AU9389998A

AU9389998A - Attenuated respiratory syncytial viruses

Info

Publication number: AU9389998A
Application number: AU93899/98A
Authority: AU
Inventors: Valerie B. Randolph; Mohinderjit S. Sidhu; Stephen A. Udem
Original assignee: American Cyanamid Co
Current assignee: Wyeth Holdings LLC
Priority date: 1997-09-19
Filing date: 1998-09-15
Publication date: 1999-04-12
Also published as: WO1999015672A1; JP2001517448A; BR9812232A; CA2302867A1; KR20010030630A; EP1015594A1; CN1273603A

Description

WO99/15672 PCTUS98/19145 ATTENUATED RESPIRATORY SYNCYTIAL VIRUSES Field Of The Invention 5 This invention relates to respiratory syncytial viruses of subgroup B 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 10 Government has certain rights in the invention. Background Of The Invention Respiratory syncytial virus (RSV) is a 15 nonsegmented, negative-sense, single stranded enveloped RNA virus. RSV belongs to the Family Paramyxoviridae, the Subfamily Pneumovirinae and the genus Pneumovirus. Pneumoviruses have 10 protein-encoding cistrons. These proteins in RSV are the nucleocapsid protein N, the 20 phosphoprotein P, the nonglycosylated virion matrix protein M, the attachment protein G, the fusion protein F, the polymerase protein L, the nonstructural proteins NS1 and NS2, the small hydrophobic protein SH, and the transcription elongation factor protein M2. 25 The genomic RNA of RSV 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. RSV encodes and packages its own RNA 30 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 35 antigenome (+) strand serves as the template for WO99/15672 PCT/US98/19145 - 2 generating further copies of the (-) strand genomic RNA. The polymerase complex actuates and achieves transcription and replication by engaging the cis 5 acting signals at the 3' end of the genome, in 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 made from the downstream genes (e.g., the polymerase 10 gene (L)) relative to their upstream neighbors (i.e., the nucleoprotein gene (N)). Therefore, there is always a gradient of mRNA abundance according to the position of the genes relative to the 3'-end of the genome. RSV is the leading cause of viral pneumonia 15 and bronchiolitis in infants and young children and is responsible for an estimated 95,000 hospitalizations and 4,500 deaths per year in the United States (Bibliography entries 1,2,3). Serious disease is most prevalent in infants 6 weeks to 6 months of age and in 20 children with certain underlying illnesses (e.g. immunodeficiencies, congenital heart disease and bronchopulmonary dysplasia). Two major subgroups of RSV have been identified, A and B, as well as antigenic variants 25 within each subgroup (4). Multiple variants of each subgroup have been found to cocirculate in epidemics which occur annually during late fall, winter, and spring months (5). Most children are infected by two years of age. Complete immunity to RSV, however, does 30 not develop and reinfections occur throughout life (6,7). These reinfections often are symptomatic, though generally confined to mild upper respiratory tract disease. A decrease in severity of disease is associated with two or more prior infections and, in 35 some studies, with high levels of serum antibody, WO99/15672 PCTIUS98/19145 - 3 suggesting that protective immunity to RSV disease will accumulate following repeated infections (2,6,8,9,10,11). There is also evidence that children infected with one of the two major RSV subgroups may be 5 somewhat protected against reinfection with the homologous subgroup (12). These observations suggest that it is both possible and worthwhile to develop an RSV vaccination regimen for infants and young children which would provide sufficient temporary immunity to 10 protect against severe disease and death. The identification of the two major subgroups of RSV has been based on reactivities of the F and G surface glycoproteins with monoclonal antibodies (4,13) and further delineated by sequence analysis (14,15). 15 Both F and G proteins elicit neutralizing antibodies and immunization with these proteins provides protection against reinfection in mouse and cotton rat models (16,17,18). Most neutralizing antibodies are directed against the F protein. Beeler and Coelingh 20 reported that out of 16 neutralization epitopes mapped to the F protein, 8 epitopes were conserved in all or all but one of 23 virus isolates tested (19). A high degree of sequence homology exists between the F protein of subgroups A and B (approximately 90% amino 25 acid and approximately 80% nucleotide) whereas a much lower degree of homology exists between the G proteins (approximately 50-60% amino acid and approximately 60 70% nucleotide) (14). Correspondingly, immunity elicited by the F protein is more crossprotective 30 between subgroups than is immunity elicited by the G protein (16,17). In mice, humoral immunity induced by both the F and G proteins is thought to be responsible for protection against reinfection with virus (20) whereas the CTL response is thought to be more 35 important in resolution of primary infections WO 99/15672 PCTIUS98/19145 - 4 (21,22,23). The M2 (or 22K) protein has been shown to be a potent inducer of cytotoxic lymphocytes (CTL) in mice, with lesser CTL recognition of F, N, and P proteins (24,25). Human CTL's have been shown to 5 recognize the F, M2, N, M, SH, and NS2 (or ib) proteins (26). These data suggest that the F protein of either virus subgroup is a crucial immunogen in any RSV vaccine and that the G, M2, N, M, SH, and NS2 proteins should also be considered potential vaccine components. 10 For RSV, no vaccines of any kind are currently available. Thus, there is a need to develop vaccines against this human pathogen. Such vaccines would have to elicit an immune response in the recipient which will prevent serious RSV disease, i.e., 15 LRD. The qualitative and quantitative features of such a favorable response are extrapolated from those seen in survivors of natural virus infection, who, though not protected from reinfection by the same or highly related viruses, are protected from serious or fatal 20 disease. A variety of approaches can be considered in seeking to develop such vaccines, including the use of: (1) purified individual viral protein vaccines (subunit vaccines); (2) inactivated whole virus preparations; 25 and (3) 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 30 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 35 of polio (IPV) and hepatitis A have proven safe and WO99/15672 PCT/US98/19145 - 5 efficacious. In contrast, immunization with similarly inactivated whole RSV elicited unfavorable immune responses and/or response profiles which predisposed vaccinees to exaggerated or aberrant disease when 5 subsequently confronted with the natural or "wild-type" virus. Early attempts (1966) to vaccinate young children used a parenterally administered formalin inactivated RSV vaccine. Unfortunately, several field 10 trials of this vaccine revealed serious adverse reactions - the development of a severe illness with unusual features following subsequent natural infection with RSV (27,28). It has been suggested that this exposure to formalinized RSV antigen elicited an 15 abnormal or unbalanced immune response profile, predisposing the vaccinee to RSV disease potentiation (29,30). Several different live, attenuated viruses have proven remarkably effective as a means of 20 achieving immunoprophylaxis. Pursuit of such vaccine candidates for RSV has been intense and long-standing. RSV temperature sensitive (ts) mutants derived by chemical mutagenesis (31) were shown to be attenuated in rodent and non-human primate models 25 (32,33). Cold adaptation, a process by which virus is adapted to growth at temperatures colder than those at which it normally optimally grows, has been used to develop attenuated ts virus mutants for use as vaccines 30 (for review see (34)). This method generally results in the accumulation of multiple genetic lesions which may help to confer phenotypic stability by reducing the probability that reversion of any one lesion will result in reversion of the relevant phenotype. Maassab 35 has used stepwise cold adaptation to successfully WO99/15672 PCT/US98/19145 - 6 develop several ts influenza vaccine candidates currently in clinical trials (35,36,37). These mutants, which bear attenuating mutations in at least four different genes, appear to be attenuated, 5 immunogenic, and phenotypically stable. Belshe and co-workers have used cold adaptation to develop attenuated, ts strains of a paramyxovirus, parainfluenza virus type 3 (38,39). In this case, cold adaptation was carried out in primary 10 African green monkey kidney cells by reducing temperatures to 20 0 C. Analysis of several virus variants cloned from this cold adapted population demonstrated that the level of attenuation and temperature sensitivity increased as the length of cold 15 adaptation increased. These variants were shown to have reduced potential for virulence in humans, however the temperature sensitive phenotype was somewhat unstable in clinical trials (40). RSV was successfully cold adapted to 25-26 0 C 20 in several laboratories in the mid 1960's, but was found to be under-attenuated in vaccine trials (34,41,42). Maassab and DeBorde (34) have suggested this may be because cold adaptation was not carried out at low enough temperatures, or clones of adequately 25 attenuated virus were not isolated from a genetically mixed population of cold-adapted virus. Nevertheless, this means for generating attenuated live RSV vaccine candidates is lengthy and, at best, unpredictable, relying largely on the 30 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 35 under- or overattenuated in the human or animal host WO 99/15672 PCT/1US98/19145 - 7 for whom they are intended as vaccine candidates. Thereafter, two live, attenuated RSV mutants were generated by cold passage or chemical mutagenesis. These RSV strains were found to have reduced virulence 5 in seropositive adults. Unfortunately, they proved either over- or underattenuated when given to seronegative infants; in some cases they were also found to lack genetic stability (43,44). Another vaccination approach using parenteral administration of 10 live virus was found ineffective and efforts along this line were discontinued (45). Notably, these live RSV vaccines were never associated with disease enhancement as observed with the formalin-inactivated RSV vaccine described above. 15 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 20 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 25 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. This favorable immune response pattern is 30 contrasted with the delimited responses elicited by inactivated or subunit vaccines, which typically are largely restricted to the humoral immune surveillance arm. Further, the immune response profile elicited by some formalin inactivated whole virus vaccines, e.g., 35 measles and respiratory syncytial virus vaccines WO99/15672 PCT/US98/19145 - 8 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 5 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 10 of the wild-type virus which has lost its disease producing potential (i.e., virulence), while retaining sufficient replication competence to infect the recipient and elicit the desired immune response profile in adequate abundance. 15 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 20 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 25 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 from the wild-type, disease-causing viruses -- i.e., changes that are associated with attenuation. 30 Rational vaccine design would be assisted by a better understanding of RSV, in particular, by the identification of the virally encoded determinants of virulence as well as those genomic changes which are responsible for attenuation. 35 WO99/15672 PCT/US98/19145 - 9 Summary Of The Invention Accordingly, it is an object of this invention to identify those regions of the polymerase 5 gene of RSV subgroup B where mutations result in attenuation of those viruses. It is a further object of this invention to produce recombinantly-generated RSV subgroup B which incorporate such attenuating mutations in their 10 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 15 discussed below are achieved by the generation and isolation of recombinantly-generated, attenuated, RSV subgroup B having at least one attenuating mutation in the RNA polymerase gene. The at least one attenuating mutation in the 20 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 25 (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 wild 30 type 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 WO99/15672 PCTIUS98/19145 - 10 complete viral nucleotide sequence (whether of wild type virus or virus attenuated by non-recombinant means) is manipulated by introducing one or more of the attenuating mutations described in this application to 5 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 10 invention, such a complete wild-type or vaccine viral nucleotide sequence (as well as a revertant sequence) is used: (1) to design PCR primers for use in a PCR assay to detect the presence of the corresponding virus in a sample; or (2) to design and select peptides for 15 use in an ELISA to detect the presence of the corresponding virus in a sample. Brief Description Of The Figures 20 Figure 1 shows a flow chart detailing the propagation of RSV 2B working seed MK7Vl4b and RSV 3A working seed MK8V17b. Figure 2 shows growth and cytopathic effect 25 of RSV 2B at temperatures from 26oC to 36 0 C in Vero cells. Figure 3 shows growth and cytopathic effect of RSV 3A at temperatures from 26 0 C to 36 0 C in Vero cells. 30 Figure 4 graphically shows titration results obtained at each passage of RSV 2B and RSV 3A. Figure 5 shows the growth curves of RSV 2B, RSV 2Bp24G, RSV 2Bp20L, RSV 3A, RSV 3Ap20E and RSV 3Ap20F in Vero cells at temperatures from 20'C to 40 0 C. 35 Figure 6 compares graphically the growth of WO99/15672 PCTIUS98/19145 - 11 RSV 2B and RSV 2Bp20L in cotton rats from 3 to 7 days post-infection. Figure 7 compares the relative growth and pathogenicity of RSV 2B and RSV 2Bp20L in four (4) year 5 old seropositive chimps. Figure 8 is a diagram showing virus titrations for monkeys infected with the RSV 2B ts mutants and subsequently challenged with the parental strain. 10 Figure 9 is a diagram showing virus growth in African green monkey cells infected with the RSV 3A ts mutants and challenged with the parental 3A strain. Figure 10 is a diagram showing a growth study in African green monkeys comparing TS-1 with RSV 2Bp33F 15 and 3Ap28F. Figure 11 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 20 between the M2 and L genes (bottom portion). The RSV 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 strain has 145 nucleotides in the 5' trailer region, as 25 compared to 149 nucleotides in the 18537 strain. The 2B strain has one more nucleotide in each of the NS-1, NS-2 and N genes, and one fewer nucleotide in each of the M and F genes, as compared to the 18537 strain. 30 Detailed Description Of The Invention The first step in the identification of attenuating mutations in the L gene of RSV subgroup B vaccine strains was the generation of those strains 35 from wild-type strains. The original RSV subgroup B WO99/15672 PCT/US98/19145 - 12 vaccine strains (as well as subgroup A vaccine strains) were generated by cold adaptation of the wild-type virus. Cold adaptation comprises obtaining live virulent virus derived from clinical isolates that have 5 been isolated in primary rhesus monkey kidney cells. These are then passed in Vero cells at 35-36 0 C and plaque purified. Preferably, the Vero cells are passage 133 to 148 of the Vero cell line CCL81, obtained from the American Type Culture Collection 10 (ATCC), 12301 Parklawn Drive, Rockville, Maryland, U.S.A. 20852. The maintenance medium is preferably MEM with 2% FBS, L-glutamine, non-essential amino acids and 20mM Hepes pH 7.5, and the freezing medium is MEM with 10% FBS and 20mM Hepes pH 7.5. 15 A confluent monolayer of Vero cells is inoculated with about 1.0 ml of virus inoculum, and virus is allowed to absorb for about one to two hours (preferably, 70 to 120 minutes, and most preferably 90 minutes) at ambient temperature (about 18 0 C to about 20 25 0 C). The virus flask is incubated at about 18'C to about 26 0 C, preferably about 20'C, for about two to fifteen days. Virus is harvested by removing the medium and replacing it with freezing medium. The 25 flask is then frozen directly at -70'C, then thawed in a 32oC water bath. A portion (about 1 ml) is removed from the freeze-thaw lysate and is used to inoculate Vero cells; the process is then repeated. The remaining freeze 30 thaw lysate is stored at -70°C. It can be used to perform virus titrations and plaque purify virus. To plaque purify virus, the freeze-thaw lysate is thawed in a 32 0 C water bath. About three to five serial dilutions of the lysate are made in the 35 maintenance medium. Six-well, twenty-four well, or WO99/15672 PCT/US98/19145 - 13 ninety-six well plates containing confluent Vero cells are rinsed with a phosphate buffered saline solution. Wells are inoculated with virus dilution, using only enough volume to cover the bottom of the well. Virus 5 inoculum is adsorbed for 90 minutes at ambient temperature. Wells are overlaid with 1% methylcellulose in MEM-maintenance medium. Plates are incubated at 32 0 C for five days. Isolated plaques are identified microscopically by looking for typical 10 syncytial plaques, and wells are marked. Plaques are picked at marked sites using small bore pipette or pipette tip and are emulsified in 0.5 ml maintenance medium for 1-3 hours at 4 0 C. Picked plaques are used to inoculate duplicates of 25 cm 2 flask or 96-well 15 plates containing Vero cell monolayers as described above. Duplicate inoculated flasks or plates are overlaid with maintenance medium. One duplicate is incubated at 32oC and the other at 39 0 C for 5-10 days. Flasks or plates incubated at 32oC are examined 20 microscopically for virus cytopathic effect (CPE). Flasks or plates incubated at 39 0 C are stained by immunoperoxidase assay for RSV specific antigen. Flasks or plates which demonstrate easily detectable CPE at 32oC and little or no detectable RSV antigen by 25 immunoperoxidase staining are selected as containing temperature sensitive (ts) mutants. Virus from the selected flask or plate described above are harvested by freeze-thaw technique. This virus represents a plaque purified mutant. 30 Cold adaptation as just described was used to develop attenuated strains of RSV from two parental strains derived from clinical isolates. Seven ts mutants were isolated, four from a subgroup B virus (RSV 2B) and three from a subgroup A virus (RSV 3A). 35 All seven mutants displayed a temperature sensitive WO99/15672 PCT/US98/19145 - 14 phenotype in Vero cell culture, each with unique characteristics. All mutants were attenuated in growth in cotton rats, but displayed different phenotypes. Growth of one of the ts mutants, RSV 2Bp20L, was shown 5 to be attenuated in seropositive chimpanzees. All seven mutants retained two major neutralization epitopes. Cold adaptation of RSV had previously been done in primary or diploid cell lines (bovine embryonic 10 kidney, WI38, and cercopithecus monkey kidney) at temperatures beginning at 34 0 C-37 0 C and decreasing to 25 0 C-26°C. No attempt had been made to isolate multiple individual mutant phenotypes from the cold adapted virus (37,46,47). 15 The approach described herein to cold adapting RSV differed in several significant ways from these previous attempts. This procedure started with a subgroup A and subgroup B virus of different strains than those used previously. These strains bore 20 distinct phenotypic differences from the reference strains and each other. These strains were passed several times to adapt virus to Vero cells and to plaque purify virus. Virus was passaged in a continuous cell line, Vero cells, rather than a diploid 25 or primary cell line. Several strategies of temperature change were used, to provide a greater potential for isolation of a variety of mutant phenotypes. Unlike previous RSV cold adaptation 30 strategies where cold adaptation had started at 34-37 0 C and gone down to 25-26 0 C, cold adaptation started at either 26 0 C (since it was found that the parental strains grew well at this temperature) or 22°C, and gradually reduced the growth temperature to 20 0 C. 35 Passage strategies attempted to cover both the WO99/15672 PCTIUS98/19145 - 15 recommendation of "slow" adaptation to very low temperatures as proposed by Massab and DeBorde (37), as well as efforts to try a faster and more aggressive approach. RNA viruses mutate at such a high frequency 5 that any population of virus will contain a number of individual virus variants (48). Therefore, a variety of virus mutants were isolated from individual flasks at various virus passage levels and from different cold adaptation strategies. 10 The results were interesting and somewhat unexpected. The rate at which virus became adapted (i.e., grew to consistently high titers at the 20 0 C temperature), was most affected by the strain of virus used, implying a significant host-related factor in 15 adaptation. RSV 2B easily adapted to the cold temperatures, even using a rapid adaptation scheme. In contrast, RSV 3A grew poorly at the low temperatures. RSV 3A was eventually cold-adapted using the slow passage scheme, but the more rapid adaptation 20 approaches did not appear promising and were discontinued. Based on cold adaptation experiences reported by other researchers, it was expected ts mutants would arise and eventually become the predominant virus variants in the cold-adapted 25 populations. For example, Belshe and Hissom (41) reported that with parainfluenza virus type 3 adapted to grow at 20'C, 80% of plaque purified virus clones were ts by passage 18 and 100% were ts by passage 45. In this study, even after 38-40 low temperature 30 passages, including up to 32 passages done at 20'C, RSV ts mutants remained a minor population. This would suggest that ts and cold-adapted phenotypes may not be as strongly linked in RSV as they are in other viruses. The level of attenuation is a critical factor 35 in developing vaccines for any target population and is WO99/15672 PCTIUS98/19145 - 16 of particular importance for vaccines intended for infants and young children. Virus must be sufficiently attenuated to not cause disease, yet grow well enough in the vaccine to elicit protective immunity. 5 Widely accepted markers for attenuation are ts phenotype and reduced growth in animal models. However, these markers are only approximate and testing must eventually be done in the target population. The RSV 3A ts mutants could be distinguished from the RSV 10 3A parental virus by reduced replication in both the nose and lungs. Also of note, although the RSV 3A parental virus grew much better in the nose than the lungs of cotton rats, virus recovery was similar in both nose and lungs of BALB/c mice. These data suggest 15 that the attenuation seen in cotton rats is due to more than one factor, and that this factor is not directly related to temperature sensitivity as measured in vitro. The cotton rat is relatively nonpermissive for growth of RSV and disease does not develop, suggesting 20 that this model is an unreliable indicator of level of attenuation in humans. In contrast, chimpanzees are highly susceptible to RSV infection and develop an upper and lower respiratory tract disease that is very similar to 25 that seen in humans. In seropositive chimps, it was found that the RSV 2B parental strain caused mild upper respiratory tract disease similar to that caused by natural RSV infections in adult humans. The RSV 2Bp20L mutant did not grow, clearly demonstrating that this ts 30 mutant was attenuated in a permissive host as well as the non-permissive cotton rat. The level of attenuation is best assessed in a seronegative chimp, as prior virus exposure will affect the host response to virus challenge. Unfortunately, testing in 35 seronegative chimps is severely hampered by the limited WO99/15672 PCT/US98/19145 - 17 availability of these animals. The mutants described herein bear the desirable traits of an attenuated, phenotypically stable, and immunogenic RSV vaccine virus in the human 5 target population. The immunopotency of the recombinantly generated RSV subgroup B vaccine is determined by monitoring the immune response of test animals following immunization with the vaccine. Test animals 10 include, but are not limited to, mice, rats (e.g., cotton rats), rabbits, primates, e.g., African green monkeys, chimps, and human subjects. Methods of introduction of the immunogen may include oral, parenteral, topical, intranasal or any other standard 15 routes of immunizations. The immune response of the test subjects is analyzed by four approaches: (a) the reactivity of the resultant immune serum to authentic RSV antigens, as assayed by known techniques, e.g., enzyme linked immunosorbant assay (ELISA), immunoblots, 20 radioimmunoprecipitations, etc.; (b) the ability of the immune serum to neutralize RSV infectivity in vitro; (c) the ability of the immune serum to inhibit virus fusion in vitro; and (d) protection from RSV infection or significant disease. 25 The cold-adapted RSV mutants are capable of eliciting an immune response when administered to a subject without causing significant disease, such as respiratory distress or otitis media. As used herein, the term "cold-adapted mutant" means an attenuated 30 virus that has been attenuated by propagation at lower than optimal temperatures. Examples of cold-adapted mutant viruses have been provided as described above. The cold-adapted mutant RSV may be a mutant of subgroup A, such as the group consisting of 3Ap20E, 3Ap20F and 35 3Ap28F. The cold-adapted mutant RSV may also be a WO99/15672 PCT/US98/19145 - 18 mutant of the subgroup B, such as the group consisting of 2Bp33F, 2Bp24G, 2Bp20L and 2Bp34L. The subgroup B viruses are then sequenced and differences between wild-type and mutant strains are identified. Those 5 mutations which contribute to the attenuated phenotype are then assessed. Transcription and replication of negative sense, single stranded RNA viral genomes such as RSV subgroup B are achieved through the enzymatic activity 10 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 15 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 20 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 25 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 30 ribonucleotide polymerization, capping and polyadenylation of mRNA transcripts, methylation and perhaps specific phosphorylation of P proteins. The L protein's central role in genomic transcription and replication is supported by its large size, sensitivity 35 to mutations, and its catalytic level of abundance in WO99/15672 PCT/US98/19145 - 19 the transcriptionally active viral complex (49). These considerations led to the proposal that L proteins consist of a linear array of domains whose concatenated structure integrates discrete functions 5 (50). 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: (1) a putative RNA template 10 recognition and/or phosphodiester bond formation domain; (2) an RNA binding element; and (3) an ATP binding domain. All prior studies of L proteins of nonsegmented negative-sense, single stranded RNA viruses have revealed these putative functional 15 elements (50). In summary, the invention comprises the identification of changes in the polymerase gene (L) which result in attenuation of the virus while retaining sufficient ability of the virus to replicate. 20 Attenuation is optimized by rational mutations of the polymerase gene, which provide the desired balance of replication efficiency: so that the virus vaccine is no longer able to produce disease, yet retains its capacity to infect the vaccinee's cells, to express 25 sufficiently abundant gene products to elicit the full spectrum and profile of desirable immune responses, and to reproduce and disseminate sufficiently to maximize the abundance of the immune response elicited. Animal studies have demonstrated a decrease 30 in viral replication sufficient to avoid illness but adequate to elicit the desired immune response. This likely represents a decrease in transcription, a decrease in gene expression of virally encoded proteins, a decrease in antisense templates and, 35 therefore, the production of fewer new genomes. The WO99/15672 PCT/US98/19145 - 20 resulting attenuated viruses are significantly less virulent than the wild-type. The attenuating mutations described herein may be introduced into viral strains by two methods: 5 (1) 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 10 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 15 model. Attenuated viruses are subjected to nucleotide sequencing of their polymerase genes to locate the sites of attenuating mutations. Once this has been done, method (2) is then carried out. (2) A preferred means of introducing 20 attenuating mutations comprises making predetermined mutations using site-directed mutagenesis. These mutations are identified either by method (1) or by reference to closely-related viruses whose attenuating mutations are already known. One or more mutations are 25 introduced into the polymerase gene. Cumulative effects of different combinations of coding and non coding changes can also be assessed. The mutations to the polymerase gene are introduced by standard recombinant DNA methods into a 30 DNA copy of the viral genome. This may be a wild-type or a modified viral genome background (such as viruses modified by method (1)), thereby generating a new virus. Infectious clones or particles containing these attenuating mutations are generated using the cDNA 35 "rescue" system, which has been applied to a variety of WO99/15672 PCT/US98/19145 - 21 viruses, including Sendai virus (51); measles virus (52); respiratory syncytial virus (53); rabies (54); vesicular stomatitis virus (VSV) (55); and rinderpest virus (56); these references are hereby incorporated by 5 reference. See, for RSV rescue, published International patent application WO 97/12032, designating the United States (57); this application is hereby incorporated by reference. Briefly, all Mononegavirales rescue systems 10 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 (e.g., the T7 RNA polymerase promoter) and a self-cleaving ribozyme sequence (e.g., the hepatitis 15 delta ribozyme) which is inserted into a propagatable bacterial plasmid. This transcription vector provides the readily manipulable DNA template from which the RNA polymerase (e.g., T7 RNA polymerase) can faithfully transcribe a single-stranded RNA copy of the viral 20 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 25 for rescue of new virus progeny are the virus-specific trans-acting proteins needed to encapsidate the naked, single-stranded viral antigenome or genome RNA transcripts into functional nucleocapsid templates: the viral nucleocapsid (N or NP) protein, the 30 polymerase-associated phosphoprotein (P) and the polymerase (L) protein. These proteins comprise the active viral RNA-dependent RNA polymerase which must engage this nucleocapsid template to achieve transcription and replication. 35 The trans-acting proteins required for RSV WO99/15672 PCT/US98/19145 - 22 rescue are the encapsidating protein N, the polymerase complex proteins, P and L, and an additional protein, M2, the RSV-encoded transcription elongation factor. Typically, these viral trans-acting proteins 5 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 10 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 15 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 20 nucleocapsid protein and engaged by the required polymerase components produced concurrently from co transfected expression plasmids encoding the required virus-specific trans-acting proteins. These events and processes lead to the prerequisite transcription of 25 viral mRNAs, the replication and amplification of new genomes and, thereby, the production of novel viral progeny, i.e., rescue. For the rescue of rabies, VSV and Sendai, T7 polymerase is provided by recombinant vaccinia virus 30 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 measles virus (MV) cDNA rescue, this 35 requirement is avoided by creating a cell line that WO99/15672 PCT/US98/19145 - 23 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 5 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 10 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 15 and PIV-3 (58,59). The rescue system thus provides a composition which comprises a transcription vector comprising an isolated nucleic acid molecule encoding a genome or antigenome of RSV subgroup B having at least one 20 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 N, P, L and M2 proteins necessary for encapsidation, transcription and 25 replication. Host cells are then transformed or transfected with the at least two 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 30 virus. The rescued infectious RSV is then tested for its desired phenotype (temperature sensitivity, cold adaptation, plaque morphology, and transcription and replication attenuation), first by in vitro means. 35 If the attenuated phenotype of the rescued WO99/15672 PCT/US98/19145 - 24 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 5 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 or intratracheal routes of inoculation (60). Protection in non-human primates is 10 measured by such criteria as disease signs and symptoms, virus shedding and 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" 15 virus is considered to be "recombinantly-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 20 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 25 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. 30 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 (4). More recently, the A and B lineages of RSV strains have been confirmed by sequence analysis 35 (14,15). Bovine, ovine, and caprine strains of this WO99/15672 PCTIUS98/19145 - 25 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 the human and the bovine/ovine strains (61,62), and may 5 be influenced, at least in part, by receptor binding. RSV is the primary cause of serious viral pneumonia and bronchiolitis in infants and young children. Serious disease, i.e., lower respiratory tract disease (LRD), is most prevalent in infants less 10 than six months of age. It most commonly occurs in the 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 15 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 20 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 25 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 30 isolated only in the winter months, e.g., 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 35 drift (such as that seen with influenza A viruses), but WO99/15672 PCT/US98/19145 - 26 sequence studies demonstrating accumulation of amino acid changes in the hypervariable regions of the G protein and SH proteins suggest that immune pressure may drive virus evolution. 5 In mouse and cotton rat models, both the F and G proteins of RSV elicit neutralizing antibodies and immunization with these proteins alone provides longterm protection against reinfection (16,17). In humans, complete immunity to RSV does not 10 develop and reinfections occur throughout life (6,7); 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 15 with one of the two major RSV subgroups may be somewhat protected against reinfection with the homologous subgroup (13), observations which suggest that a live attenuated virus vaccine may provide protection sufficient to prevent serious morbidity and mortality. 20 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. 25 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 (7,10,63). The 30 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 35 size and shape to those of all other paramyxoviruses.

