EP1169457A2 - Production de vaccins a base d'un virus respiratoire syncytial chimerique attenue a partir de sequences nucleotidiques clonees - Google Patents

Production de vaccins a base d'un virus respiratoire syncytial chimerique attenue a partir de sequences nucleotidiques clonees

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Publication number
EP1169457A2
EP1169457A2 EP00920058A EP00920058A EP1169457A2 EP 1169457 A2 EP1169457 A2 EP 1169457A2 EP 00920058 A EP00920058 A EP 00920058A EP 00920058 A EP00920058 A EP 00920058A EP 1169457 A2 EP1169457 A2 EP 1169457A2
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Prior art keywords
rsv
chimeric
gene
virus
antigenome
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English (en)
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Peter L. Collins
Brian R. Murphy
Stephen S. Whitehead
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US Department of Health and Human Services
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US Department of Health and Human Services
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Priority claimed from US09/291,894 external-priority patent/US6689367B1/en
Application filed by US Department of Health and Human Services filed Critical US Department of Health and Human Services
Priority to EP06075156A priority Critical patent/EP1690940A1/fr
Publication of EP1169457A2 publication Critical patent/EP1169457A2/fr
Ceased legal-status Critical Current

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    • C12N2760/18561—Methods of inactivation or attenuation

Definitions

  • RSV Human respiratory syncytial virus
  • RSV-specific cytotoxic T cells another effector arm of induced immunity, are also important in resolving an RSV infection.
  • the F and G surface glycoproteins are the two major protective antigens of RSV, and are the only two RSV proteins which have been shown to induce RSV neutralizing antibodies and long term resistance to challenge (Collins et al . , Fields Virology, Fields et al . eds., 2:1313-1352. Lippincot -Raven, Philadelphia. (1996); Connors et al., J. Virol. 65(3):1634-7 (1991)).
  • the third RSV surface protein, SH did not induce RSV-neutralizing antibodies or significant resistance to RSV challenge.
  • RSV vaccines One obstacle to development of live RSV vaccines is the difficulty in achieving an appropriate balance between attenuation and immunogenicity. Genetic stability of attenuated viruses also can be a problem. Vaccine development also is impeded by the relatively poor growth of RSV in cell culture and the instability of the virus particle. Another feature of RSV infection is that the immunity which is induced is not fully protective against subsequent infection.
  • RSV exists as two antigenic subgroups A and B, and immunity against one subgroup is of reduced effectiveness against the other.
  • RSV can reinfect multiple times during life, reinfections usually are reduced in severity due to protective immunity induced by prior infection, and thus immunoprophylaxis is feasible.
  • a live-attenuated RSV vaccine would be administered intranasally to initiate a mild immunizing infection. This has the advantage of simplicity and safety compared to a parenteral route . It also provides direct stimulation of local respiratory tract immunity, which plays a major role in resistance to RSV. It also abrogates the immunosuppressive effects of RSV-specific maternally- derived serum antibodies, which typically are found in the very young. Also, while the parenteral administration of RSV antigens can sometimes be associated with immunopathologic— complications (Murphy et al . , Vaccine 8(5):497-502 (1990)), this has never been observed with a live virus.
  • Formalin-inactivated virus vaccine was tested against RSV in the mid-1960s, but failed to protect against RSV infection or disease, and in fact exacerbated symptoms during subsequent infection by the virus. (Kim et al . , Am. J. Epidemiol . , 89:422-434 (1969), Chin et al . , Am J. Epidemiol. , 89:449-463 (1969); Kapikian et al . , Am. J. Epidemiol. , 89:405-421 (1969) ) . More recently, vaccine development for RSV has focused on attenuated RSV mutants. Friedewald et al., J. Amer. Med. Assoc.
  • the cpRSV mutant retained a low level virulence for the upper respiratory tract of seronegative infants.
  • tsRSV temperature sensitive RSV
  • glycoproteins induced resistance to RS virus infection in the lungs of cotton rats, Walsh et al . , J. Infect. Pis. 155:1198- 1204 (1987) , but the antibodies had very weak neutralizing activity and immunization of rodents with purified subunit vaccine led to disease potentiation (Murphy et al . , Vaccine 8:497-502 (1990) ) .
  • Vaccinia virus recombinant-based vaccines which express the F or G envelope glycoprotein have also been explored. These recombinants express RSV glycoproteins which are indistinguishable from the authentic viral counterpart, and rodents infected intradermally with vaccinia-RSV F and G recombinants developed high levels of specific antibodies that neutralized viral infectivity. Indeed, infection of cotton rats with vaccinia-F recombinants stimulated almost complete resistance to replication of RSV in the lower respiratory tract and significant resistance in the upper tract. Olmsted et al., Proc. Natl. Acad. Sci. USA 83:7462-7466 (1986).
  • Recombinant DNA technology has made it possible to recover infectious negative-stranded RNA viruses from cDNA, to genetically manipulate viral clones to construct novel vaccine candidates, and to rapidly evaluate their level of attenuation and phenotypic stability (for reviews, see Conzelmann, J. Gen . Virol . 77:381-89 (1996); Palese et al . , Proc. Natl. Acad. Sci. U.S.A. 93:11354-58, (1996)).
  • HN and F glycoproteins of human parainfluenza virus type 3 have been replaced by those of human parainfluenza virus type 1 (HPIVl) , and the resulting chimeric virus grew in cell culture and in experimental animals with an efficiency similar to its wild-type parents (Tao et al . , J. Virol . 72 (4) :2955-61 (1998), incorporated herein by reference.
  • Jin et al., Virology 251 (1) :206-14 (1998) report a subgroup A virus which expresses the G protein of a subgroup B RSV as an additional gene (Jin et al., Virology 251 (1) : 206-14 (1998)).
  • the F protein also exhibits significant subgroup-specificity, it would be preferable to express both subgroup B glycoproteins in a subgroup B-specific vaccine.
  • production of a chimeric A-B virus will not produce a viable vaccine candidate without further modifications to achieve proper attenuation and virulence.
  • the present invention provides chimeric, recombinant respiratory syncytial virus (RSV) that are infectious and elicit a propylactic or therapeutic immune response in humans or other mammals.
  • the invention provides novel methods and compositions for designing and producing attenuated, chimeric RSV suitable for vaccine use.
  • novel, isolated polynucleotide molecules and vectors incorporating such molecules that comprise a chimeric RSV genome or antigenome including a partial or complete RSV genome or antigenome of one RSV strain or subgroup virus combined with one or more heterologous gene(s) or gene segment (s) of a different RSV strain or subgroup virus.
  • methods and compositions incorporating chimeric, recombinant RSV for prophylaxis and treatment of RSV infection are also provided within the invention.
  • Chimeric RSV of the invention are recombinantly engineered to incorporate nucleotide sequences from more than one RSV strain or subgroup to produce an infectious, chimeric virus or subviral particle.
  • candidate vaccine viruses are recombinantly engineered to elicit an immune response against RSV in a mammalian host susceptible to RSV infection, including humans and non-human primates.
  • Chimeric RSV according to the invention may elicit an immune response to a specific RSV subgroup or strain, or a polyspecific response against multiple RSV subgroups or strains .
  • Exemplary chimeric RSV of the invention incorporate a chimeric RSV genome or antigenome, as well as a major nucleocapsid (N) protein, a nucleocapsid phosphoprotein (P) , a large polymerase protein (L) , and a RNA polymerase elongation factor. Additional RSV proteins may be included in various combinations to provide a range of infectious subviral particles as well as complete viral particles.
  • Chimeric RSV of the invention include a partial or complete RSV genome or antigenome from one RSV strain or subgroup virus combined with one or more heterologous gene(s) or gene segmen (s) of a different RSV strain or subgroup virus to form the chimeric RSV genome or antigenome.
  • chimeric RSV incorporate a partial or complete human RSV genome or antigenome of one RSV subgroup or strain combined with one or more heterologous gene(s) or gene segment (s) from a different human RSV subgroup or strain.
  • a chimeric RSV may incorporate a chimeric genome or antigenome comprised of a partial or complete human RSV A subgroup genome or antigenome combined with one or more heterologous gene(s) or gene segment (s) from a human RSV B subgroup virus.
  • Heterologous genes or gene segments from one RSV strain or subgroup represent "donor” genes or polynucleotides that are combined with, or substituted within, a "recipient” genome or antigenome.
  • the recipient genome or antigenome typically acts as a "backbone” or vector to import heterologous genes or gene segments to yield a chimeric RSV exhibiting novel phenotypic characteristics.
  • addition or substitution of heterologous genes or gene segments within a selected recipient RSV strain may result in attenuation, growth changes, altered immunogenicity, or other desired phenotypic changes as compared with a corresponding phenotype (s) of the unmodified recipient and/or donor.
  • Genes and gene segments that may be selected for use as heterologous inserts or additions within the invention include genes or gene segments encoding a NS1, NS2, N, P, M, SH, M2(0RF1), M2(ORF2), L, F or G protein or portion thereof.
  • chimeric RSV incorporates one or more heterologous gene(s) that encode an RSV F, G or SH glycoprotein.
  • the chimeric RSV may incorporate a gene segment encoding a cytoplasmic domain, transmembrane domain, ectodomain or immunogenic epitope of a RSV F, G or SH glycoprotein.
  • immunogenic proteins, domains and epitopes are particularly useful within chimeric RSV because they can generate novel immune responses in an immunized host.
  • one or more immunogenic gene(s) or gene segment (s) from one donor RSV subgroup or strain within a recipient genome or antigenome of a different RSV subgroup or strain can generate an immune response directed against the donor subgroup or strain or against both the donor and recipient subgroup or strain.
  • one or more human RSV subgroup B glycoprotein genes F, G and SH or a cytoplasmic domain, transmembrane domain, ectodomain or immunogenic epitope thereof, is added to, or substituted within, an RSV A genome or antigenome.
  • Attenuated, chimeric RSV are produced in which the chimeric genome or antigenome is further modified by introducing one or more attenuating point mutations specifying an attenuating phenotype. These point mutations may be generated de novo and tested for attenuating effects according to a rational design mutagenesis strategy. Alternatively, the attenuating point mutations are identified in biologically derived mutant RSV and thereafter incorporated into a chimeric RSV of the invention.
  • chimeric RSV of the invention are attenuated by incorporation of at least one, and more preferably two or more, attenuating point mutations identified from a panel of known, biologically derived mutant RSV strains.
  • Preferred mutant RSV strains described herein are cold passaged ( cp) and/or temperature sensitive (ts) mutants, for example the mutants designated "cpts RSV 248. (ATCC VR)
  • chimeric RSV with or without attenuating point mutations, are mutated by a non-point nucleotide modification to produce desired phenotypic, structural, or functional changes.
  • the selected nucleotide modification will specify a phenotypic change, for example a change in growth characteristics, attenuation, temperature-sensitivity, cold-adaptation, plaque size, host range restriction, or immunogenicity.
  • Structural or functional changes include introduction or ablation of restriction sites into RSV encoding cDNAs for ease of manipulation and identification.
  • an SH, NS1, NS2 or G gene is modified in the chimeric RSV, e.g., by deletion of the gene or ablation of its expression.
  • the nucleotide modification can include a deletion, insertion, addition or rearrangement of a cis-acting regulatory sequence for a selected RSV gene.
  • a cis-acting regulatory sequence of one RSV gene is changed to correspond to a heterologous regulatory sequence, which may be a counterpart cis-acting regulatory sequence of the same gene in a different RSV or a cis-acting regulatory sequence of a different RSV gene.
  • a gene end signal may be modified by conversion or substitution to a gene end signal of a different gene in the same RSV strain.
  • the nucleotide modification may comprise an insertion, deletion, substitution, or rearrangement of a translational start site within the chimeric genome or antigenome, e.g., to ablate an alternative translational start site for a selected form of a protein.
  • the translational start site for a secreted form of the RSV G protein is ablated to modify expression of this form of the G protein and thereby produce desired in vivo effects.
  • chimeric RSV genome or antigenome according to the invention, including modifications that introduce into the chimeric genome or antigenome a non-RSV molecule such as cytokine, a T-helper epitope, a restriction site marker, or a protein of a microbial pathogen capable of eliciting a protective immune response against the pathogen in a mammalian host .
  • chimeric RSV are constructed that incorporate a gene or gene segment from a parainfluenza virus (PIV) , for example a PIV HN or F glycoprotein or an immunogenic domain or epitope thereof .
  • PIV parainfluenza virus
  • Chimeric RSV designed and selected for vaccine use often have at least two and sometimes three or more attenuating mutations to achieve a satisfactory level of attenuation for broad clinical use.
  • at least one attenuating mutation occurs in the RSV polymerase gene (either in the donor or recipient gene) and involves one or more nucleotide substitution (s) specifying an amino acid change in the polymerase protein specifying an attenuation phenotype which may or may not involve a temperature-sensitive ( s) phenotype.
  • Exemplary chimeric RSV in this context incorporate one or more nucleotide substitutions in the large polymerase gene L resulting in an amino acid change at amino acid Phe 521 , Gln 831 , Met 1169 _ or Tyr 1321 , as exemplified by the changes, Leu for Phe 521 , Leu for Gln 831 , Val for Met 1169r and Asn for Tyr 1321 .
  • Other alternative amino acid assignments at this position can of course be made to yield a similar effect as the identified, mutant substitution.
  • it is prefarable to modify the chimeric genome or antigenome to encode an alteration at the subject site of mutation that corresponds conservatively to the alteration identified in the mutant virus.
  • substitution will involve an identical or conservative amino acid to the substitute residue present in the mutant viral protein.
  • alter the native amino acid residue at the site of mutation non-conservatively with respect to the substitute residue in the mutant protein (e.g., by using any other amino acid to disrupt or impair the identity and functiono of the wild-type residue) .
  • Chimeric RSV of the invention may incorporate a ts mutation in any additional RSV gene besides L, e.g., in the M2 gene.
  • two or more nucleotide changes are incorporated in a codon specifying an attenuating mutation, e.g., in a codon specifying a ts mutation, thereby decreasing the likelihood of reversion from an attenuated phenotype.
  • Attenuating mutations may be selected in coding portions of a donor or recipient RSV gene or in non-coding regions such as a cis-regulatory sequence.
  • Exemplary non- coding mutations include single or multiple base changes in a gene start sequence, as exemplified by a single or multiple base substitution in the M2 gene start sequence at nucleotide 7605 (nucleotide 7606 in recombinant sequence) .
  • compositions e.g., isolated polynucleotides and vectors incorporating an RSV-encoding cDNA
  • methods are provided for producing -an isolated infectious chimeric RSV.
  • infectious chimeric RSV are generated from a chimeric RSV genome or antigenome, a nucleocapsid (N) protein, a nucleocapsid phosphoprotein (P) , a large (L) polymerase protein, and an RNA polymerase elongation factor.
  • compositions and methods are provided for introducing the aforementioned structural and phenotypic changes into a recombinant chimeric RSV to yield infectious, attenuated vaccine viruses.
  • an expression vector which comprises an isolated polynucleotide molecule encoding a chimeric RSV genome or antigenome. Also provided is the same or different expression vector comprising one or more isolated polynucleotide molecules encoding N, P, L and RNA polymerase elongation factor proteins.
  • the vector (s) is/are preferably expressed or coexpressed in a cell or cell-free lysate, thereby producing an infectious chimeric RSV particle or subviral particle.
  • the RSV genome or antigenome and the N, P, L and RNA polymerase elongation factor (preferably the product of the M2(0RF1) of RSV) proteins can be coexpressed by the same or different expression vectors.
  • the N, P, L and RNA polymerase elongation factor proteins are each encoded on different expression vectors.
  • the polynucleotide molecule encoding the chimeric RSV genome or antigenome can be a chimera of different human RSV subgroups or strains, for example a polynucleotide containing sequences from a subgroup A RSV operably joined with sequences from a subgroup B RSV.
  • the chimeric genome or antigenome can be a chimera of human and non-human (e.g., bovine or murine) RSV sequences.
  • the chimeric genome or antigenome can be a chimera of RSV and non-RSV sequences, for example a polynucleotide containing sequences from a human RSV operably joined with PIV sequences.
  • the chimeric genome or antigenome can be further modified by insertion, rearrangement, deletion or substitution of one or more nucleotides, including point mutations, site- specific nucleotide changes, and changes involving entire genes or gene segments introduced within a heterologous donor gene or gene segment or the recipient, background genome or antigenome.
  • alterations typically specify one or more phenotypic change (s) in the resulting recombinant RSV, such as a phenotypic change that results in attenuation, temperature- sensitivity, cold-adaptation, small plaque size, host range restriction, alteration in gene expression, or a change in an immunogenic epitope .
  • infectious viral or subviral particles yield infectious viral or subviral particles, or derivatives thereof.
  • An infectious virus is comparable to the authentic RSV virus particle and is infectious as is. It can directly infect fresh cells.
  • An infectious subviral particle typically is a subcomponent of the virus particle which can initiate an infection under appropriate conditions.
  • a nucleocapsid containing the genomic or antigenomic RNA and the N, P, L and M2 (ORF1) proteins is an example of a subviral particle which can initiate an infection if introduced into the cytoplasm of cells.
  • Subviral particles provided within the invention include, inter alia, viral particles which lack one or more protein (s), protein segmen (s), or other viral component (s) not essential for infectivity.
  • Infectious chimeric RSV can incorporate heterologous, coding or non-coding nucleotide sequences from any RSV or RSV-like virus, e.g., human, bovine, murine (pneumonia virus of mice) , or avian (turkey rhinotracheitis virus) RSV, or from another enveloped virus, e.g., parainfluenza virus (PIV).
  • the recombinant RSV comprises a chimera of a human RSV genomic or antigenomic sequence recombinantly joined with one or more heterologous RSV sequence (s).
  • Exemplary heterologous sequences include RSV sequences from one human RSV strain combined with sequences from a different human RSV strain.
  • chimeric RSV of the invention may incorporate sequences from two or more wild-type or mutant RSV strains, for example mutant strains selected from cpts RSV 248, cpts 248/404, cpts 248/955, cpts RSV 530, cpts 530/1009, or cpt ⁇ - 530/1030) .
  • chimeric RSV may incorporate sequences from two or more, wild-type or mutant RSV subgroups, for example a combination of RSV subgroup A and subgroup B sequences.
  • one or more human RSV coding or non-coding polynucleotides are substituted with a counterpart sequence from bovine or murine RSV, alone or in combination with one or more selected attenuating point mutations, e.g., cp and/or ts mutations, to yield novel attenuated vaccine strains.
  • a chimeric bovine-human RSV incorporates a substitution of the human RSV NP gene or gene segment with a counterpart bovine NP gene or gene segment, which chimera can optionally be constructed to incorporate a SH gene deletion, one or more cp or ts point mutations, or various combinations of these and other mutations disclosed herein.
  • isolated polynucleotides, expression vectors, and methods for producing chimeric RSV are provided wherein the genome or antigenome is recombinantly altered compared to either the donor or recipient sequence.
  • mutations are incorporated within a chimeric RSV genome or antigenome based on their ability to alter the structure and/or function of a chimeric RSV clone, e.g., by altering the structure, expression and or function of a selected protein encoded or a cis-acting RNA sequence thereby yielding a desired phenotypic change. Desired phenotypic changes include, e.g., changes in viral growth in culture, temperature sensitivity, plaque size, attenuation, and immunogenicity.
  • isolated polynucleotides and expression vectors comprise a chimeric RSV genome or antigenome having at least one attenuating point mutation adopted from a biologically derived mutant RSV.
  • at least one point mutation is present in the polymerase gene L involving a nucleotide substitution that specifies a ts phenotype.
  • Exemplary RSV clones and vectors incorporate a nucleotide substitution that results in an amino acid change in the polymerase gene at Phe 521 , Gln 831 , Met 1169 ⁇ or Tyr 1321 .
  • two or three mutations are incorporated in a codon specifying the attenuating mutation in order to increase the level of genetic stability.
  • RSVs incorporate at least two attenuating ts mutations. Mutations incorporated within chimeric cDNAs, vectors and viral particles of the invention can be introduced individually or in combination into a full-length RSV cDNA and the phenotypes of rescued virus containing the introduced mutations can be readily determined.
  • amino acid changes displayed by attenuated, biologically-derived viruses versus a wild-type RSV for example changes exhibited by cpRSV or tsRSV, are incorporated in combination within recombinant RSV to yield a desired level of attenuation.
  • the present invention also provides chimeric RSV clones, vectors and particles incorporating multiple, phenotype-specific mutations introduced in selected combinations into the chimeric genome or antigenome to produce an attenuated, infectious virus or subviral particle.
  • This process coupled with routine phenotypic evaluation, provides chimeric RSV having such desired characteristics as attenuation, temperature sensitivity, altered immunogenicity, cold-adaptation, small plaque size, host range restriction, etc. Mutations thus identified are compiled into a "menu” and introduced in various combinations to calibrate a vaccine virus to a selected level of attenuation, immunogenicity and stability.
  • the invention provides for supplementation of one or more mutations adopted from biologically derived RSV, e.g., cp and ts mutations, with additional types of mutations involving the same or different genes.
  • Target genes for mutation in this context include the attachment (G) protein, fusion (F) protein, small hydrophobic (SH) , RNA binding protein (N) , phosphoprotein (P) , the large polymerase protein (L) , the transcription elongation factor (M2) , M2 0RF2, the matrix (M) protein, and two nonstructural proteins, NS1 and NS2.
  • G attachment protein
  • F small hydrophobic
  • N RNA binding protein
  • P phosphoprotein
  • L large polymerase protein
  • M2 transcription elongation factor
  • M2 transcription elongation factor
  • M2 matrix protein
  • NS1 and NS2 two nonstructural proteins
  • the SH gene is deleted in the donor or recipient context to yield a chimeric RSV having novel phenotypic characteristics, including enhanced growth in vi tro and/or attenuation in vivo .
  • this gene deletion, or another selected, non-essential gene or gene segment deletion, such as a NS1 or NS2 gene deletion is combined in a chimeric RSV with one or more separate mutations specifying an attenuated phenotype, e.g., a point mutation adopted directly (or in modified form, e.g., by introducing multiple nucleotide changes in a codon specifying the mutation) from a biologically derived attenuated RSV mutant.
  • the SH gene or NS2 gene may be deleted in combination with one or more cp and/or ts mutations adopted from cpts248/404, cpts530/l009, cpts530/1030 , or another selected mutant RSV strain, to yield a chimeric RSV having increased yield of virus, enhanced attenuation, and genetic resistance to reversion from an attenuated phenotype, due to the combined effects of the different mutations.
  • a variety of other genetic alterations can be produced in a chimeric RSV genome or antigenome, alone or together with one or more attenuating point mutations adopted from a biologically derived mutant RSV.
  • genes or gene segments from non-RSV sources may be inserted in whole or in part.
  • the order of genes can be changed, gene overlap removed, or a RSV genome promoter replaced with its antigenome counterpart .
  • Different or additional modifications in the chimeric genome or antigenome can be made to facilitate manipulations, such as the insertion of unique restriction sites in various intergenic regions (e.g., a unique Stul site between the G and F genes) or elsewhere.
  • Nontranslated gene sequences can be removed to increase capacity for inserting foreign sequences.
  • polynucleotide molecules or vectors encoding the chimeric RSV genome or antigenome can be modified to encode non-RSV sequences, e.g., a cytokine, a T-helper epitope, a restriction site marker, or a protein of a microbial pathogen (e.g., virus, bacterium or fungus) capable of eliciting a protective immune response in an intended host.
  • non-RSV sequences e.g., a cytokine, a T-helper epitope, a restriction site marker, or a protein of a microbial pathogen (e.g., virus, bacterium or fungus) capable of eliciting a protective immune response in an intended host.
  • the invention provides a cell or cell-free lysate containing an expression vector which comprises an isolated polynucleotide molecule encoding a chimeric RSV genome or antigenome as described above, and an expression vector (the same or different vector) which comprises one or more isolated polynucleotide molecules encoding the N, P, L and RNA polymerase elongation factor proteins of RSV.
