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WO2002002605A2 - Vaccins pour l'homme et les bovins a base de chimeres attenuees du virus de la parainfluenza (piv) de l'homme et du bovin - Google Patents

Vaccins pour l'homme et les bovins a base de chimeres attenuees du virus de la parainfluenza (piv) de l'homme et du bovin Download PDF

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WO2002002605A2
WO2002002605A2 PCT/US2001/021527 US0121527W WO0202605A2 WO 2002002605 A2 WO2002002605 A2 WO 2002002605A2 US 0121527 W US0121527 W US 0121527W WO 0202605 A2 WO0202605 A2 WO 0202605A2
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piv
genome
chimeric
antigenome
gene
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PCT/US2001/021527
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WO2002002605A3 (fr
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Mario H. Skiadopoulos
Peter L. Collins
Brian R. Murphy
Alexander C. Schmidt
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The Government Of The United States Of America, As Represented By The Department Of Health And Human Services
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Priority to AU2001271909A priority Critical patent/AU2001271909A1/en
Publication of WO2002002605A2 publication Critical patent/WO2002002605A2/fr
Publication of WO2002002605A3 publication Critical patent/WO2002002605A3/fr

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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/544—Mucosal route to the airways
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18611—Respirovirus, e.g. Bovine, human parainfluenza 1,3
    • C12N2760/18634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18611—Respirovirus, e.g. Bovine, human parainfluenza 1,3
    • C12N2760/18661—Methods of inactivation or attenuation

Definitions

  • HPIV3 Human parainfluenza virus type 3
  • RSV respiratory syncytial virus
  • HPIVl and HPIV2 are the principal etiologic agents of laryngotracheobronchitis (croup) and also can cause severe pneumonia and bronchiolitis (Collins et al, 1996, supra).
  • HPIVl, HPIV2, and HPIV3 were identified as etiologic agents for 6.0, 3.2, and 11.5%, respectively, of hospitalizations for respiratory tract disease accounting in total for 18% of the hospitalizations, and, for this reason, there is a need for an effective vaccine (Murphy et al, Virus Res. 11:1-15, 1988).
  • HPIVl, HPIV2, and HPIV3 are distinct serotypes that do not elicit significant cross-protective immunity.
  • a second PIV3 vaccine candidate, JS cp45 is a cold-adapted mutant of the JS wildtype (wt) strain of HPIV3 (Karron et al, 1995b, supra; and Belshe et al, J. Med. Virol. 10:235-242, 1982a; each incorporated herein by reference).
  • This live, attenuated, cold-passaged (cp) PIV3 vaccine candidate exhibits temperature-sensitive (ts), cold- adaptation (ca), and attenuation (att) phenotypes, which are stable after viral replication in vitro.
  • the cp45 virus is protective against human PIV3 challenge in experimental animals and is attenuated, genetically stable, and immunogenic in seronegative human infants and children (Belshe et al, 1982a, supra; Belshe et al, Infect. Immun. 37:160- 165, 1982b: Clements et al. J. Clin. Microbiol. 29:1175-1182. 1991; Crookshanks et al, J. Med. Virol. 13:243-249. 1984; Hall et al. Virus Res. 22:173-184. 1992; Karron et al, 1995b, supra; each incorporated herein by reference). Because these PIV3 candidate vaccine viruses are biologically derived there is no proven method for adjusting their level of attenuation as will likely be necessary for broad clinical application.
  • these disclosures allow for genetic manipulation of viral cDNA clones to determine the genetic basis of phenotypic changes in biological mutants, e.g., which mutations in the HPIV3 cp45 virus specify its ts, ca and att phenotypes, and which gene(s) or genome segment(s) of BPIV3 specify its attenuation phenotype.
  • these and related disclosures render it feasible to construct novel PIV vaccine candidates having a wide range of different mutations and to evaluate their level of attenuation, immunogenicity and phenotypic stability (see also, U.S. Patent Application Serial No. 09/586,479 and its priority Provisional Patent Application Serial No. 60/143,134, filed by Bailly et al. on July 9, 1999; and U.S. Patent Application Serial No. 09/350,821, filed by Durbin et al on July 9, 1999; each incorporated herein by reference).
  • infectious wild type recombinant PIV3 (r)PIV3, as well as a number of ts derivatives, have now been recovered from cDNA, and reverse genetics systems have been used to generate infectious virus bearing defined attenuating mutations and to study the genetic basis of attenuation of existing vaccine viruses.
  • the three amino acid substitutions found in the L gene of cp45, singularly or in combination, have been found to specify the ts and attenuation phenotypes. Additional ts and attenuating mutations are present in other regions of the PIV3 45.
  • a chimeric PIV1 vaccine candidate has been generated using the PIV3 cDNA rescue system by replacing the PIV3 HN and F open reading frames (ORFs) with those of PIV 1 in a PIV3 full-length cDNA that contains the three attenuating mutations in L.
  • the recombinant chimeric virus derived from this cDNA is designated rP ⁇ V3-l.cp45L (Skiadopoulos et al., J. Virol. 72:1762-1768, 1998: Tao et al. J. Virol. 72:2955-2961. 1998: Tao et al. Vaccine 17:1100-1108, 1999, incorporated herein by reference).
  • rPIV3-l.cp45L was attenuated in hamsters and induced a high level of resistance to challenge with PIV1.
  • a recombinant chimeric virus, designated rPIV3-l.cp45, has been produced that contains 12 of the 15 cp45 mutations, i.e., excluding the mutations that occur in HN and F, and is highly attenuated in the upper and lower respiratory tract of hamsters (Skiadopoulos et al., Vaccine 18:503-510, 1999a).
  • BPIV3 which is antigenically-related to HPIV3, offers an alternative approach to the development of a live attenuated virus vaccine for HPIVl, HPIV2, and HPIV3.
  • the first vaccine used in humans live vaccinia virus believed to be of bovine origin, was developed by Jenner almost 200 years ago for the control of smallpox. During the ensuing two centuries, vaccinia virus was successful in controlling this disease and played an essential role in the final eradication of smallpox. In this "Jennerian" approach to vaccine development, an antigenically-related animal virus is used as a vaccine for humans. Animal viruses that are well adapted to their natural host often do not replicate efficiently in humans and hence are attenuated.
  • the recently licensed quadrivalent rotavirus is an example of the Jennerian approach to vaccine development in which a nonhuman rotavirus strain, the rhesus rotavirus (RRV), was found to be attenuated in humans and protective against human serotype 3 to which it is antigenically highly related (Kapikian et al, Adv. Exp. Med. Biol. 327:59-69, 1992). Since there was a need for a nmltivalent vaccine that would induce resistance to each of the four major human rotavirus serotypes, the Jennerian approach was modified by constructing three reassortant viruses using conventional genetic techniques of gene reassortment in tissue culture.
  • RRV rhesus rotavirus
  • Each single gene reassortant virus contained 10 RRV genes plus a single human rotavirus gene that coded for the major neutralization antigen (VP7) of serotype 1, 2, or 4.
  • the intent was to prepare single gene substitution RRV reassortants with the attenuation characteristics of this simian virus and the neutralization specificity of human rotavirus serotype 1, 2, or 4.
  • the quadrivalent vaccine based on the host range restriction of the simian RRV in humans provided a high level of efficacy against human rotavirus infection in infants and young children (Perez-Schael et al, N. Enel. J. Med. 337:1181-1187, 1997).
  • the vaccine virus retains mild reactogenicity in older seronegative infants lacking maternal antibody, therefore a second generation Jennerian vaccine, based on the UK strain of bovine rotavirus, is being developed to replace the RRV vaccine (Clements-Mann et al, Vaccine 17:2715-2725. 1999).
  • the Jennerian approach also is being explored to develop vaccines for parainfluenza type 1 virus and for hepatitis A virus which are attenuated and immunogenic in non-human primates (Emerson et al, J. Infect. Pis. 173:592-597, 1996; Hurwitz et al, Vaccine 15:533-540, 1997).
  • the Jennerian approach was used for the development of a live attenuated vaccine for influenza A virus but it failed to produce a consistently attenuated vaccine for use in humans (Steinhoff et al. , J. Infect. Pis.
  • reassortant viruses that contain two gene segments encoding the hemagglutinin and neuraminidase surface glycoproteins from a human influenza A virus and the six remaining gene segments from an avian influenza A virus were attenuated in humans (Clements et al, J. Clin. Microbiol. 27:219-222, 1989; Murphy et al, J. Infect. Pis. 152:225-229, 1985; and Snyder et al, J. Clin. Microbiol. 23:852-857, 1986). This indicated that one or more of the six gene segments of the avian virus attenuated the avian-human influenza A viruses for humans.
  • the genetic determinants of this attenuation were mapped using reassortant viruses possessing a single gene segment from an attenuating avian influenza A virus and the remaining genes from a human strain. It was shown that the nonstructural (NS), polymerase (PB 1, PB2) and M genes contributed to the attenuation phenotype of avian influenza A viruses in humans (Clements et al, J. Clin. Microbiol. 30:655-662, 1992).
  • BRSV bovine respiratory syncytial virus
  • the instant invention provides a new basis for attenuating a wild type or mutant parental virus for use as a vaccine against HPF/, in which attenuation is based completely or in part on host range effects, while at least one or more of the major neutralization and protective antigenic determinant(s) of the chimeric virus is homologous to the virus against which the vaccine is directed.
  • the HN and F proteins of BPIV3 are each approximately 80% related by amino acid sequence to their corresponding HPIV3 proteins (Suzu et al, Nucleic Acids Res. 15:2945-2958, 1987, incorporated herein by reference) and 25% related by antigenic analysis (Coelingh et al, J. Virol.
  • BPIV3 has host range genes that restrict replication in the respiratory tract of rhesus monkeys, chimpanzees and humans, it remains unknown which of the bovine proteins or noncoding sequences contribute to this host range restriction of replication. It is possible that any of the BPIV3 proteins or noncoding sequences may confer a host range phenotype. It is not possible to determine in advance which genes or genome segments will confer an attenuation phenotype. This can only be accomplished by systematic substitution of BPIV3 coding and non-coding sequences for their HPIV3 counterparts and by evaluation of the recovered HPIV3/BPIV3 chimeric viruses in seronegative rhesus monkeys or humans.