WO99/15672 PCT/US98/19145 - 27 When visualized by negative staining and electron microscopy, virions are irregular in shape and range in diameter from 150-300 nm (64). The nucleocapsid of this virus is a symmetrical helix similar to that of 5 other paramyxoviruses, except that the helical diameter is 12-15 nm rather than 18nm. The envelope consists of a lipid bilayer that is derived from the host membrane and contains virally coded transmembrane surface glycoproteins. The viral glycoproteins mediate 10 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 (65). 15 Neuraminidase activity is present in members of the genera Paramyxovirus, Rubulavirus, and is absent in Morbillivirus and Pneumovirus of mice (PVM) (65). RSV possesses two subgroups, designated A and B. The wild-type RSV (strain 2B) genome is a single 20 strand of negative-sense RNA of 15,218 nucleotides (SEQ ID NO:1) that are transcribed into ten major subgenomic mRNAs. Each of the ten mRNAs encodes a major polypeptide chain: Three are transmembrane surface proteins (G, F and SH); three are the proteins 25 associated with genomic RNA to form the viral nucleocapsid (N, P and L); 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 30 and replication; one is the nonglycosylated virion matrix protein (M); and the last is M2, another nonglycosylated protein recently shown to be an RSV specified transcription elongation factor (see Figure 11). These ten viral proteins account for nearly all 35 of the viral coding capacity.

WO99/15672 PCT/US98/19145 - 28 The viral genome is encapsidated with the major nucleocapsid protein (N), and is associated with the phosphoprotein (P), and the large (L) polymerase protein. These three proteins have been shown to be 5 necessary and sufficient for directing RNA replication of cDNA encoded RSV minigenomes (66). Further studies have shown that for transcription to proceed with full processing, the M2 protein (ORF 1) is required (64). When the M2 protein is missing, truncated transcripts 10 predominate, and rescue of the full length genome does not occur (64). Both the M (matrix protein) and the M2 proteins are internal virion-associated proteins that are not present in the nucleocapsid structure. By 15 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 20 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 25 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 (3). The function of the SH protein is unknown, 30 although a recent report has implicated its involvement in the fusion function of the virus (67). The genomes of two wild-type RSV subgroup B strains (2B and 18537) have now been sequenced in their entirety (see SEQ ID NOS:1 and 3, discussed below). 35 Genomic RNA is neither capped nor polyadenylated (68).