  • an expression vector which comprises one or more isolated polynucleotide molecules encoding the N, P, L and RNA polymerase elongation factor proteins of RSV.
  • Attenuated chimeric RSV of the invention is capable of eliciting a protective immune response in an infected human host, yet is sufficiently attenuated so as to not cause unacceptable symptoms of severe respiratory disease in the immunized host.
  • the attenuated chimeric virus or subviral particle may be present in a cell culture supernatant, isolated from the culture, or partially or completely purified.
  • the virus may also be lyophilized, and can be combined with a variety of other components for storage or delivery to a host, as desired.
  • the invention further provides novel vaccines comprising a physiologically acceptable carrier and/or adjuvant and an isolated attenuated chimeric RSV as described above.
  • the vaccine is comprised of chimeric RSV having at least one, and preferably two or more attenuating mutations or other nucleotide modifications as described above.
  • the vaccine can be formulated in a dose of IO 3 to 10 s PFU of attenuated virus.
  • the vaccine may comprise attenuated chimeric virus that elicits an immune response against a single RSV strain or antigenic subgroup, e.g. A or B, or against multiple RSV strains or subgroups.
  • chimeric RSV of the invention can individually elicit a monospecific immune response or a polyspecific immune response against multiple RSV strains or subgroups.
  • Chimeric RSV can be combined in vaccine formulations with other chimeric RSV or non-chimeric RSV having different immunogenic characteristics for more effective protection against one or multiple RSV strains or subgroups.
  • the invention provides a method for stimulating the immune system of an individual to elicit an immune response against one or more strains or subgroups of RSV in a mammalian subject.
  • the method comprises administering a formulation of an i ' mmunologically sufficient amount of an attenuated, chimeric RSV as described above in a physiologically acceptable carrier and/or adjuvant.
  • the immunogenic composition is a vaccine comprised of chimeric RSV having at least one, and preferably two or more attenuating mutations or other nucleotide modifications as described above .
  • the vaccine can be formulated in a dose of IO 3 to IO 6 PFU of attenuated virus.
  • the vaccine may comprise attenuated chimeric virus that elicits an immune response against a single RSV strain or antigenic subgroup, e.g. A or B, or against multiple RSV strains or subgroups.
  • the chimeric RSV can elicit a monospecific immune response or a polyspecific immune response against multiple RSV strains or subgroups.
  • chimeric RSV having different immunogenic characteristics can be combined in a vaccine mixture or administered separately in a coordinated treatment protocol to elicit more effective protection against one RSV strain, or against multiple RSV strains or subgroups .
  • Fig. 1 is a graph demonstrating the substantially complete correlation between the replication of a series of subgroup A respiratory syncytial viruses in the lungs of mice with their replication in the chimpanzee.
  • Figs. 2 and 3 show the construction of cDNA encoding
  • FIG. 2 shows the structures of the cDNA and the encoded antigenome RNA (not to scale) .
  • the specific cDNAs and viruses used were of strain A2 of subgroup A RSV.
  • the diagram of the antigenome includes the following features: the 5' -terminal nonviral G triplet contributed by the T7 promoter, the four sequence markers at positions 1099 (which adds one nt to the length) , 1139, 5611, and 7559 (numbering referring to the first base of the new restriction site) , the ribozyme and tandem T7 terminators, and the single nonviral 3 ' -phosphorylated U residue contributed to the 3' end by ribozyme cleavage (the site of cleavage is indicated with an arrow) . Note that the nonviral 5'-GGG and 3 ' -U residues are not included in length values given here and thereafter for the antigenome.
  • nucleotide insertion at position 1099 is included, and thus the numbering for cDNA-derived antigenome is one nucleotide greater downstream of this position than for biologically derived antigenome.
  • the 5' to 3 ' positive-sense sequence of D46 (the genome itself being negative-sense) is depicted in SEQ ID NO: 1, where the nucleotide at position four can be either C or G.
  • sequence positions assigned to restriction sites in this Figure and throughout are intended as a descriptive guide and do not alone define all of the nucleotides involved.
  • the length values assigned to restriction fragments here and throughout also are descriptive, since length assignments may vary based on such factors as sticky ends left following digestion.
  • sequence 3 shows the sequence markers contained in the cDNA-encoded antigenome RNA, where sequences are positive sense and numbered relative to the first nt of the leader region complement as 1; identities between strains A2 and 18537, representing RSV subgroups A and B, respectively, ase indicated with dots; sequences representing restriction sites in the cDNA are underlined; gene-start (GS) and gene-end (GE) transcription signals are boxed; the initiation codon of the N translational open reading frame at position 1141 is italicized, and the restriction sites are shown underneath each sequence.
  • a single C residue was inserted at position 1099 to create an Aflll site in the NS2-N intergenic region, and the AG at positions 1139 and 1140 immediately upstream of the N translational open reading frame were replaced with CC to create a new Ncol site.
  • substitution of G and U at positions 5612 and 5616, respectively, created a new Stul site in the G-F intergenic region.
  • a C replacement at position 7560 created a new ⁇ p-l site in the F-M2 intergenic region .
  • Fig. 4 illustrates structures of cDNAs (approximately to scale) involved in the insertion of mutations, assembly of complete antigenome constructs, and recovery of recombinant virus.
  • Four types of mutations were inserted into the pUC118- or pUC119-borne cDNA subclones shown in the bottom row, namely six silent restriction sites in the L gene (underlined over the D53 diagram on the top) , two HEK changes in the F gene (H) , five cp changes (cp) , and the mutations specific to the various biological mutagenesis steps: 248, 404, 530, 1009, and 1030 (as indicated).
  • the mutagenized subclones were inserted into the D50 (representing the RSV antigenome from the leader to the beginning of the M2- L overlap with the T7 promoter immediately upstream of the leader) or D39 (representing the RSV antigenome from the M2-L overlap to the trailer with the ribozyme and T7 terminators immediately downstream of the trailer) intermediate plasmids shown in the middle row.
  • the appropriate D50 and D39 were assembled into full-length D53 antigenome cDNA as shown on the top row (RTT indicates the location of the hammer-head ribozyme followed by two T7 transcription terminators) .
  • Fig. 5 provides maps of six mutant antigenome cDNAs which were used to recover recombinant RSV.
  • the ts phenotypes of the recombinants are summarized on the right of the figwre.
  • Fig. 6 shows construction of D46/1024CAT cDNA encoding an RSV antigenome containing the CAT ORF flanked by RSV transcription signals (not to scale, RSV-specific segments are shown as filled boxes and CAT sequence as an open box) .
  • the source of the CAT gene transcription cassette was RSV-CAT minigenome cDNA 6196 (diagram at top) .
  • the RSV-CAT minigenome contains the leader region, GS and GE signals, noncoding (NC) RSV gene sequences, and the CAT ORF, with -Xmal restriction endonuclease sites preceding the GS signal and following the GE signal.
  • the nucleotide lengths of these elements are indicated, and the sequences (positive-sense) surrounding the Xmal sites are shown above the diagram.
  • a 8-nucleotide Xmal linker was inserted into Stul site of the parental plasmid D46 to construct the plasmid D46/1024.
  • D46 is the complete antigenome cDNA and is equivalent to D53 ; the difference in nomenclature is to denote that these represent two different preparations.
  • the Xma-Xmal fragment of the plasmid 6196 was inserted into the plasmid D46/1024 to construct the plasmid
  • the RNA encoded by the D46 cDNA is shown at the bottom, including the three 5' -terminal nonviral G residues contributed by the T7 promoter and the 3 '-terminal phosphorylated U residue contributed by cleavage of the hammerhead ribozyme; the nucleotide lengths given for the antigenome do not include these nonviral nucleotides.
  • the L gene is drawn offset to indicate the gene overlap.
  • Fig. 7 is a diagram (not to scale) of the parental wild-type D46 plasmid encoding an RSV antigenome (top) , and the D46/6368 derivative in which the SH gene has been deleted (bottom) .
  • the RSV genes are shown as open rectangles with the GS and GE transcription signals shown as filled boxes on the upstream and downstream ends, respectively.
  • the T7 phage promoter (left) and hammerhead ribozyme and T7 terminators used to generate the 3' end of the RNA transcript (right) are shown as small open boxes.
  • the Seal and Pad fragment of D46 was replaced with a short synthetic fragment, resulting in D46/6368.
  • the sequence flanking the SH gene in D46, and the sequence of the engineered region in D46/6368, are each shown framed in a box over the respective plasmid map.
  • the sequence of the Seal - Pad fragment in D46, and its replacement in D46/6368, are shown in bold and demarcated with arrows facing upward.
  • the M GE, SH GS, SH GE and G GS sites are indicated with overlining.
  • the new M-G intergenic region in D46/6368 is labeled 65 in the diagram at the bottom to indicate its nucleotide length.
  • the positive-sense T7 transcript of the SH-minus D46/6368 construct is illustrated at the bottom; the three 5' -terminal nonviral G residues contributed by the T7 promoter and the 3' -terminal U residue are shown (Collins, et al. Proc. Natl. Acad. Sci. USA 92:11563-11567 (1995), incorporated herein by reference) . These nonviral nucleotides are not included in length measurements.
  • Fig. 8 provides results of RT-PCR analysis of total intraceUular RNA from cells infected with the D46 wild-type or D46/6368 SH-minus virus to confirm the deletion in the SH locus.
  • RT was performed with a positive-sense primer that anneals upstream of the SH gene, and the PCR employed in addition a negative-sense primer that anneals downstream of the SH gene.
  • Fig. 9 shows Northern blot hybridization of RNAs encoded by the D46 wild-type and D46/6368 SH-minus virus.
  • RNA Total intraceUular RNA was isolated from infected cells and subjected to oligo(dT) chromatography without a prior denaturation step, conditions under which the selected RNA also includes genomic RNA due to sandwich hybridization. RNAs were electrophoresed on formaldehyde-agarose gels and blotted onto nitrocellulose membrane. Replicate blots were hybridized individually with [ 32 P] -labeled DNA probes of the M, SH, G, F, M2, or L genes, as indicated. Lanes: (1) D46/6368 RNA; (2) D46 ⁇ RNA; (3) uninfected HEp-2 cell RNA.
  • Positions of the genomic RNA (gen.), mRNAs (large letters) and read-through transcripts (small letters) are shown on the left.
  • the positions of the 0.24-9.5 kb RNA ladder molecular weight markers (Life Technologies) which was run in parallel and visualized by hybridization with [ 32 P] -labeled DNA of phage lambda, are shown on the right.
  • Fig. 10 shows SDS-PAGE of [ 35 S] -labeled RSV proteins synthesized in HEp-2 cells infected with the D46 wild-type or D46/6368 SH-minus virus. Proteins were subjected to immunoprecipitation with antiserum raised against purified virions and analyzed by electrophoresis in pre-cast gradient 4% - 20% Tris-glycine gels (Novex, San Diego, CA) . Positions of viral proteins are indicated to the left; positions and molecular masses (in kilodal ons) of marker proteins (Kaleidoscope Prestained Standards, Bio-Rad, Richmond, CA) , are shown to the right . Figs.
  • FIG. 11-13 provide growth curves for D46 wild-type and D46/6368 SH-minus viruses in HEp-2 cells (Fig. 11) , 293 cells (Fig. 12), and AGMK-21 cells (Fig. 13).
  • Triplicate cell monolayers in 25-cm 2 culture flasks were infected with 2 PFU per cell of either virus, and incubated at 37°C. Aliquots were taken at indicated time points, stored at -70°C, and titrated in parallel by plaque assay with antibody staining. Each point shown is the average titer of three infected cell monolayers .
  • Figs. 14 and 15 show kinetics of virus replication in the upper (Fig. 14) and lower (Fig. 15) respiratory tract of mice inoculated intranasally with the D46 wild-type virus, D46/6368 SH-minus virus, or the biologically-derived cpts248/404 virus.
  • Mice in groups of 24 were inoculated intranasally with 10 s PFU of the indicated virus.
  • Six mice from each group were sacrificed on the indicated day and the nasal turbinates and lung tissues were removed and homogenized, and levels of infectious virus were determined by plaque assay on individual specimens and mean log 10 titers were determined.
  • Fig. 16 shows a comparison of the transcription products and gene order of SH-minus virus compared to its wild-type counterpart.
  • the upper panel summarizes an analysis of the amounts of certain mRNAs produced by the SH-minus virus compared with the wild-type parent recombinant virus.
  • IntraceUular mRNAs were isolated from cells infected with the SH-minus or wild-type virus and analyzed by Northern blot hybridization with gene-specific probes. The amount of hybridized radioactivity was quantitated, and the relative abundance of each individual mRNA produced by the SH-minus virus versus its wild-type parent is shown.
  • the lower panel shows the gene order of the wild-type virus from the M gene (position 5 in the gene order) to the L gene (position 10) . This is compared to that of the SH-minus virus, in which the positions in the gene order of the G, F, M2 and L genes are altered due to deletion of the SH gene.
  • Fig. 17 depicts the D46 antigenome plasmid which was modified by deletion of the SH gene in such a way as to not insert any heterologous sequence into the recombinant virus .
  • the MGE, M-SH intergenic (IG) , SH GS, SH GE and SH-G IG sequences are shown.
  • the area which was removed by the deletion is underlined, with the deletion points indicated with upward pointing triangles.
  • the antigenome resulting from this deletion is D46/6340.
  • Fig. 18 depicts the introduction of tandem translation stop codons into the translational open reading frame (ORF) encoding the NS2 protein.
  • Plasmid D13 contains the left end of the antigenome cDNA, including the T7 promoter (shaded box), the leader region, and the NS1, NS2, N, P, M and SH genes. Only the cDNA insert of D13 is shown.
  • the Aatll - Aflll fragment containing the T7 promoter and NS1 and NS2 genes was subcloned into a pGem vector, and site-directed mutagenesis was used to modify the NS2 ORF in the region illustrated by the sequence.
  • the wild-type sequence of codons 18 to 26 is shown (the encoded amino acids are indicated below) , and the three nucleotides above are the three substitutions which were made to introduce two termination codons (ter) and an Xhol site (underlined) as a marker. Ttee resulting cDNA and subsequent recovered virus are referred to as NS2-knockout (KO) .
  • Fig. 19 compares production of infectious virus by wild-type RSV (D53) versus NS2-knockout RSV in HEp-2 cells. Triplicate monolayers were infected with either virus at an input moi of three pfu/cell, and samples were taken at the indicated intervals and quantitated by plaque assay and immunostaining.
  • Fig. 20 depicts alteration of gene-end (GE) signals of the NS1 and NS2 genes.
  • the cDNA insert of plasmid D13 representing the left hand end of the antigenome cDNA from the T7 promoter (shaded) to the Pad site at position 4623, is shown.
  • the Aatl -Aflll fragment containing the T7 promoter and the NSl and NS2 genes was subcloned into a pGem vector. It was modified by site-directed mutagenesis simultaneously at two sites, namely the NSl and NS2 GE signals were each modified to be identical to that found in nature for the N gene.
  • sequences of the wild-type NSl and NS2 GE signals are shown (and identified by sequence position relative to the complete antigenome sequence) , and the nucleotide substitutions are shown above the line.
  • the dash in the wild- type sequence of the NS2 GE signal indicates that the mutation increased the length of the GE signal by one nucleotide.
  • Fig. 21 depicts the deletion of the polynucleotide sequence encoding the NSl protein.
  • the left hand part of the D13 cDNA is shown at the bottom: D13 contains the left hand part of the antigenome cDNA, from the leader to the end of the SH gene, with the T7 promoter immediately upstream of the leader.
  • the sequence on either side of the deletion point (upward arrow) is shown on top.
  • the deletion spans from immediately before the translational start site of the NSl ORF to immediately before that of the NS2 ORF. Thus, it has the effect of fusing the NSl GS and upstream noncoding region to the NS2 ORF. This precludes the disruption of any cis-acting sequence elements which might extend into the NSl gene due to its leader-proximal location.
  • Fig. 22 depicts the deletion of the polynucleotide sequence encoding the NS2 mRNA.
  • the left hand part of the D13 cDNA is shown along with the sequence on either side of the deletion point (upward arrow) .
  • the deletion spans from immediately downstream of the NSl gene to immediately downstream of the NS2 gene.
  • the sequence encoding the NS2 mRNA has been deleted in its entirety, but no other sequence has been disrupted.
  • the resulting cDNA and subsequent recovered recombinant virus are referred to as ⁇ NS2.
  • Fig. 23 depicts the ablation of the translational start site for the secreted form of the G protein.
  • the 298- amino acid G protein is shown as an open rectangle with the signal-anchor sequence filled in.
  • the amino acid sequence for positions 45 to 53 is shown overhead to illustrate two nucleotide substitutions which change amino acid 48 from methionine to isoleucine and amino acid 49 from isoleucine to valine.
  • the former mutation eliminates the translational start site for the secreted form.
  • the two mutations also create an Mfel site, which provides a convenient method for detecting the mutation.
  • the resulting cDNA and subsequent recovered virus are referred to as M48I (methionine-48 to isoleucine-48) .
  • Fig. 24 shows the results of a comparison of production of infectious virus by wild-type RSV (D53) versus that of two isolates of recovered D53/M481 membrane G mutant virus.
  • Fig. 25A shows the negative-sense genomic RNA of RSV strain A2 (antigenic subgroup A) and illustrates replacement of the F and G genes with their counterparts of strain Bl (antigenic subgroup B) .
  • Each rectangle represents a gene encoding a single mRNA, and the grey and filled boxes at the left and right ends of each rectangle represent gene-start (GS) and gene-end (GE) transcription signals, respectively.
  • the thin lines at each end of the genome represent the leader
  • the gene replacement is done at the level of an antigenome cDNA using Pad and Sphl sites which precede the G gene and follow the F gene, respectively.
  • the L gene is drawn offset to illustrate that it overlaps with the upstream M2 gene, a detail which is not immediately germane to this example.
  • Figs. 25B and 25C illustrate the sequence (positive- sense) in the chimeric rAB virus, namely recombinant RSV strain A2 in which the F and G glycoprotein genes were replaced with those of strain Bl .
  • the sequence shown contains part of the SH-G (Part B) and F-M2 (Part C) junction. Sequence derived from the strain A2 backbone is shown in lower case, and that from the strain Bl donor is in upper case. The last A2-specific nucleotide at the junction between the A2 and Bl sequence is numbered according to the unmodified recombinant A2 genomic sequence.
  • the SH gene-end (GE) and F GE signals are boxed.
  • the Pad and Sphl recognition sites are italicized.
  • IG intergenic region.
  • Fig. 26 illustrates modification the cDNA of the strain Bl G and F genes in order to improve stability during growth in E. coli .
  • Two positive-sense sequences are shown: the upper one (labeled "new") is the modified Bl sequence, and the lower (“wt") is the wild-type Bl sequence.
  • the sequence shown includes the downstream end of the G translational open reading frame (ORF) , its encoded amino acids (shown as the single letter code below the sequence) , the G GE signal
  • Fig. 27 illustrates replication of the chimeric recombinant AB wt RSV and ABcp248/404/l030 derivative in the upper (top panel; nasopharyngeal swab) and lower (lower panel; tracheal lavage) respiratory tract of seronegative chimpanzees. This is based on data from Table 46. The light horizontal dotted line in each graph is the lower limit of detectability. Bars indicate Standard Error.
  • the present invention provides infectious, chimeric respiratory syncytial virus (RSV) that are attenuated and capable of eliciting a propylactic or therapeutic immune response in mammalian patients susceptible to RSV infection. Also provided within the invention are novel methods and compositions for designing and producing attenuated, chimeric RSV, as well as methods and compositions for prophylaxis and treatment of RSV infection.
  • RSV respiratory syncytial virus
  • Chimeric RSV of the invention are recombinantly engineered to incorporate nucleotide sequences from more than one RSV strain or subgroup to produce an infectious, chimeric virus or subviral particle.
  • candidate vaccine virus are recombinantly engineered to elicit an immune response against RSV in a mammalian host, including humans and non-human primates.
  • Chimeric RSV according to the invention may elicit an immune response to a specific RSV subgroup or strain, or they may elicit a polyspecific response against multiple RSV subgroups or strains.
  • heterologous genes, gene segments, or single or multiple nucleotides of one RSV are added to a partial or complete RSV genome or antigenome or substituted therein by counterpart sequence (s) from a heterologous RSV to produce a chimeric RSV genome or antigenome.
  • the chimeric RSV of the invention thus includes a partial or complete "recipient" RSV genome or antigenome from one RSV strain or subgroup virus combined with an additional or replacement "donor" gene or gene segment of a different RSV strain or subgroup virus.
  • chimeric RSV incorporate a partial or complete human RSV genome or antigenome of one RSV subgroup or strain combined with a heterologous gene or gene segment from a different human RSV subgroup or strain.
  • preferred chimeric RSV incorporate a chimeric genome or antigenome comprised of a partial or complete human RSV A or B subgroup genome or antigenome combined with a heterologous gene or gene segment from a different human RSV A or B subgroup virus.
  • Heterologous donor genes or gene segments from one RSV strain or subgroup are combined with or substituted within a recipient genome or antigenome that serves as a backbone for insertion or addition of the donor gene or gene segment.
  • the recipient genome or antigenome acts as a vector to import and express heterologous genes or gene segments to yield chimeric RSV that exhibit novel structural and/or phenotypic characteristics.
  • addition or substitution of a heterologous gene or gene segment within a selected recipient RSV strain yields novel phenotypic effects, for example attenuation, growth changes, altered immunogenicity, or other desired phenotypic changes, as compared with corresponding phenotypes of the unmodified recipient and/or donor.
  • Genes and gene segments that are useful as heterologous inserts or additions within a chimeric RSV genome or antigenome include genes or gene segments encoding a NSl, NS2, N, P, M, SH, M2(0RF1), M2(ORF2), L, F or G protein or a portion thereof.
  • chimeric RSV incorporate a heterologous gene encoding a RSV F, G or SH glycoprotein.
  • the chimeric RSV may incorporate a gene segment encoding only a portion of a selected protein, for example a cytoplasmic domain, transmembrane domain, ectodomain or immunogenic epitope of a RSV F, G or SH glycoprotein.
  • chimeric RSV useful in a vaccine formulation can be conveniently modified to accommodate antigenic drift in circulating virus.
  • the modification will be in the G and/or F proteins.
  • An entire G or F gene, or a gene segment encoding a particular immunogenic region thereof, from one RSV strain is incorporated into a chimeric RSV genome or antigenome cDNA by replacement of a corresponding region in a recipient clone of a different RSV strain or subgroup, or by adding one or more copies of the gene, such that several antigenic forms are represented.
  • Progeny virus produced from the modified RSV clone can then be used in vaccination protocols against emerging RSV strains .
  • chimeric RSV may also be constructed that express a chimeric protein, e.g., an immunogenic protein having a cytoplasmic tail and/or transmembrane domain specific to one RSV strain or subgroup fused to an ectodomain of a different RSV.
  • a chimeric protein e.g., an immunogenic protein having a cytoplasmic tail and/or transmembrane domain specific to one RSV strain or subgroup fused to an ectodomain of a different RSV.
  • Other exemplary recombinants of this type may express duplicate protein regions, such as duplicate immunogenic regions.
  • a variety of gene segments provide useful donor polynucleotides for inclusion within a chimeric genome or antigenome to express chimeric RSV having novel and useful properties.
  • heterologous gene segments may beneficially encode a cytoplasmic tail, transmembrane domain or ectodomain, an epitopic site or region, a binding site or region, an active site or region containing an active site, etc., of a selected protein from one RSV.
  • These and other gene segments can be added or substituted for a counterpart gene segment in another RSV to yield novel chimeric recombinants, for example recombinants expressing a chimeric protein having a cytoplasmic tail and/or transmembrane domain of one RSV fused to an ectodomain of another RSV.
  • Useful genome segments ⁇ - this regard range from about 15-35 nucleotides in the case of gene segments encoding small functional domains of proteins, e.g., epitopic sites, to about 50, 75, 100, 200-500, and 500- 1,500 or more nucleotides for gene segments encoding larger domains or protein regions.
  • heterologous genes may be added or substituted in whole or in part to a background genome or antigenome to form a chimeric genome or antigenome.
  • a selected protein or protein region e.g., a cytoplasmic tail, transmembrane domain or ectodomain, an epitopic site or region, a binding site or region, an active site or region containing an active site, etc.
  • a counterpart gene or gene segment in a different RSV genome or antigenome to yield novel recombinants having desired phenotypic changes compared to wild-type or parent RSV strains.
