these revertants have now been characterized and assessed. Correlations between phenotype ts, vaccine attenuation and sequence changes have been identified.

A summary of results is presented in Tables 7-12.

63 Table 7 Sequence comparison between RSV 2B and 2B33F strains

I

Nuci.

pos. t Nucleotide changes Gene/ 3' end RSV 2B RSV RSV 2B33F Amino acid region of vRNA 2B33F 5a changes revertant Genomic 4 C G G non-coding Promoter 6 extra A extra A non-coding M 4175 T C C non-coding 4199 T C C non-coding SH 4329 T C C Phe-Leu 4409 T C C none Ile (36) 4420 T C C Ile-Thr 4442 T C C none His (47) 4454 T C C none Cys (51) 4484 T C C none Tyr (61) 4497 T C C Stop-Gin (66) 4505 T C C none Ser (68) 4525 T C C Ile-Thr 4526 T C C Ile-Thr 4542 T C C Stop-Gin (81) 4561 T C C Leu-Pro (87) 4575 T C C Trp-Arg (92) 4598 T C C none Thr (99) L 9559 G A A Arg-Lys (353) 9853* A G A Lys-Arg (451)* 12186 G A A Asp-Asn (1229) 14587 C T T Thr-Ile (2029) 15071 A G G non-coding For 2B33F and 2B33F TS(i), nuci. pos. numibers are one larger than for 2B for M, SH L genes At pos. 9853, the Lys-Arg change has reverted back to Lys in the 2B33F strain 64 Table 8 Sequence comparison between RSV 2B and 2B20L strains Nuci.

pos. t Nucleotide changes Gene/ 3' end RSV 2B RSV RSV 2B20L Amino acid region of vRNA 2B20L RI changes revertant Genomic 4 C G G non-coding* Promoter 6 extra A extra A non-coding* L 8 963 C T T none Thr (154) 13347 A A G Asn-Asp (1616) 14587 C T T Thr-Ile(2029)* 14649 A G G Asn-Asp (2050) 14650 A A T Asn-Asp-Val 1 (2050) For 2B20L and 2B20L nuci. pos. numbers are one larger than for 2B for L gene Mutation is common in 2B33F and 2B20L strains At pos. 14650, the mutation suppresses the ts phenotype in 2B20L revertant 2004237877 10 Dec 2004 Table 9 RSV 2B, ts and Revertant Strains Sample Source In Vitro Phenotype In Vivo Growth* tsca Cotton Rat AGM 39/320C EOP 20/32 0 C Nasal Lungs Nasal Bronchial plaque morph Yield turbinates Wash Lavage RSV 2B Wild-type Parent 0.7 0.0001 5.58 5. 8a 5.86 4.7e Strain (WT) 3 9 b 5 2 b (4/4) (4/4) RSV 2B33F ca, ts mutant isolated 0.00007 0.04 1.6 a <1.58 3.00 <0.9.

from 2B cold-passaged (sp/int/wt) 1 9 b 1 2 b (0/4) RS B3 ax 33 e SV B3F S 2B33F spinner passage, 0.5 0.03 51.7' 3.5a 4.2w T()plaque picked at 39'C (WTr) (4/4) RSV 2B33F 4a 2B33F spinner passage, 0.7 0.01 <1.76a 3 .58 NDl ND TS(+ plaque picked at 39'C (WT) (4/4) RSV 2B33F 3b 2B33F spinner passage, 0.5 0.04 92.56 2.9a ND NDl TS(+ plaque picked at 39*C (4/4) AGM pp2 2B33F-infected AGM 0.3 0.00002 :5 2 Ob 16 D N #A2,d7 nasal wash (sp,int) (4/4) plaque picked at 32*C 2004237877 10 Dec 2004 Table 9 (continued) RSV 2B, ts and Revertant Strains Sample Source In Vitera Phenotype In Vivo Growth* tsca Cotton Rat AGM 39/32 0 C EOP 20/32 0 C Nasal Lungs Nasal Bronchial plaque morph Yield turbinates Wash Lavage AGM pp4 2B33F-infected AGM 0.1 0.008 1 6 b1.b NDD #A2,d7 nasal wash (sp,int) (4/4) plaque picked at 32*C AGM pp6 2B33F-infected AGM 0.000004 50.00005 51.5b 1 1 b ND ND #A4,dl2 nasal wash (wt) (0/4) plaque picked at 32*C AGM pp7 2B33F-infected AGM 0.000004 0.007 4 b b ND N #A4,d12 nasal wash (sp/int/wt) (0/4) plaque picked at 32 0

