WO1999015672A1 - Virus syncytiaux respiratoires attenues - Google Patents

Virus syncytiaux respiratoires attenues Download PDF

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Publication number
WO1999015672A1
WO1999015672A1 PCT/US1998/019145 US9819145W WO9915672A1 WO 1999015672 A1 WO1999015672 A1 WO 1999015672A1 US 9819145 W US9819145 W US 9819145W WO 9915672 A1 WO9915672 A1 WO 9915672A1
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rsv
virus
attenuated
vaccine
strains
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PCT/US1998/019145
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English (en)
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Stephen A. Udem
Mohinderjit S. Sidhu
Valerie B. Randolph
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American Cyanamid Company
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Priority to BR9812232-0A priority Critical patent/BR9812232A/pt
Priority to AU93899/98A priority patent/AU9389998A/en
Priority to CA002302867A priority patent/CA2302867A1/fr
Priority to KR1020007002922A priority patent/KR20010030630A/ko
Priority to EP98947010A priority patent/EP1015594A1/fr
Priority to JP2000512963A priority patent/JP2001517448A/ja
Publication of WO1999015672A1 publication Critical patent/WO1999015672A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18561—Methods of inactivation or attenuation
    • C12N2760/18564—Methods of inactivation or attenuation by serial passage

Definitions

  • 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 Government has certain rights in the invention.
  • Respiratory syncytial virus is a 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 phosphoprotein P, the nonglycosylated virion matrix protein , 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.
  • 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 dependent RNA Polymerase.
  • Messenger RNAs are only synthesized once the virus has been uncoated in the infected cell. Viral replication occurs after synthesis of the mRNAs and requires the continuous synthesis of viral proteins.
  • the newly synthesized antigenome (+) strand serves as the template for generating further copies of the (-) strand genomic RNA.
  • the polymerase complex actuates and achieves transcription and replication by engaging the cis- 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 1 to its 5 1 end. There is always less mRNA made from the downstream genes (e.g., the polymerase 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 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 children with certain underlying illnesses (e.g. immunodeficiencies, congenital heart disease and bronchopulmonary dysplasia) .
  • immunity elicited by the F protein is more crossprotective between subgroups than is immunity elicited by the G protein (16,17).
  • 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 important in resolution of primary infections (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).
  • CTL cytotoxic lymphocytes
  • Human CTL ' s have been shown to recognize the F, M2 , N, M, SH, and NS2 (or lb) proteins (26) .
  • Such vaccines would have to elicit an immune response in the recipient which will prevent serious RSV disease, i.e., LRD.
  • RSV disease i.e., 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 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; 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 exception of hepatitis B surface antigen, viral subunit vaccines have generally only elicited short-lived and/or inadequate immunity, particularly in naive recipients .
  • IPV polio
  • hepatitis A Formalin inactivated whole virus preparations of polio (IPV) and hepatitis A have proven safe and efficacious.
  • immunization with similarly inactivated whole RSV elicited unfavorable immune responses and/or response profiles which predisposed vaccinees to exaggerated or aberrant disease when subsequently confronted with the natural or "wild- type" virus .
  • RSV temperature sensitive (ts) mutants derived by chemical mutagenesis (31) were shown to be attenuated in rodent and non-human primate models (32,33).
  • Belshe and co-workers have used cold adaptation to develop attenuated, ts strains of a paramyxovirus, parainfluenza virus type 3 (38,39).
  • cold adaptation was carried out in primary African green monkey kidney cells by reducing temperatures to 20°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 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°C 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 attenuated virus were not isolated from a genetically mixed population of cold-adapted virus.
  • RSV mutants were generated by cold passage or chemical mutagenesis. These RSV strains were found to have reduced virulence in seropositive adults. Unfortunately, they proved either over- or underattenuated when given to seronegative infants; in some cases they were also found to lack genetic stability (43,44). Another vaccination approach using parenteral administration of 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. 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-l.
  • Appropriately attenuated live derivatives of wild-type viruses offer a distinct advantage as vaccine candidates.
  • live, replicating agents they initiate infection in recipients during which viral gene products are expressed, processed and presented in the context of the vaccinee's specific MHC class I and II molecules, eliciting humoral and cell-mediated immune responses, as well as the coordinate cytokine patterns, which parallel the protective immune profile of survivors of natural infection.
  • 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) .