  • counterpart genes, gene segments, proteins or protein regions two counterpart polynucleotides from a heterologous source, including different genes in a single RSV strain, or different variants of the same gene, including species and allelic variants among different RSV subgroups or strains .
  • Counterpart genes and gene segments share at least moderate structural similarity.
  • counterpart gene segments may encode a common structural domain of a protein of interest, such as a cytoplasmic domain, transmembrane domain, ectodomain, binding site or region, epitopic site or region, etc. Typically, they will share a common biological function as well.
  • protein domains encoded by counterpart gene segments may providing a common membrane spanning function, a specific binding activity, an immunological recognition site, etc.
  • a desired biological activity shared between the products of counterpart genes and gene segments will be substantially similar in quantitative terms, i.e., they will not differ by more than 30%, preferably by no more than 20%, more preferably by no more than 5-10%.
  • Counterpart genes and gene segments for use within , the invention embrace an assemblage of alternate, species having a range of size and sequence variation.
  • selection of counterpart genes and gene segments relies on substantial sequence identity between the subject counterparts.
  • a selected polynucleotide "reference sequence” is defined as a sequence or portion thereof present in either the donor or recipient genome or antigenome. This reference sequence is used as a defined sequence to provide a rationale basis for a sequence comparison.
  • the reference sequence may be a defined a segment of a cDNA or gene, or a complete cDNA or gene sequence .
  • a reference sequence for use in defining counterpart genes and gene segments is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length.
  • two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window”, as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J.
  • sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size) , and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25- 50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
  • the reference sequence may be a subset of a larger sequence.
  • proteins and protein regions encoded by chimeric RSV of the invention are also typically selected to have conservative relationships, i.e., to have substantial sequence identity or sequence similarity, with selected reference polypeptides.
  • sequence identity means peptides share identical amino acids at corresponding positions.
  • sequence similarity means peptides have identical or similar amino acids (i.e., conservative substitutions) at corresponding positions.
  • substantially sequence identity means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity or more (e.g., 99 percent sequence identity) .
  • substantially similarity means that two peptide sequences share corresponding percentages of sequence similarity.
  • residue positions which are not identical differ by conservative amino acid substitutions .
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a conservative group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic- hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine- leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine .
  • Stereoisomers e.g., D-amino acids
  • unnatural amino acids such as , ⁇ -disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention.
  • Examples of unconventional amino acids include: 4-hydroxyproline, ⁇ -carboxyglutamate, e-N,N- ⁇ -N- trimethyllysine, e-N-acetyllysine, O-phosphoserine, N- acetylserine, N-formylmethionine, 3-methylhistidine, 5- hydroxylysine, ⁇ -N-methylarginine, and other amino and imino acids (e.g., 4-hydroxyproline).
  • amino acids may be modified by glycosylation, phosphorylation and the like.
  • the invention disclosed herein describes cDNA-based methods that are useful to construct a large panel of recombinant, chimeric RSV viruses and subviral particles. These recombinant constructs offer improved characteristics of attenuation and immunogenicity for use as vaccine agents. Among desired phenotypic changes in this context are resistance to reversion from an attenuated phenotype, improvements in attenuation in culture or in a selected host environment, immunogenic characteristics (e.g., as determined by enhancement, or diminution, of an elicited immune response) , upregulation or downregulation of transcription and/or translation of selected viral products, etc.
  • Attenuated, chimeric RSV are produced in which the chimeric genome or antigenome is further modified by introducing one or more attenuating point mutations that specifies an attenuating phenotype. These point mutations may be generated de novo and tested for attenuating effects according to a rational design mutagenesis strategy. Alternatively, the attenuating point mutations are identified in biologically derived mutant RSV and thereafter incorporated into a chimeric RSV of the invention. Attenuating point mutations in biologically derived
  • RSV for incorporation within a chimeric vaccine strain may occur naturally or may be introduced into wild-type RSV strains by well known mutagenesis procedures.
  • incompletely attenuated parental RSV strains can be produced by chemical mutagenesis during virus growth in cell cultures to which a chemical mutagen has been added, by selection of virus that has been subjected to passage at suboptimal temperatures in order to introduce growth restriction mutations, or by selection of a mutagenized virus that produces small plaques ( sp) in cell culture, as generally described herein and in USSN 08/327,263, incorporated herein by reference.
  • biologically derived RSV any RSV not produced by recombinant means.
  • biologically derived RSV include naturally occurring RSV of all subgroups and strains, including, e.g., naturally occurring RSV having a wild-type genomic sequence and RSV having genomic variations from a reference wild-type RSV sequence, e.g., RSV having a mutation specifying an attenuated phenotype.
  • biologically derived RSV include RSV mutants derived from a parental RSV strain by, inter alia , artificial mutagenesis and selection procedures .
  • mutations are preferably introduced into a parental strain which has been incompletely or partially attenuated, such as the well known ts-1 or ts-ING or cpRSV mutants of the A2 strain of RSV subgroup A, or derivatives or subclones thereof.
  • additional mutation (s) can be generated that further attenuate the strain, e.g., to a desired level of restricted replication in a mammalian host, while retaining sufficient immunogenicity to confer protection in vaccinees.
  • Partially attenuated mutants of the subgroup A or B virus can be produced by well known methods of biologically cloning wild-type virus in an acceptable cell substrate and developing, e.g., cold-passaged mutants thereof, subjecting the virus to chemical mutagenesis to produce ts mutants, or selecting small plaque or similar phenotypic mutants (see, e.g., Murphy et al . , International Publication WO 93/21310, incorporated herein by reference) .
  • an exemplary, partially attenuated parental virus is cp 23, which is a mutant of the Bl strain of subgroup B .
  • mutations specifying attenuated phenotypes may be introduced individually or in combination in incompletely attenuated subgroup A or B virus to produce vaccine virus having multiple, defined attenuating mutations that confer a desired level of attenuation and immunogenicity in vaccinees.
  • production of a sufficiently attenuated biologically derived RSV mutant can be accomplished by several known methods .
  • On such procedure involves subjecting a partially attenuated virus to passage in cell culture at progressively lower, attenuating temperatures.
  • a partially attenuated virus is produced by passage in cell cultures (e.g., primary bovine kidney cells) at suboptimal temperatures, e.g., 20-26°C.
  • the cp mutant or other partially attenuated strain e.g., ts-1 or spRSV
  • ts-1 or spRSV is adapted to efficient growth at a lower temperature by passage in MRC-5 or Vero cells, down to a temperature of about 20-24°C, preferably 20-22°C.
  • This selection of mutant RSV during cold-passage substantially eliminates any residual virulence in the derivative strains as compared to the partially attenuated parent.
  • specific mutations can be introduced into biologically derived RSV by subjecting a partially attenuated parent virus to chemical mutagenesis, e.g., to introduce ts mutations or, in the case of viruses which are already ts, additional ts mutations sufficient to confer increased attenuation and/or stability of the ts phenotype on the attenuated derivative.
  • Means for the introduction of ts mutations into RS virus include replication of the virus in the presence of a mutagen such as 5-fluorouridine or 5-fluorouracil in a concentration of about IO "3 to 10" s M, preferably about IO "4 M, exposure of virus to nitrosoguanidine at a concentration of about 100 ⁇ g/ml, according to the general procedure described in, e.g., Gharpure et al . , J. Virol . 3:414-421 (1969) and Richardson et al . , J. Med. Virol. 3:91-100 (1978), or genetic introduction of specific ⁇ s mutations.
  • Other chemical mutagens can also be used. Attenuation can result from a ts mutation in almost any RSV- gene, although a particularly amenable target for this purpose has been found to be the polymerase (L) gene.
  • the level of temperature sensitivity of replication in exemplary attenuated RSV for use within the invention is determined by comparing its replication at a permissive temperature with that at several restrictive temperatures.
  • the lowest temperature at which the replication of the virus is reduced 100-fold or more in comparison with its replication at the permissive temperature is termed the shutoff temperature.
  • both the replication and virulence of RSV correlate with the mutant's shutoff temperature. Replication of mutants with a shutoff temperature of 39°C is moderately restricted, whereas mutants with a shutoff of 38°C replicate less well and symptoms of illness are mainly restricted to the upper respiratory tract.
  • a virus with a shutoff temperature of 35 to 37°C will typically be fully attenuated in humans.
  • Attenuated biologically derived mutant and chimeric RSV of the invention which are ts will have a shutoff temperature in the range of about 35 to 39°C, and preferably from 35 to 38°C.
  • the addition of a ts mutation into a partially attenuated strain produces multiply attenuated virus useful within vaccine compositions of the invention.
  • Attenuated RSV strains as candidate vaccines for intranasal administration have been developed using multiple rounds of chemical mutagenesis to introduce multiple mutations into a virus which had already been attenuated during cold-passage (e.g., Connors et al . , Virology 208: 478-484 (1995); Crowe et al . , Vaccine 12: 691-699 (1994); and Crowe et al . , Vaccine 12: 783-790 (1994), incorporated herein by reference) . Evaluation in rodents, chimpanzees, adults and infants indicate that certain of these candidate vaccine strains are relatively stable genetically, are highly immunogenic, and may be satisfactorily attenuated.
  • Nucleotide sequence analysis of some of these attenuated viruses indicates that each level of increased attenuation is associated with specific nucleotide and amino acid substitutions.
  • the present invention provides the ability to distinguish between silent incidental mutations versus those responsible for phenotype differences by introducing the mutations, separately and in various combinations, into the genome or antigenome of infectious RSV clones. This process coupled with evaluation of phenotype characteristics of parental and derivative virus identifies mutations responsible for such desired characteristics as attenuation, temperature sensitivity, cold-adaptation, small plaque size, host range restriction, etc.
  • chimeric RSV of the invention are attenuated by incorporation of at least one, and more preferably two or more, attenuating point mutations identified from such a menu, which may be defined as a group of known mutations within a panel of biologically derived mutant RSV strains .
  • Preferred panels of mutant RSV strains described herein are cold passaged ( cp) and/or temperature sensitive ( s) mutants, for example a panel comprised of RSV mutants designated " cpts RSV 248 (ATCC VR 2450), cpts RSV 248/404 (ATCC VR 2454), cpts RSV 248/955 (ATCC VR 2453), cpts RSV 530 (ATCC VR 2452), cpts RSV 530/1009 (ATCC VR 2451), cpts RSV 530/1030 (ATCC VR 2455), RSV B-l cp52/2B5 (ATCC VR 2542), and RSV B-l cp-23 (ATCC VR 2579)" (each deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC) of 10801
  • Attenuating mutations are provided which can each be combined with any other mutation (s) within the panel for calibrating the level of attenuation in a recombinant, chimeric RSV for vaccine use. Additional mutations may be derived from RSV having non-ts and non- cp attenuating mutations as identified, e . g . , in small plaque ( sp) , cold-adapted ( ca) or host-range restricted (hr) mutant strains. Attenuating mutations may be selected in coding portions of a donor or recipient RSV gene or in non-coding regions such as a cis-regulatory sequence. For example, attenuating mutations may include single or multiple base changes in a gene start sequence, as exemplified by a single or multiple base substitution in the M2 gene start sequence at nucleotide 7605.
  • Chimeric RSV designed and selected for vaccine use often have at least two and sometimes three or more attenuating mutations to achieve a satisfactory level of attenuation for broad clinical use.
  • at least one attenuating mutation occurs in the RSV polymerase gene (either in the donor or recipient gene) and involves a nucleotide substitution specifying an amino acid change in the polymerase protein specifying a temperature-sensitive (ts) phenotype.
  • Exemplary chimeric RSV in this context incorporate one or more nucleotide substitutions in the large polymerase gene L resulting in an amino acid change at amino acid Phe 521 ,
  • chimeric RSV of the invention may incorporate a ts mutation in a different RSV gene, e.g., in the M2 gene.
  • two or more nucleotide changes are incorporated in a codon specifying an attenuating mutation, e.g., in a codon specifying a ts mutation, thereby decreasing the likelihood of reversion from an attenuated phenotype.
  • chimeric RSV can be readily constructed and characterized that incorporate at least one and up to a full complement of attenuating point mutations present within a panel of biologically derived mutant RSV strains.
  • mutations can be assembled in any combination from a selected panel of mutants, for example, cpts RSV 248 (ATCC VR 2450) , cpts RSV
  • chimeric RSV for vaccine use incorporate at least one and up to a full complement of attenuating mutations specifying a temperature-sensitive amino acid substitution at Phe 521 , Gln 831 , Met 1169 or Tyr 1321 in the RSV polymerase gene L, or a temperature- sensitive nucleotide substitution in the gene- start sequence of gene M2.
  • chimeric RSV of claim may incorporate at least one and up to a full complement of mutations from cold-passaged attenuated RSV, for example one or more mutations specifying an amino acid substitution at Val 267 in the RSV N gene, Glu 218 or Thr 523 in the RSV F gene, Cys 319 or His 1690 in the RSV polymerase gene L.
  • the chimeric RSV of the invention features human RSV B subgroup glycoprotein genes F and G that are added or substituted within a human RSV A genome or antigenome to form a chimeric clone which is further modified to incorporate one or more attenuating point mutations adopted from biologically derived mutant RSV.
  • the chimeric RSV has both human RSV B subgroup glycoprotein genes F and G are substituted to replace counterpart F and G glycoprotein genes within an RSV A genome, which is further modified to incorporate attenuating point mutations selected from (i) a panel of mutations specifying temperature-sensitive amino acid substitutions Gln 831 to Leu, and Tyr 1321 to Asn in the RSV polymerase gene L; (ii) a temperature-sensitive nucleotide substitution in the gene- start sequence of gene M2; (iii) an attenuating panel of mutations adopted from cold-passaged RSV specifying amino acid substitutions Val 267 lie in the RSV N gene, and Cys 319 to Tyr and His 1690 Tyr in the RSV polymerase gene L; or (iv) a deletion of the SH gene.
  • attenuating point mutations selected from (i) a panel of mutations specifying temperature-sensitive amino acid substitutions Gln 831 to Leu, and Tyr 1321 to Asn in the RSV polymerase gene L; (
  • these and other examples of chimeric RSV incorporate at least two attenuating point mutations adopted from biologically derived mutant RSV, which may be derived from the same or different biologically derived mutant RSV strains. Also preferably, these exemplary mutants have one or more of their attenuating mutations stabilized by multiple nucleotide changes in a codon specifying the mutation.
  • infectious RSV permits introduction of specific engineered changes within chimeric RSV.
  • infectious, recombinant RSV are employed for identification of specific mutation (s) in biologically derived, attenuated RSV strains, for example mutations which specify ts, ca, att and other phenotypes. Desired mutations are thus identified and introduced into recombinant, chimeric RSV vaccine strains.
  • the capability of producing virus from cDNA allows for routine incorporation of these mutations, individually or in various selected combinations, into a full- length cDNA clone, whereafter the phenotypes of rescued recombinant viruses containing the introduced mutations to be readily determined.
  • the invention provides for other, site- specific modifications at, or within close proximity to, the identified mutation. Whereas most attenuating mutations produced in biologically derived RSV are single nucleotide changes, other "site specific" mutations can also be incorporated by recombinant techniques into biologically derived or recombinant RSV.
  • site-specific mutations include insertions, substitutions, deletions or rearrangements of from 1 to 3, up to about 5-15 or more altered nucleotides (e.g., altered from a wild-type RSV sequence, from a sequence of a selected mutant RSV strain, or from a parent recombinant RSV clone subjected to mutagenesis) .
  • Such site-specific mutations may be incorporated at, or within the region of, a selected, biologically derived point mutation.
  • the mutations can be introduced in various other contexts within an RSV clone, for example at or near a cis-acting regulatory sequence or nucleotide sequence encoding a protein active site, binding site, immunogenic epitope, etc.
  • Site-specific RSV mutants typically retain a desired attenuating phenotype, but may exhibit substantially altered phenotypic characteristics unrelated to attenuation, e.g., enhanced or broadened immunogenicity, or improved growth.
  • site-specific mutants include recombinant RSV designed to incorporate additional, stabilizing nucleotide mutations in a codon specifying an attenuating point mutation.
  • two or more nucleotide substitutions are introduced at codons that specify attenuating amino acid changes in a parent mutant or recombinant RSV clone, yielding a biologically derived or recombinant RSV having genetic resistance to reversion from an attenuated phenotype.
  • site-specific nucleotide substitutions, additions, deletions or rearrangements are introduced upstream (N-terminal direction) or downstream (C-terminal direction), e.g, from 1 to 3, 5-10 and up to 15 nucleotides or more 5' or 3 ' , relative to a targeted nucleotide position, e.g., to construct or ablate an existing cis-acting regulatory element.
  • changes to chimeric RSV disclosed herein include deletions, insertions, substitutions or rearrangements of whole genes or gene segments. These mutations may alter small numbers of bases (e.g., from 15-30 bases, up to 35-50 bases or more) , or large blocks of nucleotides (e.g., 50-100, 100-300, 300-500, 500-1,000 bases) in the donor or recipient genome or antigenome, depending upon the nature of the change (i.e., a small number of bases may be changed to insert or ablate an immunogenic epitope or change a small gene segment, whereas large block (s) of bases are involved when genes or large gene segments are added, substituted, deleted or rearranged.
  • bases e.g., from 15-30 bases, up to 35-50 bases or more
  • large blocks of nucleotides e.g., 50-100, 100-300, 300-500, 500-1,000 bases
  • the invention provides for supplementation of mutations adopted into a chimeric RSV clone from biologically derived RSV, e.g., cp and ts mutations, with additional types of mutations involving the same or different genes in a further modified chimeric RSV clone.
  • RSV encodes ten mRNAs and ten or eleven proteins. Three of these are transmembrane surface proteins, namely the attachment G protein, fusion F protein involved in penetration, and small hydrophobic SH protein. G and F are the major viral neutralization and protective antigens.
  • RNA binding protein N the RNA binding protein
  • phosphoprotein P the large polymerase protein L
  • transcription elongation factor M2 ORF1 The matrix M protein is part of the inner virion and probably mediates association between the nucleocapsid and the envelope.
  • NSl nonstructural proteins
  • the present invention also provides a range of additional methods for attenuating chimeric RSV based on recombinant engineering of infectious RSV clones.
  • a variety of alterations can be produced in an isolated polynucleotide sequence encoding the chimeric RSV genome or antigenome for incorporation into infectious clones.
  • the invention allows for introduction of modifications which delete, substitute, introduce, or rearrange a selected nucleotide or plurality of nucleotides from a parent chimeric genome or antigenome, as well as mutations which delete, substitute, introduce or rearrange whole gene(s) or gene segment (s), within a chimeric RSV clone.
  • Desired modifications of infectious chimeric RSV are typically selected to specify a desired phenotypic change, e.g., a change in viral growth, temperature sensitivity, ability to elicit a host immune response, attenuation, etc. These changes can be brought about either in a donor or recipient genome or antigenome by, e.g., mutagenesis of a parent RSV clone to ablate, introduce or rearrange a specific gene(s) or gene region(s) (e.g., a gene segment that encodes a protein structural domain, such as a cytoplasmic, transmembrane or extracellular domain, an immunogenic epitope, binding region, active site, etc.).
  • Genes of interest in this regard include all of the genes of the RSV genome:
  • modifications in a chimeric RSV which simply alter or ablate expression of a selected gene, e.g., by introducing a termination codon within a selected RSV coding sequence, changing the position of an RSV gene relative to an operably linked promoter, introducing an upstream start codon to alter rates of expression, modifying (e.g., by changing position, altering an existing sequence, or substituting an existing sequence with a heterologous sequence) GS and/or GE transcription signals to alter phenotype (e.g., growth, temperature restrictions on transcription, etc.), and various other deletions, substitutions, additions and rearrangements that specify quantitative or qualitative changes in viral replication, transcription of selected gene(s), or translation of selected protein (s) .
  • modifications in a chimeric RSV which simply alter or ablate expression of a selected gene, e.g., by introducing a termination codon within a selected RSV coding sequence, changing the position of an RSV gene relative to an operably linked promoter, introducing an upstream start cod
  • the ability to analyze and incorporate other types of attenuating mutations into chimeric RSV for vaccine development extends to a broad assemblage of targeted changes in RSV clones.
  • deletion of the SH gene yields a recombinant RSV having novel phenotypic characteristics, including enhanced growth.
  • an SH gene deletion (or any other selected, non-essential gene or gene segment deletion) , is combined in a chimeric RSV with one or more additional mutations specifying an attenuated phenotype, e.g., one or more point mutation(s) adopted from a biologically derived attenuated RSV mutant.
  • the SH gene or NS2 gene is deleted in combination with one or more cp and/or ts mutations adopted from cpts248/404, cpts530/l009, cpts530/1030 , or another selected mutant RSV strain, to yield a recombinant RSV having increased yield of virus, enhanced attenuation, and resistance to phenotypic reversion, due to the combined effects of the different mutations.
  • any RSV gene which is not essential for growth for example the SH, N, P, NSl and NS2 genes, can be ablated or otherwise modified in a chimeric RSV to yield desired effects on virulence, pathogenesis, immunogenicity and other phenotypic characters.
  • ablation by deletion of a non-essential gene such as SH results in enhanced viral growth in culture.
  • this effect is likely due in part to a reduced nucleotide length of the viral genome.
  • the modified viral genome is 14,825 nt long, 398 nucleotides less than wild-type.
  • the invention provides several readily obtainable methods and materials for improving chimeric RSV growth.
  • a variety of other genetic alterations can be produced in a RSV genome or antigenome for incorporation into infectious chimeric RSV, alone or together with one or more attenuating point mutations adopted from a biologically derived mutant RSV.
  • Additional heterologous genes and gene segments e.g. from different RSV genes, different RSV strains or types, or non-RSV sources
  • Different or additional modifications in the sequence can be made to facilitate manipulations, such as the insertion of unique restriction sites in various intergenic regions or elsewhere.
  • Nontranslated gene sequences can be removed to increase capacity for inserting foreign sequences.
  • a chimeric RSV which alter or ablate the expression of a selected gene or gene segment without removing the gene or gene segment from the chimeric RSV clone.
  • this can be achieved by introducing a termination codon within a selected coding sequence, changing the position of a gene or introducing an upstream start codon to alter its rate of expression, or changing GS and/or GE transcription signals to alter phenotype (e.g., growth, temperature restrictions on transcription, etc.).
  • Preferred mutations in this context include mutations directed toward cis-acting signals, which can be identified, e.g., by mutational analysis of RSV minigenomes .
  • insertional and deletional analysis of the leader and trailer and flanking sequences identified viral promoters and transcription signals and provided a series of mutations associated with varying degrees of reduction of RNA replication or transcription.
  • Saturation mutagenesis (whereby each position in turn is modified to each of the nucleotide alternatives) of these cis-acting signals also has identified many mutations which reduced (or in one case increased) RNA replication or transcription. Any of these mutations can be inserted into a chimeric antigenome or genome as described herein.