WO99/15672 PCT/US98/19145 - 29 In both the virion and intracellularly, genomic RNA is tightly associated with the N protein. The 3' end of the genomic RNA consists of a 44-nucleotide extragenic leader region that is presumed 5 to contain the major viral promoter (68; Fig. 11). The 3' genomic promoter region is followed by ten viral genes in the order 3'-NSl-NS2-N-P-M-SH-G-F-M2-L-5' (Fig. 11). The L gene is followed by a 145-149 nucleotide extragenic trailer region (see Figure 11). 10 Each gene 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 15 signal. Each gene terminates with a semi-conserved 12 14 nucleotide gene end (3'-A G U/G U/A ANNN U/A A 3

-

5 ) (where N can be any of the four bases) that directs transcription termination and polyadenylation (Fig. 11). The first nine genes are non-overlapping and are 20 separated by intergenic regions that range in size from 3 to 56 nucleotides for RSV B strains (Fig. 11). 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 25 succeeding gene expression in a minreplicon system (Fig. 11). The last two RSV genes overlap by 68 nucleotides (Fig. 11). 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 30 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 35 polyadenylated read-through transcripts are the WO99/15672 PCT/US98/19145 - 30 products of genomic transcription (64). Transcriptional mapping studies using UV light mediated genomic inactivation showed that RSV genes are transcribed in their 3' to 5' order from a single 5 promoter near the 3' end (69). Thus, RSV synthesis appears to follow the single entry, sequential transcription model proposed for all Mononegavirales (70,71). According to this model, the polymerase (L) contacts genomic RNA in the nucleocapsid form at the 3' 10 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 15 showed that synthesis of each transcript begins at the first nucleotide of the gene start signal (64). The 5' 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 20 polyadenylated at their 3' ends (72). Both of these modifications are thought to be made co transcriptionally by the viral polymerase. Three regions of the RSV 3' genomic promoter have been found to be important as cis acting elements (73). These 25 regions are the first ten nucleotides (presumably acting as a promoter), nucleotides 21-25, and the gene start signal located at nucleotides 45-53 (73). Unlike other Paramyxovirinae, such as measles, Sendai and PIV 3, the remainder of the leader and non-coding region of 30 NS1 gene of RSV was found to be highly tolerant of insertions, deletions and substitutions (73). Additionally, by saturation mutagenesis (wherein each base is replaced independently by each of the other three bases and compared for translation and 35 replication efficiencies) within the first 12 WO99/15672 PCTIUS98/19145 - 31 nucleotides of the 3' genomic promoter region, a U tract located at nucleotides 6-10 was shown to be highly inhibitory to substitutions (73). In contrast, the first five nucleotides were relatively tolerant of 5 a number of substitutions and two of them at position four were up-regulatory mutations, resulting in a four 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 10 signals for mRNA synthesis and appear to be self contained and largely independent of the nature of adjoining sequence (74). The L gene start signal lies 68 nucleotides upstream of the M2 gene-end signal, resulting in gene 15 overlap (Fig. 11) (64). 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 20 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 25 start signal or whether there is a second, internal promoter for L gene transcription (64). 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 30 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 (69). Gene overlap is a 35 second mechanism that reduces the synthesis of full WO99/15672 PCT/US98/19145 - 32 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 sequence context, while the G ORF begins at the second 5 methionyl codon in the mRNA. RSV RNA replication is thought (64) to follow the model proposed from studies with vesicular stomatitis virus and Sendai virus (70,71). This involves a switch from the stop-start mode of mRNA 10 synthesis to an antiterminator read-through mode. This results in synthesis of positive sense replication intermediate (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 15 mechanism involved in the switch to the antiterminator mode is proposed to involve cotranscriptional encapsidation of the nascent RNA by N protein (70,71). RNA replication in RSV like other nonsegmented negative-strand RNA viruses is dependent on ongoing 20 protein synthesis (75). Predicted RI RNA has been detected for the standard virus as well as RSV-CAT minigenome (64,75). RI RNA was 10-20 fold less abundant intracellularly than was the progeny genome both for the standard and the minigenome system. The 25 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 30 reference to the appropriate SEQ ID NOS. contained herein: WO99/15672 PCT/US98/19145 - 33 Virus Nucleotide Sequence L Protein Sequence Wild-Type 2B SEQ ID NO:1 SEQ ID NO:2 18537 SEQ ID NO:3 SEQ ID NO:4 5 Vaccine 2B33F SEQ ID NO:5 SEQ ID NO:6 2B20L SEQ ID NO:7 SEQ ID NO:8 10 Revertant 2B33F TS(+) SEQ ID NO:9 SEQ ID NO:10 2B20L TS(+) SEQ ID NO:11 SEQ ID NO:12 It is noted that the Sequence Listings recite "DNA"; 15 this is necessary for the antigenomic message sense RNA to be provided in full (reciting sequences as "RNA" causes each "T" to be deleted from the sequence). Each RSV virus genome encodes an L protein that is 2,166 amino acids long. Genome length and 20 other nucleotide information is as follows: Virus Genome Wild-Type Lencrth L Start Codon L Stop Codon 2B 15218 8502-8504 15000-15002 25 18537 15229 8509-8511 15007-15009 Vaccine 2B33F 15219 8503-8505 15001-15003 2B20L 15219 8503-8505 15001-15003 30 Revertant 2B33F TS(+) 15219 8503-8505 15001-15003 2B20L TS(+) 15219 8503-8505 15001-15003 35 As detailed in Example 8 (especially Tables WO99/15672 PCT/US98/19145 - 34 21 and 22) below, the key potentially attenuating sites for the L protein of RSV subgroup B are as follows: amino acid residues 353 (arginine -+ lysine), 451 (lysine -> arginine), 1229 (aspartic acid -> 5 asparagine), 2029 (threonine -+ isoleucine) and 2050 (asparagine -> aspartic acid). It is understood that the nucleotide changes responsible for these amino acid changes are not limited to those set forth in Example 8 below; all changes in nucleotides which result in 10 codons which are translated into these amino acids are within the scope of this invention. The attenuated RSV subgroup B viruses of this invention exhibit a substantial reduction of virulence compared to wild-type viruses which infect human and 15 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. 20 The attenuated RSV subgroup B 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 25 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 30 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 WO99/15672 PCT/US98/19145 - 35 with cryoprotective additives or stabilizers such as proteins (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate), saline, or other protective agents. This 5 mixture is maintained in a liquid state, or is then dessicated or lyophilized for transport and storage and mixed with water immediately prior to administration. Formulations comprising the attenuated viruses of this invention are useful to immunize a 10 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 RSV subgroup B virus 15 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 20 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, 25 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. 30 In another embodiment of this invention, an isolated nucleic acid molecule having the complete viral nucleotide sequence of the wild-type viruses, the vaccine viruses or the revertant viruses described herein is used to generate oligonucleotide probes (from 35 either positive strand antigenomic message sense or WO99/15672 PCT/US98/19145 - 36 negative strand complementary genomic sense) and to express peptides (from positive strand antigenomic message sense only), which are used to detect the presence of those wild-type viruses, vaccine strains 5 and/or revertant strains in samples of body fluids and tissues. The nucleotide sequences are used to design highly specific and sensitive diagnostic tests to detect the presence of the virus in a sample. Polymerase chain reaction (PCR) primers are 10 synthesized with sequences based on the viral wild type, vaccine or revertant 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 15 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. 20 ELISA tests are used to detect the presence of antigens of the wild-type, vaccine or revertant viral strains. Peptides are designed and selected to contain one or more distinct residues based on the wild-type, vaccine or revertant sequences described 25 herein. These peptides are then coupled to a hapten (e.g., keyhole limpet hemocyanin (KLH) and used to immunize animals (e.g., rabbits) for the production of monospecific polyclonal antibody. A selection of these polyclonal antibodies, or a combination of polyclonal 30 and monoclonal antibodies can then be used in a "capture ELISA" to detect antigens produced by those viruses. Samples of mutant RSV described herein have been deposited by Applicants' assignee on March 19, 35 1992 with the American Type Culture Collection (ATCC) WO99/15672 PCTIUS98/19145 - 37 12301 Parklawn Drive, Rockville, Maryland, U.S.A. 20852, under the provisions of the Budapest Treaty for the Deposit of Microorganisms for the Purposes of Patent Procedures ("Budapest Treaty"). The viruses were 5 accorded the following ATCC designation numbers: 2Bp33F(VR 2364), 2Bp24G(VR 2370), 2Bp20L(VR 2368), 2Bp34L(VR 2365), 3Ap20E(VR 2369), 3Ap20F(VR 2367), and 3Ap28F(VR 2366). In addition, samples of the 2B wild type RSV virus were deposited by Applicants on August 10 21, 1997 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A., under the provisions of the Budapest Treaty and have been assigned ATCC accession number VR2586. Given these deposited subgroup B strains and 15 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 subgroup B strains described herein, as well as taking 20 these strains and making additional mutations from the panel of mutations set forth in Tables 21 and 22 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 25 are not to be construed as limiting the scope of the invention. Examples 30 Standard molecular biology techniques are utilized according to the protocols described in Sambrook et al. (76).

WO99/15672 PCT/US98/19145 - 38 Example 1 Passage and Characterization of RSV 2B and RSV 3A Parental Strains 5 RSV 2B and RSV 3A parental strains were isolated and passed in qualified cell lines and under conditions consistent with use as clinical study material. Two RSV strains, 20648 and 23095, were 10 isolated by Dr. Robert Belshe (St. Louis University Health Science Center, St. Louis, MO) from nasal swab samples taken from ill children. These viruses were later recovered from the original frozen nasal swab samples, passed two to three times in primary rhesus 15 monkey kidney (PRMK) cells, and then sent to applicants. Isolate 20648 (subgroup B) was renamed RSV 2B. Virus was passed seven times in PRMK cells at 35 0 C, two times in Vero cells at 35 0 C and plaque 20 purified and amplified three times (six passages) in Vero cells at 36oC. Virus was further amplified an additional two times in Vero (36'C), stocks were filtered with a 0.2 m filter and amplified another two times in Vero cells. This was followed by production 25 of a Master Seed (RSV 2B, MK7 Vl2b), Intermediate working seed (RSV 2B, MK7 Vl3b) and Working seed (RSV 2B, MK7 Vl4b). See Figure 1. Isolate 23095 (subgroup A) was renamed RSV 3A. RSV 3A was passed eight times in PRMK cells at 30 35 0 C. This was followed by two passages in Vero at 35°C and six passages in Vero cells at 36 0 C, including three plaque purification steps. Virus was further passaged six times in Vero cells at 36 0 C including a 0.2 m filtration step. This was followed by production 35 of a Master seed (RSV 3A, MK8 Vl5b), Intermediate WO99/15672 PCT/US98/19145 - 39 working seed (RSV 3A, MK8 Vl6b), and Working seed (RSV 3A, MK8 Vl7b). See Figure 1. Subgroup specificities of RSV 2B and RSV 3A Master seeds were confirmed using subgroup specific 5 monoclonal antibodies. Virus stocks were shown to be free of microbial contaminants and adventitious agents. The F, N, and G proteins of RSV 2B and RSV 3A stocks and reference RSV strains A2, Long, and 18537 were analyzed by radioimmunoprecipitation (RIP) and 10 western blotting procedures using monoclonal antibodies. The F1 subunits of the RSV subgroup B strains, 2B and 18537, migrated faster on SDS polyacrylamide gels than did the F1 subunits of the RSV subgroup A strains, 3A and Long. No difference in 15 migration of the N proteins of the RSV 2B and 3A strains and the reference strains was seen. In RIP gels, the G protein was visible as two bands at approximately 80-90 kD and approximately 45 kD. The 80-90 kD bands of RSV 3A and Long comigrated; however, 20 the 80-90 kD band of RSV 2B also appeared to comigrate with the subgroup A species rather than with the faster RSV 18537 (subgroup Bl). This suggests that RSV 2B may be a member of the B2 subgroup as described by Akerlind (77). In western blots, the relative proportions of 25 80-90 kD and 45 kD bands were roughly equal for RSV Long, A2, 2B, and 18537 grown in Vero cells, but staining of the 80-90 kD band of RSV 3A was significantly greater, suggesting a difference in processing of the G protein for this strain when grown 30 in Vero cells. These data demonstrate that the apparent Mr for the RSV 2B and RSV 3A strains are consistent with current subgroup classifications of RSV, but confirm that these strains are not identical to the prototype RSV reference strains. 35 Growth of RSV 2B, RSV 3A and RSV A2 in mice WO99/15672 PCT/US98/19145 - 40 and in cotton rats was compared. Both RSV 2B and RSV 3A replicated poorly in Balb/c mice compared to the RSV A2 reference strain. Consistent recovery of RSV 2B and RSV 3A could only be obtained at the highest inoculum 5 dose used (106.0-6.2 PFU), and was similar in magnitude to recovery of RSV A2 at a 100-fold lower inoculum (1043 PFU). In contrast, growth of RSV 2B in cotton rat nose and lungs was similar to growth of RSV A2. Growth of RSV 3A in the nose was similar to the other 10 strains; however growth in lungs was significantly poorer. Both mouse and cotton rat growth data indicate that RSV 2B and RSV 3A have significantly different in vivo growth characteristics than the RSV A2 reference strain, as well as differing from each other. 15 Example 2 Cold Adaptation of RSV In order to select an appropriate starting 20 temperature for cold adaptation of RSV, growth of the RSV 2B and RSV 3A parental strains in Vero cells at temperatures ranging from 26°C to 36'C was compared. Cells were infected at an MOI of 0.4 and virus yield and CPE was monitored for four days. The results, 25 shown in Figures 2 and 3, demonstrated that for both virus strains, growth at 30 0 C, 32'C, and 36°C was similar in kinetics and yield. At 26'C, virus growth lagged behind growth at the higher temperatures by about 24 hours. The limiting factor in achieving 30 optimum titers appeared to be the viral CPE, which occurred earlier at higher temperatures. For both RSV 2B and RSV 3A, optimum titers were achieved by maintaining cultures at 30'C. At this temperature, a lower level of CPE allowed growth and spread of virus 35 to continue over a longer time period. The results WO99/15672 PCT/US98/19145 - 41 suggested that these strains of RSV were already well adapted to growth at 30 0 C to 36 0 C. A maximum tem perature of 26 0 C was selected as a starting temperature for cold adaptation, as virus growth at this tempera 5 ture was suboptimal and therefore some selective pressure for cold adaptation would be exerted. Cold adaptation was initiated on virus stocks RSV 2B (passage MK7 V14) and RSV 3A (passage MK8 V14). To maximize the chance of recovering appropriately 10 attenuated mutants from these cold-adapted populations, two flasks of virus were independently passed using each of three different cold adaptation strategies. This provided a total of six cold-adapted populations for RSV 2B and six for RSV 3A. Virus was passaged in 15 25 cm 2 flasks containing confluent Vero cell monolayers. At each passage, virus was harvested by replacing the maintenance medium (10 mls of MEM/2%FBS/20mMHepes) in the infected flask with a reduced volume of freezing medium (3 mls of 20 MEM/10%FBS/20mMHepes) and performing a quick freeze at -70'C followed by a thaw at 32°C. To infect the next passage, one ml of the freeze-thaw lysate was transferred to a fresh flask of confluent Vero cells, virus was allowed to adsorb at room temperature (20°C 25 22°C), and then flasks were overlaid with maintenance medium (MEM/2%FBS/20mMHepes) and incubated at the appropriate temperature in water baths (i.e., 26'C, 22 0 C, 20 0 C). Titrations were performed at 32°C on each 30 freeze-thaw lysate and the remainder of the material was stored at -70'C for future isolation of virus variants. Three passaging strategies were used. Flasks E and F were "slowly" adapted, beginning at 26 0 C with four passages every two days, followed by passage 35 once every week until titers appeared to be relatively WO99/15672 PCT/US98/19145 - 42 stable or were increasing. Virus was then passaged weekly at 22oC until consistently high titers were achieved, and finally maintained by passage every 1-2 weeks at 20'C. Flasks G, H ,and I were adapted by a 5 more moderate strategy. Virus was passaged two times at 26oC at three day intervals, then passaged weekly at 22oC five times, and finally maintained by passage every 1-2 weeks at 20 0 C. Flasks J and L were "rapidly" adapted, starting with five weekly passages at 22oC, 10 followed by passage at 1-2 week intervals at 20'C. Actual passage conditions and titration results are shown in Tables 1 and 2, and are summarized in Table 3. Titration results obtained at each passage are graphically displayed in Figure 4. The titration 15 results demonstrated an influence of strain on rate of adaptation. For RSV 2B, all three cold adaptation strategies eventually yielded high virus titers when flasks were maintained at 20'C. In contrast, RSV 3A was adapted to growth at 20 0 C using a "slow" strategy 20 (E,F), but efforts to force a more rapid adaptation resulted in a precipitous decline in virus growth. Passage of these cultures (3A:H,I,J,L) was discontinued. To screen the cold-adapted virus populations 25 for accumulation of ts variants, virus taken from each flask following 5 and 17 weeks of cold adaptation was tested for efficiency of plaquing (EOP) at 39 0 C vs. 32 0 C. As seen in Table 4, in most cases plaquing efficiency of the cold passage virus was relatively 30 high at 39 0 C ( 0.2) and was similar to values obtained with the parental virus control (>0.6). The results showed that the cold-adapted virus populations, with the possible exception of flask RSV 3A-F, had not become predominantly ts over a period of up to 17 low 35 temperature passages.