  • Attenuated virus is used to prepare vaccines which elicit a protective immune response against the wild- type form of the virus .
  • an isolated, positive strand, antigenomic message sense nucleic acid molecule (or an isolated, negative strand genomic sense nucleic acid molecule) having the complete viral nucleotide sequence (whether of 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 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.
  • 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 use in an ELISA to detect the presence of the corresponding virus in a sample.
  • Figure 1 shows a flow chart detailing the propagation of RSV 2B working seed MK7V14b and RSV 3A working seed MK8V17b.
  • Figure 2 shows growth and cytopathic effect of RSV 2B at temperatures from 26oC to 36°C in Vero cells .
  • Figure 3 shows growth and cytopathic effect of RSV 3A at temperatures from 26°C to 36°C in Vero cells.
  • 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°C.
  • Figure 6 compares graphically the growth of 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 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.
  • 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 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 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 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.
  • 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 from wild-type strains.
  • the original RSV subgroup B 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 been isolated in primary rhesus monkey kidney cells. These are then passed in Vero cells at 35-36°C and plaque purified.
  • the Vero cells are passage 133 to 148 of the Vero cell line CCL81, obtained from the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland,
  • the maintenance medium is preferably MEM with 2% FBS, L-glutamine, non-essential amino acids and 20mM Hepes pH 7.5
  • the freezing medium is MEM with 10% FBS and 20mM Hepes pH 7.5.
  • 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°C to about 25°C) .
  • the virus flask is incubated at about 18°C to about 26°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 flask is then frozen directly at -70°C, then thawed in a 32°C 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- thaw lysate is stored at -70°C. It can be used to perform virus titrations and plaque purify virus.
  • plaque purify virus the freeze- thaw lysate is thawed in a 32°C water bath. About three to five serial dilutions of the lysate are made in the maintenance medium. Six-well, twenty- four well, or 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 inoculum is adsorbed for 90 minutes at ambient temperature. Wells are overlaid with 1% methylcellulose in MEM-maintenance medium. Plates are incubated at 32°C for five days. Isolated plaques are identified microscopically by looking for typical 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°C. Picked plaques are used to inoculate duplicates of 25 cm 2 flask or 96-well plates containing Vero cell monolayers as described above.
  • Duplicate inoculated flasks or plates are overlaid with maintenance medium. One duplicate is incubated at 32°C and the other at 39°C for 5-10 days. Flasks or plates incubated at 32°C are examined microscopically for virus cytopathic effect (CPE) . Flasks or plates incubated at 39°C are stained by immunoperoxidase assay for RSV specific antigen. Flasks or plates which demonstrate easily detectable CPE at 32°C and little or no detectable RSV antigen by 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.
  • CPE virus cytopathic effect
  • RNA viruses mutate at such a high frequency 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. The results were interesting and somewhat unexpected.
  • Virus must be sufficiently attenuated to not cause disease, yet grow well enough in the vaccine to elicit protective immunity.
  • 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 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 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 vi tro . The cotton rat is relatively nonpermissive for growth of RSV and disease does not develop, suggesting that this model is an unreliable indicator of level of attenuation in humans .
  • chimpanzees are highly susceptible to RSV infection and develop an upper and lower respiratory tract disease that is very similar to that seen in humans.
  • 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 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.
  • testing in seronegative chimps is severely hampered by the limited availability of these animals.
  • mutants described herein bear the desirable traits of an attenuated, phenotypically stable, and immunogenic RSV vaccine virus in the human 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 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 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, radioimmunoprecipitations, etc.; (b) the ability of the immune serum to neutralize RSV infectivity in vi tro ; (c) the ability of the immune serum to inhibit virus fusion in vi tro; and (d) protection from RSV infection or significant disease.
  • ELISA enzyme linked immunosorbant assay
  • 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.
  • cold-adapted mutant means an attenuated 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 3Ap28F.
  • the cold-adapted mutant RSV may also be a 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 mutations which contribute to the attenuated phenotype are then assessed.
  • RNA viral genomes such as RSV subgroup B are achieved through the enzymatic activity of a multimeric protein acting on the ribonucleoprotein core (nucleocapsid) .
  • Naked genomic RNA cannot serve as a template. Instead, these genomic sequences are recognized only when they are entirely encapsidated by the N protein into the nucleocapsid structure. It is only in that context that the genomic and antigenomic terminal promoter sequences are recognized to initiate the transcriptional or replication pathways.