  • chimeric RSV of the present invention involve replacement of the 3 ' end of genome with its counterpart from antigenome, which is associated with changes in RNA replication and transcription.
  • the intergenic regions (Collins et al., Proc. Natl. Acad. Sci. USA 83:4594-4598 (1986), incorporated herein by reference) can be shortened or lengthened or changed in sequence content, and the naturally-occurring gene overlap (Collins et al . , Proc. Natl. Acad. Sci. USA 84:5134-5138 (1987), incorporated herein by reference) can be removed or changed to a different intergenic region by the methods described herein.
  • the level of expression of specific RSV proteins can be increased by substituting the natural sequences with ones which have been made synthetically and designed to be consistent with efficient translation.
  • codon usage can be a major factor in the level of translation of mammalian viral proteins (Haas et al . , Current Biol . 6:315-324 (1996)).
  • Examination of the codon usage of the mRNAs encoding the F and G proteins of RSV, which are the major protective antigens shows that the usage is consistent with poor expression.
  • codon usage can be improved by the recombinant methods of the invention to achieve improved expression for selected genes .
  • a sequence surrounding a translational start site (preferably including a nucleotide in the -3 position) of a selected RSV gene is modified, alone or in combination with introduction of an upstream start codon, to modulate chimeric RSV gene expression by specifying up- or down-regulation of translation.
  • chimeric RSV gene expression can be modulated by altering a transcriptional GS signal of a selected gene(s) of the virus.
  • the GS signal of NS2 is modified to include a defined mutation (e.g., the 404 (M2) mutation described hereinbelow) to superimpose a ts restriction on viral replication.
  • RSV clones within the invention can incorporate modifications to a transcriptional GE signal.
  • RSV clones may be generated which have a substituted or mutated GE signal of the NSl and NS2 genes for that of the N gene, resulting in decreased levels of readthrough mRNAs and increased expression of proteins from downstream genes.
  • the resulting chimeric virus will exhibit increased growth kinetics and increased plaque size, providing but one example of alteration of RSV growth properties by modification of a cis-acting regulatory element in the RSV— genome .
  • expression of the G protein is increased by modification of the G mRNA.
  • the G protein is expressed as both a membrane bound and a secreted form, the latter form being expressed by translational initiation at a start site within the G translational open reading frame .
  • the secreted form can account for as much as one-half of the expressed G protein.
  • Ablation of the internal start site e.g., by sequence alteration, deletion, etc.
  • Ablation of the secreted form of G also will improve the quality of the host immune response to exemplary, chimeric RSV, because the soluble form of G is thought to act as a "decoy" to trap neutralizing antibodies.
  • soluble G protein has been implicated in enhanced immunopathology due to its preferential stimulation of a Th2-biased response.
  • levels of chimeric RSV gene expression are modified at the level of transcription.
  • the position of a selected gene in the RSV gene map can be changed to a more promoter-proximal or promotor- distal position, whereby the gene will be expressed more or less efficiently, respectively.
  • modulation of expression for specific genes can be achieved yielding reductions or increases of gene expression from twofold, more typically four-fold, up to ten-fold or more compared to wild-type levels.
  • the NS2 gene (second in order in the RSV gene map) is substituted in position for the SH gene (sixth in order) , yielding a predicted decrease in expression of NS2.
  • Increased expression of selected RSV genes due to positional changes can be achieved up to 10-fold, 30-fold, 50-fold, 100-fold or more, often attended by a commensurate decrease in expression levels for reciprocally, positionally substituted genes.
  • the F and G genes are transpositioned singly or together to a more promoter- proximal or promoter-distal site within the chimeric RSV gene map to achieve higher or lower levels of gene expression, respectively.
  • These and other transpositioning changes yield novel chimeric RSV clones having attenuated phenotypes, for example due to decreased expression of selected viral proteins involved in RNA replication.
  • RSV is provided in which expression of a viral gene, for example the NS2 gene, is ablated at the translational level without deletion of the gene or of a segment thereof, by, e.g., introducing two tandem translational termination codons into a translational open reading frame (ORF) .
  • ORF translational open reading frame
  • These forms of "knock-out" virus will exhibit reduced growth rates and small plaque sizes in tissue culture.
  • the methods and compositions of the invention provide yet additional, novel types of attenuating mutations which ablate expression of a viral gene that is not one of the major viral protective antigens.
  • knockout virus phenotypes produced without deletion of a gene or gene segment can be alternatively produced by deletion mutagenesis, as described herein, to effectively preclude correcting mutations that may restore synthesis of a target protein.
  • gene “knock-outs” for chimeric RSV can be made using alternate designs. For example, insertion of translation termination codons into ORFs, or disruption of the RNA editing sites, offer alternatives to silencing or attenuating the expression of selected genes. Methods for producing these and other knock-outs are well known in the art (as described, for example, in Kretzschmar et al . , Virology 216:309-316 (1996); Radecke et al .
  • Infectious chimeric RSV clones of the invention can also be engineered according to the methods and compositions disclosed herein to enhance immunogenicity and induce a level of protection greater than that provided by infection with a wild-type RSV or a parent chimeric RSV.
  • an immunogenic epitope from a heterologous RSV strain or type, or from a non-RSV source such as PIV can be added to a chimeric clone by appropriate nucleotide changes in the polynucleotide sequence encoding the chimeric genome or antigenome.
  • chimeric RSV can be engineered to add or ablate (e.g., by amino acid insertion, substitution or deletion) immunogenic epitopes associated with desirable or undesirable immunological reactions.
  • additional genes or gene segments may be inserted into or proximate to the recipient RSV genome or antigenome. These genes may be under common control with recipient genes, or may be under the control of an independent set of transcription signals.
  • Genes of interest include the RSV genes identified above, as well as non-RSV genes.
  • Non-RSV genes of interest include those encoding cytokines (e.g., IL-2 through IL-15, especially IL-2, IL-6 and IL-12, etc.), gamma-interferon, and proteins rich in T helper cell epitopes. These additional proteins can be expressed either as a separate protein, or as a chimera engineered from a second copy of one of the RSV proteins, such as SH. This provides the ability to modify and improve the immune responses against RSV both quantitatively and qualitatively.
  • cytokines e.g., IL-2 through IL-15, especially IL-2, IL-6 and IL-12, etc.
  • gamma-interferon e.g.
  • insertion of foreign genes or gene segments, and in some cases of noncoding nucleotide sequences, within a chimeric RSV genome results in a desired increase in genome length causing yet additional, desired phenotypic effects. Increased genome length results in attenuation of the resultant RSV, dependent in part upon the length of the insert.
  • certain proteins e.g. a cytokine
  • IL-2 expressed in vaccinia virus e.g. Flexner et al . , Nature 33: -259-62 (1987)
  • gamma interferon e.g. Flexner et al . , Nature 33: -259-62 (1987)
  • Deletions, insertions, substitutions and other mutations involving changes of whole viral genes or gene segments within chimeric RSV of the invention yield highly stable vaccine candidates, which are particularly important in the case of immunosuppressed individuals. Many of these changes will result in attenuation of resultant vaccine strains, whereas others will specify different types of desired phenotypic changes.
  • certain viral genes are known which encode proteins that specifically interfere with host immunity (see, e.g., Kato et al . , EMBO . J. 16:578-87 (1997) , incorporated herein by reference) . Ablation of such genes in chimeric vaccine viruses is expected to reduce virulence and pathogenesis and/or improve immunogenicity.
  • the infectious chimeric RSV produced from a cDNA-expressed genome or antigenome can be any of the RSV or RSV-like strains, e.g., human, bovine, murine, etc., or of any pneumovirus, e.g., pneumonia virus of mice or turkey rhinotracheitis virus.
  • the RSV strain may be one which is endogenous to the subject being immunized, such as human RSV being used to immunize humans.
  • the genome or antigenome of endogenous RSV can be modified, however, to express RSV genes or gene segments from a combination of different sources, e.g., a combination of genes or gene segments from different RSV species, subgroups, or strains, or from an RSV and another respiratory pathogen such as PIV.
  • chimeric RSV are provided wherein genes or gene segments within a human RSV are replaced with counterpart heterologous genes or gene segments from a non-human RSV, e.g., a bovine or murine RSV.
  • chimeric RSV may incorporate genes or gene segments from a human RSV in a non-human RSV recipient or background clone, e.g., a bovine or murine RSV clone.
  • RSV genes or gene segments in this context can include part or all of one or more of the NSl, NS2 , N, P, M, SH, M2 (ORF1) , M2 (ORF2) and L genes, or non-immunogenic parts of the G and F genes.
  • human and non-human RSV cis-acting sequences such as promoter or transcription signals, can be replaced with, respectively, non-human or human counterpart sequences.
  • Chimeric human/non-human RSV bearing heterologous genes or cis-acting elements are selected for host range restriction and other desired phenotypes favorable for vaccine use.
  • bovine RSV sequences are selected for introduction into human RSV based on known aspects of bovine RSV structure and function, as provided in, e.g., Pastey et al . , J. Gen. Viol. 76:193-197 (1993); Pastey et al., Virus Res . 29:195-202 (1993); Zamora et al . , J. Gen . Virol . 73:737-741 (1992); Mallipeddi et al . , J. Gen. Virol.
  • mutations of interest for introduction within chimeric RSV are modeled after a tissue culture-adapted nonpathogenic strain of pneumonia virus of mice (the murine counterpart of human RSV) which lacks a cytoplasmic tail of the G protein (Randhawa et al., Virology 207:240-245 (1995)).
  • the cytoplasmic and/or transmembrane domains of one or more of the human RSV glycoproteins, F, G and SH are added, deleted, modified, or substituted within a chimeric RSV using a heterologous counterpart sequence (e.g., a sequence from a cytoplasmic, or transmembrane domain of a F, G, or SH protein of murine pneumonia virus) to achieve a desired attenuation.
  • a heterologous counterpart sequence e.g., a sequence from a cytoplasmic, or transmembrane domain of a F, G, or SH protein of murine pneumonia virus
  • a nucleotide sequence at or near the cleavage site of the F protein, or the putative attachment domain of the G protein can be modified by point mutations, site-specific changes, or by alterations involving entire genes or gene segments to achieve novel effects on viral growth in tissue culture and/or infection and pathogenesis.
  • infectious chimeric RSV intended for administration to humans can be a human RSV that has been modified to contain genes from, e.g., a bovine or murine RSV or a PIV, such as for the purpose of attenuation.
  • a bivalent vaccine to both PIV and RSV can be provided.
  • a heterologous RSV species, subgroup or strain, or a distinct respiratory pathogen such as PIV may be modified, e.g., to contain genes that encode epitopes or proteins which elicit protection against human RSV infection.
  • the human RSV glycoprotein genes can be substituted for the bovine glycoprotein genes such that the resulting chimeric RSV, which now bears the human RSV surface glycoproteins in a bovine background, would retain a restricted ability to replicate in a human host due to the remaining bovine genetic background, while eliciting a protective immune response in humans against human RSV strains .
  • a chimeric bovine-human RSV incorporates a substitution of the human RSV NP gene or gene segment with a counterpart bovine NP gene or gene segment, which chimera can optionally be constructed to incorporate additional genetic changes, e.g., point mutations or gene deletions.
  • additional genetic changes e.g., point mutations or gene deletions.
  • replacement of a human RSV coding sequence e.g., of NSl, NS2, NP, etc.
  • non-coding sequence e.g., a promoter, gene-end, gene-start, intergenic or other cis-acting element
  • a counterpart bovine or murine RSV sequence is expected to yield chimeric RSV having a variety of possible attenuating and other phenotypic effects.
  • host range and other desired effects are expected to arise from a non-human RSV gene imported within a human RSV background, wherein the non-human gene does not function efficiently in a human cell, e.g., from incompatibility of the heterologous sequence or protein with a biologically interactive human RSV sequence or protein (i.e., a sequence or protein that ordinarily cooperates with the substituted sequence or protein for viral transcription, translation, assembly, etc.)
  • a biologically interactive human RSV sequence or protein i.e., a sequence or protein that ordinarily cooperates with the substituted sequence or protein for viral transcription, translation, assembly, etc.
  • chimeric RSV are employed as vectors for protective antigens of other pathogens, particularly respiratory tract pathogens such as parainfluenza virus (PIV) .
  • chimeric RSV may be engineered which incorporate sequences that encode protective antigens from PIV to produce infectious, attenuated vaccine virus.
  • PIV parainfluenza virus
  • the cloning of PIV cDNA and other disclosure is provided in United States Patent Application entitled PRODUCTION OF PARAINFLUENZA VIRUS VACCINES FROM CLONED NUCLEOTIDE SEQUENCES, filed May 22, 1998, Serial No. 09/083,793 (corresponding to International Publication No. WO 98/53078) and its priority, provisional application filed May 23, 1997, Serial No.
  • a chimeric RSV which comprises a chimera of a RSV genomic or antigenomic sequence and at least one PIV sequence, for example a polynucleotide containing sequences from both RSV and PIV1, PIV2, PIV3 or bovine PIV.
  • individual genes of RSV may be replaced with counterpart genes from human PIV, such as the HN and/or F glycoprotein genes of PIV1, PIV2, or PIV3.
  • a selected, heterologous gene segment such as a cytoplasmic tail, transmembrane domain or ectodomain of HN or F of HPIVl, HPIV2, or HPIV3 can be substituted for a counterpart gene segment in, e.g., the same gene in an RSV clone, within a different gene in the RSV clone, or into a non-coding sequence of the RSV genome or antigenome.
  • a gene segment from HN or F of HPIV3 is substituted for a counterpart gene segment in RSV type A, to yield constructs encoding chimeric proteins, e.g.
  • fusion proteins having a cytoplasmic tail and/or transmembrane domain of RSV fused to an ectodomain of RSV to yield a novel attenuated virus, and/or a multivalent vaccine immunogenic against both PIV and RSV.
  • RSV clones can be made to facilitate manipulations, such as the insertion of unique restriction sites in various intergenic regions (e.g., a unique Stul site between the G and F genes) or elsewhere.
  • Nontranslated gene sequences can be removed to increase capacity for inserting foreign sequences.
  • compositions e.g., isolated polynucleotides and vectors incorporating a chimeric RSV-encoding cDNA
  • infectious chimeric RSV are generated from a chimeric RSV genome or antigenome, a nucleocapsid (N) protein, a nucleocapsid phosphoprotein (P) , a large (L) polymerase protein, and an RNA polymerase elongation factor.
  • compositions and methods are provided for introducing the aforementioned structural and phenotypic changes into a recombinant chimeric RSV to yield infectious, attenuated vaccine viruses.
  • infectious, chimeric RSV clone introduction of the foregoing defined mutations into an infectious, chimeric RSV clone can be achieved by a variety of well known methods.
  • infectious clone is meant cDNA or its product, synthetic or otherwise, which can be transcribed into genomic or antigenomic RNA capable of serving as template to produce the genome of an infectious virus or subviral particle.
  • defined mutations can be introduced by conventional techniques (e.g., site-directed mutagenesis) into a cDNA copy of the genome or antigenome.
  • antigenome or genome cDNA subfragments to assemble a complete antigenome or genome cDNA as described herein has the advantage that each region can be manipulated separately (smaller cDNAs are easier to manipulate than large ones) and then readily assembled into a complete cDNA.
  • the complete antigenome or genome cDNA, or any subfragment thereof can be used as template for oligonucleotide-directed mutagenesis.
  • mutations are introduced by using the Muta-gene phagemid in vi tro mutagenesis kit available from Bio-Rad.
  • cDNA encoding a portion of an RSV genome or antigenome is cloned into the plasmid pTZ18U, and used to transform CJ236 cells (Life Technologies) .
  • Phagemid preparations are prepared as recommended by the manufacturer.
  • Oligonucleotides are designed for mutagenesis by introduction of an altered nucleotide at the desired position of the genome or antigenome.
  • the plasmid containing the genetically altered genome or antigenome fragment is then amplified and the mutated piece is then reintroduced into the full-length genome or antigenome clone.
  • RSV infectious RSV
  • the functions of the RSV proteins including the NSl, NS2, SH, M2 (ORF1) and M2(ORF2) proteins, can be investigated and manipulated by introducing mutations which ablate or reduce their level of expression, or which yield mutant protein.
  • recombinant RSV is constructed in which expression of a viral gene, namely the SH gene, is ablated by deletion of the mRNA coding sequence and flanking transcription signals. Surprisingly, not only could this virus be recovered, but it grew efficiently in tissue culture.
  • the invention also provides methods for producing an infectious chimeric RSV from one or more isolated polynucleotides, e.g., one or more cDNAs .
  • cDNA encoding a RSV genome or antigenome is constructed for intraceUular or in vi tro coexpression with the necessary viral proteins to form infectious RSV.
  • RSV antigenome is meant an isolated positive-sense polynucleotide molecule which serves as the template for the synthesis of progeny RSV genome.
  • a cDNA is constructed which is a positive-sense version of the RSV genome, corresponding to the replicative intermediate RNA, or antigenome, so as to minimize the possibility of hybridizing with positive-sense transcripts of the complementing sequences that encode proteins necessary to generate a transcribing, replicating nucleocapsid, i.e., sequences that encode N, P, L and M2 (0RF1) protein.
  • nucleocapsid i.e., sequences that encode N, P, L and M2 (0RF1) protein.
  • genome and antigenome were equally active in rescue, whether complemented by RSV or by plasmids, indicating that either genome or antigenome can be used and thus the choice can be made on methodologic or other grounds.
  • a native RSV genome typically comprises a negative- sense polynucleotide molecule which, through complementary viral mRNAs, encodes eleven species of viral proteins, i.e., the nonstructural species NSl and NS2, N, P, matrix (M) , small hydrophobic (SH) , glycoprotein (G) , fusion (F) , M2(0RF1),
  • the genome or antigenome of the recombinant RSV of the invention need only contain those genes or portions thereof necessary to render the viral or subviral particles encoded thereby infectious. Further, the genes or portions thereof may be provided by more than one polynucleotide molecule, i.e., a gene may be provided by complementation or the like from a separate nucleotide molecule.
  • recombinant RSV is meant a RSV or RSV-like viral or subviral particle derived directly or indirectly from a recombinant expression system or propagated from virus or subviral particles produced therefrom.
  • the recombinant expression system will employ a recombinant expression vector which comprises an operably linked transcriptional unit comprising an assembly of at least a genetic element or elements having a regulatory role in RSV gene expression, for example, a promoter, a structural or coding sequence which is transcribed into RSV RNA, and appropriate transcription initiation and termination sequences.
  • the genome or antigenome is coexpressed with those RSV proteins necessary to (i) produce a nucleocapsid capable of RNA replication, and (ii) render progeny nucleocapsids competent for both RNA replication and transcription. Transcription by the genome nucleocapsid provides the other RSV proteins and initiates a productive infection. Alternatively, additional RSV proteins needed for a productive infection can be supplied by coexpression.
  • RSV antigenome may be constructed for use in the present invention by, e.g., assembling cloned cDNA segments, representing in aggregate the complete antigenome, by polymerase chain reaction (PCR; described in, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202, and PCR Protocols : A Guide to Methods and Applications, Innis et al . , eds., Academic Press, San Diego (1990) , incorporated herein by reference) of reverse-transcribed copies of RSV mRNA or genome RNA.
  • PCR polymerase chain reaction
  • cDNAs containing the lefthand end of the antigenome, spanning from an appropriate promoter (e.g., T7 RNA polymerase promoter) and the leader region complement to the SH gene are assembled in an appropriate expression vector, such as a plasmid (e.g., pBR322) or various available cosmid, phage, or DNA virus vectors.
  • a plasmid e.g., pBR322
  • the vector may be modified by mutagenesis and/or insertion of synthetic polylinker containing unique restriction sites designed to facilitate assembly.
  • a plasmid vector described herein was derived from pBR322 by replacement of the Pstl- Eco-Rl fragment with a synthetic DNA containing convenient restriction enzyme sites.
  • pBR322 as a vector stabilized nucleotides 3716-3732 of the RSV sequence, which otherwise sustained nucleotide deletions or insertions, and propagation of the plasmid was in bacterial strain DH10B to avoid an artifactual duplication and insertion which otherwise occurred in the vicinity of nt 4499.
  • the G, F and M2 genes can be assembled in a separate vector, as can be the L and trailer sequences.
  • the righthand end (e.g., L and trailer sequences) of the antigenome plasmid may contain additional sequences as desired, such as a flanking ribozyme and tandem T7 transcriptional terminators .
  • the ribozyme can be hammerhead type (e.g., Grosfeld et al . , J. Virol . 69:5677-5686 (1995)), which would yield a 3' end containing a single nonviral nucleotide, or can any of the other suitable ribozymes such as that of hepatitis delta virus (Perrotta et al . , Nature 350:434-436 (1991)) which would yield a 3' end free of non-RSV nucleotides.
  • a middle segment (e.g., G-to-M2 piece) is inserted into an appropriate restriction site of the leader-to-SH plasmid, which in turn is the recipient for the L-trailer-ribozyme-terminator piece, yielding a complete antigenome.
  • the leader end was constructed to abut the promoter for T7 RNA polymerase which included three transcribed G residues for optimal activity; transcription donates these three nonviral G's to the 5' end of the antigenome. These three nonviral G residues can be omitted to yield a 5' end free of nonviral nucleotides.
  • the trailer end was constructed to be adjacent to a hammerhead ribozyme, which upon cleavage would donate a single 3 ' -phosphorylated U residue to the 3' end of the encoded RNA.
  • complementing sequences encoding proteins necessary to generate a transcribing, replicating RSV nucleocapsid are provided by one or more helper viruses .
  • helper viruses can be wild-type or mutant.
  • the helper virus can be distinguished phenotypically from the virus encoded by the RSV cDNA.
  • monoclonal antibodies which react immunologically with the helper virus but not the virus encoded by the RSV cDNA.
  • Such antibodies can be neutralizing antibodies.
  • the antibodies can be used in affinity chromatography to separate the helper virus from the recombinant virus .
  • mutations can be introduced into the RSV cDNA to provide antigenic diversity from the helper virus, such as in the HN or F glycoprotein genes.
  • nucleotide insertions and deletions can be made in the RSV genome or antigenome to generate an attenuated, chimeric clone.
  • the nucleotide length of the genome of wild-type human RSV (15,222 nucleotides) is a multiple of six, and members of the Paramyxovirus and Morbillivirus genera typically abide by a "rule of six," i.e., genomes (or minigenomes) replicate efficiently only when their nucleotide length is a multiple of six (thought to be a requirement for precise spacing of nucleotide residues relative to encapsidating NP protein) .
  • RSV lacks the strict requirement of genome length being a multiple of six, and nucleotide insertions and deletions can be made in the RSV genome or antigenome without defeating replication of the recombinant RSV of the present invention.
  • cDNA encoding an RSV genome or antigenome include by reverse transcription-PCR using improved PCR conditions (e.g., as described in Cheng et al., Proc. Natl. Acad. Sci. USA 91:5695-5699 (1994); Samal__et al., J. Virol 70:5075-5082 (1996), each incorporated herein by reference) to reduce the number of subunit cDNA components to as few as one or two pieces .
  • different promoters can be used (e.g., T3 , SP6) or different ribozymes (e.g., that of hepatitis delta virus.
  • Different DNA vectors e.g., cosmids
  • M2(0RF1) RNA polymerase elongation factor
  • M2(0RF1) or a substantially equivalent transcription elongation factor for negative strand RNA viruses is required for the production of infectious RSV and is a necessary component of functional nucleocapsids during productive infection.
  • M2(0RF1) The need for the M2(0RF1) protein is consistent with its role as a transcription elongation factor.
  • the need for expression of the RNA polymerase elongation factor protein for negative strand RNA viruses is a feature of the present invention.
  • M2(0RF1) can be supplied by expression of the complete M2-gene, either by the chimeric genome or antigenome or by coexpression therewith, although in this form the second ORF2 may also be expressed and have an inhibitory effect on RNA replication. Therefore, for production of infectious virus using the complete M2 gene the activities of the two ORFs should be balanced to permit sufficient expression of M(ORFl) to provide transcription elongation activity yet not so much of M(ORF2) to inhibit RNA replication.