WO99/15672 PCT/US98/19145 - 43 Following further cold passaging, attempts were made to isolate temperature sensitive mutants by plaque purifying virus from each cold-adapted flask. Plaque purified mutants were initially identified by 5 relatively poor growth (lower titers or smaller plaque size) at 39 0 C vs 32 0 C. In these assays, shown in Table 5, the percentage of plaque purified virus that could be clearly identified as temperature sensitive ranged from 0% to 40% of plaques picked. Several individual 10 flasks (2B-H, 2B-L, 3A-E, 3A-F) appeared to contain a relatively higher percent of ts phenotypes, and in some cases the percentage of ts mutants increased over time. However, ts mutants did not appear to become a predominant variant over a period of up to 42 weeks of 15 cold passaging. To summarize, cold passaging of RSV 2B and RSV 3A resulted in cold adaptation of virus based on the ability of virus to grow stably at 20 0 C with consistently high yields. Analysis of EOP assays and 20 the rate of isolation of ts mutants indicated that although ts mutants did arise in the cold-adapted virus populations, they did not become a predominant species. Example 3 25 Screening for Vaccine Candidates in In Vitro Studies The ts mutants were further screened and selected for vaccine candidates based on degree of temperature sensitivity in vitro, attenuation in animal 30 models (including mice, cotton rats, and chimps), and retention of neutralizing epitopes. Over a period of 39 weeks of cold adaptation, a total of 13 RSV 2B and six RSV 3A ts mutants were plaque purified a second time and further 35 characterized. Comparison of EOP's at 37/32 0

C,

WO99/15672 PCT/US98/19145 - 44 39/32'C, and/or 40/32°C confirmed that these mutants had reduced plaquing efficiency at the higher temperatures and represented a range of temperature sensitivity (see Table 6). 5 Prior to completing the isolation of all 19 mutants described above, a group of four mutants, RSV 2Bp24G, RSV 2Bp20L, RSV 3Ap20E, and RSV 3Ap20F, were selected from the first set of plaque purified viruses for preliminary characterization. To look at actual 10 virus growth curves, Vero cells were infected with these four mutants at an MOI of 2, and incubated at 20°C, 32-C, 37-C, and 40 0 C for seven days. The results, shown in Figure 5, indicated that all four mutants were cold-adapted and temperature sensitive, as evidenced by 15 earlier and higher rises in titer in cultures incubated at 20'C, and reduced or absent growth of virus in cultures incubated at 37'C, 39 0 C, and 40 0 C. Based on the degree of temperature sensitivity seen in EOP and growth studies, one subgroup A and one subgroup B 20 mutant, RSV 2Bp20L and RSV 3Ap20E, were selected to perform additional preliminary experiments on phenotypic stability and growth in mice. The infectivity and immunogenicity of RSV 2Bp20L and RSV 3Ap20E were evaluated in Balb/c mice. 25 Virus growth was measured in nasal wash and lung samples harvested four and five days post-infection and serum neutralizing antibody titers were determined 32 days post-infection. Results are shown in Table 7. Growth and immunogenicity of the parental virus was 30 very low, but detectable. In contrast, no virus was recovered and no neutralizing antibody was detected following inoculation of the ts strains, indicating that these strains were highly attenuated in mice. Of the 19 ts mutants which were eventually 35 isolated, four RSV 2B and three RSV 3A mutants were WO99/15672 PCTIUS98/19145 - 45 selected for further in vitro and in vivo characterization. These mutants included the original four mutants described above, as well as three mutants isolated at later time points. Selection criteria 5 included demonstration of definite ts phenotype at both 37 0 C and 39oC and representation of both subgroups and varying passage strategies and passage numbers. These seven ts mutants were plaque purified a third time and amplified to make small working stocks. Their passage 10 histories are summarized in Table 8. The initial analysis of these mutant strains included comparison of plaquing efficiencies and plaque morphologies at 32 0 C, 37oC, and 39 0 C in Vero cells (Table 9), and growth at 32 0 C, 37 0 C, 39 0 C, and 40oC in Vero cells (Table 10) At 15 37 0 C and 39°C, EOP was reduced and small and intermediate plaque sizes predominated, indicating that mutants were ts. Some breakthrough of "wt" plaque size revertants was seen with all variants except RSV 2Bp34L and RSV 3Ap20F. 20 In growth studies, Vero cells were infected with the virus strains at an MOI of 0.2 and virus yield was determined four days post-infection. Comparison of virus yields in Vero cells at the various temperatures demonstrated that virus yield, expressed as PFU per 25 cell, decreased significantly at the higher temperatures (37 0 C, 39(C, 40 0 C). In some cases, virus yield was also somewhat reduced at 32 0 C relative to the parental strain, indicating attenuation in growth at 32 0 C. This is consistent with the smaller plaque sizes 30 observed in the 32oC EOP assays (Table 9). For all strains, at least one plaque was detected in cells incubated at 39 0 C or 40'C, suggesting that some revertants were present. Both EOP and virus yield studies demonstrate that these seven isolates possess 35 varying levels of temperature sensitivity and may WO99/15672 PCT/US98/19145 - 46 represent a range of levels of attenuation. Retention of neutralizing epitopes was examined by comparing reactivities of the seven mutants and parental strains with two neutralizing monoclonal 5 antibodies representing antigenic sites A and C on the F protein described by 19-Beeler and Coelingh (1989) (Table 11). Both antibodies were able to neutralize all the virus strains at similarly high dilutions, indicating that the neutralizing epitopes were intact. 10 Example 4 Growth of Mutant Strains in Animal Models Growth and immunogenicity of the seven ts 15 mutant strains was evaluated in cotton rats. Groups of rats were inoculated intranasally with each mutant and lungs and nasal turbinates were harvested four days post-infection for virus titrations. Sera were collected from an identical set of rats 20 days post 20 infection to test for neutralizing and EIA antibody responses. A summary of virus titration and immunogenicity results are shown in Table 12. RSV 2B grew well in the nose and lungs, whereas growth of all four RSV 2B ts mutants was very poor. Two of the 25 mutants, RSV 2Bp33F and RSV 2Bp24G, displayed a less attenuated phenotype than did RSV 2Bp20L and RSV 2Bp34L, as indicated by a slightly higher level of replication, as well as a 100% infection rate. The RSV 3A parental and ts mutant strains grew well in the 30 nasal turbinates, but poorly in the lungs. Titers of the RSV 3A ts mutants were lower than that of the parental strain, indicating that the ts mutants were somewhat more attenuated than the parent virus. Neu tralizing and EIA-F antibody titers on sera from rats 35 infected with the RSV 2B and RSV 3A parental and ts WO99/15672 PCT/US98/19145 - 47 mutant strains were also measured. The level of neutralizing and EIA-F antibody titer was low for the RSV 2B ts mutants, consistent with the low level of viral replication seen. Interestingly, titers from 5 animals infected with RSV 2Bp33F were higher than would be expected in view of the low titration values, and may indicate an intermediate level of attenuation for this virus. Neutralizing and EIA-F antibody titrations on all 3 RSV 3A ts mutants demonstrated that these 10 mutants were quite immunogenic, consistent with their high level of replication in nasal tissue. Growth of RSV 2Bp20L was further evaluated in cotton rats from three to seven days post-infection to determine if failure to recover virus was due to a 15 shift in timing of peak titers. RSV 2B was used as a positive control (see Figure 6). The growth kinetics of RSV 2B were typical of other strains of RSV; peak titers occurred on days 4 and 5 in nasal turbinates and on day 4 in lungs. These results substantiate the use 20 of day 4 as the optimal harvest day for the parental strain. RSV 2Bp20L was not detected in lungs and rare plaques were seen in nasal turbinate titrations on days 3, 5, 6, and 7, demonstrating that attenuation of this virus was not simply due to an early or late growth 25 peak. Relative growth and immunogenicity of RSV 2B and RSV 2Bp20L were also compared in four year old seropositive chimps. Two chimps were infected intranasally with 104.0 and 10 s . PFU of RSV 2B, and two 30 chimps were similarly infected with RSV 2Bp20L. The results are shown in Figure 7 and Table 13. Both chimps infected with RSV 2B developed a mild upper respiratory infection, consisting of nasal discharge and cough. Both chimps shed virus from three through 35 seven days post-infection. The amount of virus shed WO99/15672 PCT/US98/19145 - 48 was higher and shedding occurred earlier in the chimp infected with the higher dose of RSV 2B. Neither chimp inoculated with RSV 2Bp20L showed clinical signs of disease or shed virus. Chemistry and hematology 5 workups on all four chimps revealed no significant findings. Serum neutralizing and EIA-F, Ga, and Gb antibody titers were substantially increased 14 and 21 days post-infection with RSV 2B. No rises in antibody titers were seen in chimps inoculated with RSV 2Bp20L. 10 The results indicated that, in seropositive chimps, the parental RSV 2B strain was infectious and immunogenic, whereas the RSV 2Bp20L mutant was highly attenuated. 15 Example 5 Challenge Experiments in Cotton Rat Model Additional experiments were done in cotton 20 rats to evaluate the efficacy of the RSV 2B and 3A ts mutants in preventing infection when challenged with a reference strain of the homologous subgroup (RSV 18537/subgroup B and RSV A2/subgroup A). Cotton rats (eight per group) were inoculated intranasally with 25 each RSV ts mutant. Nasal turbinates and lungs were harvested four days post-infection, from four rats per group, for virus titrations. Six weeks post-infection, the remaining rats were bled for neutralizing and EIA-F titers, then challenged with the appropriate reference 30 RSV strain. Four days post-challenge, nasal turbinates and lungs were removed for virus titration. Results are shown in Tables 14 and 15. As discussed previously and shown in Table 12, growth of all four RSV 2B TS mutants was very poor 35 compared to the parental RSV 2B strain. Neutralizing WO99/15672 PCT/US98/19145 - 49 and EIA antibody titers elicited by RSV 2Bp33F and RSV 2Bp24G were relatively high despite poor virus recovery, possibly indicating an intermediate level of attenuation for both mutants. Level of protection 5 against virus challenge reflected the level of neutralizing antibody response and was high for RSV 2Bp33F and 2Bp24G, moderate for RSV 2Bp20L, and ineffective for RSV 2Bp34L. All RSV 3A strains grew in the nasal turbinates but demonstrated a high level of 10 attenuation in growth in the lungs. Titers of neutralizing and EIA antibodies were high and all rats were completely protected against virus challenge. The results demonstrate that growth of the attenuated strains elicited protective immunity against 15 virus challenge, suggesting that these strains may be useful as vaccine. Failure of vaccination with the RSV 2Bp34L strain to protect was most likely due to failure of virus to grow due to its high level of attenuation. Since cotton rats are a less susceptible host than 20 humans, failure of this strain to protect does not imply that 2Bp34L would be an ineffective vaccine in humans. Example 6 25 Challenge Experiments in African Green Monkey Model Growth, immunogenicity, and efficacy of ts mutant strains RSV 2Bp33F, 2Bp24G, 2Bp20L, 3Ap20E, 3Ap20F, and 3Ap28F were evaluated in African green 30 monkeys (AGMs). AGMs are more susceptible to infection with human RSV than are the cotton rats, and characteristics of infection may be more relevant to that seen in humans because of the closer phylogenetic relationship. Two AGMs each were inoculated with 106 35 PFU of each mutant virus by combined intranasal and WO99/15672 PCT/US98/19145 - 50 intratracheal route. Virus growth was evaluated by nasal wash and bronchial lavage. Neutralizing and EIA antibody responses were tested at approximately 0, 1, 2, 3, 4, 6, and 8 weeks post-infection. Eight weeks 5 post-infection, animals were challenged with 106 PFU of the parental strain by intranasal and intratrachial route. Virus growth and antibody response was evaluated as described above. Growth of the parental RSV 2B and 3A strains 10 can be seen in Figures 8 and 9: vaccine controls. Both virus strains grew to high titers in both the nose and lung. Nasal discharge and radiographic evidence of viral pneumonia was seen in one control monkey (032B) infected with RSV 2B, demonstrating that RSV is capable 15 of causing disease in AGMs. These results confirm differences in these characteristics of infection in AGMs vs the cotton rat model, in which disease was not observed and RSV 3A was unable to replicate in the lung. Failure of the parental strains of RSV to cause 20 disease in three of four monkeys suggests that the AGMs are not as susceptible a host as are humans. Virus titrations for each monkey infected with the RSV 2B ts mutants and then challenged with the parental strain are shown in Figure 8. RSV 2Bp33F grew 25 to low levels in the nasal wash in one of two monkeys, RSV 2Bp24G grew to low levels in nasal wash or in lungs in both monkeys. RSV 2Bp20L failed to grow. In those AGMs where the RSV 2B ts mutants grew, monkeys were partially to fully protected against challenge with 30 parental strain. Tables 16 and 17 give antibody titration results obtained for each monkey post vaccination (Table 16) and post-virus challenge (Table 17). Results show that in monkeys where virus grew, low levels of neutralizing and EIA antibody titers were 35 seen by 2.5 weeks post-infection. Following challenge WO99/15672 PCT/US98/19145 - 51 with the parental strain, antibody titers boosted one full week earlier in vaccinated monkeys with antibody titers prior to challenge, than in vaccinated animals which failed to seroconvert or in unvaccinated 5 controls. This demonstrated that vaccination with these ts mutants was sufficient to both prime the immune system and to elicit protection against virus challenge. Because these monkeys are not as susceptible to infection as humans, failure of 10 attenuated virus to grow and to effectively immunize does not imply that virus would not be effective in a fully susceptible host (i.e. seronegative human infant). Virus growth in AGMs infected with the RSV 3A 15 ts mutants and challenged with the parental 3A strain are shown in Figure 9. All three RSV 3A ts mutant strains were attenuated in growth, in the order of most to least attenuated: 3Ap28F>3Ap20E>3Ap20F. Vaccination with all three ts mutants afforded excellent protection 20 against virus challenge. Antibody response for monkeys vaccinated with RSV 3A ts mutants is shown in Table 18, and response following virus challenge is shown in Table 19. In all vaccinated AGMs, with the exception of one monkey given RSV 3Ap28F, low levels of 25 neutralizing and EIA antibody titers were detected beginning three weeks post-vaccination. Following challenge with the parental strain, all vaccinated monkeys boosted a full week earlier than the unvaccinated controls and were protected either fully 30 or partially from infection, demonstrating that vaccination primed the immune response and was protective. This included the one AGM in which antibody response was not detected following vaccination, indicating that measure of serum antibody 35 response may not be fully representative of level of WO99/15672 PCT/US98/19145 - 52 protective immunity. The results from the AGM studies again demonstrate that all six ts mutants tested were attenuated. Vaccination with those mutants which were 5 able to replicate in these monkeys was efficacious in preventing infection with challenge virus. Example 7 Degree of Attenuation 10 An RSV ts mutant, TS-1, was obtained from Dr. Brian Murphy, NIH. This ts mutant was originally derived from the RSV A2 strain by chemical mutagenesis and was tested in clinical trials in seronegative human 15 infants in the 1970's. The outcome of these trials suggested that TS-1 was underattenuated and caused an unacceptable level of disease (rhinitis and otitis media) in infants. In addition, the ts phenotype of TS-1 partially reverted following growth in humans. 20 Experiments have been carried out which compare growth of the RSV 2B and 3A ts mutants with that of the TS-1 mutant in an attempt to assess the relative in vivo attenuation level of the RSV 2A and 3B mutants, and to demonstrate differences between these mutants and what 25 had been used by others in previous clinical trials. The results of the cotton rat study are shown in Table 20, and may be compared directly with the cotton rat data shown in Tables 14 and 15. The TS-1 mutant was less attenuated than the RSV 2B and 3A ts mutants, as 30 can most clearly be seen by comparing growth in the lung. A growth study in African green monkeys (AGMs) comparing TS-1 with RSV 2Bp33F and 3Ap28F was carried out and the results are shown in Figure 10. 35 Monkeys were infected with virus either intranasally WO99/15672 PCT/US98/19145 - 53 (TS-1 and 2Bp33F) or intranasally plus intratracheally (3Ap28F). Virus was recovered in one of four monkeys infected with 2Bp33F and two of four monkeys infected with 3Ap28F. Titers were relatively low in both cases, 5 indicating that virus was attenuated. In contrast, relatively high titers of virus were recovered in all four monkeys inoculated with TS-1. In two of four monkeys, the levels of TS-1 titers were equivalent to those seen in monkeys infected with wild type virus. 10 TS-1 did not spread to the lungs, as would be expected for wild type virus, indicating that TS-1 was somewhat attenuated. The results clearly show that RSV 2Bp33F and 3Ap28F have different phenotypic characteristics than TS-1 and are significantly more attenuated. This 15 higher level of attenuation is a property that is desirable for a vaccine to be administered to human infants. Example 8 20 Sequence Analyses of RSV Subqroup B Strains The temperature-sensitive (ts) phenotype is strongly associated with attenuation in vivo; in addition, some non-ts mutations may also be 25 attenuating. Identification of ts and non-ts attenuating mutations was achieved by sequence analysis and evaluation of ts, cold-adapted (ca), and in vivo growth phenotypes of RSV mutants and revertants. The genomes of the following five RSV 2B 30 strains have now been completely sequenced: 2B parent, 2B33F, one revertant designated 2B33F TS(+), 2B20L and one revertant designated 2B20L TS(+). The 2B33F and 2B20L strains are ts and ca and are described in U.S. Serial No. 08/059,444 (78), which is hereby 35 incorporated by reference. After identifying regions WO99/15672 PCTIUS98/19145 - 54 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 5 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 10 between phenotype ts, vaccine attenuation and sequence changes have been identified. A summary of results is presented in Tables 21-26. Several significant observations can be drawn 15 from these data: a. As shown in Tables 21 (for 2B33F) and 22 (for 2B20L), there are relatively few sequence changes identified in the two mutant strains: RSV 2B33F 20 differs from parental RSV 2B by two changes at the 3' genomic promoter region, two changes at the non-coding 5'-end 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 25 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 30 2B20L virus mapped to the coding region of the L gene. Potentially attenuating mutations at the RNA dependent RNA polymerase gene have been identified. b. Two ts mutations can be identified in the L 35 gene of the attenuated virus strains 2B33F and 2B20L: WO99/15672 PCTIUS98/19145 - 55 (i) In 2B33F, a mutation at nucleotide position 9853 (A -+ G) leading to a coding change in L protein at amino acid 451 (Lys -> Arg) is clearly associated 5 with the ts and attenuation phenotypes. Reversion at this site alone in the 2B33F TS(+) 5a strain is responsible for complete restoration of growth at 39 0 C (Table 23) and partial reversion in attenuation in animals. This association of ts was also supported by 10 partial sequence analyses of six additional "full ts revertants" (designated 4a, 3b, pp2, 3A, 5a, 5A) isolated from cell culture and from chimps, in which only the nucleotide 9853 mutation reverted (Tables 24 26) (note that one AGM (African Green Monkey) isolate 15 which reverted at 9853 only partially reverted in ts phenotype). This amino acid 451 mutation (Lys -+ Arg) is amenable to stabilization in cDNA infectious clone constructs, by inserting a second mutation to stabilize the codon, thereby lessening the likelihood that it 20 will revert back to Lys. (ii) In 2B20L, a mutation at base 14,649 (A -+ G) leading to a coding change in the L protein (amino acid position 2,050, Asn -> Asp) appears to be associated 25 with the ts and attenuation phenotypes. This aspartic acid at the amino acid 2050 invariably reverts back (Asp -+ Asn) in TS(+) revertants or changes to a different amino acid (Asp -> Val) by nucleotide substitution at position 14,650 (A -> T) (Tables 22, 30 25). The above observation is based on complete sequence analysis on the TS(+) revertant R1 and partial sequence of several additional TS(+) revertants (R2, R4A, R7A, R8A) at selected regions (Table 25). An WO99/15672 PCT/US98/19145 - 56 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 5 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: 10 (i) A change at position 14,587 (C - T) with a corresponding change (Thr -+ Ile) at amino acid 2029 is present in both 2B33F and 2B20L (Tables 21,22). This nucleotide "T" substitution was found to be present in 15 10% of the population of the progenitor RSV2B strain and may have been preferred during the attenuation process. No wildtype base "C" was found in the 2B33F and 2B20L virus. 20 (ii) Two mutations are seen in the 2B33F and 2B20L 3' genomic promoter region: nucleotide 4 (C -> G) 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 TS(+) revertants were 25 analyzed, these mutations were seen to have been retained in the 2B33F TS(+)5a (Table 21) and the 2B20L TS(+)R1 (Table 22) revertants. These non-coding, cis acting mutations remained associated with partial viral attenuation. 30 Expression using the minireplicon RSV-CAT system for the analysis of these cis-acting changes has shown the 3' genomic promoter nucleotide 4 (C -* G) change to be an upregulation of transcription/replication in this in vitro system when WO99/15672 PCT/US98/19145 - 57 the 2B progenitor virus or either of the 2B33F or 2B33F TS(+) provided helper L gene functions (the N, P and M2 genes are identical in these viruses). Complementation analysis of the 2B33F 3' 5 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 10 transcription/replication functions. However, the 2B33F and 2B33F TS(+) viruses preferred their 2B33F 3' genomic promoters. This analysis clearly shows co evolution of 3' genomic promoter changes during the vaccine attenuation process, along with the RNA 15 dependent RNA polymerase gene. Reversion of ts phenotype in the 2B33F mutant 5a by reversion of the single L protein amino acid 451 (Arg -+ Lys) by sequence analysis was clearly demonstrated by support of transcription/replication functions of RSV-CAT 20 minireplicon at 37 0 C. The 2B33F virus did not provide helper functions to the RSV-CAT minireplicon (with 2B or 2B33F 3' genomic promoters) at 37 0 C. d. A biased hypermutation of SH seen in 2B33F is 25 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. 30 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 wild type strains were identical. For 2B, the codon ACA at 35 nucleotides 14586-14588 encodes a Thr at amino acid WO99/15672 PCT/US98/19145 - 58 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). 5 Example 9 PCR Assay to Detect RSV A PCR assay is used to detect the presence of 10 RSV. PCR primers are designed and selected based on homologies to the RSV sequences described herein to be specific for all subgroup B strains, or for the individual wild-type, vaccine or revertant RSV subgroup B strains described herein. The assay is conducted by 15 subjecting the sample to reverse transcription of RNA, followed by PCR amplification of selected cDNA regions corresponding to RSV nucleotide sequence. Amplified PCR products are identified on gels and their specificity confirmed by hybridization with specific 20 RSV nucleotide probes. Example 10 ELISA to Detect RSV 25 An ELISA test is used to detect the presence 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 wild-type, vaccine or revertant RSV subgroup B strains 30 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 35 ELISA" to detect the presence of an RSV antigen.