  • All paramyxoviruses require the two viral proteins, L and P, for these polymerase pathways to proceed.
  • the pneumoviruses, including RSV, also require the transcription elongation factor M2 for the transcriptional pathway to proceed efficiently.
  • Additional cofactors may also play a role, including perhaps the virus-encoded NSl and NS2 proteins, as well as perhaps host-cell encoded proteins.
  • L protein which performs most if not all the enzymatic processes associated with transcription and replication, including initiation, and termination of ribonucleotide polymerization, capping and 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 to mutations, and its catalytic level of abundance in the transcriptionally active viral complex (49) .
  • L proteins consist of a linear array of domains whose concatenated structure integrates discrete functions (50) .
  • 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 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 elements (50) .
  • 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. 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 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.
  • the attenuating mutations described herein may be introduced into viral strains by two methods: (1) Conventional means such as chemical mutagenesis during virus growth in cell cultures to which a chemical utagen has been added, selection of virus that has been subjected to passage at suboptimal temperature in order to select temperature sensitive and/or cold-adapted mutations, identification of mutant virus that produce small plaques in cell culture, and passage through heterologous hosts to select for host range mutations . These viruses are then screened for attenuation of their biological activity in an animal model. Attenuated viruses are subjected to nucleotide sequencing of their polymerase genes to locate the sites of attenuating mutations. Once this has been done, method (2) is then carried out.
  • Conventional means such as chemical mutagenesis during virus growth in cell cultures to which a chemical utagen has been added
  • selection of virus that has been subjected to passage at suboptimal temperature in order to select temperature sensitive and/or cold-adapted mutations identification of mutant virus that produce small plaques in cell culture
  • a preferred means of introducing 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 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 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 "rescue" system, which has been applied to a variety of 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 reference. See, for RSV rescue, published
  • RNA polymerase promoter e.g., the T7 RNA polymerase promoter
  • ribozyme sequence e.g., the hepatitis delta ribozyme
  • 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 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.
  • 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 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.
  • the trans-acting proteins required for RSV rescue are the encapsidating protein N, the polymerase complex proteins, P and L, and an additional protein, M2 , the RSV-encoded transcription elongation factor.
  • these viral trans-acting proteins are generated from one or more plasmid expression vectors encoding the required proteins, although some or all of the required trans-acting proteins may be produced within mammalian cells engineered to contain and express these virus-specific genes and gene products as stable transformants.
  • the typical (although not necessarily exclusive) circumstances for rescue include an appropriate mammallian cell milieu in which T7 polymerase is present to drive transcription of the antigenomic (or genomic) single-stranded RNA from the viral genomic cDNA-containing transcription vector. Either cotranscriptionally or shortly thereafter, this viral antigenome (or genome) RNA transcript is encapsidated into functional templates by the nucleocapsid protein and engaged by the required polymerase components produced concurrently from co- transfected expression plasmids encoding the required virus-specific trans-acting proteins. These events and processes lead to the prerequisite transcription of viral mRNAs, the replication and amplification of new genomes and, thereby, the production of novel viral progeny, i.e., rescue.
  • T7 polymerase is provided by recombinant vaccinia virus VTF7-3.
  • This system 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 .
  • measles virus (MV) cDNA rescue this requirement is avoided by creating a cell line that expresses T7 polymerase, as well as viral N and P proteins. Rescue is achieved by transfecting the genome expression vector and the L gene expression vector into the helper cell line.
  • MVA-T7 which expresses the T7 RNA polymerase, but does not replicate in mammalian cells, are exploited to rescue RSV, Rinderpest virus and MV.
  • synthetic full length antigenomic viral RNA are encapsidated, replicated and transcribed by viral polymerase proteins and replicated genomes are packaged into infectious virions.
  • genome analogs have now been successfully rescued for Sendai 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 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 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 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 vi tro means. If the attenuated phenotype of the rescued virus is present, challenge experiments are conducted with an appropriate animal model.
  • Non-human primates provide the preferred animal model for the pathogenesis of human disease. These primates are first immunized with the attenuated, recombinantly-generated virus, then challenged with the wild-type form of the virus. Monkeys are infected by various routes, including but not limited to intranasal or intratracheal routes of inoculation (60) .
  • protection in non-human primates is 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" virus is considered to be “recombinantly-generated", as are the progeny and later generations of the virus, which also incorporate the attenuating mutations .