C

Chimp pplA 2B33F-infected Chimp 0.5 ND ND ND NED ND #1552, d4 tracheal

(T

lavage

(T

plaque picked at 32'C Chimp pp3A 2B33F-infected Chimp 0.7 ND 2.4c -3.Oc ND ND #1560, d6 tracheal

(WT)

1 avage(44(3) plaque picked at 32'C chimp p-p5A 2B33F-infected Chimp 0.7 ND <-2.3c 3.0c ND ND #1563, dlO nasal swab (T plaque picked at 32*C (4/4) 2004237877 10 Dec 2004 Table 9 (continued) RSV 2B, ts and Revertant Strains 1 -nmpe Source In Vitro Phenotype In Vivo Growth* Cotton Rat AGM i I RSV 2B20L RSV 2B20L Ri TS (t) RSV 2B20L R2 TS RSV 2B20L R9 TS RSV 2B20L i TS ca, ts mutant isolated from 2B cold-passaged x 20 2B20L spinner passage, plaque picked at 39"C 2B20L spinner passage, plaque picked at 39*C 2B20L spinner passage, plaque picked at 39*C 2E20L spinner passage, plaque picked at 39 0

C

39/32 0 C EOP plaque morph 0. 0002 (int/wt) 0.6

(WT)

0.6

(WT)

0.8

(WT)

20/32-C Yield 0.02 Nasal turbinates (0/4) Lungs 1 3 d (0/4) NslBronchial Wash Lavage I(0/2) ND 12.3c 13.5- 1J1D tND (4/4) (4/4) ND f:52. 5C 12.7' IND IND (3/4) (4/4) I /4) LND 1:52.2c 14.0c 1 ND ND 0.7

ND

(WT)

(3/4) 2. 6- (4/4) (4/4) 3.20 ND

N

(4/4) L I. I I In Vivo growth measured in log,, mean virus t ND not done WT wild-type plaque size a Dose .106.7 PFU IN D08b1"PUI d Dose i= n 159pFU IN *Dose 106.6 PFU IN+IT iter infected/# total) sp small plaque size mnt intermediate plaque size cDose Z 106,3 PFU IN 2 Dose 106-0 PFU IN+IT 68 Table 2B33F Revertants Its Xn vi tro AGN Chimp a 4 a 3b jpp2 pp4 pp6 pp7I1A 3A base no.t

M

4176,4200 S S S S S S S S S S

SH

14 bases* s S S S S S S S S S

L

9560 S S S S S S S S S S 9854 2B 2B 2B 2B s S S ND 2B 2B 12187 S S S S S S S S S SI 14588 5 S S S S S S ND S S 15072 S S S S S S S S S S Phenotype ts 2B 2B 2B1 r r S S 12B 2B 2B caSS S 2B S 2B S ND ND ND Attenuated Jr r rJ S S JND r r t These 2E33F revertant base nos. are one larger than for 2B for M, SH and L genes *bases 4330,4410,4421,4443,4455,4485,44984506,45264527,4543, 4562,4576,4599 S samte base as 2B33F 2B reversion to 2B base or complete reversion in phenotype r moderate reversion in phenotype slight reversion in phenotype ND not done 69 Table 11 2B20L Revertants In vitro Isolates base no.t Rl R2 R3A R4A R5A R6A R7A RSA R9A R1OA