WO 99/15672 PCT/US98/19145 - 59 Table 1 RSV 2B Cold Adaptation E,F Cumm. Time Incubation Virus Yield Passage Passage Temp Time IOoPFU # Weeks oC Days E F 1 0.2 26 2 6.9 6.7 2 0.4 26 2 6.0 6.1 3 0.6 26 2 5.5 5.6 4 0.8 26 2 4.5 4.7 5 1.0 26 7 4.9 5.0 6 2.0 26 7 6.2 6.3 7 3.0 26 7 7.9* 7.6* 8 4.0 22 7 7.5 7.6 9 5.0 22 7 7.3 7.3 10 6.0 22 7 7.2* 7.2* 11 7.0 22 7 7.5* 7.7* 12 8.0 22 7 8.0* 7.9* 13 9.0 22 7 8.0* 7.9* 14 10.0 20 7 7.6 7.7 15 11.0 20 7 7.0 5.9 16 12.0 20 7 7.2 7.1 17 13.0 20 7 6.7 6.3 18 15.0 20 14 5.5 5.2 19 17.0 20 14 6.3 6.0 20 18.0 20 7 6.1 5.8 21 19.0 20 7 5.4 5.7 22 20.0 20 7 5.9 5.7 23 21.0 20 7 6.3 5.5 24 22.0 20 7 6.9 6.3 25 23.0 20 7 6.8 6.6 26 24.0 20 8 6.6 6.2 27 25.0 20 7 6.3 6.0 28 26.0 20 6 6.5 6.2 29 27.0 20 7 6.2 6.3 30 28.0 20 7 7.0 7.2 31 29.0 20 7 7.3 7.1 32 30.0 20 7 6.8 6.5 33 31.0 20 7 6.9 6.7 34 32.0 20 7 6.9 7.0 35 33.0 20 7 7.4 7.0 36 34.0 20 7 7.2 7.1 37 35.0 20 7 7.4 7.0 38 36.0 20 7 7.4 7.1 39 37.0 20 7 7.5 7.0 40 38.0 20 7 7.2 6.7 *Syncytial CPE seen at harvest SUBSTITUTE SHEET (RULE 26) WO 99/15672 PCT/US98/19145 - 60 Table lb RSV 2B Cold Adaptation G,H Cumm. Time Incubation Virus Yield Passage Passage Temp Time looPFU # Weeks oC Days G H 1 0.3 26 3 7.1 7.1 2 0.7 26 3 6.9 6.9 3 1.0 22 7 6.4 6.4 4 2.0 22 7 6.4 6.3 5 3.0 22 7 6.6 6.4 6 4.0 22 7 6.9 6.8 7 5.0 20 7 6.9 6.7 8 6.0 20 7 6.3 6.3 9 7.0 20 7 6.2 6.3 10 8.0 20 7 6.6 6.9 11 9.0 20 7 7.0 7.0 12 10.0 20 7 7.0 7.4 13 11.0 20 7 6.3 7.3 14 12.0 20 7 7.7 7.9 15 13.0 20 7 7.2 7.4 16 15.0 20 14 6.4 6.3 17 16.0 20 8 6.8 6.9 18 17.0 20 6 6.9 7.0 19 18.0 20 7 6.9 7.1 20 19.0 20 7 6.7 7.0 21 20.0 20 7 6.4 6.8 22 21.0 20 7 6.5 7.0 23 22.0 20 7 6.9 7.1 24 23.0 20 7 6.8 6.7 25 24.0 20 8 6.4 6.2 26 25.0 20 7 6.0 5.5 27 26.0 20 6 6.3 5.5 28 27.0 20 7 6.5 5.9 29 28.0 20 7 7.1 6.4 30 29.0 20 7 6.1 7.1 31 30.0 20 7 6.4 5.5 32 31.0 20 7 6.2 5.9 33 32.0 20 7 6.4 6.2 34 33.0 20 7 6.4 6.9 35 34.0 20 7 6.9 6.5 36 35.0 20 7 7.0 6.7 38 37.0 20 7 7.1 7.2 SUBSTITUTE SHEET (RULE 26) WO 99/15672 PCT/US98/19145 - 61 Table ic RSV 2B Cold Adaptation J,L Cummn. Time Incubation Virus Yield Passage Passage Temp Time loQI 0 PFU # Weeks oC Days J L 1 1.0 22 7 6.8 2 2.0 22 7 7.1 3 3.0 22 7 6.7 4 4.0 22 7 5.9 6.1 5 5.0 22 7 4.8 5.7 6 6.0 20 7 4.9 5.0 7 7.0 20 7 4.8 4.9 8 9.0 20 14 6.0 6.0 9 11.0 20 14 6.6 6.3 10 12.0 20 7 6.9 6.9 11 13.0 20 7 6.6 6.7 12 15.0 20 14 6.0 6.0 13 16.0 20 8 6.3 6.2 14 17.0 20 6 6.2 6.5 15 18.0 20 7 6.6 6.7 16 19.0 20 7 6.4 6.9 17 20.0 20 7 6.5 6.9 18 21.0 20 7 6.9 7.0 19 22.0 20 7 7.4 7.4 20 23.0 20 7 7.2 7.4 21 24.0 20 8 7.0 7.1 22 25.0 20 7 6.8 6.9 23 26.0 20 6 6.9 7.0 24 27.0 20 7 7.0 7.0 25 28.0 20 7 7.8 7.4 26 29.0 20 7 7.5 7.3 27 30.0 20 7 6.8 6.7 28 31.0 20 7 6.9 6.8 29 32.0 20 7 7.0 6.9 30 33.0 20 7 7.4 7.2 31 34.0 20 7 7.3 6.7 32 35.0 20 7 7.3 6.9 33 36.0 20 7 7.3 7.0 34 37.0 20 7 7.2 6.9 35 38.0 20 7 6.6 6.3 SUBSTITUTE SHEET (RULE 26) WO 99/15672 PCT/US98/19145 - 62 Table 2a RSV 3A Cold Adaptation E Cumm. Time Incubation Virus Yield Passage Passage Temp Time 1o 0 OPFU # Weeks oC Days E 1 0.2 26 2 6.2 2 0.4 26 2 5.1 3 0.6 26 2 4.7 4 0.8 26 2 3.8 5 1.0 26 7 4.0 6 2.0 26 7 5.0 7 3.0 26 7 6.1 8 4.0 22 7 6.0 9 5.0 22 7 5.6 10 6.0 22 7 5.8 11 7.0 22 7 5.7 12 8.0 22 7 5.9 13 9.0 22 7 5.9 14 11.0 20 14 5.8 15 13.0 20 14 6.1 16 15.0 20 14 4.8 17 17.0 20 14 4.9 18 19.0 20 14 4.8 19 20.0 20 7 4.3 20 22.0 20 14 4.9 21 24.0 20 14 5.2 22 26.0 20 15 5.6 23 28.0 20 13 6.3 24 30.0 20 14 6.3 25 32.0 20 14 7.3 26 34.0 20 14 7.8 27 36.0 20 14 7.2 28 38.0 20 14 7.4 29 40.0 20 14 6.8 30 42.0 20 14 7.3 SUBSTITUTESHEET(RULE 26) WO 99/15672 PCT/US98/19145 - 63 Table 2b RSV 3A Cold Adaptation F Cumm. Time Incubation Virus Yield Passage Passage Temp Time log 10 PFU # Weeks oC Days F 1 0.2 26 2 6.1 2 0.4 26 2 5.1 3 0.6 26 2 4.7 4 0.8 26 2 3.6 5 1.0 26 7 4.3 6 2.0 26 7 5.3 7 3.0 26 7 6.4 8 4.0 22 7 6.3 9 5.0 22 7 5.2 10 6.0 22 7 5.8 11 7.0 22 7 5.7 12 8.0 22 7 6.0 13 9.0 22 7 5.6 14 11.0 20 14 5.5 15 13.0 20 14 5.4 16 15.0 20 14 3.9 17 17.0 20 14 3.7 18 19.0 20 14 3.5 19 21.0 20 14 3.8 20 23.0 20 14 4.2 21 25.0 20 15 3.2 22 27.0 20 13 3.9 23 29.0 20 14 4.5 24 31.0 20 14 4.7 25 33.0 20 14 4.8 26 35.0 20 14 5.3 27 37.0 20 14 5.5 28 39.0 20 14 5.8 SUBSTITUTE SHEET (RULE 26) WO 99/15672 PCT/US98/19145 - 64 Table 2c RSV 3A Cold Adaptation H,I Cumm. Time Incubation Virus Yield Passage Passage Temp Time log 10 PFU # Weeks oC Days H I 1 0.3 26 3 6.9 7.0 2 0.7 26 3 6.1 6.4 3 1.0 22 7 5.8 5.8 4 2.0 22 7 5.8 5.9 5 3.0 22 7 5.9 5.7 6 4.0 22 7 5.6 5.5 7 5.0 22 7 5.1 5.1 8 6.0 20 7 4.0 3.8 9 7.0 20 7 3.3 2.8 10 9.0 20 14 3.9 3.2 11 11.0 20 14 3.9 3.1 12 13.0 20 14 4.0 3.0 J,L Cumm. Time Incubation Virus Yield Passage Passage Temp Time log 10 PFU # Weeks oC Days J L 1 1.0 22 7 6.7 2 2.0 22 7 6.7 3 3.0 22 7 6.0 4 4.0 22 7 5.7 5.6 5 5.0 22 7 4.2 4.9 6 6.0 20 7 3.7 3.7 7 7.0 20 7 3.1 3.0 8 9.0 20 14 2.8 3.2 9 11.0 20 14 2.3 3.3 10 13.0 20 14 3.0 2.8 SUBSTITUTE SHEET (RULE 26) WO 99/15672 PCT/US98/19145 - 65 Table 3 Summary of Cold Adaptation Passage History #Parental Virus Passage # Cold Adaptation Passage PRMK Vero Vero Virus 35 0 C 35 0 C 36 0 C Total Flask 26 0 C 22 0 C 20 0 C Total 2B 7 2 12 21 E,F 7 6 27 40 G,H 2 5 32 39 J,L 0 5 30 35 3A 8 2 12 22 E 7 6 17 30 F 7 6 15 28 H,I 2 5 5 12 J,L 0 5 5 10 SUBSTITUTE SHEET (RULE 26) WO 99/15672 PCT/US98/19145 - 66 Table 4 Efficiency of Plaquing of Cold Passaged Virus Virus Week 5 Week 17 2B 0.8 0.6 2B-E 0.6 0.6 2B-F 0.8 0.7 2B-G 0.6 0.8 2B-H 0.7 0.4 2B-J ND 0.9 2B-L 0.7 0.6 3A 0.6 0.6 3A-E 0.6 0.4 3A-F 0.8 0.2 3A-H 0.6 ND 3A-I 0.9 ND 3A-J 0.6 ND 3A-L 0.6 ND SUBSTITUTE SHEET (RULE 26) WO 99/15672 PCT/US98/19145 - 67 Table 5 TS Mutants Plaque Purified from Cold Adapted Virus #TS/#Total #TS/#Total Cumm./Passage Plaques Cumm./Passage Plaques Weeks # Isolated Weeks # Isolated E F E wk23/p25 0/10 1/10 wk22/p20 2/10 wk31/p33 0/10 1/10 wk32/p25 1/10 wk38/p40 0/10 1/10 wk42/p30 3/9 G H F wk23/p24 1/10 2/10 wk23/p20 1/9 wk31/p32 0/10 2/10 wk31/p24 0/10 wk38/p39 1/10 4/10 wk37/P27 1/10 wk39/p28 2/5 J L wk23/p20 0/9 1/10 wk31/p28 0/10 0/10 wk37/p34 0/8 2/9 wk38/p35 1/20 3/8 SUBSTITUTE SHEET (RULE 26) WO 99/15672 PCT/US98/19145 - 68 Table 6 Summary of EOP Data on Twice Plaque Purified RSV TS Mutants EOP RSV Isolate 37/32 0 C 39/32 0 C 40/32 0 C 2B (parent) 0.7-1.0 0.6-0.8 0.4 2Bp33F (ppl0-1) 0.5 0.002 ND 2Bp40F (pp7-20. 0.0008 ND 2Bp24G (pp2-1) 0.2 0.00001 <0.00001 2Bp39G (pp7-3) 1.0 0.009 ND 2Bp24H (pp 3