  • the ORF1 protein is provided from a cDNA engineered to lack ORF2 or which encodes a defective ORF2.
  • Efficiency of virus production may also be improved by co-expression of additional viral protein genes, such as those encoding envelope constituents (i.e., SH, M, G, F proteins).
  • Isolated polynucleotides e.g., cDNA
  • encoding the RSV genome or antigenome and, separately or in cis, the N, P, L and M2(0RF1) proteins are inserted by transfection, electroporation, mechanical insertion, transduction or the like, into cells which are capable of supporting a productive RSV infection, e.g., HEp-2, FRhL-DBS2, MRC, and Vero cells.
  • Transfection of isolated polynucleotide sequences may be introduced into cultured cells by, for example, calcium phosphate-mediated transfection (Wigler et al .
  • the N, P, L and M2(0RF1) proteins are encoded by one or more expression vectors which can be the same or separate from that which encodes the genome or antigenome, and various combinations thereof. Additional proteins may be included as desired, encoded by its own vector or by a vector encoding a N, P, L, or M2 (ORF1) protein and/or the complete genome or antigenome.
  • each cDNA being under the control of a promoter for T7 RNA polymerase, which in turn is supplied by infection, transfection or transduction with an expression system for the T7 RNA polymerase, e.g., a vaccinia virus MVA strain recombinant which expresses the T7 RNA polymerase (Wyatt et al., Virology, 210:202-205 (1995), incorporated herein by reference) .
  • the viral proteins, and/or T7 RNA polymerase can also be provided from transformed mammalian cells, or by transfection of preformed mRNA or protein.
  • antigenome or genome can be conducted in vi tro (cell-free) in a combined transcription- translation reaction, followed by transfection into cells.
  • antigenome or genome RNA can be synthesized in vi tro and transfected into cells expressing RSV proteins.
  • vi tro cell-free
  • antigenome or genome RNA can be synthesized in vi tro and transfected into cells expressing RSV proteins.
  • the criteria of viability, attenuation and immunogenicity are determined according to well known methods. Viruses which will be most desired in vaccines of the invention must maintain viability, have a stable attenuation phenotype, exhibit replication in an immunized host (albeit at lower levels) , and effectively elicit production of an immune response in a vaccinee sufficient to confer protection against serious disease caused by subsequent infection from wild-type virus.
  • viruses of the invention are not only viable and more attenuated then previous mutants, but are more stable genetically in vivo than those previously studied mutants- -retaining the ability to stimulate a protective immune response and in some instances to expand the protection afforded by multiple modifications, e.g., induce protection against different viral strains or subgroups, or protection by a different immunologic basis, e.g., secretory versus serum immunoglobulins, cellular immunity, and the like.
  • genetic instability of the ts phenotype following replication in vivo has been the rule for ts viruses (Murphy et al . , Infect . Immun. 37:235-242 (1982)).
  • RSV grows in a variety of human and animal cells.
  • Preferred cell lines for propagating attenuated RS virus for vaccine use include DBS-FRhL-2, MRC-5, and Vero cells. Highest virus yields are usually achieved with epithelial cell lines such as Vero cells.
  • Cells are typically inoculated with virus at a multiplicity of infection ranging from about 0.001 to 1.0 or more, and are cultivated under conditions permissive for replication of the virus, e.g., at about 30-37°C and for about 3-5 days, or as long as necessary for virus to reach an adequate titer.
  • Virus is removed from cell culture and separated from cellular components, typically by well known clarification procedures, e.g., centrifugat on, and may be further purified as desired using procedures well known to those skilled in the art.
  • Chimeric RSV which has been attenuated as described herein can be tested in various well known and generally accepted in vi tro and in vivo models to confirm adequate attenuation, resistance to phenotypic reversion, and immunogenicity for vaccine use.
  • the modified virus e.g., a multiply attenuated, biologically derived or recombinant RSV
  • ts phenotype i.e. ts phenotype
  • Modified viruses are further tested in animal models of RSV infection. A variety of animal models have been described and are summarized in Meignier et al . , eds .
  • Fig. 1 is a graph correlating the replication of a spectrum of respiratory syncytial subgroup A viruses in the lungs of mice with their replication in chimpanzees.
  • the relative level of replication compared to that of wt RSV is substantially identical, allowing the mouse to serve as a model in which to initially characterize the level of attenuation of the vaccine RSV candidate.
  • the mouse and cotton rat models are especially useful in those instances in which candidate RS viruses display inadequate growth in chimpanzees.
  • the RSV subgroup.-. B viruses are an example of the RS viruses which grow poorly in chimpanzees .
  • the therapeutic effect of RSV neutralizing antibodies in infected cotton rats has been shown to be highly relevant to subsequent experience with immunotherapy of monkeys and humans infected with RSV.
  • the cotton rat appears to be a reliable experimental surrogate for the response of infected monkeys, chimpanzees and humans to immunotherapy with RSV neutralizing antibodies.
  • the amount of RSV neutralizing antibodies associated with a therapeutic effect in cotton rats as measured by the level of such antibodies in the serum of treated animals i.e., serum RSV neutralization titer of 1:302 to 1:578 is in the same range as that demonstrated for monkeys (i.e., titer of 1:539) or human infants or small children (i.e., 1:877).
  • a therapeutic effect in cotton rats was manifest by a one hundred fold or greater reduction in virus titer in the lung (Prince et al . , J. Virol . 61:1851-1854) while in monkeys a therapeutic effect was observed to be a 50-fold reduction in pulmonary virus titer. (Hemming et al . , J. Infect. Dis. 152:1083-1087 (1985)). Finally, a therapeutic effect in infants and young children who were hospitalized for serious RSV bronchiolitis or pneumonia was manifest by a significant increase in oxygenation in the treated group and a significant decrease in amount of RSV recoverable from the upper respiratory tract of treated patients. (Hemming et al . , Antimicrob. Agents Chemother.
  • the cotton rat constitutes a relevant model for predicting success of chimeric RSV vaccines in infants and small children.
  • Other rodents, including mice, will also be similarly useful because these animals are permissive for RSV replication and have a core temperature more like that of humans (Wright et al . , J.
  • the invention also provides isolated, infectious chimeric RSV compositions for vaccine. use.
  • the attenuated chimeric virus which is a component of a vaccine is in an isolated and typically purified form.
  • isolated is meant to refer to RSV which is in other than a native environment of a wild-type virus, such as the nasopharynx of an infected individual. More generally, isolated is meant to include the attenuated virus as a component of a cell culture or other artificial medium.
  • Attenuated RSV of the invention may be produced by an infected cell culture, separated from the cell culture and added to a stabilizer which contains other non-naturally occurring RS viruses, such as those which are selected to be attenuated by means of resistance to neutralizing monoclonal antibodies to the F-protein.
  • Chimeric RSV vaccines of the invention contain as an active ingredient an immunogenically effective amount of RSV produced as described herein.
  • Biologically derived or recombinant RSV can be used directly in vaccine formulations, or lyophilized. Lyophilized virus will typically be maintained at about 4°C.
  • the lyophilized virus When ready for use the lyophilized virus is reconstituted in a stabilizing solution, e.g., saline or comprising SPG, Mg ++ and HEPES, with or without adjuvant, as further described below.
  • a stabilizing solution e.g., saline or comprising SPG, Mg ++ and HEPES, with or without adjuvant, as further described below.
  • the biologically derived or recombinantly modified virus may be introduced into a host with a physiologically acceptable carrier and/or adjuvant.
  • Useful carriers are well known in the art, and include, e.g., water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
  • the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration, as mentioned above.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.
  • Acceptable adjuvants include incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide . , or alum, which are materials well known in the art.
  • Preferred adjuvants also include StimulonTM QS-21 (Aquila Biopharmaceuticals, Inc., Farmingham, MA), MPLTM (3-0- deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamilton, MT) , and interleukin-12 (Genetics Institute, Cambridge, MA) .
  • the immune system of the host Upon immunization with a chimeric RSV vaccine composition as described herein, via aerosol, droplet, oral, topical or other route, the immune system of the host responds to the vaccine by producing antibodies specific for one or more RSV virus proteins, e.g., F and/or G glycoproteins. As a result of the vaccination the host becomes at least partially or completely immune to RSV infection, or resistant to developing moderate or severe RSV disease, particularly of the lower respiratory tract .
  • RSV virus proteins e.g., F and/or G glycoproteins
  • Chimeric RSV vaccines of the invention may comprise attenuated chimeric virus that elicits an immune response against a single RSV strain or antigenic subgroup, e.g. A or B, or against multiple RSV strains or subgroups.
  • the chimeric RSV can elicit a monospecific immune response or a polyspecific immune response against multiple RSV strains or subgroups.
  • chimeric RSV having different immunogenic characteristics can be combined in a vaccine mixture or administered separately in a coordinated treatment protocol to elicit more effective protection against one RSV strain, or against multiple RSV strains or subgroups.
  • the host to which the vaccine is administered can be any mammal susceptible to infection by RSV or a closely related virus and capable of generating a protective immune response to antigens of the vaccinizing virus.
  • suitable hosts include humans, non-human primates, bovine, equine, swine, ovine, caprine, lagamorph, rodents, etc. Accordingly, the invention provides methods for creating vaccines for a variety of human and veterinary uses .
  • the vaccine compositions containing the attenuated chimeric RSV of the invention are administered to a patient susceptible to or otherwise at risk of RS virus infection in an "immunogenically effective dose" which is sufficient to. induce or enhance the individual's immune response capabilities against RSV.
  • the attenuated virus of the invention is administered according to well established human RSV vaccine protocols, as described in, e.g., Wright et al . , Infect Immun. 37:397-400 (1982), Kim et al., Pediatrics 52:56-63 (1973), and Wright et al . , J. Pediatr. 88:931-936 (1976), which are each incorporated herein by reference.
  • RSV vaccine typically in a volume of 0.5 ml of a physiologically acceptable diluent or carrier.
  • RSV vaccine typically in a volume of 0.5 ml of a physiologically acceptable diluent or carrier.
  • This has the advantage of simplicity and safety compared to parenteral immunization with a non-replicating vaccine. It also provides direct stimulation of local respiratory tract immunity, which plays a major role in resistance to RSV. Further, this mode of vaccination effectively bypasses the immunosuppressive effects of RSV-specific maternally-derived serum antibodies, which typically are found in the very young. Also, while the parenteral administration of RSV antigens can sometimes be associated with immunopathologic complications, this has never been observed with a live virus.
  • the precise amount of chimeric RSV vaccine administered and the timing and repetition of administration will be determined based on the patient's state of health and weight, the mode of administration, the nature of the formulation, etc. Dosages will generally range from about IO 3 to about IO 6 plaque forming units (PFU) or more of virus per patient, more commonly from about 10 4 to 10 s PFU virus per patient.
  • the vaccine formulations should provide a quantity of attenuated RSV of the invention sufficient to effectively stimulate or induce an anti-RSV immune response, e.g., as can be determined by complement fixation, plaque neutralization, and/or enzyme-linked immunosorbent assay, among other methods. In this regard, individuals are also monitored for signs and symptoms of upper respiratory illness.
  • the attenuated virus of the vaccine grows in the nasopharynx of vaccinees at levels approximately 10-fold or more lower than wild-type virus, or approximately 10-fold or more lower when compared to levels of incompletely attenuated RSV.
  • multiple administration may be required to elicit sufficient levels of immunity.
  • Administration should begin within the first month of life, and at intervals throughout childhood, such as at two months, six months, one year and two years, as necessary to maintain sufficient levels of protection against native (wild-type) RSV infection.
  • adults who are particularly susceptible to repeated or serious RSV infection such as, for example, health care workers, day care workers, family members of young children, the elderly, individuals with compromised cardiopulmonary function, may require multiple immunizations to establish and/or maintain protective immune responses .
  • Levels of induced immunity can be monitored by measuring amounts of neutralizing secretory and serum antibodies, and dosages adjusted or vaccinations repeated as necessary to maintain desired levels of protection. Further, different vaccine viruses may be indicated for administration to different recipient groups.
  • an engineered chimeric RSV strain expressing a cytokine or an additional protein rich in T cell epitopes may be particularly advantageous for adults rather than for infants.
  • RSV vaccines produced in accordance with the present invention can be combined with viruses expressing antigens of another subgroup or strain of RSV to achieve protection against multiple RSV subgroups or strains.
  • the vaccine virus may incorporate protective epitopes of multiple RSV strains or subgroups engineered into one RSV clone as described herein.
  • vaccine viruses typically when different vaccine viruses are used they will be administered in an admixture simultaneously, but they may also be administered separately.
  • the chimeric RSV vaccines of the invention elicit production of an immune response that is protective against serious lower respiratory tract disease, such as pneumonia and bronchiolitis when the individual is subsequently infected with wild-type RSV. While the naturally circulating virus is still capable of causing infection, particularly in the upper respiratory tract, there is a very greatly reduced possibility of rhinitis as a result of the vaccination and possible boosting of resistance by subsequent infection by wild-type virus. Following vaccination, there are detectable levels of host engendered serum and secretory antibodies which are capable of neutralizing homologous (of the same subgroup) wild-type virus in vi tro and in vivo . In many instances the host antibodies will also neutralize wild-type virus of a different, nonvaccine subgroup.
  • Preferred chimeric RSV of the present invention exhibit a very substantial diminution of virulence when compared to wild-type virus that is circulating naturally in humans.
  • the chimeric virus is sufficiently attenuated so that symptoms of infection will not occur in most immunized individuals. In some instances the attenuated virus may still be capable of dissemination to unvaccinated individuals.
  • the level of attenuation of chimeric vaccine virus may be determined by, for example, quantifying the amount of virus present in the respiratory tract of an immunized host and comparing the amount to that produced by wild-type RSV or other attenuated RSV which have been evaluated as candidate vaccine strains.
  • the attenuated chimeric virus of the invention will have a greater degree of restriction of replication in the upper respiratory tract of a highly susceptible host, such as a chimpanzee, compared to the levels of replication of wild-type virus, e.g., 10- to 1000-fold less.
  • the level of replication of the attenuated RSV vaccine strain in the upper respiratory tract of is particularly useful for the level of the attenuated.
  • the chimpanzee should be less than that of the RSV A2 ts-1 mutant, which was demonstrated previously to be incompletely attenuated in seronegative human infants.
  • an ideal vaccine candidate virus should exhibit a restricted level of replication in both the upper and lower respiratory tract.
  • the attenuated viruses of the invention must be sufficiently infectious and immunogenic in humans to confer protection in vaccinated individuals. Methods for determining levels of RS virus in the nasopharynx of an infected host are well known in the literature.
  • Specimens are obtained by aspiration or washing out of nasopharyngeal secretions and virus quantified in tissue culture or other by laboratory procedure. See, for example, Belshe et al . , J. Med. Virology 1:157-162 (1977), Friedewald et al . , J. Amer. Med. Assoc. 204:690-694 (1968); Gharpure et al . , J. Virol. 3:414-421 (1969); and Wright et al . , Arch. Ges. Virusforsch. 41:238-247 (1973), each incorporated herein by reference.
  • the virus can conveniently be measured in the nasopharynx of host animals, such as chimpanzees .
  • a chimeric RSV vaccine of the present invention can be administered simultaneously with parainfluenza virus vaccine, such as described in Clements et al . , J. Clin. Microbiol. 29:1175-1182 (1991), which is incorporated herein by reference .
  • the chimeric RSV can be employed as a vector for protective antigens of other respiratory tract pathogens, such as parainfluenza, by incorporating the sequences encoding those protective antigens into the chimeric RSV genome or antigenome which is used to produce infectious chimeric RSV, as described herein.
  • a chimeric RSV is employed as a vector for transient gene therapy of the respiratory tract.
  • the chimeric RSV genome or antigenome incorporates a sequence which is capable of encoding a gene product of interest .
  • the gene product of interest is under control of the same or a different promoter from that which controls RSV expression.
  • the infectious RSV produced by coexpressing the recombinant RSV genome or antigenome with the N, P, L and M2 (ORF1) proteins and containing a sequence encoding the gene product of interest is administered to a patient.
  • Chimeric RSV is administered in an amount sufficient to result in the expression of - therapeutic or prophylactic levels of the desired gene product.
  • representative gene products which are administered in this method include those which encode, for example, those particularly suitable for transient expression, e.g., interleukin-2, interleukin-4, gamma-interferon, GM-CSF, G-CSF, erythropoietin, and other cytokines, glucocerebrosidase, phenylalanine hydroxylase, cystic fibrosis transmembrane conductance regulator (CFTR) , hypoxanthine-guanine phosphoribosyl transferase, cytotoxins, tumor suppressor genes, antisense RNAs, and vaccine antigens.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • a parent stock of cold-passaged RSV (cpRSV) was prepared.
  • Flow Laboratories Lot 3131 virus the cpRSV parent virus that is incompletely attenuated in humans, was passaged twice in MRC-5 cells at 25°C, terminally diluted twice in MRC- 5 cells at 25°C, then passaged three times in MRC-5 to create cpRSV suspension for mutagenesis .
  • the cpRSV was mutagenized by growing the parent stock in MRC-5 cells at 32°C in the presence of 5-fluorouracil in the medium at a concentration of 4 x 10 " M. This concentration was demonstrated to be optimal in preliminary studies, as evidenced by a 100-fold decrease in virus titer on day 5 of growth in cell culture, compared to medium without 5-fluorouracil .
  • the mutagenized stock was then analyzed by plaque assay on Vero cells that were maintained under an agar overlay, and after an appropriate interval of incubation, plaques were stained with neutral red dye. 854 plaques were picked and the progeny of each plaque were separately amplified by growth on fresh monolayers of Vero cells.
  • strains were biologically cloned by serial plaque-purification on Vero cells three times, then amplified on Vero cells.
  • the cloned strains were titered at 32°, 37°, 38°, 39° and 40°C (in an efficiency of plaque formation (EOP) assay) to confirm their sp and ts phenotypes . Because titers of some cloned strains were relatively low even at the permissive temperature (32°), these viruses were passaged once in HEp-2 cells to create virus suspensions for in vi tro analysis.
  • the phenotypes of the progeny of the mutagenized cpRSV are presented on Table 1.
  • Shut -off temperature is defined as the lowest restrictive temperature at which a 100-fold or greater reduction of plaque titer is observed (bold figures in table) .
  • RSV cpspl43 (sp refers to the small plaque (sp) phenotype)
  • the remaining mutant progeny had the ts phenotype.
  • the RSV cpts mutants exhibit a variation in ability _to produce plaques in monolayer cultures in vitro " over the temperature range 37°C to 40°C, with cpts368 retaining the ability to produce plaques at 40 °C, whereas the most temperature -sensitive (ts) virus, cpts248, failed to produce plaques at 38°C.
  • ts temperature -sensitive virus
  • mice The level of replication of the cpRSV derived mutants in the upper and lower respiratory tracts of BALB/c mice was studied next (Table 2) . It was found that cpts530 and cpts248, two of the mutants exhibiting the greatest temperature sensitivity (see Table 1) , were about 7- to 12 -fold restricted in replication in the nasal turbinates of the mice (Table 2) . However, none of the viruses was restricted in replication in the lungs compared to the cpRSV parent virus. This greater restriction of replication in the nasal turbinates than in the lungs is not characteristic of ts mutants, which generally are more restricted in replication in the warmer lower respiratory tract (Richman and Murphy, Rev. Infect. Dis. 1:413-433 (1979).
  • the virus produced in the lungs and nasal turbinates retained the ts character of the input virus (data not presented) .
  • the present findings suggested that the combination of the ts mutations on the background of the mutations of the cp parent virus has resulted in cpRSV ts progeny with a higher level of stability of the ts phenotype after replication in vivo than had been seen with previously studied ts mutants.
  • the virus present on day 12 (NOTE: in normal mice, virus is no longer detectable at this time) was characterized and found to retain an unaltered ts phenotype (Table 3) .
  • the ts-1 mutant included in the test as a positive control exhibited an unstable ts phenotype in vivo.
  • the results show that a high level of stability of the ts phenotype of the cpRSV derived mutants following prolonged replication in rodents was achieved, which represents a significant and heretofore unattained very desirable property in the viruses of the invention.
  • Table 2 Replication of cpts and cpsp- RSV mutants in BALB/c mice 1
  • A2 wild- type >40 5. .0 + 0.16 5, .8 ⁇ 0.20 5.0 ⁇ 0.11 5.8 ⁇ 0.19 cpRSV >40 4, .7 ⁇ 0.07 5. .3 ⁇ 0.18 4.8 + 0.16 5.3 ⁇ 0.21 ts-1 38 4.
  • mice were administered 10 6 - 3 p.f.u. intranasally in a 0.1 ml inoculum on day 0, then sacrificed on day 4 or 5.
  • Plaque titers shown represent the mean log, ⁇ pfu/gram tissue of 19 or 20 samples ⁇ standard error
  • Virus is isolated from nasal and throat swab specimens and tracheal lavage fluids by inoculation into
  • RSV-sensitive HEp-2 cells as described above. Quantities of virus can also be determined directly by the plaque technique using HEp-2 cells as described in Schnitzer et al . , J. Virol. 17:431-438 (1976), which is incorporated herein by reference. Specimens of serum are collected before administration of virus and at 3 to 4 weeks post-inoculation for determination of RSV neutralizing antibodies as described in Mills et al . , J. Immununol . 107:123-130 (1970), which is incorporated herein by reference. The most ts and attenuated of the cpRSV derivative
  • cpts248 was studied and compared to wild-type RSV and the cpRSV parent virus (Table 4) .
  • Replication of the cpRSV parent virus was slightly reduced in the nasopharynx compared to wild-type, there was a reduction in the amount of rhinorrhea compared to wild-type virus, and there was an approximate 600-fold reduction in virus replication in the lower respiratory tract compared to wild-type.
  • the cp virus was significantly restricted in replication in the lower respiratory tract of chimpanzees, a very desirable property not previously identified from prior evaluations of cpRSV in animals or humans.
  • the cpts 248 virus was 10-fold restricted in replication in the nasopharynx compared to wild-type, and this restriction was associated with a marked reduction of rhinorrhea.
  • the cpts248 virus has many properties desirable for a live RSV vaccine, including: 1) attenuation for the upper and lower respiratory tract; 2) increased genetic stability after replication in vivo, even after prolonged replication in immunosuppressed animals; 3) satisfactory immunogenicity; and 4) significant protective efficacy against challenge with wild-type RSV.
  • the cpts530 virus shares with cpts248 similar temperature sensitivity of plaque formation, a similar degree of restriction of replication in the nasal turbinates of mice, and a high level of genetic stability in immunodeficient nude mice, whereby it also represents an RS virus vaccine strain.
  • Peak titer virus Inoculation number (days) (log-oPfu/ml) (days) (log 10 pfu/ml) Mean 0 Peak cpts-248 IN + IT 1 10 4.6 8 d 5.4 0.2 1
  • Mean rhinorrhea score represents the sum of daily scores for a period of eight days surrounding the peak day of virus shedding, divided b eight. Four is the highest score; zero is ⁇ e lowest score, d Virus isolated only on day indicated.
  • NP nasopharyngeal
  • TL tracheal lavage
  • NT 3 ⁇ 0. NT 4 ⁇ 0. _NT 5 ⁇ 0. NT 6 ⁇ 0. NT 7 ⁇ 0. NT 8 ⁇ 0. NT 9 ⁇ 0. NT
  • Serum antibody titers (reciprocal mean log.,)
  • Virus used to Chimpanzee Duration Peak titer Duration Peak titer Day 42 immunize animal number (days) (log 10 pfu/ml) (days) (log 10 pfu/ml) Mean* Peak Day 28 or 56 cpts-248 1 5 2.7 0 ⁇ 0.7 0 0 10.1 11.0 2 9 1.8 0 ⁇ 0.7 0 0 10.3 14.5 cp-RSV 5 5 1.0 0 ⁇ 0.7 0 0 11.1 13.3 6 8 0.7 0 ⁇ 0.7 0 0 11.4 12.9 none 9 9 5.1 13 5.4 1.0 1 ⁇ 3.3 12.4
  • Mean rhinorrhea score represents the sum of scores during the eight days of peak virus shedding divided by eight.