  • a codon containing an attenuating point mutation may be stabilized by introducing a second or a second plus a third mutation in the codon without changing the amino acid encoded by the codon bearing only the attenuating point mutation.
  • Infectious virus clones containing the attenuating and stabilizing mutations are also generated using the cDNA "rescue" system described above.
  • 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 (14,15). Bovine, ovine, and caprine strains of this virus have also been isolated. The host specificity of the virus is most clearly associated with the G attachment protein, which is highly divergent between the human and the bovine/ovine strains (61,62), and may 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)
  • LFD lower respiratory tract disease
  • RSV additionally is associated with asthma and hyperreactive airways and it is a significant cause of mortality in "high risk" children with bronchopulmonary dysplasia and congenital heart disease (CHD) .
  • CHD congenital heart disease
  • RSV In adults, RSV generally presents as uncomplicated upper respiratory illness; however, in the elderly it rivals influenza as a predisposing factor in the development of serious LRD, particularly bacterial bronchitis and pneumonia. Disease is always confined to the respiratory tract, except in the severely immunocompromised, where dissemination to other organs can occur. Virus is spread to others by fomites contaminated with virus-containing respiratory secretions, and infection initiates through the nasal, oral, or conjunctival mucosa.
  • RSV disease is seasonal and virus is usually isolated only in the winter months, 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 drift (such as that seen with influenza A viruses) , but sequence studies demonstrating accumulation of amino acid changes in the hypervariable regions of the G protein and SH proteins suggest that immune pressure may drive virus evolution. In mouse and cotton rat models, both the F and G proteins of RSV elicit neutralizing antibodies and immunization with these proteins alone provides longter protection against reinfection (16,17).
  • the RSV virion consists of a ribonucleoprotein core contained within a lipoprotein envelope.
  • the virions of pneumoviruses are similar in size and shape to those of all other paramyxoviruses . When visualized by negative staining and electron microscopy, virions are irregular in shape and range in diameter from 150-300 nm (64) .
  • the nucleocapsid of this virus is a symmetrical helix similar to that of 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 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) . 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 strand of negative-sense RNA of 15,218 nucleotides (SEQ ID NO:l) 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 associated with genomic RNA to form the viral nucleocapsid (N, P and L) ; two are nonstructural proteins (NSl and NS2) which accumulate in the infected cells but are also present in the virion in trace amounts and may play a role in regulating transcription and replication; one is the nonglycosylated virion matrix protein (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 of the viral coding capacity.
  • 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 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 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.
  • the M protein is thought to render the nucleocapsid transcriptionally inactive before packaging and to mediate its association with the viral envelope.
  • the NSl and NS2 proteins have only been detected in very small amounts in purified virions, and at this time are considered non-structural . Their functions are uncertain, though they may be regulators of transcription and replication.
  • Three transmembrane surface glycoproteins are present in virions: G, F, and SH.
  • G and F are envelope glycoproteins that are known to mediate attachment and penetration of the virus into the host cell.
  • these glycoproteins represent major independent immunogens (3) .
  • the function of the SH protein is unknown, although a recent report has implicated its involvement in the fusion function of the virus (67) .
  • Genomic RNA is neither capped nor polyadenylated (68) . 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 to contain the major viral promoter (68; Fig. 11) .
  • the 3 ' genomic promoter region is followed by ten viral genes in the order 3 ' -NS1-NS2-N-P-M-SH-G-F-M2-L-5 • (Fig. 11) .
  • the L gene is followed by a 145-149 nucleotide extragenic trailer region (see Figure 11) .
  • 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) .
  • transcription begins at the first nucleotide of the 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 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 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 last 68 nucleotides of the M2 mRNA (exclusive of the Poly-A tail) , as well as the first 68 nucleotides of the L mRNA.
  • the L gene start signal lies 68 nucleotides upstream of the M2 gene-end signal, resulting in gene 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 M2 gene-end motif. This results in much lower transcription of L mRNA.
  • the gene overlap seems incompatible with a model of linear sequential transcription. It is not known whether the polymerase that exits the M2 gene jumps backward to the L gene- start signal or whether there is a second, internal promoter for L gene transcription (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 down the M2 gene to the L gene-start signal.
  • 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 second mechanism that reduces the synthesis of full length L mRNA.
  • 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 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 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 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 protein synthesis (75) .