L

8964 S S S S S S S S S S 13348 C* S ND S S ND S S S S 14588 S S S S S S S S S S 14650 S S 2B S 2B 2B S S 2B 2B 14651 A* A* S A* S S A* A* S S Phenotype ts 2B 2B ND ND ND ND ND ND 2B 2B Attenuated r r ND ND ND ND ND ND r r t These 2B20L revertant base genes S =same base as 2B20L 2B =reversion to 2B base nos. are one larger than for 2B for L r moderate reversion in phenotype base change, different from 2B or 2B20L ND not done 70 Table 12 RSV 2B, ts and Revertant Strains: Phenotype Sunumary Virus Isolate Source In V!itro in Vi va Phenotype Attenuation ts ca Cotton AGM Rat RSV 2B IWild-type Parent Strain j- I I I RSV 2B33F ca, ts mutant isolated from 2B, cold-passaged x 331 RSV 2B33F 5a 2B33F spinner passage plaque picked at 39*C RSV 2B33F 4a 2B33F spinner passage

ND

TS plaque picked at 39'C RSV 2B33F 3b 2B33F spinner passage

ND

plaque picked at 39*C AGM pp2 2B33F-infected AGM A2, ND d7 nasal wash plaque picked at 32 0

C

AGM pp4 2B33F-infected AGM A2, ND d7 nasal wash plaque picked at 32*C AGM pp6 2B33F-infected AGM A4, ND d12 nasal wash plaque picked at 32*C AGM pp7 2B33F-infected AGM A4, ND d12 nasal wash plaque picked at 32*C Chimp pplA 2B33F-infected chim ND ND ND #1552, d4 tracheal lavage, plaque picked at 32 0

C

Chimp pp3A 2B33F-infected chimp ND N #1560, d6 tracheal lavage, plaque picked at 32*C Chimp pp5A 2B33F-infected chimp ND ND #1563, dlO tracheal lavage, plaque picked at 32 0

C

71 RSV 2B, Table 12 (continued) ts and Revertant Strains: Phenotype Summary Virus Isolate Source In Vitro In Viva Phenotype Attenuation ts ca Cotton AGM RSV 2E20L ca, ts mutant isolated from 2B, cold-passaged x RSV 2B20L Ri 2B20L spinner passage ND

ND

plaque picked at 39 0

C

RSV 2B20L R2 2B20L spinner passage ND

ND

plaque picked at 39 0

C

IRSV 2B20L R9 2B20L spinner passage ND

ND

plaque picked at 39 0

C

RSV 2B20L R10 2B20L spinner passage ND

ND

plaque picked at 39'C ND =not done -=wild-type phenotype, not temperature sensitive, not cold adapted, not attenuated +o to increasing levels of temperature sensitivity, coldadaptation or attenuation 0 CM 72- 0 Several significant observations can be drawn from these data: 00 a. As shown in Tables 7 (for 2B33F) and 8 (for 2B20L), there are relatively few sequence changes identified in the two mutant strains: RSV 2B33F differs from parental RSV 2B by two changes at the 3' C genomic promoter region, two changes at the non-coding of the M gene, and four coding changes plus one non-coding (poly(A) motif) change in the RNA dependent RNA polymerase coding L gene. In addition, 14 changes mapped to the SH gene alone. RSV 2B20L differs from its RSV 2B parent only at seven nucleotide positions, of which three are common with 2B33F virus, including two changes at the 3' genomic promoter and one coding change in the L gene. Two additional unique changes of 2B20L virus mapped to the coding region of the L gene.