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2 ) ND 0.003 0.001 2Bp32H (pp6-2) 0.9 0.03 ND 2Bp39H (pp6-5) 1.0 0.04 ND 2Bp35J (pp2-1) 0.4 0.2 ND 2Bp20L (pp5-1) 0.02 ND <0.00001 2Bp34L (pp2-2) 0.005 0.0005 ND 2Bp35L (ppl-l) 0.3 0.02 ND 2Bp35L (pp 2 -1) 0.5 0.1 ND 2Bp35L (pp8-3) 0.2 0.05 ND EOP RSV Isolate 37/32 0 C 39/32 0 C 40/32 0 C 3B (parent) 1.0 0.5-0.9 0.6 3Ap20E (pp3-1) 0.6 0.006 0.000009 3Ap25E (pp7-5) 0.5 0.2 ND 3Ap30E (pp 3 -1) 0.4 0.08 3Ap20F (pp 4

-

3 ) 0.8 >0.1 0.000004 3Ap27F (ppl-2) 0.3 0.003 ND 3Ap28F (ppl0-1) 0.2 0.002 ND ND = Not Done SUBSTITUTE SHEET (RULE 26) WO 99/15672 PCTIUS98/19145 - 69 1 vI v H~ v a) 4J. 44I ri 0 4J1 E-1 V 0 :3cnI N c v v iCq4 a) ) . ' d 03 0 U (D Ac * ol tfq ri H 4 4J (n~ EU r4 4 - r i (d m~ eqZ 1.4 EUE'H 4 m0 0) 'o- ( .rIr 4'. z rda t ru4 Ln.r a4 H, 4a) E~ (D o (d .o 00W 124I -r (DI HZ 0 p~ i4 C-4 -au 9) .r X m J I P) a) .) V4 o 0 w. .r4I U o + H- ,( . -EJ r-I o 4.) ) -. -1 - -. 1-. Fi 2 .rq 4.)~ co 0 0OC 0 rI >4 4.) 4 44 .4.) a)E .4.) (drI 1) 0 V. 0 * ) H 0 a1)

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4 4' a) a)O0H-4) V) M P)4I C4C M o I wL Ai"( 14 (D -) 4-) 0 ~ (Eda) ri to0 (d H t ) '4 ) H H)( Idd (d0 ( ) r 414.. 4UP ) z > z 0 M 'L -'L) 0 1 1.4 P4 C~l CN m M > SUBSTITUTE SHEET (RULE 26) WO 99/15672 PCTIUS98/1 9145 -70 cw, mx 0 0 T4 4J~ ~ (dLn Ln U) Ln Ln 4-4 ..g + $4 :3 >1 34 o0 0 C**4 H- H- C4H 0 0 N 0 %D L n nk (d 0 P4 m tid (40 C14 E-C I I C 0 C414 F-d 0 N CI cI C4 N CIq NI N $4 C-l rl H 44 H. CI LA CId 0 co --. Pd PdPd Pd P $4 (d1 l '4 00 (d~~ ~ 4 dn q cq C r 4~C C C4 C7 r-4 r- C4CI SUBSTTUTESHEE (RUE 26 WO 99/15672 PCTIUS98/1 9145 - 71 0 > 0 0 H H1 0 .Im pO H) H 00 HlNz4)ul% X 4) m r a%0 0 CIO1 ,- .0 0 CO HO 3% MHO%00 04 HnU 0n cr-I C cr- roi ~-o 0 00 00 rZI 0) + +4 + (+ P4I COa.C H .J 0r -0C4 C I cn o LAI0 14 C'4 > 14CN P4 C1 4 C' 40c 4 SUBSTITUTE SHEET (RULE 26) WO 99/15672 PCTIUS98/19145 _ 72 00 0 4 T4 4H00 (U 0H oH M4 P- HHiHE a% 0 0 0O 0 00 000 CD 4JJ rdo c% n 0~0 C) 0 r-H O- " C 0~0 0 (0 0 'taCecO C14 4- ~ I E- )a rdl (d 0 0 C1 lC 1 - O N - 0) C4r q N4)$

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ro Ic M +~ SUSITT SHE (RL>6 WO 99/1 5672 PCTiUS98/19145 - 73 wn 0 -00 0 0 C0 0) 0 0 0 0 0 0) 0JC 0 0 0 0 0 0 D Ui 0 0 0 0D LA 0 0 0 0 H- 0D 0 0 0) 0 0 0) 0 m~ M vi V1 v V rdH OD Co C)O L C CD4 en 0 C 0 CA 0 >0 0 0 0D 0 0 0 wn > d 0 0 0 0D 00 0 0 0 0 C) 0C 0 0 0 0 0 5 D 0 0 U m 0 kA 10r-4 Q I0 0 o 0 H 0 e i 0 0 0 a)(~0 0 0 0 0 00 0 n 0 (D 00 H 0 440 O n U) 0 o n C14 e4 C . 0D -0 0 s NC m en' N'4 N'H en C~ N 4 'H C'4' ('4 Vl-H Q0 i 04 C,4 en P4' r- P4 P 4 I' mH m m 4 ' 4 n 4 1 > C40C N'4 ('4' e1n en CVoC H (' LA en H P4 P4 Pd P P4 P4 P4 P4 P 4 P4 SUBSTITUTE SHEET (RULE 26) WO99/15672 PCT/US98/19145 - 74 Table 11 Monoclonal Antibody Neutralization of RSV 2B and RSV 3A Parental and TS Mutants Neutralization Titers Challenge Strains 143-6C 133-1H 2B 15,091 46,775 2Bp33F 23,364 32,690 2Bp24G >25,600 32,571 2Bp20L 25,972 32,790 2Bp34L 16,757 77,172 3A 99,814 46,493 3Ap20E 76,203 >25,600 3Ap20F 69,513 13,743 3Ap28F 80,436 34,136 Neutralizations were done by a standard 60% plaque reduction neutralization assay on Vero cell monolayers in 96-well microtiter plates. Challenge with a 1:400 dilution of non neutralizing monoclonal antibody 131-2G showed no reduction in titer in any of the nine strains. SUBSTITUTE SHEET (RULE 26) WO 99/15672 PCT/US98/19145 - 75 0 0 I l C C-4 0 0N C- -4 C1 C) -c C)ca Lii -V V vi- 0 I 0 1000 C -) * 0 D)CC01C 0 )C'N N-DN N-) w m 11' CD _*6- .C-oj C) N .~C)ic1 C10C- )C L VI-V V it V V I_ LL 'T( CO 04 1 C) u' 10~0cC CC)CD< r-N CO a M r,- 'Nr- 10CD IC'4 -"CO c) ci C oCNI CN co CD 0C~ 6 mC fOCO LU -V *V -1 0E 4 o-------------------C

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CJ) C ) C) C- C= ICC) )c) ) C 0- = C%4 COOO0 > (0 CD 0 0 q0 00 w oCD > cl uc:,) It CD0 CDC O "-mCIT ~ -v CCo > N~J ' LOJ N Cj C) C) m CD C) qC CD C) 4- C -+ ,a- 0e co 0) C 0 cc 0~~~4 C-wql N-- v C a)L +- EC 60 - a)c 4D t U) a) CN 0)~C 0- CD CT) C0N)1 E u OC) - C N ) '- C 0 6D >D C D U ~o. 0C (n 0~ 0 '0) 00 CD-0 C? ) C) 04 m? C (N (N ( L6 CO C C r:v CO .4 < i i: <' -"U >0~~C (1N cN ( N ( ? O C) C) 0 .u SUSzrT cHE (RL06 WO 99/15672 PCT/US98/19145 -76 two < miccot Co + E CO <OO ~ % O o LU < N . -"C C "- , , , - ~ qC N C H A z '- 0 0 < 0 0< C3 Oo 0) ou oo o z ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~- Ml) qI r- C : ()V )C 0 t - )C4 0 o"-l oo o 0 m C14 C C 0. SUSITT SHE (RUL 26 WO 99/15672 PCT/US98/19145 -77 $4 0) $4 ~ $4 M M 2 1 Ha Vaj~ V V .4 41 Ca 4J~z 4o VH 0d p 0 H 0 0+4 .41 -v.4 v.40 k4 r-4 H.. $( Ca $4 4 (h o w vV1V 0 0 $4 44d -H r) (d mA 00 0a 04~C rj 0 C1i - r -I uI r-i , 4 > 2' 4.) -4 k V (d OD 4 U) (d . d NTUS W4 0 N0 r ( r '-I 4-4 r- *1- H >L n 0 1 '14 Ln -40 u-I u-V i .)4. (1)m 0) 0$'A C1 V j H > Hm * V 4 (U 4.) 4 EA4 rj $4 V) $4 4-4 r- mm vHr H >(d > 4.) (U U) r4- H 4) -l C v.4 4 (d - r ii. 4 .) 4U J V d H 4.)- vd .1.) T-1 H ( 4 .H ( VH -v4 rT4 V1 v .) (U Ca (d .) V4 4.)(U C (U . 14 a V.H .0 -.4i 14 M .............. '1 .......... o'd 0 r(U Ca 0 rl 0 m H 0 OW 0 0 a V >'VVV V H~ (U (U H .4 m0 0 0 V aa 4(U tmU k4 ty 4 - i m >4 :3 0 0 a' Vo (U ix -I -r t) i m4 V (U a w Vd V 44I Ea _! C $4 0-r4 0 r 4 ia I Ca t~C 'I~~ i-v4( 0 0............. 4 i--i V) Ca 4) V 0)OA~~L 0 U 1 H- V 4.) Via H. 0 0 A ) 0 mCa w '44 L0 u-- 04 u 0 kD u) mm mq M N >4C'C'4 ol C -4 C SUBSTITUTE SKEET (RULE 26) WO 99/15672 PCT/US98/19145 - 78 $4 40) 0 4.) X4 r74 043 0. H C 4. 4.) V.. 0'00 r 0D 4) 4.)r4 >D Vo 0) 14H H LA iiT M) V- Vb) ~ ( ~ $ U0 $40 0 r: r o4 4.) 4 I (4 D C C 0H CUl .I0 44 0 .j C L l$ (d' I D tq H V0 00 U) rzH (' o - 14 v 0 0 0 C14 1)I H 0 4 ) rN V.~' *n k n H4.i 0H HLA C' 0n k n 0) -r4 m) 0 V. $4H r- v > 4 ' - >0 m 0)14 ) )H N. $4' 04 0) 0 00)r m (U 14 - (U m) 4) 4).)r 0 $4w w >- (U 4 o 4 4)H- ('L C -~ C v J) 4. 4) -ri 4. $ r 4 N VI V44 V. . ) - 4 A~ :j m ) 4) id :J M 2 0 0 4) (U 4 '0H$4) 0 >4 $ 44 M~lV V H) H1 v CDmo 0 W A 0) 4.) 4i V, CD ( 4) (d U j 0 (d HH D 041 v vi 0W $)4'd t 0 k E-~~ (U Hd M U i 0) ) H .A m r4.) (U >1 cd u :j$4 0 T-4 0 r 0 cq mD 0)0 (U 00 A 0 4 H0 0.4 $4. t ~ -H 0 $ d c 0 4.) -H) (')* (m r- Q '4 SUSITT SHEE (tUL 26)Tj 0 0 WO99/15672 PCT/US98/19145 - 79 Table 16 RSV Growth and Immunogenicity in African Green Monkeys: RSV 2B ts Mutants' ts Mutant Immunogenicity Virus Growth Neutralization EIA Peak Virus Titer Titers 2 Titers 3 (log01O PFU/ml) (x10 3 ) Virus AGM Nasal Lung Day 2B A2 3A anti-F 2Bp33F SK034 <0.7 <0.7 0 <10 <10 <10 <0.15 7 <10 <10 <10 <0.15 14 <10 <10 <10 0.88 24 <10 <10 <10 0.49 27 <10 <10 <10 0.92 41 <10 <10 <10 0.26 2Bp33F SKO28 2.9 <0.7 0 <10 <10 <10 <0.15 7 <10 <10 <10 <0.15 14 <10 <10 <10 <0.15 24 28 <10 <10 2.92 27 33 <10 <10 3.56 41 19 <10 11 1.74 2Bp24G SK012 <0.7 1.9 0 <10 <10 <10 <0.15 7 <10 <10 <10 <0.15 14 <10 <10 <10 <0.15 24 12 <10 <10 14.91 27 10 <10 <10 13.18 41 <10 <10 <10 11.05 1 = All monkeys were inoculated with 106 PFU of RSV 2B ts virus, IN+IT. 2 = 60% plaque reduction neutralization test. 3 = Source of coating protein is RSV A2 F protein.