  • 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 nucleotide sequences (in positive strand, antigenomic, message sense) of various wild-type, vaccine and revertant RSV strains, as well as the deduced amino acid sequences of the RNA polymerase (L protein) of these RSV viruses, are set forth as follows with reference to the appropriate SEQ ID NOS . contained herein: Virus Nucleotide Sequence L Protein Sequence Wild-Type 2B SEQ ID N0:1 SEQ ID NO: 2 18537 SEQ ID NO: 3 SEQ ID NO: 4
  • Each RSV virus genome encodes an L protein that is 2,166 amino acids long. Genome length and other nucleotide information is as follows:
  • 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 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 animal hosts.
  • the extent of attenuation is such that symptoms of infection will not arise in most immunized individuals, but the virus will retain sufficient replication competence to be infectious in and elicit the desired immune response profile in the vaccinee.
  • the attenuated 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 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 MPLTM (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamilton, MT) and IL-12 (Genetics Institute, Cambridge, MA) .
  • the formulation including the attenuated virus is intended for use as a vaccine.
  • the attenuated virus may be mixed 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.
  • 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 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 human or animal subject to induce protection against infection by the wild-type counterpart of the attenuated virus.
  • this invention further provides a method of immunizing a subject to induce protection against infection by an RSV subgroup B virus by administering to the subject an effective immunizing amount of a vaccine formulation incorporating an attenuated version of that virus as described hereinabove .
  • a sufficient amount of the vaccine in an appropriate number of doses must be administered to the subject to elicit an immune response.
  • Persons skilled in the art will readily be able to determine such amounts and dosages.
  • Administration may be by any conventional effective form, such as intranasally, parenterally, orally, or topically applied to any mucosal surface such as intranasal, oral, eye, vaginal or rectal surface, such as by an aerosol spray.
  • the preferred means of administration is by intranasal administration.
  • 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 oiigonucleotide probes (from either positive strand antigenomic message sense or 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 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.
  • PCR primers are 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 described herein which have nucleotides which are distinct for a defined strain of virus.
  • Amplified PCR products are identified on gels and their specificity confirmed by hybridization with specific nucleotide probes .
  • ELISA tests are used to detect the presence of antigens of the wild-type, 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 herein.
  • 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.
  • KLH keyhole limpet hemocyanin
  • a selection of these polyclonal antibodies, or a combination of polyclonal and monoclonal antibodies can then be used in a hapten (e.g., keyhole limpet hemocyanin (KLH) and used to immunize animals (e.g., rabbits) for the production of monospecific polyclonal antibody.
  • KLH keyhole limpet hemocyanin
  • capture ELISA to detect antigens produced by those viruses .
  • 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 .
  • Isolate 20648 (subgroup B) was renamed RSV 2B.
  • Virus was passed seven times in PRMK cells at 35°C, two times in Vero cells at 35°C and plaque purified and amplified three times (six passages) in Vero cells at 36°C.
  • 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 of a Master Seed (RSV 2B, MK7 V12b) , Intermediate working seed (RSV 2B, MK7 V13b) and Working seed (RSV 2B, MK7 V14b) . See Figure 1.
  • Isolate 23095 (subgroup A) was renamed RSV 3A.
  • RSV 3A was passed eight times in PRMK cells at 35°C. This was followed by two passages in Vero at
  • Subgroup specificities of RSV 2B and RSV 3A Master seeds were confirmed using subgroup specific 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 western blotting procedures using monoclonal antibodies.
  • the FI subunits of the RSV subgroup B strains, 2B and 18537 migrated faster on SDS- polyacrylamide gels than did the FI subunits of the RSV subgroup A strains, 3A and Long.
  • virus was harvested by replacing the maintenance medium (10 mis of MEM/2%FBS/20mMHepes) in the infected flask with a reduced volume of freezing medium (3 mis of MEM/10%FBS/20mMHepes) and performing a quick freeze at -70°C followed by a thaw at 32°C.
  • Flasks E and F were "slowly" adapted, beginning at 26°C with four passages every two days, followed by passage once every week until titers appeared to be relatively stable or were increasing. Virus was then passaged weekly at 22°C 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 more moderate strategy.
  • Flasks J and L were "rapidly" adapted, starting with five weekly passages at 22°C, followed by passage at 1-2 week intervals at 20°C.
  • Plaque purified mutants were initially identified by relatively poor growth (lower titers or smaller plaque size) at 39°C vs 32°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 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 cold passaging.