Potentially attenuating mutations at the non-coding 3' genomic promoter region and the RNA dependent RNA polymerase gene have been identified.

b. Two ts mutations can be identified in the L gene of the attenuated virus strains 2B33F and 2B20L: In 2B33F, a mutation at nucleotide position 9853 (A leading to a coding change in L protein at amino acid 451 (Lys ->Arg) is clearly associated with the ts and attenuation phenotypes. Reversion at this site alone in the 2B33F 5a strain is responsible for complete restoration of growth at 39°C (Table 9) and partial reversion in attenuation in animals. This association with the ts and attenuation phenotypes was also supported by partial sequence analyses of six additional "full TS revertants" (designated 4a, 3b, pp2, 3A, 5a, 5A) isolated from cell 73 0 culture and from chimps, in which only the nucleotide 9853 mutation reverted (Tables 10-12) (note that one 0 AGM (African Green Monkey) isolate which reverted at 00 9853 only partially reverted in ts phenotype). This C 5 amino acid 451 mutation (Lys is amenable to stabilization in cDNA infectious clone constructs, by inserting a second mutation to stabilize the codon, C-i thereby lessening the likelihood that it will revert back to Lys.

(ii) In 2B20L, a mutation at base 14,649 (A leading to a coding change in the L protein (amino acid position 2,050, Asn ->Asp) appears to be associated with the ts and attenuation phenotypes. This aspartic acid at the amino acid 2050 invariably reverts back (Asp ->Asn) in revertants or changes to a different amino acid (Asp Val) by nucleotide substitution at position 14,650 (A (Tables 8, 11). The above observation is based on complete sequence analysis on the revertant R1 and partial sequence of several additional revertants (R2, R4A, R7A, R8A) at selected regions (Table 11). An additional mutation is seen in the R1 revertant at nucleotide postion 13,347 (amino acid 1616, Asn Asp) associated with the above reversion. However, the effect of this mutation on the ts phenotype is not known; the L gene of other revertants has not been sequenced completely.

c. Three base changes are common to 2B33F and 2B20L strains of virus: A change at position 14,587 (C T) with a corresponding change (Thr lie) at amino acid 2029 is c- -74 present in both 2B33F and 2B20L (Tables This nucleotide substitution was found to be present in of the population of the progenitor RSV2B strain 00 and may have been preferred during the attenuation process. No wildtype base was found in the 2B33F C and 2B20L virus.

C (ii) Two mutations are seen in the 2B33F and 2B20L 3' genomic promoter region: nucleotide 4 (C and the insertion of an extra A in the stretch of A's at positions 6-11 (in antigenomic, message sense). When the sequences of selected revertants were analyzed, these mutations were seen to have been retained in the 2B33F TS(+)5a (Table 7) and the 2B20L TS(+)R1 (Table 8) revertants. These non-coding, cisacting mutations remained associated with partial viral attenuation.

Expression using the minireplicon RSV-CAT system for the analysis of these cis-acting changes has shown the 3' genomic promoter nucleotide 4 (C change to be an upregulation of transcription/replication in this in vitro system when the 2B progenitor virus or either of the 2B33F or 2B33F provided helper L gene functions (the N, P and M2 genes are identical in these viruses).

Complementation analysis of the 2B33F 3' genomic promoter and the helper functions provided by the progenitor RSV2B virus or the 2B33F and 2B33F TS(+) viruses by this RSV-CAT minireplicon system has also been conducted. All three viruses supported both the 2B and 2B33F 3' genomic promoter mediated transcription/replication functions. However, the 2B33F and 2B33F viruses preferred their 2B33F 3' genomic promoters. This analysis clearly shows coevolution of 3' genomic promoter changes during the S- 75 0 vaccine attenuation process, along with the RNA dependent RNA polymerase gene. Reversion of ts phenotype in the 2B33F mutant 5a by reversion of the 00 single L protein amino acid 451 (Arg ->Lys) by 5 sequence analysis was clearly demonstrated by support of transcription/replication functions of RSV-CAT minireplicon at 37°C. The 2B33F virus did not provide c helper functions to the RSV-CAT minireplicon (with 2B or 2B33F 3' genomic promoters) at 37°C.

d. A biased hypermutation of SH seen in 2B33F is present in all 2B33F revertants, regardless of phenotype, and is not seen in 2B20L, which is ts, ca, and attenuated. Thus, there are no data at this time that associate this mutation with any biological phenotype.