  • the present invention provides human-bovine chimeric parainfluenza viruses (PIVs) that are infectious and attenuated in humans and other mammals.
  • the invention provides novel methods for designing and producing attenuated, human-bovine chimeric PIVs that are useful in various compositions to generate a desired immune response against PIV in a host susceptible to PIV infection.
  • methods and compositions incorporating human-bovine chimeric PIV for prophylaxis and treatment of PIV infection are also provided within the invention.
  • the invention thus involves a method for developing live attenuated PIV vaccine candidates based on chimeras between HPIVs and BPIV3.
  • Chimeras are constructed through a cDNA-based virus recovery system. Recombinant viruses made from cDNA replicate independently and are propagated in the same manner as if they were biologically-derived viruses.
  • Chimeric human-bovine PIV of the invention are recombinantly engineered to incorporate nucleotide sequences from both human and bovine PIV strains to produce an infectious, chimeric virus or subviral particle.
  • candidate vaccine viruses are recombinantly engineered to elicit an immune response against PIV in a mammalian host susceptible to PIV infection, including humans and non-human primates.
  • Human-bovine chimeric PIV according to the invention may elicit an immune response to a specific PTV, e.g., HPIV3, or a polyspecific response against multiple PIVs, e.g., HPIVl and HPIV3.
  • Additional chimeric viruses can be designed in accordance with the teachings herein which serve as vectors for antigens of non-PIV pathogens, for example respiratory syncytial virus (RSV) or measles virus.
  • RSV respiratory syncytial virus
  • Exemplary human-bovine chimeric PIV of the invention incorporate a chimeric PIV genome or antigenome comprising both human and bovine polynucleotide sequences, as well as a major nucleocapsid (N) protein, a nucleocapsid phosphoprotein (P), and a large polymerase protein (L). Additional PIV proteins may be included in various combinations to provide a range of infectious subviral particles, up to a complete viral particle or a viral particle containing supernumerary proteins, antigenic determinants or other additional components.
  • Chimeric human-bovine PIV of the invention include a partial or complete "background" PIV genome or antigenome derived from or patterned after a human or bovine PIV strain or subgroup virus combined with one or more heterologous gene(s) or genome segment(s) of a different PIV strain or subgroup virus to form the human-bovine chimeric PTV genome or antigenome.
  • chimeric PIV incorporate a partial or complete human PIV background genome or antigenome combined with one or more heterologous gene(s) or genome segment(s) from a bovine PIV.
  • the partial or complete background genome or antigenome typically acts as a recipient backbone or vector into which are imported heterologous genes or genome segments of the counterpart, human or bovine PIV.
  • Heterologous genes or genome segments from the counterpart, human or bovine PIV represent "donor" genes or polynucleotides that are combined with, or substituted within, the background genome or antigenome to yield a human-bovine chimeric PIV that exhibits novel phenotypic characteristics compared to one or both of the contributing PIVs.
  • heterologous genes or genome segments within a selected recipient PIV strain may result in an increase or decrease 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 genome segments that may be selected for use as heterologous substitutions or additions within human-bovine chimeric PIV of the invention include genes or genome segments encoding a PIV N, P, C, D, V, M, F, HN and/or L protein(s) or portion(s) thereof.
  • genes and genome segments encoding non-PIV proteins may be incorporated within human-bovine PIV of the invention.
  • Regulatory regions such as the extragenic 3' leader or 5' trailer regions, and gene-start, gene-end, intergenic regions, or 3' or 5' non-coding regions, are also useful as heterologous substitutions or additions.
  • Preferred human-bovine chimeric PIV vaccine candidates of the invention bear one or more of the major antigenic determinants of HPIV3 in a background which is attenuated by the substitution or addition of one or more BPIV3 genes or genome segments.
  • the major protective antigens of PIVs are their HN and F glycoproteins, although other proteins can also contribute to a protective immune response.
  • the background genome or antigenome is an HPIV genome or antigenome, e.g., an HPIV3, HPIV2, or HPIVl background genome or antigenome, to which is added or into which is substituted one or more BPIV gene(s) or genome segment(s), preferably from BPIV3.
  • an ORF of the N gene of a BPIV3 is substituted for that of an HPIV.
  • the background genome or antigenome may be a BPIV genome or antigenome which is combined with one or more genes or genome segments encoding a HPIV3, HPIV2, or HPIVl glycoprotein, glycoprotein domain or other antigenic determinant.
  • any BPIV gene or genome segment can be combined with HPIV sequences to give rise to a human-bovine chimeric PIV vaccine candidate.
  • HPIV including different strains of a particular HPIV serotype, e.g. ,
  • HPIV3 will be a reasonable acceptor for attenuating BPIV gene(s).
  • the HPIV3 gene(s) or genome segment(s) selected for inclusion in a human-bovine chimeric PIV for use as a vaccine against human PJV will include one or more of the HPIV protective antigens such as the HN or F glycoproteins.
  • human-bovine chimeric PIVs bearing one or more bovine gene(s) or genome segment(s) exhibit a high degree of host range restriction, e.g., in the respiratory tract of mammalian models of human PIV infection such as non-human primates.
  • the human PIV backbone is attenuated by the addition or substitution of one or more bovine gene(s) or genome segment(s), for example to a partial or complete human, e.g., HPIV3, PIV background genome or antigenome.
  • the partial or complete HPIV background genome or antigenome is combined with one or more heterologous gene(s) or genome segment(s) of a N, P and/or M gene of a BPIV to form a human-bovine chimeric PIV genome or antigenome.
  • the N gene of HPIV3, or a genome segment of N is substituted by the BPIV3 N gene or a corresponding genome segment to yield a novel human-bovine chimeric PIV vaccine candidate.
  • one or more heterologous genes or genome segments encoding a partial or complete open reading frame (ORF) of HPIV P and/or M protein(s) is/are substituted by one or more BPIV3 counterpart gene(s) or genome segment(s).
  • the heterologous gene or genome segment encodes a BPIV3 M protein substituted for the counterpart M protein in a partial HPIV, e.g., HPIV3, background genome or antigenome.
  • Exemplary recombinant viruses of this type described herein include rHPIV3-M ⁇ .
  • the heterologous gene or genome segment encodes a BPIV3 P protein substituted for the counterpart M protein in a partial HPIV, e.g., HPIV3, background genome or antigenome.
  • Exemplary recombinant viruses in this context described herein include rHPrV3-P B .
  • the heterologous gene or genome segment encodes a BPIV3 L protein substituted for the counterpart L protein in a partial HPIV, e.g., HPIV3, background genome or antigenome.
  • Exemplary recombinant viruses in this context described herein include ⁇ HPIV3-L B .
  • the degree of host range restriction exhibited by human-bovine chimeric PTV vaccine candidates of the invention is comparable to the degree of host range restriction exhibited by the respective BPIV parent or "donor" strain.
  • the restriction should have a true host range phenotype, i.e., it should be specific to the host in question and should not restrict replication and vaccine preparation in vitro in a suitable cell line.
  • human-bovine chimeric PIV bearing one or more bovine gene(s) or genome segment(s) elicit a high level of resistance in hosts susceptible to PIV infection.
  • the invention provides a new basis for attenuating a live virus vaccine against PrV, one which is based on host range effects due to the introduction of one or more gene(s) or genome segment(s) from a heterologous PIV, e.g., between HPIV3 and BPIV3.
  • human-bovine chimeric PIV incorporates one or more heterologous gene(s) that encode an HPIV HN and/or F glycoprotein(s).
  • the chimeric PTV may incorporate one or more genome segment(s) encoding an ectodomain (and alternatively a cytoplasmic domain and/or transmembrane domain), or immunogenic epitope of an HPIV HN and/or F glycoprotein(s).
  • immunogenic proteins, domains and epitopes are particularly useful within human-bovine chimeric PIV because they generate novel immune responses in an immunized host, hi particular, the HN and F proteins, and immunogenic domains and epitopes therein, provide major protective antigens.
  • addition or substitution of one or more immunogenic gene(s) or genome segment(s) from a human PIV subgroup or strain to or within a bovine background, or recipient, genome or antigenome yields a recombinant, chimeric virus or subviral particle capable of generating an immune response directed against the human donor virus, including one or more specific human PIV subgroups or strains, while the bovine backbone confers an attenuated phenotype making the chimera a useful candidate for vaccine development.
  • one or more human PIV glycoprotein genes e.g., HN and/or F, are added to or substituted within a partial or complete bovine genome or antigenome to yield an attenuated, infectious human-bovine chimera that elicits an anti-human PIV immune response in a susceptible host.
  • human-bovine chimeric PIV additionally incorporate a gene or genome segment encoding an immunogenic protein, protein domain or epitope from multiple human PIV strains, for example two HN or F proteins or immunogenic portions thereof each from a different HPIV, e.g., HPIVl or HPIV2.
  • one glycoprotein or immunogenic determinant may be provided from a first HPIV, and a second glycoprotein or immunogenic determinant may be provided from a second HPIV by substitution without the addition of an extra glycoprotein- or determinant- encoding polynucleotide to the genome or antigenome.
  • HPIV glycoproteins and antigenic determinants may also be achieved by construction of a genome or antigenome that encodes a chimeric glycoprotein in the recombinant virus or subviral particle, for example having an immunogenic epitope, antigenic region or complete ectodomain of a first HPIV fused to a cytoplasmic domain of a heterologous HPIV.
  • a heterologous genome segment encoding a glycoprotein ectodomain from a HPIVl or HPIV2 HN or F glycoprotein may be joined with a genome segment encoding a corresponding HPIV3 HN or F glycoprotein cytoplasmic/endodomain in the background genome or antigenome.
  • a human-bovine chimeric PIV genome or antigenome may encode a substitute, extra, or chimeric glycoprotein or antigenic determinant thereof in the recombinant virus or subviral particle, to yield a viral recombinant having both human and bovine glycoproteins, glycoprotein domains, or immunogenic epitopes.