  • a score of zero indicates no rhinorrhea detected on any day of the ten-day observation period.
  • Progeny viruses that exhibited a greater degree of temperature-sensitivity in vi tro than cpts248 or that had the small plaque phenotype were selected for further study. Mutant derivatives of the cpts248 that possessed one or more additional ts mutations were produced by 5-fluorouracil mutagenesis (Table 8) . Ts mutants that were more temperature-sensitive ( ts) than the cpts248 parental strain were identified, and some of these had the small plaque ( sp) phenotype. These cpts248 derivatives were administered to mice.
  • cpts248/804, 248/955, 248/404, 248/26, 248/18, and 248/240 mutants were more restricted in replication in the upper and lower respiratory tract of the mouse than their cpts248 parental virus (Table 9) .
  • viable mutants of cpts248 which were more attenuated than their cpts248 parental virus were identified, and these derivatives of cpts248 exhibited a wide range of replicative efficiency in mice, with cpts248/26 being the most restricted.
  • the ts phenotype of the virus present in nasal turbinates and lungs of the mice was almost identical to that of the input virus, indicating genetic stability.
  • this virus exhibited a high degree of reduction in replication compared to wild-type, being 60, 000-fold reduced in the nasopharynx and 100, 000-fold in the lungs.
  • ts derivatives of the cpts530 virus were also generated (Table 13) .
  • the cpts-530 derivatives were more restricted in replication in mice than the cpts530 parental strain.
  • One mutant, cpts-530/1009 was 30 times more restricted in replication in the nasal turbinates of mice.
  • This cpts530 derivative is also highly restricted in replication in the upper and lower respiratory tract of seronegative chimpanzees (Table 14) .
  • cpts530 was 30-fold restricted in replication, while cpts530/1009 was 100-fold restricted compared to wild-type virus.
  • cpts mutants were highly restricted (20,000 to 32, 000-fold) in the lower respiratory tract compared with wild-type virus, even when the mutants were inoculated directly into the trachea.
  • chimpanzees previously infected with cpts530/l009, cpts530 or cpRSV exhibited significant restriction of virus replication in the nasopharynx and did not develop significant rhinorrhea following subsequent combined intranasal and intratracheal challenge with wild-type RSV (Table 15) .
  • chimpanzees previously infected with any of the mutants exhibited complete resistance in the lower respiratory tract to replication of wild-type challenge virus.
  • Table 8 The efficiency of plaque formation of ten mutants derived from RSV cpts248 by additional 5F mutagenesis .
  • Shut-off temperature is defined as the lowest restrictive temperature at which a 100-fold or greater reduction of plaque titer in Hep-2 cells is observed (bold figures in table) .
  • Pinpoint-plaque phenotype ( ⁇ 10% wild-type plaque size) .
  • Virus titer (mean log, 0 pfu/g tissue of six animals ⁇ standard error)
  • Virus used to temperature infect animal of 32°C 36°C 37°C 38°C 32°C 36°C 37°C 38°C virus (°C)
  • A2 wild-type >40 5.1 ⁇ 0.15 5.2 ⁇ 0.23 5.2 ⁇ 0.14 5.2 ⁇ 0.27 6.1 ⁇ 0.14 5.8 ⁇ 0.23 6.0 ⁇ 0.12 5.9 ⁇ 0.1 cp-RSV >40 4.9 ⁇ 0.20 5.1 ⁇ 0.16 4.9 ⁇ 0.24 4.9 ⁇ 0.22 6.0 ⁇ 0.16 5.9 ⁇ 0.23 5.6 ⁇ 0.15 5.6 ⁇ 0.1 ts-1 38 3.9 ⁇ 0.25 2.7 ⁇ 0.27 2.4 ⁇ 0.42 2.5 ⁇ 0.29 4.1 ⁇ 0.21 3.5 ⁇ 0.23 2.6 ⁇ 0.18 2.0 ⁇ 0.2 cpts-248 38 4.0 ⁇ 0.16 2.5 ⁇ 0.34 ⁇ 2.0 ⁇ 2.0 4.4 ⁇ 0.37 1.8 ⁇ 0.15 ⁇ 1.7 ⁇ 1.7
  • the level of replication of the cpts-248/404 virus was found to be 2.4 ⁇ 0.24 and 2.6 ⁇ 0.31 in the nasal turbinates and lungs, respectively.
  • Peak titer Duration b Peak titer with indicated virus Inoculation number (days) (log 10 pfu/ml) (days) (log 10 pfu/ml) Mean c Peak cprs-248/404 IN + IT 13 0 ⁇ 0.7 0 ⁇ 0.7 0 0
  • Mean rhinorrhea score represents the sum of daily scores for a period of eight days surrounding the peak day of virus shedding, divided by eight. Four is the highest score; zero is the lowest score. d Virus isolated only on day indicated.
  • Table 12 The efficiency of plaque formation and replication of Balb/c mice of five small-plaque derivatives of RSV cpts-248/404.
  • Shut-off temperature is defined as the lowest restrictive temperature at which at 100-fold or greater reduction of plaque titer is observed (bo figures in table).
  • mice were administered 10 63 p.f.u. intranasally under light anesthesia on day 0, then sacrificed by C0 2 asphyxiation on day 4 when tissues were harvested for virus titer.
  • Pinpoint-plaque phenotype ( ⁇ 10% wild-type plaque size).
  • mice 2 (mean log 10 pfu/g
  • Shut-off temperature is defined as the lowest restrictive temperature at which a 100-fold or greater reduction of plaque titer is observed (bold figures in table) .
  • mice were administered 10 63 p.f.u. intranasally under light anesthesia on day 0, then sacrificed by C0 2 asphyxiation on day 4.
  • the neutralizing/ELISA F ratio of the antibodies produced in the infused immunized animals post-challenge was about 10- to 20-fold higher than in the non-infused animals and was consistent in all groups, regardless of mutant used to immunize (Table 17) .
  • the presence of passively-acquired antibodies at the time of immunization with a live virus vaccine might alter the immune response to vaccine in three distinct ways.
  • a significant decrease in the level of replication of vaccine virus might occur that results in decreased immunogenicity.
  • the passively-transferred RSV antibodies could restrict the replication of the vaccine viruses, especially the most defective mutants, and greatly decrease their immunogenicity.
  • the results presented herein indicate that replication of the least attenuated mutant (cpts248) in the lower respiratory tract was indeed abrogated by the presence of passively-acquired serum IgG RSV antibodies, whereas replication in the upper respiratory tract did not appear to be significantly affected.
  • the replication of the least attenuated mutant tested, cpts248, was ⁇ 200-fold more
  • passive antibodies can alter the immune response to infection by causing a decrease in the amount and functional activity of antibodies that are induced. For this reason the magnitude and the character of the antibody response to live virus immunization in the presence of passive antibodies was analyzed.
  • Postimmunization serum ELISA IgG F antibody titers of immunized, infused animals were 10-fold lower than the postimmunization F titers of non-infused seronegative animals.
  • the serum RSV neutralizing antibody response was also slightly decreased in those animals, on average being 2 -fold lower than in non- infused animals.
  • the human immune globulin preparation used contained a low level of antibodies to the G glycoprotein of RSV (Table 17) . This petted an examination of the IgG RSV G glycoprotein antibody response of the chimpanzees to infection with the candidate vaccine viruses. The G antibody responses demonstrated at least a 4-fold or greater increase, indicating that each of the passively-immunized animals was infected by vaccine virus, including chimpanzees immunized with cpts248/404 which did not shed virus.
  • the magnitude of the G antibody response to immunization did not appear to be adversely influenced by the passively transferred antibodies.
  • the antibody response to RSV wild-type virus challenge of animals immunized in the presence of passively-acquired antibodies could be altered.
  • Chimpanzees immunized in the absence of infused antibodies exhibited significant resistance to subsequent RSV challenge.
  • these animals failed to develop an appreciable antibody response to challenge virus.
  • each of the 6 infused, immunized animals also exhibited significant resistance to RSV, a greatly enhanced antibody response to challenge was observed.
  • Post-challenge F or G antibody levels in the treated animals immunized with cpts530/l009 or cpts248/404 were increased at least 10-fold, while the neutralizing antibody response represented as much as an 800- fold increase.
  • the day on which postimmunization titer was determined was also the day on which challenge was performed, i.e., day 28 for animals not infus with antibody, day 42 for animals infused.
  • Virus used for antibodies Chimpanzee Duration Peak Titer Duration Titer immunization present Number (days) (lofl in pfu/ml) (days) (log, n pfu/ml) Mean b Peak cpts-248/404 no 17 c 0 ⁇ 0.7 0 ⁇ 0.7 0 0 no 18 c 8 3.4 0 ⁇ 0.7 0 0 yes 21 6 2.7 0 ⁇ 0.7 0.5 2 yes 22 0 ⁇ 0.7 0 ⁇ 0.7 0 0 cpts-530/1009 no 1 7 2.1 0 ⁇ 0.7 0 0 no 2 0 ⁇ 0.7 0 ⁇ 0.7 0 0 yes 23 6 2.5 0 ⁇ 0.7 0.5 1 yes 24 7 2.0 0 ⁇ 0.7 0.2 1 cpts-248 no 25 c 5 2.7 0 ⁇ 0.7 0 0 no 26 c 9 1.8 0 ⁇ 0.7 0 0 yes 29 0 ⁇ 0.7 0 ⁇ 0.7 0
  • a parent stock of cold-passaged RSV A2 obtained from Flow Laboratories was prepared by passage in MRC-5 cells at 25°C as described in Example 1. Briefly, cold-passaged virus was inoculated into MRC-5 or Vero cell monolayer culture at a multiplicity of infection of ⁇ 0.01 and the infected cells were incubated for 3 to 14 days before subsequent passage. Virus was passaged over 20 times at 20-22°C to derive more attenuated virus. The technique of rapid passage, as soon as the first evidence of virus replication is evident (i.e., 3 to 5 days), was preferable for selection of mutants able to replicate efficiently at low temperatures . Additionally, an RSV subgroup B strain, St.
  • clone 1A1 was isolated in primary African Green monkey kidney cells, passaged and cloned in MRC-_ cells (1A1-MRC14) , and cold-passaged 52 times in MRC-5 or Vero cells at 32 to 22°C.
  • a second strategy employed a biologically cloned derivative of the uncloned parental cpRSV 3131 virus.
  • This virus was biologically cloned in bovine embryonic kidney (BEK) cells [the tissue used to originally derive the cpRSV 3131 virus--see Friedewald et al., J. Amer. Med. Assoc. 204:690-694 (1968)] .
  • BEK bovine embryonic kidney
  • This cloned virus was then passaged at 10 day intervals in Vero cells at low temperature.
  • the cpRSV 3131 virus was cloned by two serial terminal dilutions
  • T2P4 in MRC-5 cells and passaged at 10-day intervals in MRC-5 or Vero cells .
  • the third strategy involved selection of mutants that produce large plaques at low temperature.
  • An RSV 3131 derivative virus designated plaque Dl that produces large plaques at 25°C has been identified.
  • This virus was derived from the third passage (P3) level of the cp3131-l (BEK) lineage cp3131-17 (BEK) lineage.
  • the largest plaque produced by P3 virus was amplified at 32°C, then re-plaqued at 25°C. Once again the largest plaque was selected, amplified, and re-plaqued. After five such cycles, large placque mutant virus Dl was obtained. Dl was biologically cloned by two additional cycles of plaque-to-plaque purification at 25°C.
  • Biologically cloned virus Dl produces distinctly and uniformly larger plaques at 25°C than cp3131 or wild-type virus A2.
  • Dl is cold adapted by the criterion of large plaque size at 25°C.
  • Efficiency of plaque formation studies demonstrated that Dl is not temperature sensitive.
  • Dl plaques are indistinguishable from those of wild-type RSV or cp3131, suggesting that Dl is not restricted in growth at this temperature. Consistent with this, Dl produces extensive cytopathic effects in Vero cell monolayers at 37°C and 40°C (i.e. the highest temperatures tested) .
  • RSV ts mutants were passaged at low temperature to select RSV nts mutants with the ca phenotype, i.e., increased capacity to replicate at suboptimal temperature compared to wild-type parental virus.
  • a parent stock of ts-1 NG1 virus was prepared from Flow Laboratories Lot M4 of live Respiratory Syncytial Virus (A-2) ts-1 NG-1 mutant, MRC-5 grown virus.
  • This mutant derived from the ts-1 mutant by a second round of mutagenesis using nitrosoguanidine, possesses two or more independent ts mutations, but still induces substantial rhinorrhea in susceptible chimpanzees.
  • This virus was passaged twice in Vero cells at 32°C to create a ts-1 NG-1 suspension for mutagenesis.
  • the virus was then grown in the presence of 4xlO" 4 M 5-fluorouracil to induce additional mutations during replication or was exposed to 5-azacytidine at 36°C after 5-fluorouracil treatment.
  • the mutagenized stock was then analyzed by plaque assay on Vero cells that were maintained under an agar overlay, and, after an appropriate interval of incubation, plaques were identified microscopically. 586 plaques were picked, and the progeny of each plaque were separately amplified by growth on fresh monolayers of Vero cells.
  • the ts-1 NGl/A-20-4 virus is attenuated for chimpanzees (Table 21) and infection of chimpanzees with ts-1 NGl/A-20-4 induced resistance to wild-type virus challenge (Table 22) . Significantly, rhinorrhea does not occur.
  • Mutagenesis of the ts-4 virus was also performed, using the same method as for mutagenesis of ts-1 NG1, virus. Mutations were also introduced into the ts-4 viruses by cold-passage. The ts-4 virus replicates to high titer at 22°C after 43 cold-passages. Six plaque progeny that were more ts than the RSV ts-4 parent virus were identified (Table 23) . The ts-4 cp-43 is even further restricted in replication in Balb/c mice (Table 24) .
  • TslNGl 5 .8 4 4. .31 ⁇ 0. .17 ⁇ 2.0 2 .82 + 0 .25 ⁇ 2.0
  • Peak titer Duration Peak titer virus Inoculation 3 number (Days) ⁇ log 10 pfu/ml) (Days) (log 10 pfu/ml) Mean 6 Peak ts-1 NG1/A-20-4 IN + IT 15 0 ⁇ 0.7 0 ⁇ 0.7 0 0
  • IN + IT 12 9 5.4 8 5.6 1.0 3 mean 10 mean 5.5 mean 9.3 mean 5.7 mean 1.4 mean 2.8
  • b Indicates last day post-infection on which virus was recovered.
  • c Mean rhinorrhea score represents the sum of daily scores for a period of eight days surrounding the peak day of virus shedding, divided by eight.
  • Mean rhinorrhea score represents the sum of scores during the eight days of peak virus shedding divided by eight Four is the highest score; zero is the lowest score.
  • d Serum neutralizing titers in this table were determined in a new assay simultaneously with other specimens represented in the table
  • Table 23 The efficiency of plaque formation of six mutants derived from RSV ts-4 and tested in HEp-2 cells at permissive and restrictive temperatures, compared with controls.
  • Shut-off temperature is defined as the lowest restrictive temperature at which a 100-fold or greater reduction of plaque titer is observed (bold figures in table).
  • Pinpoint-plaque phenotype ( ⁇ 10% wild-type plaque size)
  • Virus titer (mean log 10 pfu/g tissue of six animals + standard error)
  • mice were administered 10 S 3 p. f .u. intranasally under light anesthesia on day 0, then sacrificed by C0 2 asphyxiation on day 4.
  • RSV Subgroup B Vaccine Candidates This Example describes the development of RSV subgroup B virus vaccine candidates. The same approach used for the development of the subgroup A mutants of the invention was utilized for the subgroup B viruses.
  • a parent stock of wild-type B-l RS virus was cold-passaged 52 times in Vero cells at low temperature (20-25°C) and the virus was subjected to plaque purification at passages 19 and 52.
  • Three of the clones derived from the passage 52 suspension were evaluated independently, and one clone, designated RSV B-lcp52/2B5, was selected for further evaluation because it was highly attenuated in the upper and lower respiratory tract of the cotton rat (Table 25) .
  • the RSV B-152/2B5 mutant has been further mutagenized with 5-fluorouracil and the resulting plaques picked and screened at 32° vs. 38°C for the ts phenotype.
  • the selected cpts mutants were plaque-purified three times in Vero cells and then amplified twice in Vero cells.
  • seven cpts mutants of RSV B-lcp52/2B5 have been identified (Table 30) and their level of replication in cotton rats has been studied (Table 31) .
  • One of these mutants, namely cptsl76 was further mutagenized and a series of mutant derivatives were obtained that were more ts in vitro than the RSV B-lcpts 176 parent virus (Table 32) .
  • the subgroup B mutants are infectious and exhibit a significant degree of attenuation for cotton rats, monkeys, and chimpanzees.
  • the RSV B-l cp mutant viruses induced resistance in monkeys against wild-type challenge.
  • the ts mutants of the RSV B-l cpts52/2B5 virus are attenuated and demonstrate a more restricted level of replication in the nasopharynx and lungs of the cotton rat than the RSV B-l 52/2B5 parent virus.
  • Table 25 Replication in cotton rats of RSV B-l wild-type compared with five plaque-purified cold-passaged mutants derived from RSV B-l, in two separate experiments .
  • Virus titer on day 4 in indicated tissue (mean log 10 pfu/g tissue ⁇ RSV B-l wild-type standard error of the mean) locr-nPfu/q)
  • Isolates were virus suspensions obtained following amplification by one Vero cell tissue culture passage of virus present in the original nasal turbinate homogenate on day 14 of an immunosuppressed cotton rat.
  • () indicates the numbers of animals from which virus was detected at 1.2 log 10 pfu/g or greater.
  • 0 indicates the numbers of animals from which virus was detected at 1.5 log 10 pfu/g or greater.
  • NP swab Tracheal Lava ⁇ e NP swab Lava ⁇ e
  • B-1 (6) B-1 (4) ⁇ 10 3,327 3,822 ⁇ 10 2,048 2,521 A2(2) 30,574 35,120
  • Mean rhinorrhea score represents the sum of daily scores for a period of eight days surrounding the peak day of virus shedding, divided by eight.
  • Shut-off temperature is defined as the lowest restrictive temperature at which a 100-fold or greater reduction of plaque titer is observed
  • the complete nucleotide sequence of the wild-type B-l virus has been determined [SEQ ID NO: 2] .
  • This sequence was compared with the sequence of the attenuated B-l derivative, cp-52/2B5 (cp-52), described above.
  • This sequence analysis revealed a large deletion in cp-52 spanning most of the SH and G genes, with no predicted ORF for either gene. More specifically, most of the region spanning the SH and G genes of the cp-52 virus was deleted, retaining only the SH gene-start signal and a portion of the 3' (downstream) end of the G gene and its gene-end signal.
  • the remaining SH:G region could encode a chimeric transcript of -91 nucleotides with no ORF.
  • Northern blot analysis of cp-52 confirmed that multiple unique polytranscripts contained SH:G read-through mRNAs, consistent with a deletion mutation spanning the SH:G gene junction.
  • subgroup B mutants isolated at different passage levels in the cp-52 passage history incorporate various of the cp-52 mutations, depending on passage level (Table 34) .
  • Exemplary subgroup B mutants in this context include RSV B-l cp- 12, RSV B-l cp-23, RSV B-l cp-32, RSV B-l cp-42.
  • Table 34 depicts (as negative sense) the distribution of these specific mutations among exemplary B subgroup mutants. This varied distribution of mutations allows for more refined characterization of the attenuating effects of these mutations in the designated strains.
  • cp-23 incorporates the mutations at nucleotide positions 5626, 6460, 14164 and 14596 found in cp-52 (Table 34) , but has no differences from the parental B-l wild-type strain in the SH and G gene region that is deleted in cp-52.
  • cp-42 incorporates the same SH and G deletion as cp-52, while cp32 presents a distinct deletion of sequences within the SH and G genes .
  • G/F 5626 G T T *ND T non-coding lGr F 6318 T ND T T ND C Glu ⁇ Gly (218) 6460 A A G G *ND G silent (265)
  • cpts248 strain was subjected to chemical mutagenesis to further reduce residual reactogenicity, yielding a series of mutants with increased temperature sensitivity or small plaque phenotype, including cpts248/404.
  • cp ⁇ s530/l009 was derived from cpts530.
  • the genetic bases for increased attenuation and ts phenotype of cpts248 and cpts530 were determined by comparing the complete genomic sequence of these viruses with that of the previously determined sequence of the cpRSV parental virus .
  • the complete nucleotide sequence of cpRSV was determined and compared with that of RSV A2 wild-type virus (a laboratory strain which was not part of the direct passage lineage) , as well as with the sequence of its low passaged wild-type parent virus (RSV A2/HEK7) .
  • the cpRSV differs from its RSV A2/HEK7 parent virus by five nucleotide changes, one in the nucleoprotein (N) , two in the fusion protein (F) gene and two in the polymerase (L) gene.
  • N nucleoprotein
  • F fusion protein
  • L polymerase
  • Example 1 The derivation of the RSV mutants cpts248 and cpts530 from their cpRSV parent by random chemical mutagenesis was described in Example 1.
  • the virus suspension used for infecting cells for production of virus to be used as a source of purified viral RNA was a clarified tissue culture supernatant containing virus that had been passaged four times in liquid medium in Vero cell monolayer culture following biological cloning (i.e., three plaque-to-plaque passages in Vero cell monolayers under agar) .
  • the resulting cDNA was separated from primers using TE-100 spin columns (Clontech, Palo Alto, CA) and used as template in polymerase chain reactions (PCR) to generate a series of ten overlapping cDNA clones spanning the entire RSV genome.
  • the oligonucleotide primers for PCR were designed from the nucleotide sequence of the prototype A2 virus (Mink et al . ,
  • vRNA viral RNA
  • the polyadenylated RNA product was purified by extraction with phenol chloroform and ethanol precipitation, and then reverse transcribed into cDNA, amplified by PCR, and cloned using a rapid amplification by the 3' RACE system (Life Technologies) .
  • vRNA was reverse transcribed into cDNA, tailed using terminal deoxynucleotidyl transferase and dCTP, column purified, made double-stranded and amplified by PCR, and cloned using a 5' RACE system (Life Technologies) .
  • Cloned cDNAs for cpts248 were sequenced from double stranded plasmids by the dideoxynucleotide method using synthetic oligonucleotide primers (either plasmid primers or RSV specific primers), [ ⁇ 35 S] dATP and Sequenase 2.0 (United States Biochemicals, Cleveland, OH) . Differences between, the observed sequences and those of the previously published parental virus cpRSV were confirmed by sequencing two or more clones in both forward and reverse directions, and by sequencing uncloned PCR products .
  • Nucleotide sequences of the cpts248/404, cpts530, cpts530/l009, and cpts530/l030 were determined using a different technique .
  • Three to ten overlapping cDNA clones representing the genome of cptsRSV mutant virus were generated by RT-PCR of total infected-cell RNA or vRNA.
  • the complete nucleotide sequence of each clone was determined by automated DNA sequence at the NCI Frederick Cancer (Frederick, MD) using Taq kit (ABI, Foster City, CA) on a M13 sonicated random library constructed in phage M13 for each plasmid insert .
  • the cpRSV actually consisted of a mixed population of viruses . This accounts for the initial failure to detect the change at position 1938 in Conners et al., supra.
  • a virus with the A mutation at position 1,938 was the immediate parent of the cpts248 derivative as well as of the cpts530 sister clone.
  • the cpRSV which was ttie. immediate parent virus of the cpts derivative contains five amino acid differences from its A2/HEK parent.
  • a single nucleotide difference between the cpRSV and cpts248 mutants was found at nucleotide position 10,989 (an A to T change) (Table 36) .
  • This mutation occurred within the polymerase (L) translation open reading frame, encoding a predicted amino acid change of gin to leu at amino acid position 831 in the 2,165 amino acid L protein.
  • the cpts248/404 mutant possesses two nucleotide differences from its cpts248 parent, one at nucleotide 12,046 (T to A) in the L gene, and one at nucleotide 7605 (T to C) in the transcription start signal sequence of the M2 gene.
  • the nucleotide substitution in the L gene resulted in an amino acid change from asp to glu at position 1183 in the L protein.
  • the cpts248/404 virus acquired two nucleotide changes in the L gene (corresponding to two amino acid substitutions) and one nucleotide change in the transcription start signal of the M2 gene .
  • the cpts248/404 mutant differs in seven amino acids (four in L, two in F, and one in N) and in one nucleotide in the transcription start signal of the M2 gene.
  • the cpts530 differs from the parental strain cpRSV by the single additional nucleotide substitution of C to A at position 10,060 and results in a phe to leu amino acid change at position 521 of the L protein (Table 37) .
  • the cpts530/1009 mutant has a single nucleotide substitution at nucleotide 12,002 (A to G) resulting in a met to val substitution at amino acid 1169 of the L protein.
  • the cpts530/1009 mutant differs in seven amino acids (four in L, two in F, and one in N) .
  • the cpts530/1030 mutant has a single additional nucleotide substitution at nucleotide 12458 (T to A) resulting in a Tyr to Asn substitution at amino acid 1321 of the L protein. Compared to its wt progenitor, A2/HEK, the cpts530/1030 mutant differs in seven amino acids (four in L, two in F, and one in N) . Table 37. Nucleotide Sequence Differences among cp-RSV, cpts-530, cpts- 530/1009 mutant viruses
  • the ts and attenuation phenotypes of the cpts248 and cpts530 each are associated with a single nucleotide change in the polymerase gene.
  • the incremental increase in ts and attenuation phenotypes between the cpts530, and cpts530/1009 or cpts530/l030 was also each associated with a one amino change in L.
  • cpts248, cpts530, cpts530/l009, and cpts530/l030 a single, but different, amino acid substitution in L conferred the ts and attenuation phenotypes on the progenitor strains. These amino . acid substitutions are acting in cooperation with the five cpRSV mutations to enhance the stability of the ts phenotype following replication in animals.
  • the incremental increase in attenuation and temperature sensitivity observed between the cpts248 and cpts248/404 was associated with two nucleotide changes, either or both of which could contribute to the ts and attenuation phenotypes.
  • the four specific sites in L i.e., those specific for the cpts530, cpts530/l009, cpts530/1030 and cpts248 viruses
  • L The four specific sites in L (i.e., those specific for the cpts530, cpts530/l009, cpts530/1030 and cpts248 viruses) that are singly associated with the ts and attenuation phenotypes and one or both sites in cpts248/404 are identified by the findings summarized herein as core regions of the RSV genome or L protein at which mutation can lead to attenuation.
  • the specific mutations at the four sites in the L protein were specific amino acid substitutions, it is likely that other amino acid substitutions as well as in frame insertions or deletions at these sites and at contiguous amino acids within about five amino acids of a specific site can also result in attenuation.
  • the encoded amino acid changes in L do not appear to involve the regions of highest sequence conservation among the paramyxovirus polymerase proteins, the proposed ATP binding site (Stec et al., Virology 183:273-287 (1991)), nor the regions suggested to be homologous to motifs of RNA-dependent RNA and DNA polymerases (Poch et al . , EMBO J. 8:3867-3874 (1989)). It is more likely that the effect of these mutations is at amino acid level rather than nucleotide level, given that the mutation does not lie within the 3 ' and 5 ' genome termini nor the short gene-start and gene-end sequences.