Another wild-type RSV designated 18537 was also sequenced and compared to the sequence of the wild-type RSV 2B strain. With one exception, at all the critical residues described above, the two wildtype strains were identical. For 2B, the codon ACA at nucleotides 14586-14588 encodes a Thr at amino acid 2029 of the L protein, while for 18537, the codon ATT at nucleotides 14593-14595 encodes an Ile at amino acid 2029 (the L gene start codon is at nucleotides 8509- 8511 in 18537, compared to 8502-8504 in 2B).

Example 4 PCR Assay to Detect Measles Virus A 21 year old patient was admitted to a hospital with a three week history of progressive nonproductive cough, shortness of breath, and fever. His symptoms failed to improve following treatment with clarithromycin for seven days or after a similar course c 76 0 of treatment with atovaquone. Concomitant complaints of right upper quadrant abdominal pain proved recalciltrant to omeprazole and antacids. Relevant 0 past medical history included Factor VIII deficiency and HIV infection diagnosed 3-4 years prior to this hospital admission. One year earlier, he had received O a booster immunization of measles-mumps-rubella

(MMR)

C- vaccine as required for college enrollment.

Bronchoalveolar lavage and transbronchial biopsies performed two days after admission to the hospital demonstrated reactive hyperplasia and alveolar lining cell desquamation with minimal chronic inflammation. No microorganisms were revealed by Gram, methenamine silver, or PAS stains. CT scans of the chest showed multiple, ill-defined, confluent nodules at the left lung base. Despite administration of empiric antimicrobials for opportunistic bacterial, mycobacterial, and fungal pathogens commonly responsible for pulmonary complications of advanced HIV disease, the patient became and remained febrile to 39 0 C. A left-sided pleural effusion developed; diagnostic thoracentesis showed it to be exudative but otherwise non-diagnostic. Bronchoalveolar lavage performed three weeks later only demonstrated alveolar histiocytes, some of which were hemosiderin laden, a few lymphocytes, and neutrophils. FITE, AFB, and methanamine silver stains again were negative.

Two weeks thereafter, a wedge resection of the left lung was performed through CT-guided minithoracotomy. Multiple tissue sections revealed nodular areas of acute and chronic inflammation with regions of necrosis and fibrosis. Numerous multinuclated giant cells were present, some of which contained both intracytoplasmic and intranuclear inclusions suggestive of measles virus giant cell 77 0 pneumonia. Special stains for bacteria, fungi, P.

carinii, and acid fast organisms again gave negative results. Electron microscopic examination of sections 00 of this lung biopsy revealed particles morphologically CM 5 consistent with paramyxoviruses such as measles virus.

SSerum anti-measles IgM titers determined by a solid Sphase hemadsorbant assay were negative, as was a c subsequent IgM capture immunoassay.

Two weeks later, Rhesus monkey kidney (RMK) tissue culture cells inoculated with the patient's lung biopsy material revealed cytopathic changes characteristic of measles virus infection.

Confirmation was obtained using an immunofluorescence assay with monoclonal antibodies directed to measles virus. Based upon this diagnosis, oral ribavirin 1000mg B.I.D. was given for 14 days. Unfortunately, the patient progressively deteriorated, eventually dying two months later.

In order to ascertain the nature of the measles virus present in the patient, reverse transcription and PCR amplification of virus obtained from infected tissues were performed, followed by sequence analysis. The measles virus isolated from Rhesus monkey kidney cells inoculated with tissue from this patient's lung biopsy was propagated by two serial passages in the continuous Vero (monkey kidney) tissue culture cell line. Total infected cell RNA was extracted at the second Vero cell passage using TRIzol reagent (Life Technologies, Grand Island, NY) according to the manufacturer's protocol. Total RNA was similarly extracted from the patient's lung biopsy material. The measles virus vaccine strain (Moraten) currently used in the United States as a component of the trivalent MMR vaccines, was obtained in its univalent form (Attenuvax

M

Merck, Sharpe, Dohme).