  • a heterologous genome segment encoding a glycoprotein ectodomain from a human PIV HN or F glycoprotein may be joined with a genome segment encoding a corresponding bovine HN or F glycoprotein cytoplasmic/endodomain in the background genome or antigenome.
  • human-bovine chimeric amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids
  • PIV may be constructed by substituting the heterologous gene or genome segment for a counterpart gene or genome segment in a partial PIV background genome or antigenome.
  • the heterologous gene or genome segment may be added as a supernumerary gene or genome segment in combination with a complete (or partial if another gene or genome segment is deleted) PIV background genome or antigenome.
  • two human PIV HN or F genes or genome segments can be included, one each from HPIV2 and HPIV3.
  • a heterologous gene or genome segment is added at or near an intergenic position within a partial or complete PIV background genome or antigenome.
  • the gene or genome segment can be placed in other noncoding regions of the genome, for example, within the 5' or 3' noncoding regions or in other positions where noncoding nucleotides occur within the partial or complete genome or antigenome.
  • noncoding regulatory regions contain cis-acting signals required for efficient replication, transcription, and translation, and therefore represent target sites for modification of these functions by introducing a heterologous gene or genome segment or other mutation as disclosed herein.
  • Attenuating mutations are introduced into cis-acting regulatory regions to yield, e.g., (1) a tissue specific attenuation (Gromeier et al, J. Virol. 73:958-964, 1999; Zimmermann et al, J. Virol. 71 :4145-4149, 1997), (2) increased sensitivity to interferon (Zimmermann et al, 1997, supra), (3) temperature sensitivity (Whitehead et al, 1998a, supra), (4) a general restriction in level of replication (Men et al, J. Virol. 70:3930-3937, 1996; Muster et al, Proc. Natl. Acad. Sci.
  • Attenuating mutations can be achieved in various ways to produce an attenuated human-bovine chimeric PIV of the invention, for example by point mutations, swaps of sequences between related viruses, or single or multiple nucleotide deletions.
  • a heterologous gene or genome segment may be added or substituted at a position corresponding to a wild- type gene order position of a counterpart gene or genome segment within the partial or complete PIV background genome or antigenome.
  • the heterologous gene or genome segment is added or substituted at a position that is more promoter-proximal or promotor-distal compared to a wild-type gene order position of a counterpart gene or genome segment within the background genome or antigenome, to enhance or reduce expression, respectively, of the heterologous gene or genome segment.
  • bovine genes or genome segments may be added to or substituted within a human PIV background to form an attenuated, human- bovine chimeric PIV.
  • the chimera may be comprised of one or more human gene(s) or genome segment(s) added to or substituted within a bovine PIV background to form an attenuated PIV vaccine candidate.
  • a chimeric PIV genome or antigenome is formed of a partial or complete bovine PIV background genome or antigenome combined with a heterologous gene or genome segment from a human PIV.
  • one or more bovine PIV gene(s) or genome segment(s) is substituted for a counterpart gene(s) or genome segment(s) within a human PIV background genome or antigenome.
  • one or more human PIV glycoprotein genes e.g., HN and/or F or a genome segment encoding a cytoplasmic domain, transmembrane domain, ectodomain or immunogenic epitope of a human PIV glycoprotein gene is substituted for a counterpart gene or genome segment within the bovine PIV background genome or antigenome.
  • both human PIV glycoprotein genes HN and F may be substituted to replace counterpart HN and F glycoprotein genes in a bovine PIV background genome or antigenome.
  • the chimeric human-bovine PIV of the invention can be readily designed as "vectors" to incorporate antigenic determinants from different pathogens, including more than one PIV strain or group (e.g., both human PIV3 and human PIV1), respiratory syncytial virus (RSV), measles and other pathogens (see, e.g., U.S. Provisional Patent Application Serial No. 60/170,195, filed December 10, 1999 by Murphy et al, incorporated herein by reference).
  • RSV respiratory syncytial virus
  • human-bovine chimeric PIV are comprised of a partial or complete BPIV background genome or antigenome combined with one or more heterologous gene(s) or genome segment(s) from a human PIV.
  • one or more of the HPIV glycoprotein genes HN and F, or one or more genome segments encoding a cytoplasmic domain, transmembrane domain, ectodomain or immunogenic epitope of the HN and/or F genes may be added to a BPIV background genome or antigenome or substituted for one or more counterpart genes or genome segments within the BPIV background genome or antigenome to yield the chimeric construct.
  • both HPIV glycoprotein genes HN and F will be substituted to replace counterpart HN and F glycoprotein genes in the BPIV background genome or antigenome, as exemplified by the recombinant chimeric virus ⁇ BPIV3-FH H N H described in related U.S. Patent Application Serial No: 09/586,479, filed June 1, 2000 (incorporated herein by reference).
  • Attenuated, human-bovine chimeric PIV are produced in which the chimeric genome or antigenome is further modified by introducing one or more attenuating mutations specifying an attenuating phenotype in the resultant virus or subviral particle.
  • mutations for example, in RNA regulatory sequences or in encoded proteins.
  • Attenuating mutations may be generated de novo and tested for attenuating effects according to a rational design mutagenesis strategy.
  • the attenuating mutations may be identified in existing biologically derived mutant PIV and thereafter incorporated into a human-bovine chimeric PIV of the invention.
  • Introduction of attenuating and other desired phenotype-specifying mutations into chimeric bovine-human PIV of the invention may be achieved by transferring a heterologous gene or genome segment, e.g., a gene encoding an L protein or portion thereof, into a bovine or human PIV background genome or antigenome.
  • a heterologous gene or genome segment e.g., a gene encoding an L protein or portion thereof
  • the mutation may be present in the selected background genome or antigenome, and the introduced heterologous gene or genome segment may bear no mutations or may bear one or more different mutations.
  • the human bovine background or "recipient" genome or antigenome is modified at one or more sites corresponding to a site of mutation in a heterologous virus (e.g., a heterologous bovine or human PIV or a non-PIV negative stranded RNA virus) to contain or encode the same or a conservatively related mutation (e.g., a conservative amino acid substitution) as that identified in the donor virus (see, PCT/USOO/09695 filed April 12, 2000 and its priority U.S. Provisional Patent Application Serial No. 60/129,006, filed April 13, 1999, incorporated herein by reference).
  • a heterologous virus e.g., a heterologous bovine or human PIV or a non-PIV negative stranded RNA virus
  • a bovine background or "recipient” genome or antigenome is modified at one or more sites corresponding to a site of mutation in HP1V3 JS cp45, as enumerated below, to contain or encode the same or a conservatively related mutation as that identified in the cp45 "donor.”
  • Preferred mutant PIV strains for identifying and incorporating attenuating mutations into bovine-human chimeric PIV of the invention include cold passaged (cp), cold adapted (ca), host range restricted (hr), small plaque (sp), and/or temperature sensitive (ts) mutants, for example the JS HPIV3 cp45 mutant strain.
  • one or more attenuating mutations identical or conservative to a known mutation in cp45 occur in the polymerase L protein, e.g., at a position corresponding to Tyr 942 , Leu 992 , or Thr 1558 of JS wild type HPIV3.
  • Attenuating mutations in the N protein may be selected and incorporated in a human-bovine chimeric PIV, for example which encode amino acid substitution(s) at a position corresponding to residues Val 96 or Ser 389 of JS.
  • Alternative or additional mutations may encode amino acid substitution(s) in the C protein, e.g., at a position corresponding to Ile 96 of JS, and/or in the M protein at a position corresponding to Pro ⁇ 99 (for example a Pro ⁇ 99 to Thr mutation).
  • Attenuating mutations from biologically derived PIV mutants for incorporation into human-bovine chimeric PIV of the invention also include mutations in noncoding portions of the PJV genome or antigenome, for example in a 3 ' leader sequence.
  • Exemplary mutations in this context may be engineered at a position in the 3' leader of a recombinant virus at a position corresponding to nucleotide 23, 24, 28, or 45 of JS.
  • Yet additional exemplary mutations may be engineered in the N gene start sequence, for example by changing one or more nucleotides in the N gene start sequence, e.g., at a position corresponding to nucleotide 62 of JS.
  • a large "menu" of attenuating mutations is provided, each of which mutations can be combined with any other mutation(s) for adjusting the level of attenuation in a recombinant PIV bearing a genome or antigenome that is a chimera of human and bovine gene(s) or genome segment(s).
  • mutations within recombinant PIV of the invention include one or more, and preferably two or more, mutations of JS cp45.
  • Desired human-bovine chimeric PIV of the invention 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.
  • recombinant human-bovine chimeric PIV incorporate one or more attenuating mutation(s) stabilized by multiple nucleotide substitutions in a codon specifying the mutation.
  • Additional mutations which can be adopted or transferred to human- bovine chimeric PIV of the invention may be identified in non-PIV nonsegmented negative stranded RNA viruses and incorporated in PIV mutants of the invention. This is readily accomplished by mapping the mutation identified in a heterologous negative stranded RNA virus to a corresponding, homologous site in a recipient PIV genome or antigenome and mutating the existing sequence in the recipient to the mutant genotype (either by an identical or conservative mutation), as described in PCT/USOO/09695 filed April 12, 2000 and its priority U.S. Provisional Patent Application Serial No. 60/129,006, filed April 13, 1999, incorporated herein by reference.
  • the invention provides related cDNA clones, vectors and particles, each of which incorporate HPIV and BPIV sequences and, optionally, one or more of the additional, phenotype-specific mutations set forth herein. These are introduced in selected combinations, e.g., into an isolated polynucleotide which is a recombinant cDNA genome or antigenome, to produce a suitably attenuated, infectious virus or subviral particle upon expression, according to the methods described herein.
  • This process provides human-bovine chimeric PIV having such desired characteristics as attenuation, temperature sensitivity, altered immunogenicity, cold-adaptation, small plaque size, host range restriction, genetic stability, etc.
  • vaccine candidates are selected which are attenuated and yet are sufficiently immunogenic to elicit a protective immune response in the vaccinated mammalian host.