WO99/15672 PCT/US98/19145 - 80 Table 16 (continued) RSV Growth and Immunogenicity in African Green Monkeys: RSV 2B ts Mutants 1 ts Mutant Immunogenicity Virus Growth Neutralization EIA Peak Virus Titer Titers 2 Titers 3 (loglO PFU/ml) (x10 3 ) Virus AGM Nasal Lung Day 2B A2 3A anti-F 2Bp24G SKO30 3.2 <0.7 0 <10 <10 <10 <0.15 7 <10 <10 <10 <0.15 14 <10 <10 <10 <0.15 24 440 10 204 34.13 27 404 21 190 37.64 41 256 <10 98 17.74 2Bp20L SK033 <0.7 <0.7 0 <10 <10 <10 0.24 7 <10 <10 <10 0.30 14 <10 <10 <10 1.48 24 <10 <10 <10 1.64 27 <10 <10 <10 1.24 41 <10 <10 <10 0.34 2Bp20L SK042 <0.7 <0.7 0 <10 <10 <10 <0.15 7 <10 <10 <10 <0.15 14 <10 <10 <10 <0.15 24 <10 <10 <10 1.64 27 <10 <10 <10 0.20 41 <10 <10 <10 0.15 1 = All monkeys were inoculated with 106 PFU of RSV 2B ts virus, IN+IT. 2 = 60% plaque reduction neutralization test. 3 = Source of coating protein is RSV A2 F protein.

WO99/15672 PCT/US98/19145 - 81 Table 17 RSV Growth and Immunogenicity in African Green Monkeys: RSV 2B Challenge of Monkeys 8 Weeks Post-Vaccinated with RSV 2B ts Mutants' Challenge Immunogenicity Virus Growth Neutralization EIA Peak Virus Titers 2 Titers 3 Titer Vaccine (loglO0 PFU/ml) (x10 3 ) Virus AGM Nasal Lung Day 2B A2 3A anti-F 2Bp33F SK034 5.7 5.0 0 <10 <10 <10 0.52 7 <10 <10 <10 0.91 14 451 10 472 303.84 21 6797 33 829 592.61 28 4353 50 815 135.85 42 1978 44 264 52.76 2Bp33F SK028 <0.8 3.8 0 13 <10 <10 2.50 7 208 65 576 43.64 14 2868 443 2051 131.70 21 1883 404 2344 144.39 28 1127 227 2797 53.22 42 941 79 729 43.30 2Bp24G SK012 3.9 3.2 0 <10 <10 <10 21.58 7 281 111 323 258.89 14 604 431 731 551.49 21 698 298 692 668.50 28 357 325 985 189.81 42 272 82 895 104.16 1 = AGMs were previously vaccinated with RSV 2B ts strains. All monkeys were challenged 8 weeks post-vaccination with 106 PFU of RSV 2B, IN+IT. 2 = 60% plaque reduction neutralization test. 3 = Source of coating protein is RSV A2 F protein.

WO99/15672 PCT/US98/19145 - 82 Table 17 (continued) RSV Growth and Immunogenicity in African Green Monkeys: RSV 2B Challenge of Monkeys 8 Weeks Post-Vaccinated with RSV 2B ts Mutants' Challenge Immunogenicity Virus Growth Neutralization EIA Peak Virus Titers 2 Titers 3 Titer Vaccine (logl0 PFU/ml) (x10 3 ) Virus AGM Nasal Lung Day 2B A2 3A anti-F 2Bp24G SK030 <0.8 <0.7 0 91 <10 69 34.55 7 628 322 1120 174.07 14 1617 397 1953 203.58 21 1184 256 968 145.71 28 851 276 1313 49.28 42 637 48 329 27.36 2Bp20L SK033 5.3 4.5 0 <10 <10 <10 1.78 7 <10 <10 <10 1.88 14 516 <10 325 289.94 21 500 22 550 418.91 28 783 56 525 148.25 42 518 48 442 91.39 2Bp20L SK042 5.4 3.3 0 <10 <10 <10 0.21 7 <10 <10 <10 0.32 14 36 <10 116 135.80 21 213 21 284 116.99 28 256 30 300 30.06 42 516 40 289 19.30 1 = AGMs were previously vaccinated with RSV 2B ts strains. All monkeys were challenged 8 weeks post-vaccination with 106 PFU of RSV 2B, IN+IT. 2 = 60% plaque reduction neutralization test. 3 = Source of coating protein is RSV A2 F protein.

WO99/15672 PCT/US98/19145 - 83 Table 17 (continued) RSV Growth and Immunogenicity in African Green Monkeys: RSV 2B Challenge of Monkeys 8 Weeks Post-Vaccinated with RSV 2B ts Mutants' Challenge Immunocrenicity Virus Growth Neutralization EIA Peak Virus Titers 2 Titers 3 Titer Vaccine (loglO PFU/ml) (x10 3 ) Virus AGM Nasal Lun Day 2B A2 3A anti-F Control SK046 5.9 4.7 0 <10 <10 <10 0.10 7 <10 <10 <10 0.13 14 272 50 594 275.33 21 488 98 1140 587.93 28 1377 75 1393 190.49 42 1659 47 573 183.97 Control 032B 5.5 3.9 0 <10 <10 <10 0.25 7 <10 <10 <10 0.24 14 2462 201 3458 626.57 21 1546 303 1279 482.31 28 1162 104 1729 164.53 42 1044 83 689 75.1 1 = AGMs were previously vaccinated with RSV 2B ts strains. All monkeys were challenged 8 weeks post-vaccination with 106 PFU of RSV 2B, IN+IT. 2 = 60% plaque reduction neutralization test. 3 = Source of coating protein is RSV A2 F protein.

WO99/15672 PCT/US98/19145 - 84 Table 18 RSV Growth and Immunogenicity in African Green Monkeys: RSV 3A ts Mutants' ts Mutant Immunogenicity Virus Growth Neutralization EIA Peak Virus Titers 2 Titers 3 Titer (logl0 PFU/ml) (x10 3 ) Virus AGM Nasal Lung Day 2B A2 3A anti-F 3Ap20E 01128 1.7 1.3 0 <10 <10 <10 <0.05 7 <10 <10 <10 <0.05 14 <10 <10 <10 3.72 21 38 <10 39 23.12 28 21 <10 13 27.42 40 14 <10 <10 31.11 3Ap20E 0L1161 2.3 2.9 0 <10 <10 <10 <0.05 7 <10 <10 <10 <0.05 14 <10 <10 30 5.84 21 14 12 57 19.51 28 56 18 126 27.53 40 44 31 108 38.90 3Ap20F 90B037 3.2 <1.0 0 <10 <10 <10 <0.05 7 <10 <10 <10 <0.05 14 <10 <10 11 1.17 21 12 <10 47 34.32 28 20 17 86 31.58 40 49 26 123 35.05 1 = All monkeys were inoculated with 106 PFU of RSV 3A ts virus, IN+IT. 2 = 60% plaque reduction neutralization test. 3 = Source of coating protein is RSV A2 F protein.

WO99/15672 PCTIUS98/19145 - 85 Table 18 (continued) RSV Growth and Immunogenicity in African Green Monkeys: RSV 3A ts Mutants' ts Mutant Immunogenicity Virus Growth Neutralization EIA Peak Virus Titers 2 Titers 3 Titer (loglO PFU/ml) (x10 3 ) Virus AGM Nasal Lung Day 2B A2 3A anti-F 3Ap20F 90B045 3.9 1.0 0 <10 <10 <10 0.35 7 <10 <10 <10 0.30 14 <10 <10 19 3.16 21 11 11 22 12.61 28 12 13 24 16.82 40 24 <10 39 14.92 3Ap28F 91B027 2.0 <0.8 0 <10 <10 <10 <0.05 7 <10 <10 <10 <0.05 14 <10 <10 <10 0.18 21 <10 <10 <10 1.29 28 <10 <10 <10 2.63 40 <10 11 27 3.83 3Ap28F 91B043 4 2.9 <0.9 0 <10 <10 <10 <0.05 7 <10 <10 <10 <0.05 14 <10 11 27 3.83 1 = All monkeys were inoculated with 106 PFU of RSV 3A ts virus, IN+IT. 2 = 60% plaque reduction neutralization test. 3 = Source of coating protein is RSV A2 F protein. 4 = Monkey died on day 15. Cause of death unrelated to RSV infection.

WO99/15672 PCT/US98/19145 - 86 Table 19 RSV Growth and Immunogenicity in African Green Monkeys: RSV 3A Challenge of Monkeys 8 Weeks Post-Vaccinated with RSV 3A ts Mutants' Challenge Immunogenicity Virus Growth Neutralization EIA Peak Virus Titers 2 Titers 3 Titer Vaccine (loglO PFU/ml) (x10 3 ) Virus AGM Nasal Lung Day 2B A2 3A anti-F 3Ap20E 01128 1.4 0.7 0 14 12 <10 35.75 7 216 281 602 699.10 14 417 265 784 611.24 21 289 307 573 247.16 28 263 289 731 463.57 42 145 141 426 285.47 3Ap20E 0L1161 2.2 <0.8 0 21 <10 56 25.37 7 526 412 2735 535.05 14 516 521 2382 252.93 21 581 473 1840 275.32 28 478 437 1651 244.01 42 250 239 753 141.33 3Ap20F 90B03 <1.1 <0.8 0 84 56 221 41.17 7 2374 2093 6051 435.25 14 3701 2916 8652 450.55 21 2933 2224 6561 481.99 28 1849 1588 4031 287.28 42 3086 967 3950 207.98 1 = AGMs were previously vaccinated with RSV 3A ts strains. All monkeys were challenged 8 weeks post vaccination with 106 PFU of RSV 3A, IN+IT. 2 = 60% plaque reduction neutralization test. 3 = Source of coating protein is RSV A2 F protein.

WO99/15672 PCT/US98/19145 - 87 Table 19 (continued) RSV Growth and Immunogenicity in African Green Monkeys: RSV 3A Challenge of Monkeys 8 Weeks Post-Vaccinated with RSV 3A ts Mutants' Challenge Immunogenicity Virus Growth Neutralization EIA Peak Virus Titers 2 Titers 3 Titer Vaccine (loglO PFU/ml) (x10 3 ) Virus AGM Nasal Lung Day 2B A2 3A anti-F 3Ap20F 90B045 <1.0 <0.8 0 24 12 56 13.44 7 644 627 1381 182.99 14 1024 549 2174 223.70 21 1835 699 2130 273.87 28 831 318 1499 177.21 42 534 258 1073 127.80 3Ap28F 91B027 <1.0 0.8 0 <10 <10 <10 5.50 7 408 229 521 150.97 14 585 560 2016 234.25 21 449 311 1161 359.53 28 316 400 714 184.91 42 242 217 436 142.67 Control 91K041 5.0 4.7 0 <10 <10 <10 <0.05 7 <10 <10 <10 0.13 14 19 <10 205 213.29 21 106 33 423 602.54 28 123 99 278 562.05 42 107 80 277 252.99 1 = AGMs were previously vaccinated with RSV 3A ts strains. All monkeys were challenged 8 weeks post vaccination with 106 PFU of RSV 3A, IN+IT. 2 = 60% plaque reduction neutralization test. 3 = Source of coating protein is RSV A2 F protein.

WO99/15672 PCTIUS98/19145 - 88 Table 19 (continued) RSV Growth and Immunogenicity in African Green Monkeys: RSV 3A Challenge of Monkeys 8 Weeks Post-Vaccinated with RSV 3A ts Mutants' Challenge Immunogenicity Virus Growth Neutralization EIA Peak Virus Titers 2 Titers 3 Titer Vaccine (lo0gl0 PFU/ml) (x10 3 ) Virus AGM Nasal Lung Day 2B A2 3A anti-F Control 91K059 5.1 4.6 0 <10 <10 <10 <0.05 7 <10 <10 <10 0.09 14 97 34 384 166.59 21 288 158 1259 268.47 28 <160 <160 575 286.52 42 290 178 1448 218.74 1 = AGMs were previously vaccinated with RSV 3A ts strains. All monkeys were challenged 8 weeks post vaccination with 106 PFU of RSV 3A, IN+IT. 2 = 60% plaque reduction neutralization test. 3 = Source of coating protein is RSV A2 F protein.

WO 99/15672 PCT/US98/19145 -89 W N ) (D 0) ~4) 0 4.) OtOd ~~P 0) 0 7Ll M) W 0 (U 0 () 0 >- 1tO * 4.) t~ V Vm (d 0 p HY 41S i (U 0 0U 41 0 0 0)M . r74 >1 H C', 0, V 0 ~H C1 C) v E-1 'i( COlM 4 u co H 0 (d 0 44 ~ ~ ~ ~ ~ ( -4 00lc ) 0 0) ) N N- (Ul (Ud -) > O) PU P (U C' t] 4. d 0. oH 4 H N 0 04 0)rl r L )( (U m) S .rl~~t 0) 0 Wo : -jk W p4 rl 4 0 (U0m Q) o a) Vt* 0 0) 4j ki N ) q C14 cn 0 rl (U 0 ~ (U 01 0 Si rl 4. >~ )~l ( 4.) r'iN p ) H w N P4 -'-4 N 1 0S - 0) -'- r-O N 0l E4.) C (U 4.) 4 ) 0 (d rq OWl . ) 'dH-'4 (U 0 V4p I w C4 0' d 0 4UQ J +10)) (U N ) 0 i 0) u r- ,I (U d 0 N r to, oi C)C rl 0 (d I >i CiV)a ' Pd 0) 0)N 0 -I :J (U C.7' ,Qf 0' 4.) 4. 4J (U P M P Si14 0) WH 41~ ) 3 HJ W N P4 C4) Sd 0l ) Q) H o p 44 O -r r 0 (d i 1 d0 N0 I iiC 4 m qP 4 4j HD cm SUBSTITUTE SHEET (RULE 26) WO 99/15672 PCT/US98/19145 - 90 Table 21 Sequence comparison between RSV 2B and 2B33F strains Nucl. Nucleotide changes pos.t Gene/ 3' end RSV 2B RSV RSV 2B33F Amino acid region of vRNA 2B33F TS(+), 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 (10) 4409 T C C none Ile (36) 4420 T C C Ile-Thr (40) 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-Gln (66) 4505 T C C none Ser (68) 4525 T C C Ile-Thr (75) 4526 T C C Ile-Thr (75) 4542 T C C Stop-Gln (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 t For 2B33F and 2B33F TS(+), nucl. pos. numbers 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 TS(+) strain WO 99/15672 PCT/US98/19145 - 91 Table 22 Sequence comparison between RSV 2B and 2B20L strains Nucl. Nucleotide changes pos.t Gene/ 3' end RSV 2B RSV RSV 2B20L Amino acid region of vRNA 2B20L TS(+), R1 changes revertant Genomic 4 C G G non-coding* Promoter 6 - extra A extra A non-coding* L 8963 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 (2050)** t For 2B20L and 2B20L TS(+), nucl. 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 TS(+) revertant WO 99/15672 PCT/US98/1 9145 - 92 r-4 .00 0go om ~- V- 4 0 -d w to 4J-M c(U W i c 00i A' n 'w i 'w '4' JJ 44 DO~44~.44 .. w vf4 4 Ln Ln v *'. .- -' -1 . 0 (D3 0 01