  • RNA regions are thought to contain all of the cis-acting RNA sequences required for efficient encapsidation, transcription, replication, and packaging into virions (Collins et al., Proc. Natl. Acad. Sci . USA 88:9663-9667 (1991); Collins et al., Proc. Natl. Acad. Sci. USA 93:81-85 (1996), each incorporated herein by reference) .
  • the cpts248/404, cpts530/l009, and cpts530/1030 contain at least three mutations that contribute to the attenuation phenotype, two ts and one non-ts (e.g., the five cp mutations) , and this is a partial non-limiting explanation for the high level of stability of these viruses in vitro and in vivo. Determination of the complete sequence of RSV vaccine virus strains and of their parental viruses permits analysis at the genetic level of the stability of vaccine viruses during vaccine virus production and during shedding by volunteers in clinical trials.
  • a cDNA clone of, e.g., the cpts248/404 virus one or both of the ts mutations at amino acid position 521 (in the cpts530 mutant) or 1169 (in the cpts530/1009 mutant) or other attenuating or stabilizing mutations as desired.
  • the level of attenuation of the cpts248/404 virus can be increased in an incremental fashion and a vaccine strain that has the specific level of attenuation desired for both safety and immunogenicity can be generated in a rational way.
  • the level of attenuation of the cpts530/1009 and cpts530/1030 mutants can be increased by the specific introduction of one or more of the attenuating mutations in the cpts248/404 virus.
  • the stability of the attenuation phenotype can be augmented by site- directed mutagenesis of full-length RSV cDNA.
  • a cDNA clone encoding the antigenome of RSV strain A2 was constructed, as illustrated in Fig. 2.
  • the cDNA was synthesized in segments by reverse transcription (RT) and polymerase chain reaction (PCR) using synthetic oligonucleotides as primers and intraceUular RSV mRNA or genome RNA isolated from purified virions as template.
  • the final cDNA was flanked on the leader end by the promoter for T7 RNA polymerase, which included three transcribed G residues for optimal activity; transcription would result in the donation of these three nonviral G's to the 5' end of the antigenome.
  • the cDNA trailer end was constructed to be adjacent to a previously-described hammerhead ribozyme, which upon cleavage would donate a single 3 ' -phosphorylated U residue to the 3 ' end of the encoded RNA (Grosfeld et al . , J. Virol. 69:5677-5686 (1995), incorporated herein by reference).
  • the ribozyme sequence was followed by a tandem pair of terminators of T7 RNA polymerase.
  • Fig. 2 shows the structures of the cDNA and the encoded antigenome RNA.
  • the diagram of the antigenome includes the following features: the 5' -terminal nonviral G triplet contributed by the T7 promoter, the four sequence markers at positions 1099 (which adds one nt to the length) , 1139, 5611, and 7559, the ribozyme and tandem T7 terminators, and the single nonviral 3 ' -phosphorylated U residue contributed to the 3' end by ribozyme cleavage (the site of cleavage is indicated with an arrow) .
  • cDNA segments (Fig. 2, middle) representing in aggregate the complete antigenome were constructed by RT-PCR of RSV mRNA or genome RNA.
  • the complete antigenome cDNA is called D46 or D53; the different names referring to different preparations of the same plasmid.
  • Fig. 3 restriction site markers
  • sequence markers were inserted into the cDNA-encoded antigenome RNA as shown in Fig. 3. Sequences are positive sense and numbered relative to the first nt of the leader region complement as 1; identities between strains A2 and 18537 (Johnson and Collins, J. Gen Virol. 69:2901-2906 (1988), incorporated herein by reference) , representing subgroups A and B, respectively, are indicated with dots; sequences representing restriction sites in the cDNA are underlined; GS and GE transcription signals are boxed; the initiation codon of the N translational open reading frame at position 1141 is italicized, and the sequence markers are shown underneath each sequence .
  • a single C residue was inserted at position 1099 to create an Aflll site in the NS2-N intergenic region, and the AG at positions 1139 and 1140 immediately upstream of the N translational open reading frame were replaced with CC to create a new Ncol site.
  • substitution of G and U at positions 5612 and 5616, respectively created a new Stul site in the G-F intergenic region.
  • a C replacement at position 7560 created a new Sptil site in the F-M2 intergenic region.
  • the method of the invention for producing infectious RSV from cD ⁇ A-expressed antigenome involves its coexpression with those RSV proteins which are sufficient to (i) produce an_ antigenome nucleocapsid capable of R ⁇ A replication, and (ii) render the progeny genome nucleocapsid competent for both R ⁇ A replication and transcription. Transcription by the genome nucleocapsid provides all of the other RSV proteins and initiates a productive infection.
  • Plasmid-borne cD ⁇ A encoding the antigenome was transfected, together with plasmids encoding proteins ⁇ , P, L and M2 (ORF1) , into HEp-2 cells which had been infected with a recently-described vaccinia virus MVA strain recombinant which expresses the T7 R ⁇ A polymerase ( yatt et al., Virol . 210:202- 205 (1995) , incorporated herein by reference) .
  • the MVA strain is a host range mutant which grows permissively in avian cells whereas in mammalian cells there is a block at a late stage in virion maturation that greatly reduces the production of infectious virus.
  • the MVA recombinant was similar to the more commonly-used R-based recombinant (Fuerst et al., Proc. Natl. Acad. Sci. USA 83: 8122-8126 (1986)) with regard to the level of expression of T7 polymerase and cytopathogenicity, but the level of progeny produced was sufficiently low that supernatants could be passaged to fresh cells with minimal cytopathogenicity. This should facilitate the recovery of any recombinant RSV which might be produced in transfected, vaccinia virus-infected cells.
  • Transfection and recovery of recombinant RSV was performed as follows. Monolayer cultures of HEp-2 cells received, per single well of a six-well dish, one ml of infection-transfection medium prepared by mixing five plasmids in a final volume of 0.1 ml Opti-MEM (Life Technologies) medium, namely 0.4 ⁇ g each of antigenome, N and P plasmids, and 0.1 ⁇ g each of L and M2 (ORF1) plasmids. This was combined with 0.1 ml of Opti-MEM containing 12 ⁇ l LipofectACE (Life Technologies) .
  • the RSV-like plaques were picked from plates which were incubated under agarose and stained with neutral red. They were propagated and compared to a laboratory strain of RSV strain A2 by plaque assay and antibody staining. The plaques derived from the transfected cultures closely resembled those of the laboratory strain. One difference was that the plaques derived from the transfected cultures appeared to be slightly smaller than those from the laboratory strain, with centers which were less well cleared.
  • the recombinant virus may differ phenotypically from this particular wild-type isolate, possibly being slightly more restricted in cell-to-cell spread and exhibiting a reduced rate of cell killing.
  • the yields of the recombinant versus laboratory virus in HEp-2 cells were essentially identical at 32° or 37°C.
  • the recombinant and laboratory viruses were indistinguishable with regard to the accumulation of intraceUular RSV mRNAs and proteins .
  • Plaque-purified, thrice-passaged recombinant RSV was analyzed in parallel with laboratory virus by RT-PCR using three primer pairs flanking the four inserted markers .
  • Three independent plaque-purified recombinant RSV isolates were propagated in parallel with an uninfected control culture.
  • Clarified medium supernatants were treated with polyethylene glycol and high salt (Zoller and Smith, DNA 3:479-488 (1984)) to precipitate virus and RNA was extracted from the pellets with TrizolTM (Life Technologies) .
  • TrizolTM Life Technologies
  • These RNAs in parallel with additional controls of no added RNA or 0.1 ⁇ g of RNA from a laboratory isolate of strain A2, were treated with DNAse, repurified, annealed each with 50 ng of random hexamers and incubated under standard RT conditions (40 ⁇ l reactions) with or without reverse transcriptase (Connors et al . , Virol . 208:478- 484 (1995) , incorporated herein by reference) .
  • Primer pair (A) positive-sense, positions 925-942 and negative-sense, positions 1421-1440, yielding a predicted product of 516 bp (517 bp in the case of the recombinant viruses) that included the Alll and Ncol sites inserted at, respectively, the junction of the NS2 and N genes and in the N gene.
  • Primer pair (B) positive-sense, positions 5412-5429 and negative-sense, 5930-5949, yielding a predicted product of 538 bp spanning the Stul site inserted at the junction between the G and F genes.
  • Primer pair (C) positive-sense, 7280-7297 and negative-sense, 7690-7707, yielding a 428 bp fragment spanning the Sphl site inserted at the junction between the F and M2 genes.
  • PCR products were analyzed by electrophoresis on neutral gels containing 1% agarose and 2% low-melting agarose in parallel with HaelII-digested X174 DNA molecular length markers and visualized by staining with ethidium bromide. PCR products of the expected sizes were produced. The production of each was dependent on the RT step, indicating that each was derived from RNA rather than contaminating cDNA.
  • PCR products were analyzed by digestion with restriction enzymes. Digestion of products of primer pair A with Aflll or Ncol yielded fragments corresponding, to the predicted 177 and 340 bp (Aflll) or 217 and 300 bp (Ncol ) . Digestion of products of primer pair B with Stul yielded fragments comparable to the predicted 201 and 337 bp. Digestion of products from reactions with primer pair C with Sphl yielded products corresponding to the predicted 147 and 281 bp. The digests were analyzed by gel electrophoresis as above. The presence of residual undigested PCR product with Aflll was due to incomplete digestion, as was confirmed by redigestion. Thus, the restriction enzyme digestion showed that the PCR products representing recombinant virus contained the expected restriction site markers while those representing the laboratory strain did not . Nucleotide sequence analysis of cloned PCR product confirmed the sequences spanning the restriction site markers .
  • M2 (ORFl) protein in addition to N, P and L. Furthermore, it showed that the optimal method of expression of M2(0RF1) was from an engineered cDNA in which ORF2 had been deleted, although the complete cDNA containing both ORFs also supported the production of RSV.
  • the present invention demonstrates that transcription by RSV differs from that of from previously- described nonsegmented negative strand RNA viruses in requiring a fourth protein designated here as M2(0RF1), and previously called 22K or M2 (Collins et al . , J. Virol. 54:65-71 (1985)) .
  • M2 (ORFl) protein was found to be an RNA polymerase elongation factor that is important for processive, sequential transcription, and therefore must be provided (e.g., endoded by the genome or antigenome or expressed in trans by a separate plasmid or sequence within a shared vector) . (see, e.g., Collins et al . , Proc. Natl. Acad. Sci. USA 93:81-85 (1996)).
  • This requirement provides the capability, as part of this invention, for introducing specific, predetermined changes into infectious RSV.
  • This Example illustrates the introduction of specific predetermined mutations into infectious RSV using the recombinant methods described herein.
  • the complete nucleotide sequence of cpts530 RSV was determined and 5 mutations known to be present in the parent cpRSV were retained in cpts530, the further attenuated derivative.
  • One additional nucleotide change was identified at nucleotide (nt) position 10060, which resulted in a phenylalanine to leucine change at amino acid position 521 in the large polymerase (L) protein (see Tables 37, 39) .
  • This single amino acid substitution was introduced alone or in combination with the cp mutations into the full-length cDNA clone of wild-type A2 RSV.
  • This nucleotide substitution at position 12002 resulted in an amino acid change in L at position 1169 at which a methionine in the wild-type virus was replaced by a valine in the cpts530/1009 mutant.
  • This mutation has also been introduced into recombinant RSV, and the recovered virus was temperature sensitive.
  • the levels of temperature sensitivity among the 530, 530/1009 and 530/1030 recombinant virus have been confirmed with RSV-CAT or RSV-Luciferase minigenome (see above) monitored by enzyme assay or Northern blot analysis.
  • RSV-CAT or RSV-Luciferase minigenome monitored by enzyme assay or Northern blot analysis.
  • the luciferase activity generated by selected mutants relative to wild-type L protein was 18.4%, 1.5% and 0.4% for 1009, 530, and the double mutant pTMl-L support plasmid.
  • These exemplary mutations also decreased L function at 32°C with 70% activity for 1009, 40% activity for 530, and 12.5% activity for 530/1009 L protein compared to wt L protein.
  • the effects of these mutations on transcription and replication can also be determined using the minigenome system, alone or in combination with the recombinant viral methods disclosed herein.
  • nucleotide differences between wild-type and mutants are underlined. Recognition sites of restriction endonucleases are in italics. Codons in which the introduced nucleotide change(s) results in amino acid substitution are in bold. Asterisk identifies the single nucleotide change that was present in the biologically-derived mutant virus. Numbering system reflects the one nucleotide insertion in the full lengt cDNA.
  • the cDNA-based recovery system described herein was employed as follows.
  • the previously-described RSV A2 wild-type full-length cDNA clone (Collins et al., supra. , (1995)) was designed in the original construction to contain a single nucleotide insertion of C in the cDNA clone at nt position 1099 (which creates an Aflll site) and a total of 6 additional nucleotide substitutions at 4 loci.
  • nucleotide numbering system for the naturally occurring virus and for the recombinant viruses derived from cDNA are out of register by one nt after position 1099.
  • the nucleotide numbering in Tables 36 and 37, above, represents the positions for the naturally occurring viruses, while those for the cDNA clones (Table 39) and recombinant viruses derived from cDNA clones are one nucleotide more.
  • One of the 6 nucleotide substitutions is a G to C change in genome sense at nt position 4 in the leader sequence.
  • the D50 plasmid contains the RSV genome from the leader to the M2-L overlap downstream of a T7 promoter, while the D39 plasmid encodes the full-length L gene and the trailer followed by the hammerhead ribozyme and two T7 terminators (approximately 7 kb in length) bordered by BamHI and Alul restriction sites.
  • the full-length RSV cDNA clone (D53) used in transfections to rescue infectious virus was assembled by inserting the BamHI-Mlui fragment of the D39 plasmid into the D50 plasmid (see U.S. Patent Application No. 08/720,132; and published PCT Application No. PCT/US96/15524, each incorporated herein by reference) .
  • D50 was further separated into several pieces each placed in a phagemid plasmid for the purposes of facilitating mutagenesis : one piece was an Xbal-.Eco.RI fragment containing the N gene (cDNA pUC118.D50N) , and one was a StuI-BamHI fragment containing the F and M2 genes (pUC118.F-M2) .
  • D39 was further separated into two pieces each placed in a separate phagemid plasmid: one piece
  • cDNA pUC119.Ll runs from the BamHI site to the P ll site at nucleotide 12255 (note that the sequence positions assigned to restriction site locations here and throughout are intended as a descriptive guide and do not alone precisely define all of the nucleotides involved) , and the other (right hand half, cDNA pUC119.L2) from the Pm/I site to the end of the T7 terminator.
  • Mutations were placed into the pUCll ⁇ - and pUC119- based constructs illustrated in the bottom row of Fig. 4 following standard procedures (see, e.g., Kunkel et al . , Methods Enzymol, 54:367-382 (1987), incorporated herein by reference).
  • the plasmids were propagated in a dut ung strain of E. coli . , in this case CJ236, and single stranded DNA was prepared by infection with a helper phage, in this case M13K07.
  • Phosphorylated synthetic oligonucleotides each containing one or more nucleotide changes of interest were prepared, annealed to the single stranded template singly or in combination, and used to direct DNA synthesis by T4 DNA polymerase.
  • the products were ligated and transformed into a non-dut ung strain of E. coli, in this case DH5alpha or DH10B.
  • Miniprep DNA of the transformanl- colonies was screened for the presence of the mutation by restriction enzyme digestion or by nucleotide sequence analysis of the mutagenized region.
  • the first group of mutations involves six translationally silent new restriction site markers introduced into the L gene, collectively called the "sites" mutations.
  • the six sites are Bsu36I, SnaBI , Pmel , RsrII , BstEII and a second, downstream SnaBI site, and are underlined in Fig. 4 above the
  • D53 diagram D53 diagram. These six changes, collectively referred to as the "sites" mutations, were inserted for the purpose of facilitating cDNA construction. Also, it is known that recombination can occur during transfection between the D53 plasmid and the support plasmids, i.e., the N, P, M2(0RF1) and L plasmids
  • the second group of mutations involves two amino acid changes in the F gene (Fig. 4, Table 39) .
  • the cpRSV, and hence all of its derivatives, is derived from a wild-type virus called HEK-7.
  • the sequence of the original D53 cDNA differs from that of HEK-7 by single nucleotide substitutions at seven positions.
  • One is at nucleotide 4, which is a C (in negative sense) in the original D53 and G in the HEK virus.
  • biologically- derived viruses have been shown to contain either assignment, and can fluctuate between the two, and so this difference is considered incidental and not considered further here.
  • the third group of mutations involved the five amino acid substitutions found in the cpRSV virus, collectively called the "cp" mutations. These are present in all of the biologically-derived cpts viruses and contribute to the attenuation phenotype. In biologically-derived cpRSV, each of these amino acid changes is due to a single nucleotide change . As shown in Table 39, when the amino acid coding change was introduced into cDNA to make recombinant virus in four of the five cases each coding change was made to involve two nucleotide substitutions, which renders the recombinant RSV highly resistant to reversion to wild-type.
  • the fourth group of mutations involves point mutations specific to individual, biologically derived cpts viruses (Table 39, Fig. 4) , which are named after the biological step at which they were acquired.
  • derivation of the cpts248/404 virus from cpRSV in the following Example involved two steps of mutagenesis. The first yielded the cpts248 virus, which sustained a single amino acid change that is therefore called the 248 mutation.
  • the 404 (M2) mutation is noteworthy because it involves the GS transcription signal (and does not involve a protein- coding sequence) and because this mutation was shown in a minigenome system to be important for synthesis of the mRNA. Kuo et al., J. Virol. 71:4944-4953 (1977) (incorporated herein by reference) .
  • the amino acid coding changes of the 248, 404 (L) , and 1009 mutations were inserted into recombinant virus using two nucleotide substitutions for the purpose of improved genetic stability. Also, the 248, 404 (M2), 404 (L) , and 1009 mutations for recombinant virus were each designed to introduce a new restriction site for monitoring purposes, while the 1030 mutation was designed to ablate an existing site.
  • Mutagenesis was performed using the Muta-Gene ® Phagemid in vitro Mutagenesis kit (Bio-Rad, Hercules, CA) as recommended by the manufacturer.
  • the mutagenized constructs were transformed into competent E. coli DH10B (Life Technologies) .
  • Miniprep DNA of the transformant colonies was screened for the presence of the mutation by restriction enzyme digestion (see below) or by nucleotide sequence analysis of the mutagenized region.
  • the six translationally silent restriction site markers, the 530 mutation ( 521 phe ⁇ leu) , and the 5 cp mutations (Table 39) were introduced into the pUC-based constructs and subcloned into the D50 and D39 plasmids as indicated in Figs. 4 and 5.
  • the various full-length cDNA constructs were assembled using D50 and D39 constructs containing different combinations of the above mentioned mutations .
  • each D53-based construct was analyzed using various restriction enzymes (e.g. Hpal, Accl, Hi-ndlll, Pstl) , and the restriction patterns of the newly generated full-length cDNA clones were compared with that of the previously rescued wild-type full-length cDNA clone. This restriction analysis was used to determine if an insertion or deletion of 100 nt or more had occurred during the bacterial amplification of the full-length plasmids.
  • various restriction enzymes e.g. Hpal, Accl, Hi-ndlll, Pstl
  • Transfection was performed as described previously (Collins, et al., Proc. Natl. Acad. Sci. USA. 92:11563-11567 (1995) , incorporated herein by reference) . Briefly, monolayers of HEp-2 cells were infected at an MOI of 1 with recombinant vaccinia virus MVA strain expressing T7 RNA polymerase (MVA-T7) and were transfected using LipofectACE (Life Technologies) with a D53 antigenomic construct plus the N, P, L and M2 (ORFl) pTMl support plasmids. On day three, supernatants (clarified medium) were passaged onto fresh HEp-2 cells for amplification of rescued virus.
  • Virus suspensions from this first amplification were harvested 5 days after infection and, following inoculation at various dilutions onto monolayers of HEp-2 cells, were overlayed with methylcellulose for plaque enumeration or with agarose for plaque harvest and biological cloning. Plaque enumeration was performed using a monoclonal antibody-horseradish peroxidase staining procedure as previously described (Murphy et al., Vaccine, 8:497-502 (1990), incorporated herein by reference) . The recovered recombinant viruses were biologically cloned by three successive plaque purifications, and then used to generate virus suspensions following two passages on HEp-2 cells.
  • the biological cloning was important to ensure a homogeneous population of the recovered viruses, as recombination may arise during the first step of the rescue between the plasmid representing the full-length cDNA of RSV and the support plasmids containing RSV genes (Garcin et al., EMBO J. , 14:6087-6094 (1995), incorporated herein by reference) .
  • These biologically cloned and amplified virus suspensions were used in further molecular genetic or phenotypic characterization of the recombinant viruses.
  • Two biologically cloned recombinant viruses were generated for each of the cDNA constructs (Fig.
  • the recombinant RSVs generated as described above were genetically characterized to determine if they indeed contained each of the introduced mutations.
  • Monolayers of HEp-2 cells were infected with biologically cloned recombinant virus and total RNA was harvested 4 to 5 days post infection as described above.
  • RT was performed using random hexamer primers and the generated cDNA was used as template in PCR using the AdvantageTM cDNA PCR Kit (Clontech Lab. Inc., Palo Alto, CA) to generate three fragments representing almost the full-length of the recombinant RSV genomes.
  • the PCR fragments corresponded to the RSV genome between nt positions 1-5131, 5949 -10751 and 8501-15179.
  • a 544 bp fragment representing a portion of the L gene in the region of the 530 mutation between nt positions 9665 and 10209 was generated.
  • This short PCR fragment was used in cycle sequencing (using 71001 delta TAQTM* Cycle Sequencing Kit, USB, Cleveland, OH) to confirm the presence or the absence of the 530 mutation in the recovered recombinant virus, whereas the large PCR products were used in restriction enzyme digestion to confirm the presence of the silent restriction site markers and the cp mutations which were marked with specific restriction sites.
  • the efficiency of plaque formation (EOP) of the recombinant RSVs and the nonrecombinant control viruses was determined. Specifically, plaque titration at 32, 37, 38, 39 and 40°C using HEp-2 monolayer cultures in temperature controlled water baths was conducted, as described previously
  • Table 40 Comparison of the Efficiency of Plaque Formation 3 of Recombinant and Biologically Derived Viruses in HEp-2 Cells at Various Temperatures .
  • Virus titers are the average of two tests, except for r-sites and r cp- sites where data were derived from a single test .
  • ts phenotype of the biologically derived cpts530 virus is specified by the single mutation identified above as being unique to this attenuated RSV strain .
  • Genetic analysis of the cpts530 strain was confirmed in this context by the introduction of the 530 mutation into a full-length cDNA clone of the A2 wild- type ts* parent virus , followed by the recovery of a ts recombinant virus bearing the
  • the above findings illustrate several important advantages of the recombinant methods and RSV clones of the invention for developing live attenuated RSV vaccines .
  • the insertion of a selected mutation into recombinant RSV, as well as the recovery of mutations from the RSV A2 cDNA clone were relatively efficient.
  • the antigenome cDNA clone used in this example had been modified in the original construction to contain changes at five different loci, involving 6 nucleotide substitutions and one nucleotide insertion.
  • Mutagenized virus are also described containing mutations at an additional twelve loci involving 24 additional nucleotide substitutions .
  • This Example illustrates additional designs for introducing predetermined attenuating mutations into infectious RSV employing the recombinant procedures and materials described herein.
  • the biologically-derived attenuated RSV strain cpts248/404 was reconstructed as a recombinant virus (rA2cp/248/404) according to the above described methods.
  • cDNA D53 encoding rA2cp/248/404 virus was constructed by insertion of the sites, HEK, cp, 248 and 404 changes (Table 39) .
  • Recombinant virus (rA2cp/248/404) was recovered, plaque purified and amplified. The presence of the mutations in recombinant virus was analyzed by RT-PCR of viral RNA followed by restriction enzyme digestion or nucleotide sequencing or both.
  • the rA2cp/248/404 recombinant was recovered using either the pTMLwt or pTML248/404 support plasmid, the latter of which contains all of the mutations in L present in the biologically derived cpts248/404 mutant (Table 39, not including the mutations specific to the 530, 1009, or 1030 viruses) .
  • pTML248/404 as a support plasmid precludes loss of 248/404 mutations present in a full length clone by homologous recombination with the support plasmid.