  • human-bovine chimeric PIV with or without additional mutations adopted, e.g., from a biologically derived attenuated mutant virus, are constructed to have additional nucleotide modification(s) to yield a desired phenotypic, structural, or functional change.
  • 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 changes in this context include introduction or ablation of restriction sites into PIV encoding cDNAs for ease of manipulation and identification.
  • nucleotide changes within the genome or antigenome of a human-bovine chimeric PIV include modification of a viral gene by partial or complete deletion of the gene or reduction or ablation (knock-out) of its expression. These modifications can be introduced within the human or bovine background genome or antigenome, or may be introduced into the chimeric genome or antigenome by incorporation within the heterologous gene(s) or genome segment(s) added or substituted therein.
  • Target genes for mutation in this context include any of the PJV genes, including the nucleocapsid protein N, phosphoprotein P, large polymerase subunit L, matrix protein M, hemagglutimn-neuraminidase protein HN, small hydrophobic SH protein, where applicable, fusion protein F, and the products of the C, D and V open reading frames (ORFs).
  • N nucleocapsid protein
  • phosphoprotein P large polymerase subunit L
  • matrix protein M matrix protein M
  • HN hemagglutimn-neuraminidase protein
  • SH protein small hydrophobic SH protein
  • fusion protein F the products of the C, D and V open reading frames
  • one or more of the C, D, and/or V genes may be deleted in whole or in part, or its expression reduced or ablated (e.g., by introduction of a stop codon, by a mutation in an RNA editing site, by a mutation that alters the amino acid specified by an initiation codon, or by a frame shift mutation in the targeted ORF(s).
  • a mutation can be made in the editing site that prevents editing and ablates expression of proteins whose mRNA is generated by RNA editing (Kato et al, EMBO J. 16:578-587, 1997a and Schneider et al, Virologv 227:314- 322, 1997, each incorporated herein by reference).
  • one or more of the C, D, and/or V ORF(s) can be deleted in whole or in part to alter the phenotype of the resultant recombinant clone to improve growth, attenuation, immunogenicity or other desired phenotypic characteristics (see, U.S. Patent Application Serial No. 09/350,821, filed by Durbin et al on July 9, 1999, incorporated herein by reference).
  • Alternative nucleotide modifications in human-bovine chimeric PIV of the invention include a deletion, insertion, addition or rearrangement of a cis-acting regulatory sequence for a selected gene in the recombinant genome or antigenome. As with other such modifications described herein, these modifications can be introduced within the human or bovine background genome or antigenome, or may be introduced into the chimeric genome or antigenome by incorporation within the heterologous gene(s) or genome segment(s) added or substituted therein.
  • a cis-acting regulatory sequence of one PIV 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 PIV, or a cis-acting regulatory sequence of a different PIV gene.
  • a gene end signal may be modified by conversion or substitution to a gene end signal of a different gene in the same PIV strain.
  • the nucleotide modification may comprise an insertion, deletion, substitution, or rearrangement of a translational start site within the recombinant genome or antigenome, e.g., to ablate an alternative translational start site for a selected form of a protein.
  • genes or genome segments from non-PIV sources may be inserted in whole or in part.
  • the order of genes can be changed, or a PIV genome promoter replaced with its antigenome counterpart.
  • Different or additional modifications in the recombinant genome or antigenome can be made to facilitate manipulations, such as the insertion of unique restriction sites in various intergenic or non-coding regions or elsewhere.
  • Nontranslated gene sequences can be removed to increase capacity for inserting foreign sequences.
  • polynucleotide molecules or vectors encoding the human-bovine chimeric PIV genome or antigenome can be modified to encode non-PIV sequences, e.g., a cytokine, a T-helper epitope, a restriction site marker, or a protein or immunogenic epitope of a microbial pathogen (e.g., virus, bacterium, parasite, or fungus) capable of eliciting a protective immune response in an intended host.
  • a microbial pathogen e.g., virus, bacterium, parasite, or fungus
  • human-bovine chimeric PIV are constructed that incorporate a gene or genome segment from a respiratory syncytial virus (RSV), for example a gene encoding an antigenic protein (e.g., an F or G protein), immunogenic domain or epitope of RSV.
  • RSV respiratory syncytial virus
  • compositions e.g., isolated polynucleotides and vectors incorporating a PrV-encoding cDNA
  • methods for producing an isolated infectious human-bovine chimeric PIV.
  • novel, isolated polynucleotide molecules and vectors incorporating such molecules that comprise a human-bovine chimeric PIV genome or antigenome are also provided.
  • the same or different expression vector comprising one or more isolated polynucleotide molecules encoding N, P, and L proteins. These proteins also can be expressed directly from the genome or antigenome cDNA.
  • the vector(s) is/are preferably expressed or coexpressed in a cell or cell-free lysate, thereby producing an infectious human-bovine chimeric PIV viral particle or subviral particle.
  • infectious viral or subviral particles or derivatives thereof.
  • a recombinant infectious virus is comparable to the authentic PIV 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, and L 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 viral particles which lack one or more protein(s), protein segment(s), or other viral component(s) not essential for infectivity.
  • the invention provides a cell or cell-free lysate containing an expression vector which comprises an isolated polynucleotide molecule comprising a human-bovine chimeric PIV 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, and L proteins of PIV.
  • an expression vector (the same or different vector) which comprises one or more isolated polynucleotide molecules encoding the N, P, and L proteins of PIV.
  • One or more of these proteins also can be expressed from the genome or antigenome cDNA.
  • the genome or antigenome and N, P and L combine to produce an infectious human- bovine chimeric PIV virus or subviral particle.
  • the human-bovine chimeric PIVs of the invention are useful in various compositions to generate a desired immune response against PIV in a host susceptible to PIV infection.
  • Human-bovine chimeric PIV recombinants are capable of eliciting a protective immune response in an infected mammalian host, yet are sufficiently attenuated so as not to cause unacceptable symptoms of severe respiratory disease in the immunized host.
  • the human-bovine chimeric PIV recombinants should replicate with sufficient efficiency in vitro to make vaccine preparation feasible.
  • the attenuated 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 human- bovine chimeric PIV virus or subviral particle.
  • the vaccine is comprised of a human-bovine chimeric PIV having at least one, and preferably two or more additional mutations or other nucleotide modifications as described above to achieve a suitable balance of attenuation and immunogenicity.
  • the vaccine can be formulated in a dose of 10 3 to 10 7 PFU of attenuated virus.
  • the vaccine may comprise attenuated human-bovine chimeric PIV that elicits an immune response against a single PIV strain or against multiple PIV strains or groups.
  • human-bovine chimeric PIV can be combined in vaccine formulations with other PIV vaccine strains, or with other viral vaccine viruses such as RSV.
  • the invention provides a method for stimulating the immune system of an individual to elicit an immune response against PIV in a mammalian subject.
  • the method comprises administering a formulation of an immunologically sufficient amount a human-bovine chimeric PIV in a physiologically acceptable carrier and/or adjuvant.
  • the immunogenic composition is a vaccine comprised of a human-bovine chimeric PIV having at least one, and preferably two or more attenuating mutations or other nucleotide modifications specifying a desired phenotype as described above.
  • the vaccine can be formulated in a dose of 10 3 to 10 7 PFU of attenuated virus.
  • the vaccine may comprise attenuated human-bovine chimeric PIV virus that elicits an immune response against a single PIV, against multiple PIVs, e.g., HPIVl and HPIV3, or against one or more PIV(s) and a non-PIV pathogen such as RSV.
  • human-bovine chimeric PIV can elicit a monospecific immune response or a polyspecific immune response against multiple PIVs, or against one or more PIV(s) and a non-PIV pathogen such as RSV.
  • human-bovine chimeric PIV 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 PIV, against multiple PIVs, or against one or more PIV(s) and a non-PIV pathogen such as RSV.
  • the immunogenic composition is administered to the upper respiratory tract, e.g., by spray, droplet or aerosol.
  • Figures 1A-1G set forth the complete nucleotide sequence of the bovine PTV3 Ka (SEQ ID NO: 35) strain.
  • Figures 2A-2G set forth the complete nucleotide sequence of the bovine PTV3 SF (SEQ ID NO: 36) strain.
  • Figure 3 illustrates cloning of the N coding region of bovine PIV strains Ka or SF into HPIV3.
  • panel A the BPTV3 N open reading frame (ORF) is replaced for its corresponding HPIV3 sequence in the full-length rJS antigenomic cDNA (Durbin et al, 1997a, supra).
  • BPTV3 Ka and SF N genes were first amplified by RT-PCR using standard molecular biological techniques from virion RNA and subcloned as 1.9 kb fragments into pBluescript to give pBS-KaN or pBS-SFN, respectively.
  • HPIV3 rJS N gene was subcloned as a 1.9 kb Mlul/EcoRI fragment into pUC 119 from a plasmid containing the 5' half of the rJS HPIV3 antigenome (Durbin et al., 1997a, supra; U.S. Patent Application Serial No. 09/083,793, filed May 22, 1998, (corresponding to
  • FIG. 3 panel B ⁇ Following Aflll/Ncol digestion, a 1.5 kb fragment from pBS-KaN or pBS-SFN representing the BPIV3 N coding region was introduced into the Ncol/Aflll window of the HPIV3 N subclone pUC 119 JSN-Ncol Aflll as a replacement for its HPIV3 counterpart.
  • Figure 3 panel C— Each chimeric subclone was then subjected to site- directed mutagenesis to restore the sequence present in HPIV3 rJS before the start codon or after the stop codon and BPrV3 coding sequence immediately after the start codon and before the stop codon. This yielded pUCl 19B/HKaN and pUCl 19B/HSFN, which were used to import the BPTV3 N gene into the HPIV3 cDNA clone as shown in Figure 4.
  • Figure 4 illustrates insertion of the HPIV3/BPIV3 (strain Ka or SF) chimeric N gene into the HPIV3 antigenomic cDNA.
  • panel A the BPIV3 N ORF of Ka or SF flanked by HPIV3 sequence was subcloned as an Mlul/EcoRI fragment from pUCl 19B/HKaN or pUCl 19B/HSFN and inserted into pLeft+2G (Durbin et al, 1997a, supra).