S- 78 0 This virus was passaged once in Vero cells and total vaccine infected cellular RNA then was extracted as described above.

00 Each of these RNA preparations was reverse 5 transcribed (RT) to cDNA using random hexameric primers Sand Maloney murine leukemia virus reverse transcriptase S:(Perkin-Elmer/Cetus RT-PCR kit reagents, Perkin-Elmer- CI Cetus, Branchburg, NJ). The cDNA then was amplified by PCR using measles virus-specific oligodeoxynucleotide primer pairs whose design was based on the Edmonston measles virus sequence described above. These PCR products comprised a set of overlapping DNA fragments spanning the entire 15,894 nucleotide long measles genome. A consensus genomic sequence was established by direct analysis of each PCR product, without cloning, using the dideoxy terminator cycle-sequencing method established by the manufacturer (ABI PRISM 377 sequencer and ABI PRISM DNA sequencing kit; Perkin- Elmer/Cetus, Foster City, CA). Both strands of the PCR-amplified DNA products were analyzed to eliminate possible sequencing ambiguities.

The nucleotide sequences of selected regions of the measles virus genomes present in the patient's viral isolate, as well as in the diseased lung tissue, were compared with that of the Moraten vaccine virus, as well as with the nucleotide sequences of other measles virus wild-type and vaccine strains. This sequence analysis revealed identity to the Moraten vaccine strain rather than demonstrating relatedness to past or currently circulating wild-type viruses or other measles vaccine strains.

0 79 Example ELISA to Detect RSV 00 An ELISA test is used to detect the presence c 5 of RSV. Peptides are designed and selected based on homologies to the RSV sequences described herein to be specific for all subgroup B strains, or for individual C\l wild-type, vaccine or revertant RSV subgroup B strains described herein. These peptides are then coupled to KLH and used to immunize rabbits for the production of monospecific polyclonal antibody. A selection of these polyclonal antibodies, or a combination of polyclonal and monoclonal antibodies is then used in a "capture ELISA" to detect the presence of an RSV antigen.

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U.S. Patent Application No. 08/059,444.

Claims (24)

1. An isolated, recombinantly-generated, attenuated, nonsegmented, negative-sense, single -00 stranded RNA virus of the Order Mononegavirales having at least one attenuating mutation in the 3' genomic O promoter region and having at least one attenuating Smutation in the RNA polymerase gene.

2. The virus of Claim 1 wherein the virus is from the Family Paramyxoviridae.

3. The virus of Claim 2 wherein the virus is from the Subfamily Paramyxovirinae.

4. The virus of Claim 3 wherein the virus is from the Genus Morbillivirus. The virus of Claim 4 wherein the virus is measles virus.

6. The measles virus of Claim 5 wherein: the at least one attenuating mutation in the 3' genomic promoter region is selected from the group consisting of nucleotide 26 (A nucleotide 42 (A T or A and nucleotide 96 (G where these nucleotides are presented in positive strand, antigenomic, message sense; and the at least one attenuating mutation in the RNA polymerase gene is selected from the group consisting of nucleotide changes which produce changes in an amino acid selected from the group consisting of residues 331 (isoleucine -+threonine), 1409 (alanine threonine), 1624 (threonine ->alanine), 1649 (arginine ->methionine), 1717 417 (aspartic acid ->alanine), 1936 (histidine tyrosine), 2074 00 (glutamine ->arginine) and 2114 S(arginine ->lysine).

7. The virus of Claim 3 wherein the virus Sis from the Genus Paramyxovirus.

8. The virus of Claim 7 wherein the virus is human parainfluenzae virus type 3 (PIV-3).

9. The PIV-3 of Claim 8 wherein: the at least one attenuating mutation in the 3' genomic promoter region is selected from the group consisting of nucleotide 23 (T nucleotide 24 (C nucleotide 28 (G and nucleotide 45 (T where these nucleotides are presented in positive strand, antigenomic, message sense; and the at least one attenuating mutation in the RNA polymerase gene is selected from the group consisting of nucleotide changes which produce changes in an amino acid selected from the group consisting of residues 942 (tyrosine histidine), 992 (leucine phenylalanine), 1292 (leucine phenylalanine), and 1558 (threonine isoleucine). The virus of Claim 3 wherein the virus is from the Genus Rubulavirus.