  • the ts mutants were further screened and selected for vaccine candidates based on degree of temperature sensitivity in vi tro , attenuation in animal models (including mice, cotton rats, and chimps) , and retention of neutralizing epitopes.
  • Vero cells were infected with these four mutants at an MOI of 2 , and incubated at 20°C, 32°C, 37°C, and 40°C for seven days.
  • one subgroup A and one subgroup B mutant, RSV 2Bp20L and RSV 3Ap20E were selected to perform additional preliminary experiments on phenotypic stability and growth in mice.
  • RSV 2Bp20L and RSV 3Ap20E were evaluated in Balb/c mice. 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 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.
  • virus yield expressed as PFU per cell
  • virus yield was also somewhat reduced at 32°C relative to the parental strain, indicating attenuation in growth at 32°C. This is consistent with the smaller plaque sizes observed in the 32°C EOP assays (Table 9) .
  • EOP and virus yield studies demonstrate that these seven isolates possess varying levels of temperature sensitivity and may represent a range of levels of attenuation.
  • 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 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 .
  • Neutralizing and EIA-F antibody titers on sera from rats infected with the RSV 2B and RSV 3A parental and ts 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.
  • titers from 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 mutants were quite immunogenic, consistent with their high level of replication in nasal tissue.
  • 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 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 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 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 10 40 and 10 50 PFU of RSV 2B, and two 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 seven days post-infection. The amount of virus shed was higher and shedding occurred earlier in the chimp infected with the higher dose of RSV 2B.
  • AGMs African green monkeys
  • 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 10 6 PFU of each mutant virus by combined intranasal and 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 post-infection, animals were challenged with 10 6 PFU of the parental strain by intranasal and intratrachial route. Virus growth and antibody response was evaluated as described above.
  • 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 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.
  • monkeys were partially to fully protected against challenge with 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 seen by 2.5 weeks post-infection. Following challenge 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 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 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 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 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 neutralizing and EIA antibody titers were detected beginning three weeks post-vaccination.
  • TS-1 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 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.
  • 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 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.
  • Monkeys were infected with virus either intranasally (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, indicating that virus was attenuated. In contrast, relatively high titers of virus were recovered in all four monkeys inoculated with TS-1.
  • the temperature-sensitive (ts) phenotype is strongly associated with attenuation in vivo; in addition, some non-ts mutations may also be attenuating. Identification of ts and non-ts attenuating mutations was achieved by sequence analysis and evaluation of ts, cold-adapted (ca) , and in vivo growth phenotypes of RSV mutants and revertants .
  • RSV 2B33F 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.
  • RSV 2B20L differs from its RSV 2B parent only at seven nucleotide positions, of which three are common with 2B33F virus, including two changes at the 3 ' genomic promoter and one coding change in the L gene. Two additional unique changes of 2B20L virus mapped to the coding region of the L gene. Potentially attenuating mutations at the RNA dependent RNA polymerase gene have been identified.
  • Two ts mutations can be identified in the L gene of the attenuated virus strains 2B33F and 2B20L: (i) In 2B33F, a mutation at nucleotide position
  • 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) .
  • 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 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 minireplicon at 37°C.
  • the 2B33F virus did not provide helper functions to the RSV-CAT minireplicon (with 2B or 2B33F 3' genomic promoters) at 37°C.
  • the codon ACA at nucleotides 14586-14588 encodes a Thr at amino acid 2029 of the L protein
  • the codon ATT at nucleotides 14593-14595 encodes an lie at amino acid 2029 (the L gene start codon is at nucleotides 8509- 8511 in 18537, compared to 8502-8504 in 2B) .
  • a PCR assay is used to detect the presence of 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 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 RSV nucleotide probes .
  • 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 described herein. These peptides are then coupled to KLH and used to immunize rabbits for the production of monospecific polyclonal antibody. A selection of these polyclonal antibodies, or a combination of polyclonal and monoclonal antibodies is then used in a "capture ELISA" to detect the presence of an RSV antigen. Table 1
  • EOP SV iso:late 37/32°C 39/32°C 40/32°C
  • 3Ap20F (pp4-3) 0.8 >0.1 0.000004 3Ap27F (ppl-2) 0.3 0.003 ND 3Ap28F (pplO-1) 0.2 0.002 ND
  • m ro + Sera was taken 32 days post-infection.