  • the pLeft+2G plasmid contains the 5' half of the HPIV3 rJS antigenome from nt 1-7437 (genome sense) behind a T7 promoter.
  • Figure 5 provides nucleotide sequences of HPIV3, BPIV3 and chimeric viruses of the invention around N start (panel A) and stop (panel B) codons.
  • the position of the individual ORFs is described in the respective GenBank reports (#AF 178654 for BPIV3 Ka, #AF178655 for BPIV3 SF and #Z11515) (each incorporated herein by reference).
  • sequences (positive-sense) flanking the translational start (panel A) and stop (panel B) codons (each underlined) in the N gene are shown for the parental recombinant HPIV3 JS (rJS), the parental biologically-derived BPIV3 Ka and SF viruses (Ka and SF), and the chimeric cKa and cSF viruses.
  • Host-specific residues in the cKa and cSF virus sequences and their counterparts in rJS (before the start codon and after the stop codon) and SF or Ka (start codon through stop codon, inclusive) are in boldface type.
  • Plaque-purified chimeric virus was amplified by RT-PCR from virion RNA and sequenced using the Taq Dye Deoxy Terminator Cycle kit (ABI, Foster City, CA). This confirmed that the predicted sequences were present in each chimeric virus.
  • Figure 6 details the structure of the BPIV3/HPIV3 chimeric viruses of the invention, and their confirmation by Taql digestion of RT-PCR products generated from virus RNA.
  • panel A the genomes of the chimeric cKa and cSF viruses are shown schematically (not to scale) relative to that of HPIV3 and BPIV3 parent viruses. Ka- and SF-specific regions are indicated by light and dark shading respectively. Arrows above the rJS genome indicate the locations of primers used for RT-PCR amplification of chimeric and parent viruses for the purposes of diagnostic Taql digestion.
  • Taql fragments unique to each virus and which therefore serve in virus identification are indicated with an asterisk.
  • Figure 6 panel C provides Taql profiles of PCR products containing the PIV3 N coding region of chimeric cKa (left) or cSF (right) are shown flanked by those of the HPIV3 and BPIV3 parent viruses.
  • Unique Taql fragments diagnostic of virus identity and corresponding to those identified in panel B are indicated with an asterisk. Calculated lengths (bp) of DNA gel bands are indicated.
  • Figure 7 provides multicycle growth curves of parental and chimeric viruses in MDBK (panel A) or LLC-MK2 (panel B) cells.
  • Monolayers of bovine MDBK (panel A) or simian LLC-MK2 (panel B) cells in wells (9.6 cm 2 each) of a 6 well plate were infected individually at a multiplicity of infection of 0.01 with the indicated parental or chimeric virus.
  • Three replicate infections were performed for each virus. Samples were taken at the indicated time points, stored at -70°C, and titered by TCID50 assay in parallel. Growth curves are constructed using the average of 3 replicate samples at each time point. The lower limit of virus detectability was lO ⁇ TCIDso/ml, which is indicated by a dotted line.
  • panel A provides a diagrammatic representation of the rHPIV3, rHPIV3-P B , and BPTV3 genomes (not drawn to scale).
  • the position of the BPJV3 P ORF t ⁇ ⁇ ) and the PCR primers (-») used to generate an RT-PCR product spanning the P ORF are indicated relative to the rHPIV3-P ⁇ sequence.
  • panel B provides a diagrammatic representation of the rHPIV3, rHPIV3-M B , and BPIV3 genomes (not drawn to scale).
  • the position of the BPIV3 M ORF ( ⁇ ) and the PCR primers ( ⁇ ) used to generate an RT-PCR product spanning the M ORF are indicated relative to the rHPIV3- M ⁇ sequence.
  • panel A presents an agarose gel electrophoresis of an RT-PCR product (2156 bp) of rHPrV3-P B (lane 1) generated with a set of HPIV3 -specific primers flanking the P ORF, as indicated in Figure 8, panel A.
  • RT-PCR product (2156 bp) of rHPrV3-P B (lane 1) generated with a set of HPIV3 -specific primers flanking the P ORF, as indicated in Figure 8, panel A.
  • the absence of a PCR product of the appropriate size in the reaction lacking reverse transcriptase (-RT; lane 2) shows that viral RNA, not contaminating cDNA, served as a template.
  • lane 2 the unincorporated primers are evident (*).
  • M marker consisting of lambda phage DNA digested with
  • FIG. 9 Panel B presents an agarose gel electrophoresis of products from a restriction enzyme digestion (Stul) of the 2156 bp rHPIV3-P B RT-PCR product from Figure 9, panel A. Digestion of the 2156 bp fragment yields the expected (see Fig. 9, panel C) 726 bp and 1430 bp fragments demonstrating the presence of the BPIV3 P ORF in rHPIV3-P B .
  • M marker consisting of lambda phage DNA digested with Hind ⁇ I and ⁇ X174 phage DNA digested with Hae Tl.
  • FIG. 9 The nucleotide length of several size markers are indicated in bp.
  • panel C provides a diagram (not to scale) of the indicated StuI(S) site in the RT-PCR product spanning the P ORF in rHP ⁇ V3-P B (top line), compared to the corresponding region in the genome of the rHPIV3 (middle line) and BPTV3 (bottom line) parents.
  • the nucleotide positions of the primers and the Stul site, and the length of the resulting ⁇ HPIV3-P B digestion fragments are indicated.
  • the position of the P ORF is shown as a rectangle (HP ⁇ V3 ⁇ ; BP1V3 ⁇ S).
  • panel A presents an agarose gel electrophoresis of RT-PCR product (3445 bp) of rHPrV3-M B generated with a set HPIV3-specific primers amplifying the BPrV3 M ORF (lane 1) as indicated in Figure 8, panel B.
  • a PCR product of the appropriate size in the reaction lacking reverse transcriptase shows that viral RNA, not contaminating cDNA, served as a template.
  • M marker consisting of lambda phage DNA digested with HindTQ. and ⁇ X174 phage DNA digested with H ⁇ lll. The nucleotide length of several size markers are indicated.
  • panel B presents an agarose gel electrophoresis of products from restriction enzyme digestions of the 3445 bp rHPrV3-M B RT-PCR product.
  • Lane 1 the undigested 3445 bp RT-PCR product;
  • lane 2 Spe ⁇ digestion products consisting of the expected 1825 bp and 1620 bp fragments;
  • lane 3 EcoRV digestion products consisting of the expected 666 bp, 1396 bp, 221 bp, and 1162 bp fragments;
  • lane 4 Xbal digestion products consisting of the expected 2354 bp, 441 bp and 650 bp fragments, demonstrating the presence of the BPIV3 M gene in rHPLV3-M B .
  • panel C provides diagrams (not to scale) of the indicated restriction sites in the RT-PCR product of the M ORF in rHPIV3-M B that was shown in Figure 10, panels A and B.
  • the first set of diagrams illustrates the Spel (S) sites in the RT-PCR product of rHPrV3-M B (top) and the corresponding region in the genome of the rHPIV3 (middle) and BPIV3 (bottom) parents.
  • the next set of diagrams shows the EcoRV (E) sites in the rHPIV3-M B , rHPIV3, and BPIV3 segments.
  • the bottom set of diagrams shows the sites for t .eXbal(X) sites.
  • the nucleotide positions of the primers and restriction sites, and the length of the digestion products are indicated for rHPIV3-M B .
  • the position of the M ORF is shown as a rectangle (HPTV3 L_l ; BPIV3 ⁇ 1 ).
  • Figure 11A provides a schematic depiction of the genomes of chimeric rHPIV3-F B HN B and rBPrV3-F H HN H viruses, and of their parent viruses, rHPIV3 JS and BPIV3 Ka (not to scale).
  • the F and HN genes were exchanged in a single restriction fragment between rHPIV3 and rBPIV3 using Sgr Al and BsiWl sites that had been introduced in front of the M and HN gene end, respectively.
  • Figure 1 IB depicts one of the steps in assembly of an antigenomic cDNA for BPrV3 Ka.
  • a full length cDNA was constructed to encode the complete antigenomic sequence of BPIV3 Ka (GenBank accession #AF178654) with the exception of nt 21 and 23.
  • the cDNA was assembled from subclones derived from reverse transcription (RT) of viral (v)RNA and polymerase chain reaction (PCR) amplification.
  • Figure 11C illustrates features of parental and chimeric bovine-human PIV genomes.
  • the genomes of the chimeric rHPIV3 F B HN B and rBPTV3 F H HN H viruses and those of their parent viruses rHPIV3 JS and BPIV3 Ka are shown schematically (not to scale).
  • Panel 1 rPIV3JS was constructed as described elsewhere herein.
  • Panel 2 Two unique restriction enzyme recognition sites, SgrAI and BsiWI, were introduced near the M and HN gene ends, respectively. The recombinant HPIV3 and BPIV3 viruses bearing these introduced restriction sites were designated rHPIV3s and rBPIV3s as indicated.
  • Panel 3 Glycoprotein genes were exchanged between rHPIV3 JS and rBPIV3 Ka. The nucleotide sequence that was mutagenized is shown below each cDNA construct. The position of the first nucleotide of each sequence indicated. Restriction sites are underlined and nucleotides that differ Figure 12 provides a confirmation of the identity of recombinant viruses by RT-PCR of viral RNA and Eco RI digestion. RT-PCR products of viral RNA were prepared with a primer pair that recognized conserved regions on either side of the F and HN genes in both BPIV3 and HPIV3. Digestion with Eco RI resulted in a unique pattern of restriction fragments for each of the four viruses.
  • Figure 13 depicts multicycle replication of chimeric and parental viruses in simian LLC-MK2 cells.
  • Multicycle replication MOI of 0.01
  • rBPrV3-F H HN H and rHPIV3-N B is compared with the replication of their parental viruses BPIV3 Ka and rHPIV3.
  • the virus titers are shown as mean loglO TCIDsQ/ml ⁇ standard error of triplicate samples.
  • the lower limit of detection of this assay is 10 TCID 50 , as indicated by the dotted horizontal line.