11. The virus of Claim 2 wherein the virus is from the Subfamily Pneumovirinae.

12. The virus of Claim 11 wherein the virus is from the Genus Pneumovirus. 418 0 <d

13. The virus of Claim 12 wherein the virus is human respiratory syncytial virus (RSV) subgroup B. 00 14. The virus of Claim 13 wherein: the at least one attenuating mutation in the 3' genomic promoter region is selected from the group consisting of CI nucleotide 4 (C and the insertion of an additional A in the stretch of A's at nucleotides 6-11, where these nucleotides are presented in positive strand, antigenomic, message sense; and the at least one attenuating mutation in the RNA polymerase gene is selected from the group consisting of nucleotide changes which produce changes in an amino acid selected from the group consisting of residues 353 (arginine -4 lysine), 451 (lysine arginine), 1229 (aspartic acid asparagine), 2029 (threonine -f isoleucine) and 2050 (asparagine aspartic acid). The virus of Claim 1 wherein the virus is from the Family Rhabdoviridae.

16. The virus of Claim 1 wherein the virus is from the Family Filoviridae.

17. A vaccine comprising an isolated, recombinantly-generated, attenuated, nonsegmented, negative-sense, single stranded RNA virus of the Order Mononegavirales according to Claim 1 and a physiologically acceptable carrier.

18. The vaccine of Claim 17 comprising a measles virus according to Claim 5 and a physiologically acceptable carrier.

19. The vaccine of claim 18 comprising a measles virus according to claim 6 and a Sphysiologically acceptable carrier. o 20. The vaccine of claim 17 comprising a PIV-3 according to claim 8 and a physiologically acceptable carrier.

21. The vaccine of claim 20 comprising a PIV-3 according to claim 9 and a physiologically acceptable carrier.

22. The vaccine of claim 17 comprising an RSV subgroup B according to claim 13 and a physiologically acceptable carrier.

23. The vaccine of claim 22 comprising an RSV subgroup B according to claim 14 and a physiologically acceptable carrier.

24. A method for immunising an individual to induce protection against a nonsegmented, 0 negative-sense, single stranded RNA virus of the Order Mononegavirales which comprises administering to the individual the vaccine of any one of claims 17 to 23. A vaccine of any one of claims 17 to 23 when used for immunising an individual to induce protection against a nonsegmented, negative-sense, single stranded RNA virus of the Order Mononegavirales.

26. Use of a vaccine of any one of claims 17 to 23 in the manufacture of a medicament for immunising an individual to induce protection against a nonsegmented, negative-sense, single stranded RNA virus of the Order Mononegavirales.

27. An isolated nucleic acid molecule comprising a measles virus sequence in positive strand, antigenomic message sense selected from the group consisting of 1977 wild-type strain (SEQ ID NO:3), 1983 wild-type strain (SEQ ID NO:5) where the nucleotide 2499 is G or C, Montefiore wild- type strain (SEQ ID NO:7), Rubeovax T M vaccine strain (SEQ ID NO:9), where the nucleotide 2143 is T or C, Moraten vaccine strain (SEQ ID NO:11), Schwarz vaccine strain (SEQ ID NO:11), where the nucleotide 4917 is C and the nucleotide 4924 is C, and Zagreb vaccine strain (SEQ ID NO:13), and the complementary genomic sequences thereof.

28. An isolated nucleic acid molecule comprising a PIV-3 sequence in positive strand, antigenomic message sense selected from the group consisting of cp45 vaccine strain grown in foetal rhesus lung cells (SEQ ID NO:19) and cp45 vaccine strain grown in Vero cells (SEQ ID NO:21), and the complementary genomic sequences thereof.