  • x- Neutralization results are expressed as the reciprocal of the dilution giving 60% plaque reduction neutralization, again RSV 2B, 3A, and A2
  • 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.
  • PRINT is a 60% plaque reduction neutralization test.
  • EIA-F, Ga, Gb are enzyme immunoassays testing reactivity of sera with purified F protein (from RSV A2), purified Ga (from
  • Source of protein F(RSV A2), Ga(RSV A2), Gb(RSV 18537). Rise in titer day - 1 to day 21.
  • lungs and nasal turbinates were harvested for virus titrations.
  • blood was taken for neutralization and EIA titrations and rats were challenged intranasally with 10 6 PFU of RSV 18537.
  • Lungs and nasal turbinates were harvested 4 days post-challenge.
  • Virus and antibody titers are reported as geometric mean titers.
  • Source of coating protein is RSV A2 F protein.
  • m 1 Cotton rats were inoculated with virus by intranasal route. Four days post- ro cn infection, lungs and nasal turbinates were harvested for virus titrations. Six weeks post-infection, blood was taken for neutralization and EIA titrations and rats were challenged intranasally with 10 6 PFU of RSV A2.
  • Lungs and nasal turbinates were harvested 4 days post-challenge. Virus and antibody titers are reported as geometric mean titers.
  • Source of coating protein is RSV A2 F protein.
  • Peak Virus Titer Titers 2 Titers 3 (loglO PFU/ l) (xlO 3 )
  • AGMs were previously vaccinated with RSV 2B ts strains.
  • AGMs were previously vaccinated with RSV 2B ts strains.
  • AGMs were previously vaccinated with RSV 2B ts strains.
  • Source of coating protein is RSV A2 F protein.
  • AGMs were previously vaccinated with RSV 3A ts strains. All monkeys were challenged 8 weeks post- vaccination with 10 6 PFU of RSV 3A, IN+IT.
  • AGMs were previously vaccinated with RSV 3A ts strains. All monkeys were challenged 8 weeks post- vaccination with 10 6 PFU of RSV 3A, IN+IT.
  • AGMs were previously vaccinated with RSV 3A ts strains. All monkeys were challenged 8 weeks post- vaccination with 10 6 PFU of RSV 3A, IN+IT.
  • Source of coating protein is RSV A2 F protein.
  • Lungs and nasal turbinates were harvested 4 days post-challenge. Virus and antibody titers are reported as geometric mean titers.
  • Source of coating protein is RSV A2 F protein.
  • 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 Table 22 Sequence comparison between RSV 2B and 2B20L strains
  • nucl. pos. numbers are one larger than for 2B for L gene

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Abstract

Virus syncytiaux respiratoires atténués, isolés, générés par recombinaison, appartenant au sous-groupe B et possédant au moins une mutation d'atténuation dans le gène de polymérase d'ARN. Formulations de vaccins comprenant ces virus, ainsi qu'un véhicule acceptable sur le plan physiologique. On utilise ces vaccins dans le but d'immuniser un individu afin d'induire une protection contre le virus syncytial respiratoire.
PCT/US1998/019145 1997-09-19 1998-09-15 Virus syncytiaux respiratoires attenues WO1999015672A1 (fr)

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BR9812232-0A BR9812232A (pt) 1997-09-19 1998-09-15 Vìrus sincicial respiratório (rsv) humano subgrupo b, isolado, recombinantemente gerado , atenuado, vacina, processo para imunizar um indivìduo para induzir proteção contra o rsv subgrupo b, composição, processo para produzir rsv subgrupo b infeccioso atenuado, e, molécula isolada de ácido nucleico
AU93899/98A AU9389998A (en) 1997-09-19 1998-09-15 Attenuated respiratory syncytial viruses
CA002302867A CA2302867A1 (fr) 1997-09-19 1998-09-15 Virus syncytiaux respiratoires attenues
KR1020007002922A KR20010030630A (ko) 1997-09-19 1998-09-15 약독화 호흡 신시티아 바이러스
EP98947010A EP1015594A1 (fr) 1997-09-19 1998-09-15 Virus syncytiaux respiratoires attenues
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EP1015594A1 (fr) 2000-07-05
CN1273603A (zh) 2000-11-15
KR20010030630A (ko) 2001-04-16
AU9389998A (en) 1999-04-12
JP2001517448A (ja) 2001-10-09

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