  • Figure 14 documents mean titers of chimeric and parental viruses in nasopharyngeal swabs of infected rhesus monkeys over the course of infection.
  • Virus titers are shown as mean TCID 5 o/ml in LLC-MK2 cells ⁇ standard error for groups of 4 or 6 monkeys infected with the same virus.
  • the rHPIV3 group contained two animals infected with rHPIV3s, the virus containing restriction enzyme recognition sites for the glycoprotein swap. This illustrates the same experiment as shown in Table 3.
  • mean titers of rHPIV3-F B HN B are compared to rHPIV3 and BPIV3 Ka titers.
  • rBPIV3-F H HN H titers are compared to those of BPIV3 Ka and rHPIV3, which, for the last two viruses, are the same values in panel A but are presented separately to facilitate comparison.
  • Day 5 titers were excluded from the figures because they were much lower than day 4 and day 6 titers, most likely due to technical problems during the sample collection.
  • Figure 15 provides a comparison of the nucleotide sequence of a genomic region of rHPIV3-L B flanking the translation initiation codon (ATG, in bold type) and the translation termination codon (TAA, in bold type) of the L ORF.
  • the nucleotide sequencing of an RT-PCR fragment generated from vRNA of rHPIV3-L B around the junctions of the BPIV3 L ORF (BPIV3 GenBank accession #AF178654) and the HPIV3 (JS strain HPIV3 GenBank accession #Z11575) flanking sequences was determined using a Perkin Elmer ABI 3100 automated sequencer, and the engineered sequences for the rHPrV3 L B were confirmed to be present.
  • the determined sequence of rHPIV3-L B is indicated in comparison to that of its two parents.
  • the introduced BsiWl restriction enzyme recognition sequence following the L ORF stop codon in the chimera is italicized and the BPIV3 sequences are underlined.
  • the present invention provides recombinant parainfluenza virus (PIV) cloned as a chimera of human and bovine PIV genomic or antigenomic sequences to yield a human-bovine chimeric PIV.
  • PIV parainfluenza virus
  • the chimeric construction of human-bovine PIV yields a viral particle or subviral particle that is infectious in mammals, particularly humans, and useful for generating immunogenic compositions for clinical or veterinary use.
  • novel methods and compositions for designing and producing attenuated, human-bovine chimeric PIV, as well as methods and compositions for the prophylaxis and treatment of PIV infection are also provided within the invention.
  • Human-bovine chimeric PIV and immunogenic compositions according to the invention may elicit an immune response to a specific PIV, or they may elicit a polyspecific response against multiple PIVs, e.g., multiple human PIVs such as HPIVl and HPIV3.
  • Chimeric human-bovine PIV of the invention are recombinantly engineered to incorporate nucleotide sequences from both human and bovine PIV strains to produce an infectious, chimeric virus or subviral particle.
  • candidate vaccine viruses are recombinantly engineered to elicit an immune response against PIV in a mammalian host susceptible to PIV infection, including humans and non-human primates.
  • Human-bovine chimeric PIV according to the invention may elicit an immune response to a specific PIV, e.g., HPIV3, or a polyspecific response against multiple PIVs, e.g., HPIVl and HPIV3.
  • Exemplary human-bovine chimeric PIV of the invention incorporate a chimeric PIV genome or antigenome comprising both human and bovine polynucleotide sequences, as well as a major nucleocapsid (N) protein, a nucleocapsid phosphoprotein (P), and a large polymerase protein (L). Additional PIV proteins may be included in various combinations to provide a range of infectious subviral particles, up to a complete viral particle or a viral particle containing supernumerary proteins, antigenic determinants or other additional components.
  • Chimeric human-bovine PIV of the invention include a partial or complete "background" PIV genome or antigenome derived from or patterned after a human or bovine PIV strain or serotype virus combined with one or more heterologous gene(s) or genome segment(s) of a different PIV strain or serotype virus to form the human-bovine chimeric PIV genome or antigenome.
  • chimeric PIV incorporate a partial or complete human PIV (HPIV) background genome or antigenome combined with one or more heterologous gene(s) or genome segment(s) from a bovine PIV.
  • chimeric PIV incorporate a partial or complete bovine PIV (BPIV) background genome or antigenome combined with one or more heterologous gene(s) or genome segment(s) from a human PIV.
  • the partial or complete background genome or antigenome typically acts as a recipient backbone or vector into which are imported heterologous genes or genome segments of the counterpart, human or bovine PIV.
  • Heterologous genes or genome segments from the counterpart, human or bovine PIV represent "donor" genes or polynucleotides that are combined with, or substituted within, the background genome or antigenome to yield a human-bovine chimeric PIV that exhibits novel phenotypic characteristics compared to one or both of the contributing PIVs.
  • heterologous genes or genome segments within a selected recipient PIV strain may result in an increase or decrease 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 genome segments that may be selected for use as heterologous inserts or additions within human-bovine chimeric PIV of the invention mclude genes or genome segments encoding a PIV N, P, C, D, V, M, F, HN and/or L protein(s) or portion(s) thereof. Regulatory regions, such as the extragenic leader or trailer or intergenic regions, are also useful as heterologous inserts or additions.
  • the heterologous gene(s) or genome segment(s) may be added or substituted at a position corresponding to a wild-type gene order position of the counte ⁇ art gene(s) or genome segment(s) within the partial or complete PIV background genome or antigenome, which counte ⁇ art gene or genome segment is thereby replaced or displaced (e.g., to a more promotor-distal position).
  • the heterologous gene or genome segment is added or substituted at a position that is more promoter-proximal or promotor-distal compared to a wild-type gene order position of the counte ⁇ art gene or genome segment within the background genome or antigenome, which enhances or reduces, respectively, expression of the heterologous gene or genome segment.
  • heterologous immunogenic proteins, domains and epitopes to produce human-bovine chimeric PIV is particularly useful to generate novel immune responses in an immunized host.
  • Addition or substitution of an immunogenic gene or genome segment from one, donor PIV within a recipient genome or antigenome of a different PIV can generate an immune response directed against the donor subgroup or strain, the recipient subgroup or strain, or against both the donor and recipient subgroup or strain.
  • human-bovine chimeric PIV may also be constructed that express a chimeric protein, e.g., an immunogenic glycoprotein having a cytoplasmic tail and/or transmembrane domain specific to one PIV fused to an ectodomain of a different PIV to provide, e.g., a human-bovine fusion protein, or a fusion protein inco ⁇ orating domains from two different human PIVs.
  • a human-bovine chimeric PIV genome or antigenome encodes a chimeric glycoprotein in the recombinant virus or subviral particle having both human and bovine glycoprotein domains or immunogenic epitopes.
  • a heterologous genome segment encoding a glycoprotein ectodomain from a human PIV HN or F glycoprotein may be joined with a polynucleotide sequence (i.e., a genome segment) encoding the corresponding bovine HN or F glycoprotein cytoplasmic and transmembrane domains to form the human-bovine chimeric PIV genome or antigenome.
  • human-bovine chimeric PIV useful in a vaccine formulation can be conveniently modified to accommodate antigenic drift in circulating virus. Typically the modification will be in the HN and/or F proteins.
  • This may involve, for example, introduction of one or more point mutations or, alternatively, may involve an entire HN or F gene, or a genome segment encoding a particular immunogenic region thereof, from one PIV strain or group is inco ⁇ orated into a chimeric PIV genome or antigenome cDNA by replacement of a corresponding region in a recipient clone of a different PIV strain or group, or by adding one or more copies of the gene, such that multiple antigenic forms are represented.
  • Progeny virus produced from the modified PIV clone can then be used in vaccination protocols against emerging PIV strains.
  • bovine or murine PIV protein, protein domain, gene or genome segment imported within a human PIV background, wherein the bovine or murine gene does not function efficiently in a human cell, e.g., from incompatibility of the heterologous sequence or protein with a biologically interactive human PIV sequence or protein (i.e., a sequence or protein that ordinarily cooperates with the substituted sequence or protein for viral transcription, translation, assembly, etc.) or, more typically in a host range restriction, with a cellular protein or some other aspect of the cellular milieu which is different between the permissive and less permissive host.
  • bovine PIV sequences are selected for introduction into human PIV based on known aspects of bovine and human PIV structure and function.
  • HPIV3 is a member of the Respirovirus genus of the Paramyxoviridae family in the order Mononegavirales (Collins et al, 1996, supra).
  • a protein containing the V ORF in the P gene might also be produced (Durbin et al, Virologv 261:319-333. 1999, inco ⁇ orated herein by reference).
  • HPIV3 is the best characterized of the HPIVs and represents the prototype HPIV. Its genome is a single strand of negative-sense RNA 15462 nucleotides (nt) in length (Galinski et al, Virology 165:499-510, 1988; and Stokes et al, Virus Res. 25:91- 103, 1992; each inco ⁇ orated herein by reference).
  • At least eight proteins are encoded by the PIV3 genome: the nucleocapsid protein N, the phosphoprotein P, the C and D proteins of unknown functions, the matrix protein M, the fusion glycoprotein F, the hemagglutinin-neuraminidase glycoprotein HN, and the large polymerase protein L (Collins et al, 1996, supra).
  • the M, HN, and F proteins are envelope-associated, and the latter two are surface glycoproteins which, as is the case with each PIV, are the major neutralization and protective antigens (Collins et al, 1996, supra).
  • the significant sequence divergence between comparable PIV HN or F proteins among the PIVs is thought to be the basis for the type specificity of the protective immunity (Collins et al, 1996, supra; Cook et al, Amer. Jour. Hvg. 77:150-159, 1963: Rav et al. J. Infect. Pis. 162:746-749. 1990; each inco ⁇ orated herein by reference).
  • the HPIV3 genes are each transcribed as a single mRNA that encodes a single protein, with the exception of the P mRNA which contains four ORFs, namely P, C, P and V (Galinski et al, Virology 186:543-550, 1992; and Spriggs et al, J. Gen. Virol. 67:2705-2719, 1986; each inco ⁇ orated herein by reference).
  • the P and C proteins are translated from separate, overlapping ORFs in the mRNA. Whereas all paramyxo viruses encode a P protein, only members of the genus Respirovirus and Morbillivirus encode a C protein. Individual viruses vary in the number of proteins expressed from the C ORF and in its importance in replication of the virus in vitro and in vitro.