29. A composition which comprises a transcription vector comprising an isolated nucleic acid molecule encoding a genome or antigenome of a nonsegmented, negative-sense, single stranded RNA virus of the Order Mononegavirales having at least one attenuating mutation in the 3 genomic promoter region and having at least one attenuating mutation in the RNA polymerase gene, together with at least one expression vector which comprises at least one isolated nucleic acid molecule encoding the trans-acting proteins necessary for encapsidation, transcription and replication, whereby upon expression an infectious attenuated virus is produced. The composition of claim 29 wherein the transcription vector comprises an isolated nucleic acid molecule which encodes a measles virus according to claim 5 and the at least one

04407.doc expression vector comprises at least one isolated nucleic acid molecule encoding the trans-acting Sproteins N, P and L. o 31. The composition of claim 30 wherein the transcription vector comprises an isolated nucleic acid molecule which encodes a measles virus according to claim 6. 32. The composition of claim 29 wherein the transcription vector comprises an isolated nucleic acid molecule which encodes a PIV-3 according to claim 8 and the at least one expression vector comprises at least one isolated nucleic acid molecule encoding the trans-acting proteins NP, P and L. 00-. 33. The composition of claim 32 wherein the transcription vector comprises an isolated nucleic acid molecule which encodes a PIV-3 according to claim 9. 34. The composition of claim 29 wherein the transcription vector comprises an isolated Cnucleic acid molecule which encodes an RSV subgroup B according to claim 13 and the at least one expression vector comprises at least one isolated nucleic acid molecule encoding the trans-acting proteins N, P, L and M2. 35. The composition of claim 34 wherein the transcription vector comprises an isolated nucleic acid molecule which encodes an RSV subgroup B according to claim 14. 36. A method for producing infectious attenuated nonsegmented, negative-sense, single stranded RNA virus of the Order Mononegavirales which comprises transforming or transfecting host cells with the at least two vectors of claim 29 and culturing the host cells under conditions which permit the co-expression of these vectors so as to produce the infectious attenuated virus. 37. The method of claim 36 wherein the virus is the measles virus of claim 38. The method of claim 37 wherein the virus is the measles virus of claim 6. 39. The method of claim 36 wherein the virus is the PIV-3 of claim 8. The method of claim 39 wherein the virus is the PIV-3 of claim 9. 41. The method of claim 36 wherein the virus is the RSV subgroup B of claim 13. 42. The method of claim 41 wherein the virus is the RSV subgroup B of claim 14. 43. An isolated, recombinantly generated, attenuated, nonsegmented, negative sense, single stranded RNA virus of the Order Mononegavirales, substantially as hereinbefore described with reference to any one of the examples. 44. A vaccine comprising an isolated, recombinantly generated, attenuated, nonsegmented, negative sense, single stranded RNA virus of the Order Mononegavirales, substantially as hereinbefore described with reference to any one of the examples. An isolated nucleic acid molecule comprising a measles virus sequence in positive strand, antigenomic message sense substantially as hereinbefore described with reference to any one of the examples. 46. An isolated nucleic acid molecule comprising a PIV-3 sequence in positive strand, antigenomic message sense, substantially as hereinbefore described with reference to any one of the examples. 47. A composition which comprises a transcription vector comprising an isolated nucleic acid molecule encoding a genome or antigenome of a nonsegmented, negative-sense, single stranded 04407.doc RNA virus of the Order Mononegavirales substantially as hereinbefore described with reference to Sany one of the examples. o 48. A method for producing infectious attenuated nonsegmented, negative-sense, single stranded RNA virus of the Order Mononegavirales substantially as hereinbefore described with reference to any one of the examples. Dated 7 December, 2004 r The Government of the United States of America as represented by 0o The Department of Health and Human Services Wyeth Holdings Corporation SPatent Attorneys for the Applicant/Nominated Person C SPRUSON FERGUSON 04407.doc