O$

4 Ii r4r - n N .4.4 rs) 4) .3 u -f 0*- On > ~ 0-> 0 0 0' 0 0 00) P4a oo) C; C; C 0 E-1 Wto 04. 0U~ $4)~ t 04 t-0 4.) 41 0 N3 0 V oy 0 Ol ot tno 0 P4I c04-IiX 0 ( C4 h e.Q l C140 0 m m m 9 4J m m Id 4J (44J c (41 4M . m03 $4 (d a 4) w (4) (D4) 43r ) 0~~ ~ r4) 0 i .0 Ti) 04 (1 Q 04i 0 V SUSITT SHEE (RUE6 WO 99/15672 PCT/US98/19145 -93 H 4 ~u V H-l 0 1 0 .j c (UU U 4.) m 4.) z cn CIH :j 0 a- 0d 0 VI 4.) N . 4.) - ) 0 04. 0Q4 > 0 0 >4 0D (U~ ~ $4 0-. V a) H0 04. 0 4 43 0 0 0- O OrOlO 04U at4 H4UJ 04u. 0 4) 0 40) Lo E N 0 0- 0- U; t > 00 r Pd 0' 4j' d 'Od 1. JHO J0 0 .)00 0 W 0 ~ 04& UmM C1o4 C .4 U4. 3 CU 3 id r. l4 4 ~ d .4 ) -rCO4) ( 4C ) U )1 u a) 4.)U4 4) 't-. (H a 2Hr c .0 0 toW A ( 0c 114u 04UC M9 E 04J 04) 049 44 (d- 493-0 4 - 1 L4 V014 D 4 H0 4 04 a S 04 .r 4V y .V 0 P4 V 4 V 4 &4 NO: q l: 4 0 )& -4fn: -'4 Ch. P4 U SUBSTITUTE SHEET (RULE 26) WO 99/15672 PCT/US98119145 - 94 4) 00 -i 0r 0 m 4.) to r-Cl ~ 44 0 0 00 4. o n qv 0 0 11 -.4 * t V1J v- q Vi- Vi- ci4 0 4 4.) H 4.) rc j t 0 ) 0) 0m 0j H 0 _ 0' _ _ _ _ _ _: 04 00 0U 0 4 O to ~ 0 01 i0 4 ii) 4-i N 00 E-4 9 - 0 J 0 H i ODg E-LO- ' ~ ~ j4 r- E C14 C, * o -iV 0) C..-4 (DWt 4)W l O Cl UII, 0 U4tJ r:3 4 Q rO W& P CQ0) c 1 4~ r-)4. .:3 4. 4 ( 4 9 4 + +4 9 0 0 :3 w -r 4J -1 4) -i 4. -1 4J w10 01 to cd 4 - (d P - (d 04- t " SUSITT SHE (RUL 26) m ) 43Mr- WO 99/15672 PCT/US98/19145 - 95 Table 24 2B33F Revertants ts (+) In vitro AGM Chimp 5a 4a 3b pp2 pp4 pp6 pp7 1A 3A 5A 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 S S S S 2B 2B 2B 12187 S S S S S S S S S S 14588 S S S S S S S S S S 15072 S S S S S S S S S S Phenotype ts 2B 2B 2B r r S S 2B 2B 2B ca S S S 2B S 2B S ND ND ND Attenuated r r r (r) (r) S S ND r r t These 2B33F revertant base nos. are one larger than for 2B for M, SH and L genes * bases 4330,4410,4421,4443,4455,4485,4498,4506,4526,4527,4543, 4562,4576,4599 S = same base as 2B33F 2B = reversion to 2B base or complete reversion in phenotype r = moderate reversion in phenotype (r) = slight reversion in phenotype ND = not done WO 99/15672 PCT/US98/19145 - 96 Table 25 2B20L Revertants TS(+) In vitro Isolates base no.t R1 R2 R3A R4A R5A R6A R7A R8A R9A R10A 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 nos. are one larger than for 2B for L genes S = same base as 2B20L 2B = reversion to 2B base r = moderate reversion in phenotype * = base change, different from 2B or 2B20L ND = not done WO 99/15672 PCT/US98/19145 - 97 Table 26 RSV 2B, ts and Revertant Strains: Phenotype Sunmmary Virus Isolate Source In Vitro In Vivo Phenotype Attenuation ts ca Cotton AGM Rat RSV 2B Wild-type Parent Strain I - I - RSV 2B33F ca, ts mutant isolated ++++ ++ ++++ +++ from 2B, cold-passaged x 33 RSV 2B33F - 5a 2B33F spinner passage - ++ ++ + TS(+) plaque picked at 39 0 C RSV 2B33F - 4a 2B33F spinner passage - ++ ++ ND TS(+) plaque picked at 39 0 C RSV 2B33F 3b 2B33F spinner passage - ++ ++ ND TS(+) plaque picked at 39 0 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 0 C AGM pp6 2B33F-infected AGM A4, ++++ - ++++ ND d12 nasal wash plaque picked at 32 0 C AGM pp7 2B33F-infected AGM A4, ++++ ++ ++++ ND d12 nasal wash plaque picked at 32 0 C Chimp pplA 2B33F-intected chimp - ND ND ND #1552, d4 tracheal lavage, plaque picked at 32 0 C Chimp pp3A 2B33F-intected chimp - ND ++ ND #1560, d6 tracheal lavage, plaque picked at 32 0 C Chimp pp5A 2B33F-infected chimp - ND ++ ND Chimp #1563, dlO0 tracheal lavage, plaque picked at 32 0

C

WO 99/15672 PCT/US98/19145 - 98 Table 26 (continued) RSV 2B, ts and Revertant Strains: Phenotype Summary Virus Isolate Source In Vitro In Vivo Phenotype Attenuation ts ca Cotton AGM Rat RSV 2B20L ca, ts mutant isolated ++++ ++ ++++ ++++ from 2B, cold-passaged x 20 RSV 2B20L R1 2B20L spinner passage - ND ++ ND TS(+) plaque picked at 39 0 C RSV 2B20L R2 2B20L spinner passage - ND ++ ND TS(+) plaque picked at 39 0 C RSV 2B20L R9 2B20L spinner passage - ND ++ ND TS(+) plaque picked at 39 0 C RSV 2B20L RI0 2B20L spinner passage - ND ++ ND TS(+) plaque picked at 39 0 C ND = not done - = wild-type phenotype, i.e., not temperature sensitive, not cold adapted, not attenuated + to ++++. = increasing levels of temperature sensitivity, cold adaptation or attenuation WO99/15672 PCT/US98/19145 - 99 Bibliography 1. Institute of Medicine, Committee on Issues and Priorities for New Vaccine Development, pages 397-409 of Volume 1, Diseases of importance in the United States (National Academy Press, Washington, D.C. (1985)). 2. Hall, C.B., and McBride, J.T., New Encrg. J. Med., 325, 57-58 (1991). 3. McIntosh, K., and Chanock, R.M., pages 1045-1072 of Virology, B.N. Fields, et al., Eds. (2nd ed., Raven Press, 1990). 4. Anderson, L.J., et al., J. Inf. Dis., 151, 626-633 (1985). 5. Anderson, L.J., et al., J. Inf. Dis., 163, 687-692 (1991). 6. Henderson, F.W., et al., N. Encl. J. Med., 300, 530-534 (1979). 7. Hall, S.L., et al., J. Infect. Dis., 163, 693-698 (1991). 8. Lamprecht, C.L., et al., J. Infect. Dis., 134, 211-217 (1976). 9. Glezen, W.P., et al., J. Ped., 98, 706 715 (1981). 10. Glezen, W.P., et al., Am. J. Dis. Child., 140, 543-546 (1986). 11. Kasel, J.A., et al., Viral Immunology, 1, 199-205 (1987/1988). 12. Mufson, M.A., et al., J. Clin. Microbiol., 25, 1535-1539 (1987). 13. Mufson, M.A., et al., J. Gen. Virol., 66, 2111-2124 (1985). 14. Collins, P.L., pages 103-162 of The Paramyxoviruses, D.W. Kingsbury, Ed. (Plenum Press, NY and London, 1991).

WO 99/15672 PCT/US98/19145 - 100 15. Sullender, W.M., J. Virology, 65, 5425 5434 (1991). 16. Johnson, P.R., et al., J. Virolocgy, 61, 3163-3166 (1987). 17. Stott, E.J., et al., J. Virology, 61, 3855-3861 (1987). 18. Walsh, E.E., et al., J. Inf. Dis., 155, 1198-1204 (1987). 19. Beeler, J.A., and Coelingh, K.W., J. Virology, 63, 2941-2950 (1989). 20. Connors, M., et al., J. Virology, 65, 1634-1637 (1991). 21. Sun, C.S., et al., Am. Rev. Resp. Dis., 128, 668-672 (1983). 22. Anderson, J.J., et al., J. Gen. Virology, 71, 1561-1570 (1990). 23. Graham, B.S., et al., J. Clin. Invest., 88, 1026-1033 (1991). 24. Oppenshaw, P.J.M., et al., J. Virology, 64, 1683-1689 (1990). 25. Nicholas, J.A., et al., J. Virology, 64, 4232-4241 (1990). 26. Cherrie, A.H., et al., J. Virology, 66, 2102-2110 (1992). 27. Kapikian, A.Z., et al., Am. J. Epidemiology, 89, 405-421 (1969). 28. Chin, J., et al., Am. J. Epidemiology, 89, 449-463 (1969). 29. Fulginiti, V.A., et al., Am. J. Epidemiology, 89, 435-448 (1969). 30. Prince, G.A., et al., J. Virology, 57, 721-728 (1986). 31. Gharpure, M.A., et al., J. Virology, 3, 414-421 (1969). 32. Wright, P.F., et al., J. Inf. Dis., 122, WO99/15672 PCT/US98/19145 - 101 501-512 (1970). 33. Richardson, L.S., et al., J. Med. Virology, 3, 91-100 (1978). 34. Maassab, H.F., and DeBorde, D.C., Vaccine, 3, 355-369 (1985). 35. Maassab, H.F., et al., "Cold-adapted influenza viruses for use as live vaccines for man", in Viral Vaccines (Wiley-Liss, Inc. (1990)). 36. Obrosova-Serova, N.P., et al., Vaccine, 8, 57-60 (1990). 37. Edwards, K.M., et al., J. Inf. Dis., 163, 740-745 (1991). 38. Belshe, R.B., and Hissom, F.K., J. Med. Viroloqy, 10, 235-242 (1982). 39. Murphy, B.R., et al., pages 91-95 of Vaccines 90 (Cold Spring Harbor Laboratory Press (1990)). 40. Clements, M.L., et al., J. Clin. Microbiol., 29, 1175-1182 (1991). 41. Kim, H.W., et al., Pediatrics, 48, 745 755 (1971). 42. Forsyth, B.R., and Phillips, C.A., "Laboratory and antigenic studies of respiratory syncytial virus; evaluation of respiratory syncytial, meningococcal, and pneumococcal polysaccharide vaccines", NIH contract report number IDB-VDP-07-067 (1973). 43. Kim, H.W., et al., Pediatrics, 52, 56-63 (1973). 44. Hodes, D.S., et al., Proc. Soc. Exp. Biol. Med., 145, 1158-1164 (1974). 45. Belshe, R.B., et al., J. Inf. Dis., 145, 311-319 (1982). 46. Friedewald, W.T., et al., J. Amer. Med. Assn., 204, 690-694 (1968).

WO99/15672 PCT/US98/19145 - 102 47. Frickey, P.H., "Markers differentiating a 'cold' variant of respiratory syncytial virus from its parent line" (Internal report - Lederle Laboratories, Pearl River, NY (1969)). 48. Holland, J., et al., Science, 215, 1577 1585 (1982). 49. Lamb, R.A., and Kolakosky, D., pages 1177-1204 of Volume 1, Fields Virology, B.N. Fields, et al., Eds. (3rd ed., Raven Press, 1996). 50. Sidhu, M.S., et al., Virology, 193, 50 65 (1993). 51. Garcin, D., et al., EMBO J., 14, 6087 6094 (1995). 52. Radecke, F., et al., EMBO J., 14, 5773 5783 (1995). 53. Collins, P.L., et al., Proc. Natl. Acad. Sci., USA, 92, 11563-11567 (1995). 54. Published European Patent Application No. 702,085. 55. Rota, J.S., et al., Virus Res., 31, 317 330 (1994). 56. Baron, M.D., and Barrett, T., J. Virolory, 71, 1265-1271 (1997). 57. Published International Application No. WO 97/12032. 58. Kato, A., et al., Genes to Cells, 1, 569-579 (1996). 59. U.S. Provisional Patent Application No. 60/047575. 60. Shaffer, M.F., et al., J. Immunol., 41, 241-256 (1941). 61. Lerch, R.A., et al., J. Virology, 64, 5559-5569 (1990). 62. Mallipeddi, S.K., and Samal, S.K., J. Gen Virol., 74, 2787-2791 (1993).

WO99/15672 PCT/US98/19145 - 103 63. Hemming, V.G., et al., Clin. Microbiol. Res., 8, 22-33 (1995). 64. Collins, P. L. et. al., pages 1313-1351 of volume 1, Fields Virology, B. N. Fields, et al., Eds. (3rd ed., Raven Press, 1996). 65. Ling, R., and Pringle, C.R., J. Gen. Virol., 70, 1427-1440 (1989). 66. Yu, Q., et al., J. Virology, 69, 2412 2419 (1995). 67. Heminway, B.R., et al., page 167 of Abstracts of the IX International Congress of Virology, P17-2, (1993). 68. Mink, M.A., et al., Virology, 185, 615 624 (1991). 69. Dickens, L.E., et al., J. Virology., 52, 364-369 (1990). 70. Lamb, R.A., and Kolakofsky, D., pages 1177-1204 of volume 1, Fields Virology, B.N. Fields, et al., Eds. (3rd ed., Raven Press, 1996). 71. Wagner, R.R., and Rose, J.K., pages 1121-1135 of volume 1, Fields Virology, B.N. Fields, et al., Eds. (3rd ed., Raven Press, 1996). 72. Barik, S., J. Gen. Virol., 74, 485-490 (1993). 73. Collins, P.L., et al., pages 259-264 of Vaccines 93: modern approaches to new vaccines including prevention of AIDS, F. Brown et al., Eds. (Cold Spring Harbor Laboratory Press, NY, 1993). 74. Kuo, L., et al., J. Virology., 70, 6892 6901 (1996). 75. Huang, Y.T., and Wertz, G.W., J. Viroloogy, 43, 150-157 (1982). 76. Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

WO99/15672 PCT/US98/19145 - 104 77. Akerlind, B., et al., J. Gen. Viroloqgy, 69, 2145-2154 (1988). 78. U.S. Patent Application No. 08/059,444.

Claims

1. An isolated, recombinantly-generated, attenuated, human respiratory syncytial virus (RSV) subgroup B having at least one attenuating mutation in the RNA polymerase gene.

2. The virus of Claim 1 wherein 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 -> lysine), 451 (lysine -> arginine), 1229 (aspartic acid -+ asparagine), 2029 (threonine -+ isoleucine) and 2050 (asparagine -> aspartic acid).

3. A vaccine comprising an isolated, recombinantly-generated, attenuated RSV subgroup B according to Claim 1 and a physiologically acceptable carrier.

4. A vaccine comprising an isolated, recombinantly-generated, attenuated RSV subgroup B according to Claim 2 and a physiologically acceptable carrier.

5. A method for immunizing an individual to induce protection against RSV subgroup B which comprises administering to the individual the vaccine of Claim 3.

6. A method for immunizing an individual to induce protection against RSV subgroup B which comprises administering to the individual the vaccine of Claim 4.

7. A composition which comprises a trancription vector comprising an isolated nucleic acid molecule encoding a genome or antigenome of an RSV subgroup B having at least one attenuating mutation in WO99/15672 PCT/US98/19145 - 106 the RNA polymerase gene, together with at least one expression vector which comprises at least one isolated nucleic acid molecule encoding the trans-acting N, P, L and M2 proteins of the virus necessary for encapsidation, transcription and replication, whereby upon expression an infectious attenuated virus is produced.

8. The composition of Claim 7 wherein the transcription vector comprises an isolated nucleic acid molecule which encodes an RSV subgroup B according to Claim 2.

9. A method for producing infectious attenuated RSV subgoup B which comprises transforming or transfecting host cells with the at least two vectors of Claim 7 and culturing the host cells under conditions which permit the co-expression of these vectors so as to produce the infectious attenuated virus.

10. The method of Claim 9 wherein the virus is the RSV subgroup B of Claim 2.

11. An isolated nucleic acid molecule comprising a RSV subgroup B sequence in positive strand, antigenomic message sense selected from the group consisting of 2B wild-type strain (SEQ ID NO:1), 18537 wild-type strain (SEQ ID NO:3), 2B33F vaccine strain (SEQ ID NO:5), 2B20L vaccine strain (SEQ ID NO:7), 2B33F TS(+) revertant strain (SEQ ID NO:9), and 2B20L TS(+) revertant strain (SEQ ID NO:11), and the complementary genomic sequences thereof.