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  • Plant Pathology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicinal Preparation (AREA)

Abstract

Selon la présente invention, on produit un virus respiratoire syncytial (VRS) chimérique ainsi que des compositions vaccinales à base dudit virus en introduisant un ou plusieurs gènes ou segments de gène hétérologues tirés d'un sous-groupe ou d'une souche du virus VRS dans un environnement VRS receveur d'un sous-groupe ou d'une souche différente. Le virus VRS ou la particule sous-virale chimérique obtenu est infectieux et atténué, de préférence, par introduction de mutations sélectionnées spécifiant des phénotypes atténués dans un génome ou antigénome chimérique, ce qui permet d'obtenir, par exemple, des souches de vaccins sensibles à la température (ts) et/ou adaptées au froid (ca). Selon un autre mode de réalisation, le virus VRS chimérique et les compositions vaccinales à base dudit virus comportent d'autres mutations spécifiant des caractéristiques structurelles et/ou phénotypiques voulues dans un virus VRS chimérique infectieux. Un tel virus VRS infectieux comporte des mutations voulues spécifiées par insertion, délétion, substitution ou réagencement d'une ou plusieurs séquences nucléotidiques, de un ou plusieurs gènes ou segments de gènes voulus dans un clone de VRS chimérique. L'invention concerne donc une méthode de mise au point de nouveaux vaccins dirigés contre diverses souches de VRS utilisant un squelette commun atténué comme vecteur d'expression des antigènes protecteurs des souches hétérologues. Le système immunitaire d'un individu est stimulé de façon à induire une protection contre une infection à VRS naturel, de préférence de façon polyvalente, et ce en vue de l'obtention d'une protection contre plusieurs souches et/ou sous-groupes de VRS.
EP00920058A 1999-04-13 2000-03-31 Production de vaccins a base d'un virus respiratoire syncytial chimerique attenue a partir de sequences nucleotidiques clonees Ceased EP1169457A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP06075156A EP1690940A1 (fr) 1999-04-13 2000-03-31 Production de vaccins à base d'un virus respiratoire syncytial chimérique attenue à partir de séquences nucléotidiques clonées

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US09/291,894 US6689367B1 (en) 1995-09-27 1999-04-13 Production of attenuated chimeric respiratory syncytial virus vaccines from cloned nucleotide sequences
US291894 1999-04-13
PCT/US2000/008802 WO2000061611A2 (fr) 1999-04-13 2000-03-31 Production de vaccins a base d'un virus respiratoire syncytial chimerique attenue a partir de sequences nucleotidiques clonees

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EP06075156A Ceased EP1690940A1 (fr) 1999-04-13 2000-03-31 Production de vaccins à base d'un virus respiratoire syncytial chimérique attenue à partir de séquences nucléotidiques clonées
EP00920058A Ceased EP1169457A2 (fr) 1999-04-13 2000-03-31 Production de vaccins a base d'un virus respiratoire syncytial chimerique attenue a partir de sequences nucleotidiques clonees

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EP (2) EP1690940A1 (fr)
JP (1) JP2002541785A (fr)
KR (1) KR100746979B1 (fr)
CN (1) CN100491530C (fr)
AU (1) AU4065500A (fr)
BR (1) BR0011160A (fr)
CA (1) CA2369200A1 (fr)
IL (1) IL145796A0 (fr)
MX (1) MXPA01010320A (fr)
WO (1) WO2000061611A2 (fr)

Families Citing this family (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2342237T3 (es) * 1995-09-27 2010-07-02 The Usa, As Represented By The Secretary Of The Department Of Health And Human Services Produccion de virus sinciciales respiratorios infecciosos a partir de secuencias clonadas de nucleotidos.
US7485440B2 (en) 1995-09-27 2009-02-03 The United States Of America As Represented By The Department Of Health And Human Services Production of attenuated respiratory syncytial virus vaccines involving modification of M2 ORF2
JP4413999B2 (ja) 1996-07-15 2010-02-10 アメリカ合衆国 クローニングされたヌクレオチド配列からの弱毒化呼吸シンシチウムウィルスワクチンの製造
US6699476B1 (en) 1996-07-15 2004-03-02 Peter L. Collins Production of recombinant respiratory syncytial viruses expressing immune modulatory molecules
US7201907B1 (en) 1997-05-23 2007-04-10 The United States Of America, Represented By The Secretary, Department Of Health And Human Services Attenuated human-bovine chimeric parainfluenza virus(PIV) vaccines
US20030082209A1 (en) 2000-07-05 2003-05-01 Skiadopoulos Mario H. Attenuated human-bovine chimeric parainfluenza virus (PIV) vaccines
US7951383B2 (en) 1997-05-23 2011-05-31 The United States Of America As Represented By The Department Of Health And Human Services Attenuated parainfluenza virus (PIV) vaccines
US7632508B2 (en) 1997-05-23 2009-12-15 The United States Of America Attenuated human-bovine chimeric parainfluenza virus (PIV) vaccines
AU2006200619B2 (en) * 1997-09-26 2007-12-13 Medimmune, Llc Recombinant RSV virus expression systems and vaccines
WO2001004335A2 (fr) 1999-07-09 2001-01-18 The Government Of The United States Of America, As Represented By The Department Of Health And Humanservices Production de vaccins a virus respiratoires syncitiaux chimeres humains-bovins attenues
WO2001004321A1 (fr) * 1999-07-09 2001-01-18 The Government Of The United States Of America, As Represented By The Department Of Health And Human Services Production de vaccins attenues contre le virus syncytial respiratoire impliquant une modification de m2 orf2
EP1572947B1 (fr) 2002-09-18 2014-06-25 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Recuperation du virus parainfluenza humain de type 2 (hpiv2) recombine a partir d'adnc et utilisation de ce virus hpiv2 recombine dans des compositions immunogenes et en tant que vecteurs pour induire des reponses immunitaires contre le virus parainfluenza et d'autres pathogenes humains
JP2007524372A (ja) 2003-02-28 2007-08-30 ザ ガバメント オブ ザ ユナイテッド ステイツ オブ アメリカ アズ リプレゼンテッド バイ ザ セクレタリー オブ ザ デパートメント オブ ヘルス アンド ヒューマン サービシーズ 組換えヒトメタニューモウイルスおよびその使用
WO2004113517A2 (fr) 2003-06-09 2004-12-29 Wyeth Ameliorations apportees a un procede de recuperation de virus a arn de polarite negative non segmente a partir d'adnc
US8597637B1 (en) * 2010-05-18 2013-12-03 Weidong Zhang Breast cancer therapy using an engineered respiratory syncytial virus
PL2954049T3 (pl) 2013-02-08 2023-10-16 The U.S.A. As Represented By The Secretary, Department Of Health And Human Services Atenuacja ludzkiego syncytialnego wirusa oddechowego przez deoptymalizację par kodonów w skali genomu
JP7198759B2 (ja) 2016-09-23 2023-01-04 ザ・ユナイテッド・ステイツ・オブ・アメリカ・アズ・リプリゼンテッド・バイ・ザ・セクレタリー・デパートメント・オブ・ヘルス・アンド・ヒューマン・サービシーズ 弱毒化表現型を有するヒト呼吸器多核体ウイルス（ｒｓｖ）のためのワクチン候補
CN107384958B (zh) * 2017-07-14 2020-04-10 北京交通大学 基于反向遗传学构建的rsv反基因组质粒及其应用
CN113462656B (zh) * 2021-03-24 2022-09-30 兰州生物制品研究所有限责任公司 一种人三型副流感病毒冷适应温度敏感株及其应用

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US708395A (en) 1901-04-15 1902-09-02 Oscar Peterson Device for digging ditches.
US2177396A (en) 1936-04-18 1939-10-24 Borden Co Milk bottle
US4614197A (en) 1984-10-15 1986-09-30 Deere & Company Chaff spreading arrangement for a combine
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4763497A (en) 1986-09-03 1988-08-16 Clover Co., Ltd. Lock device for double sliding doors
US4800078A (en) 1987-05-28 1989-01-24 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Immunotherapeutic method of treating respiratory disease by intranasal administration of Igb
IL105456A (en) 1992-04-21 1996-12-05 American Home Prod Vaccines of attenuated respiratory syncytial virus
US20030054505A1 (en) * 1997-09-26 2003-03-20 Hong Jin Recombinant rsv expression systems and vaccines
DK0780475T4 (da) 1995-08-09 2006-10-23 Schweiz Serum & Impfinst Fremgangsmåde til fremstilling af infektiöse negativ-streng RNA-virus
JPH11512609A (ja) 1995-09-27 1999-11-02 アメリカ合衆国 クローン化されたヌクレオチド配列からの感染性ＲＳウイルス（ｒｅｓｐｉｒａｔｏｒｙ ｓｙｎｃｙｔｉａｌ ｖｉｒｕｓ）の生産
US6077514A (en) * 1996-04-04 2000-06-20 The Regents Of The University Of Michigan Attenuated respiratory syncytial virus
JP4413999B2 (ja) * 1996-07-15 2010-02-10 アメリカ合衆国 クローニングされたヌクレオチド配列からの弱毒化呼吸シンシチウムウィルスワクチンの製造
CN1250725C (zh) 1997-05-23 2006-04-12 美国政府健康及人类服务部 从克隆的核苷酸序列制备减毒副流感病毒疫苗的方法
US7208161B1 (en) 1997-05-23 2007-04-24 The United States Of America, Represented By The Secretary, Department Of Health And Human Services Production of attenuated parainfluenza virus vaccines from cloned nucleotide sequences
UA67845C2 (uk) * 1999-04-23 2004-07-15 Smithkline Beecham Plc Поліморф 5-[4-[2- (n-метил-n-(2-піридил)аміно)етокси]бензил]тіазолідин-2,4-діону солі малеїнової кислоти

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0061611A2 *

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CA2369200A1 (fr) 2000-10-19
MXPA01010320A (es) 2003-09-15
BR0011160A (pt) 2002-10-08
CN100491530C (zh) 2009-05-27
AU4065500A (en) 2000-11-14
KR20020013526A (ko) 2002-02-20
JP2002541785A (ja) 2002-12-10
IL145796A0 (en) 2002-07-25
WO2000061611A2 (fr) 2000-10-19
WO2000061611A9 (fr) 2002-03-28
EP1690940A1 (fr) 2006-08-16
KR100746979B1 (ko) 2007-08-07
CN1364195A (zh) 2002-08-14
WO2000061611A3 (fr) 2001-01-04

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