  • Sendai virus (SeV) expresses four independently initiated proteins from the C
  • ORF C, C, Yl, and Y2, whose translational start sites appear in that order in the mRNA (Curran et al, Enzyme 44:244-249, 1990; Lamb et al, in The Paramyxoviruses. P. Kingsbury, ed., pp. 181-214, Plenum Press, New York, 1991; inco ⁇ orated herein by reference), whereas HPIV3 and measles virus (MeV) express only a single C protein (Bellini et al. J. Virol. 53:908-919. 1985; Sanchez et al. Virology 147:177-86, 1985; and Spriggs et al, 1986, supra; each inco ⁇ orated herein by reference).
  • the PIV3 P protein is a fusion protein of the P and P ORFs, and is expressed from the P gene by the process of co-transcriptional RNA editing in which two nontemplated G residues are added to the P mRNA at the RNA editing site (Galinski et al, 1992, supra; and Pelet et al, EMBO J. 10:443-448, 1991 ; each inco ⁇ orated herein by reference).
  • BPIV3 the only other paramyxovirus which expresses a O protein, uses RNA editing to express this protein as well as a second protein, the V protein. Nearly all members of the genera Respirovirus, Rubulavirus, and Morbillivirus express a V protein.
  • the one member which clearly does not is HPIVl, which lacks an intact V ORF (Matsuoka et al, J. Virol. 65:3406-3410, 1991, inco ⁇ orated herein by reference).
  • the V ORF is characterized by the presence of a cysteine-rich domain that is highly conserved (Cattaneo et al, Cell 56:759-764, 1989; Park et al, Virol. 66:7033-7039, 1992; Thomas et al, Cell 54:891-902, 1988; and Vidal et al, I Virol. 64:239-246, 1990; each inco ⁇ orated herein by reference).
  • the BPIV3 V protein is expressed when one nontemplated G residue is added at the RNA editing site (Pelet et al, 1991, supra; inco ⁇ orated herein by reference).
  • two translation stop codons lie between the editing site and the V ORF, and it is not clear whether HPIV3 represents another example in which this ORF is not expressed, or whether it is expressed by some other mechanism.
  • HPIV3 editing also occurs at a second, downstream site in the P gene, although this did not appear to occur in cell culture (Galinski et al, 1992, supra).
  • MV expresses C, P, and V proteins, but also expresses a novel R protein which is synthesized by frameshifting from the P ORF to the V ORF (Liston et al, J. Virol. 69:6742-6750, 1995, inco ⁇ orated herein by reference). Genetic evidence suggests that the V ORF of HPIV3 is functional (Ourbin et al, 1999, supra).
  • the viral genome of PIV also contains extragenic leader and trailer regions, possessing all or part of the promoters required for viral replication and transcription, as well as non-coding and intergenic regions.
  • the PIV genetic map is represented as 3' leader-N-P/C/D/V-M-F-HN-L-5 ' trailer.
  • Some viruses such as simian virus 5 and mumps virus, have a gene located between F and HN that encodes a small hydrophobic (SH) protein of unknown function. Transcription initiates at the 3' end and proceeds by a sequential stop-start mechanism that is guided by short conserved motifs found at the gene boundaries.
  • each gene contains a gene-start (GS) signal, which directs initiation of its respective mRNA.
  • the downstream terminus of each gene contains a gene-end (GE) motif which directs polyadenylation and termination.
  • GS gene-start
  • GE gene-end motif
  • Exemplary sequences have been described for the human PIV3 strains JS (GenBank accession number Zl 1575, inco ⁇ orated herein by reference) and Washington (Galinski M.S., in The Paramyxoviruses. Kingsbury, D.W., ed., pp. 537-568, Plenum Press, New York, 1991, inco ⁇ orated herein by reference), and for the bovine PIV3 strain 9 ION (GenBank accession number D80487, inco ⁇ orated herein by reference).
  • PIV gene generally refers to a portion of the PIV genome encoding an mRNA and typically begins at the upstream end with a gene-start (GS) signal and ends at the downstream end with the gene-end (GE) signal.
  • the term PIV gene also embraces what is referred to as a "translational open reading frame", or ORF, particularly in the case where a protein, such as C, is expressed from an additional ORF rather than from a unique mRNA.
  • ORF translational open reading frame
  • one or more PIV gene(s) or genome segment(s) may be deleted, inserted or substituted in whole or in part.
  • deletions, insertions and substitutions may include open reading frames and/or cis-acting regulatory sequences of any one or more of the PIV genes or genome segments.
  • gene segment is meant any length of continuous nucleotides from the PIV genome, which might be part of an ORF, a gene, or an extragenic region, or a combination thereof.
  • the instant invention involves a method for developing live attenuated PIV vaccine candidates based on chimeras between HPIVs and BPIV3.
  • Chimeras are constructed through a cDNA-based virus recovery system. Recombinant viruses made from cDNA replicate independently and are propagated in the same manner as if they were biologically-derived viruses.
  • Preferred human-bovine chimeric PIV vaccine candidates of the invention bear one or more of the major antigenic determinants of one or more human PIV(s), e.g., HPIVl, HPIV2, and/or HPIV3, in a background which is attenuated by the substitution or addition of one or more BPIV genes or genome segments.
  • the major protective antigens of PIVs are their HN and F glycoproteins, although other proteins can also contribute to a protective immune response.
  • the invention provides a new basis for attenuating a wild type or mutant parental virus for use as a vaccine against PIV, one which is based on host range effects due to the introduction of one or more gene(s) or genome segment(s) between HPIV and BPIV.
  • host range effects due to the introduction of one or more gene(s) or genome segment(s) between HPIV and BPIV.
  • nucleotide and amino acid sequence differences between BPIV and HPIV, which are reflected in host range differences.
  • the percent amino acid identity for each of the following proteins is: N (86%), P (65%), M (93%), F (83%), HN (77%), and L (91%).
  • the host range difference is exemplified by the highly permissive growth of HPIV3 in rhesus monkeys, compared to the restricted replication of two different strains of BPIV3 in the same animal (van Wyke Coelingh et al, 1988, supra).
  • HPIV3 and BPIV3 remain to be determined, it is likely that they will involve more than one gene and multiple amino acid differences.
  • the involvement of multiple genes and possibly cis-acting regulatory sequences, each involving multiple amino acid or nucleotide differences gives a very broad basis for attenuation, one which cannot readily be altered by reversion. This is in contrast to the situation with other live attenuated HPIV3 viruses which are attenuated by one or several point mutations. In this case, reversion of any individual mutation may yield a significant reacquisition of virulence or, in a case where only a single residue specified attenuation, complete reacquisition of virulence.
  • the background genome or antigenome is an HPIV3 genome or antigenome
  • the heterologous gene or genome segment is a N ORF derived from, alternatively, a Ka or SF strain of BPIV3 (which are 99% related in amino acid sequence).
  • the N ORF of the HPIV3 background antigenome is substituted by the counte ⁇ art BPIV3 N ORF — yielding a novel recombinant human-bovine chimeric PIV cDNA clone.
  • Replacement of the HPIV3 N ORF with that of BPIV3 Ka or SF results in a protein with approximately 70 amino acid differences (depending on the strain involved) from that of HPIV3 N.
  • N is one of the more conserved proteins, and substitution of other proteins such as P, singly or in combination, would result in many more amino acid differences.
  • the involvement of multiple genes and genome segments each conferring multiple amino acid or nucleotide differences provides a broad basis for attenuation which is highly stable to reversion.
  • This mode of attenuation contrasts sha ⁇ ly to HPIV vaccine candidates that are attenuated by one or more point mutations, where reversion of an individual mutation may yield a significant or complete reacquisition of virulence.
  • several known attenuating point mutations in HPIV typically yield a temperature sensitive phenotype.
  • One problem with attenuation associated with temperature sensitivity is that the virus can be overly restricted for replication in the lower respiratory tract while being under attenuated in the upper respiratory tract. This is because there is a temperature gradient within the respiratory tract, with temperature being higher (and more restrictive) in the lower respiratory tract and lower (less restrictive) in the upper respiratory tract.
  • the ability of an attenuated virus to replicate in the upper respiratory tract can result in complications including congestion, rhinitis, fever and otitis media, whereas overattenuation in the lower respiratory tract can reduce immunogenicity.
  • attenuation achieved solely by temperature sensitive mutations may not be ideal.
  • host range mutations present in human-bovine chimeric PIV of the invention will not in most cases confer temperature sensitivity. Therefore, the novel method of PIV attenuation provided by the invention will be more stable genetically and phenotypically and less likely to be associated with residual virulence in the upper respiratory tract compared to other known PIV vaccine candidates.
  • both the Ka and SF HPIV3/BPIV3 chimeric recombinants were viable, since the N gene of Ka or SF strain BPIV3 differs in 70 of 515 amino acid residues, respectively, from that of the JS strain of HPIV3. It was therefore unexpected that a bovine N protein with this level of amino acid sequence divergence could efficiently interact with the HPIV3 RNA, or with other HPIV3 proteins that constitute the functional replicase/transcriptase. Equally su ⁇ rising was the finding that the Ka and SF chimeric viruses replicated as efficiently in cell culture as either HPIV3 or BPIV3 parent indicating that the chimeric recombinants did not exhibit gene incompatibilities that restricted replication in vitro.
  • Attenuation marked by replication in the lower and/or upper respiratory tract in an accepted animal model for PIV replication in humans may be reduced by at least about two-fold, more often about 5-fold, 10-fold, or 20-fold, and preferably 50-100-fold and up to 1,000-fold or greater overall (e.g., as measured between 3-8 days following infection) compared to growth of the corresponding wild-type or mutant parental PIV strain.
  • both the cKa and cSF induced a high level of protection against HPIV3 challenge in the respiratory tract of rhesus monkeys, despite the exceptional degree of restriction of replication exhibited by these viruses in this model for human PIV infection and protection.
  • previous infection with either chimeric virus induced a high level of resistance to replication of the rJS challenge virus in both the upper and lower respiratory tract.
  • human-bovine chimeric PIV of the invention provides a high level of protection in the upper and lower respiratory tract of monkeys, and both chimeric viruses represent promising vaccine candidates.
  • the immunogenic activity of human-bovine chimeric PIV will be balanced against the level of attenuation to achieve useful vaccine candidates, and will typically be marked by a reduction of replication of challenge virus, e.g., rJS in the lower and/or upper respiratory tract by about 50-100-fold, 100-500-fold, preferably about 500-2,000-fold and up to 3,000-fold or greater overall (e.g., as measured between 3-8 days post-challenge).
  • challenge virus e.g., rJS in the lower and/or upper respiratory tract
  • the recombinant vaccine viruses of the invention maintain immunogenicity while exhibiting concomitant reductions in replication and growth. This su ⁇ rising assemblage of phenotypic traits is highly desired for vaccine development.
  • any BPIV gene or genome segment singly or in combination with one or more other BPIV gene(s) or genome segment(s), can be combined with HPIV sequences to produce an attenuated HPIV3/BPIV3 chimeric recombinant virus suitable for use as a vaccine virus.
  • all HPIVs including HPIVl, HPIV2, HPIV3 and variant strains thereof, are useful recipients for attenuating BPIV gene(s) and or genome segment(s).
  • the HPIV genes selected for inclusion in a HPIV3/BP ⁇ V3 chimeric virus will include one or more of the protective antigens, such as the HN or F glycoproteins.
  • human-bovine chimeric PIVs bearing one or more bovine gene(s) or genome segment(s) exhibit a high degree of host range restriction, e.g., in the respiratory tract of mammalian models of human PIV infection such as non-human primates.
  • the human PIV backbone is attenuated by the addition or substitution of one or more bovine gene(s) or genome segment(s), for example to a partial or complete human, e.g., HPIV3, PIV background genome or antigenome.
  • the partial or complete HPIV background genome or antigenome is combined with one or more heterologous gene(s) or genome segment(s) of a N, P and/or M gene of a BPIV to form a human-bovine chimeric PIV genome or antigenome.
  • the background genome or antigenome is an HPIV3 genome or antigenome and the heterologous gene or genome segment is a gene or genome segment of a BPIV3 M or P gene.
  • the M ORF of a HPIV3 background antigenome is substituted by a counte ⁇ art BPIV3 M ORF — yielding a novel recombinant human- bovine chimeric PIV cDNA clone designated rHPIV3M B .
  • the P ORF of a HPIV3 background antigenome is substituted by a counte ⁇ art BPIV3 P ORF — yielding a novel recombinant human-bovine chimeric PIV cDNA clone designated rHPIV3M P .
  • the invention also provides human-bovine chimeric PIV which have one or more substituted genome segment(s) from BPIV3 N, P and/or M gene(s) substituted or added within a partial or complete HPIV3 genome or antigenome.
  • the ORFs for the C, D, and V proteins, as well as the sequence for the RNA editing site are all contained within the sequence containing the P ORF. Therefore transfer of only a genome segment within the P ORF can selectively transfer one or a plurality of these genetic elements without transferring the entire P ORF.
  • Alternative human-bovine chimeric PIV of the invention will contain protective antigenic determinants of HPIVl or HPIV2. This may be achieved, for example, by expression of an HN and/or F gene of HPIVl or HPIV2 as an extra gene(s) in an attenuated HPIV3/BPIV3 chimeric recombinant.
  • a HPTV3 HPIV1 or a HPIV3/HPIV2 antigenic chimeric virus in which the HPIVl or HPIV2 HN and/or F genes replace their PIV3 counte ⁇ art(s) (Skiadopoulos et al., 1999a, supra; Tao et al, 1999, supra; and U.S. Patent Application Serial No.
  • a chimeric PIV1 vaccine candidate has been generated using the PIV3 cDNA rescue system by replacing the PF 3 HN and F open reading frames (ORFs) with those of PIV1 in a PIV3 full-length cDNA that contains the three attenuating mutations in L.
  • rPIV3-l .cp45 The recombinant chimeric virus derived from this cDNA is designated rPIV3-l .cp45 (Skiadopoulos et al, 1998, supra; Tao et al, 1998, supra; Tao et al, 1999, supra).
  • rPrV3-l.cp45L was attenuated in hamsters and induced a high level of resistance to challenge with PIV1.
  • a recombinant chimeric virus designated rPIV3-l.cp45, has also been produced that contains 12 of the 15 cp45 mutations, i.e., excluding the mutations in HN and F, and is highly attenuated in the upper and lower respiratory tract of hamsters (Skiadopoulos et al, 1999a, supra).
  • HPIV/BPIV chimeric recombinants will inco ⁇ orate two or more BPIV genes or genome segments, in any combination, up to and including all of the BPIV genome other than selected genes or antigenic determinants selected from HN or F gene(s) and genome segment(s), which could come from a human HPIVl, HPIV2, or HPIV3 virus.
  • Yet additional embodiments of the invention are directed to human-bovine chimeric PIV inco ⁇ orating attenuating genes from other animal PIVs, such as murine PIV1, the canine SV5 PIV2 virus, or another avian or mammalian PIV in combination with a HPIV backbone, alternatively including a chimeric HPIV backbone, from HPIVl , HPIV2, and/or HPIV3.
  • human-bovine chimeric PIV are employed as vectors for protective antigens of heterologous pathogens, including other PIVs and non-PIV viruses and non-viral pathogens.
  • the bovine- human chimeric genome or antigenome comprises a partial or complete PIV "vector genome or antigenome" combined with one or more heterologous genes or genome segments encoding one or more antigenic determinants of one or more heterologous pathogens (see, e.g., U.S. Provisional Patent Application Serial No. 60/170,195, filed December 10, 1999 by Mu ⁇ hy et al, inco ⁇ orated herein by reference).
  • the heterologous pathogen in this context may be a heterologous PIV and the heterologous gene(s) or genome segment(s) can be selected to encodes one or more PIV N, P, C, P, V, M, F, SH, where applicable, HN and/or L protein(s), as well as protein domains, fragments, and immunogenic regions or epitopes.
  • PIV vector vaccines thus constructed may elicit a polyspecific immune response and may be administered simultaneously or in a coordinate administration protocol with other vaccine agents.
  • human-bovine chimeric PIV may comprise a vector genome or antigenome that is a partial or complete HPIV genome or antigenome, which is combined with or is modified to inco ⁇ orate one or more heterologous genes or genome segments encoding antigenic determinant(s) of one or more heterologous PIV(s), including heterologous HPIVs selected from HPIVl, HPIV2, or HPIV3.
  • the vector genome or antigenome is a partial or complete HPIV3 genome or antigenome and the heterologous gene(s) or genome segment(s) encoding the antigenic determinant(s) is/are of one or more heterologous HPIV(s).
  • the chimeric genome or antigenome inco ⁇ orates one or more gene(s) or genome segment(s) of a BPIV that specifies attenuation.
  • the bovine-human chimeric PIV inco ⁇ orates one or more HPIVl or HPIV2 genes or genome segments that encode(s) one or more HN and/or F glycoproteins or antigenic domains, fragments or epitopes thereof within a partial or complete HPIV3 vector genome or antigenome.
  • both HPIVl genes encoding HN and F glycoproteins are substituted for counte ⁇ art HPIV3 HN and F genes to form a chimeric HPIV3-1 vector genome or antigenome.
  • Such recombinant constructs can be used to produce vaccine virus directly, or can be further modified by addition or inco ⁇ oration of one or more genes or gene segments encoding one or more antigenic determinants.
  • Such constructs for the production of vaccine viruses typically inco ⁇ orate one or more heterologous gene(s) or genome segment(s) of a BPIV that specifies attenuation, for example an open reading frame (ORF) encoding an attenuating BPIV protein, such as N.
  • ORF open reading frame
  • Certain human-bovine chimeric PIV of the invention may be employed as vectors for generating specific vaccines to HPIV2, for example wherein a transcription unit comprising an open reading frame (ORF) of an HPIV2 HN gene is added to or inco ⁇ orated within a chimeric HPIV3- 1 vector genome or antigenome and the chimeric construct is attenuated by inco ⁇ oration of a BPIV gene or genome segment.
  • the vector genome or antigenome is a partial or complete BPIV genome or antigenome
  • the heterologous genes or genome segments encoding the antigenic determinant(s) is/are of one or more HPIV(s).
  • the determinant(s) is/are selected from HPIVl, HPIV2 or HPIV3 HN and F glycoproteins, but antigenic domains, fragments and epitopes of these and other antigenic proteins are also useful.
  • one or more genes or genome segments encoding one or more antigenic determinant(s) of HPIV2 is/are added to or substituted within the partial or complete BPIV vector genome or antigenome.
  • a plurality of heterologous genes or genome segments encoding antigenic determinants of multiple HPIVs may be added to or inco ⁇ orated within the partial or complete BPIV vector genome or antigenome.
  • human-bovine chimeric PIV are provided as vectors for a range of non-PIV pathogens (see, e.g., U.S. Provisional Patent Application Serial No. 60/170,195, filed Pecember 10, 1999 by Mu ⁇ hy et al, inco ⁇ orated herein by reference).
  • the vector genome or antigenome for use within these aspects of the invention may comprise a partial or complete BPIV or HPIV genome or antigenome, and the heterologous pathogen may be selected from measles virus, subgroup A and subgroup B respiratory syncytial viruses, mumps virus, human papilloma viruses, type 1 and type 2 human immunodeficiency viruses, he ⁇ es simplex viruses, cytomegalovirus, rabies virus, Epstein Barr virus, filoviruses, bunyaviruses, flaviviruses, alphaviruses and influenza viruses.
  • the heterologous pathogen may be selected from measles virus, subgroup A and subgroup B respiratory syncytial viruses, mumps virus, human papilloma viruses, type 1 and type 2 human immunodeficiency viruses, he ⁇ es simplex viruses, cytomegalovirus, rabies virus, Epstein Barr virus, filoviruses, bunyaviruses, flaviviruses, alpha
  • a HPIV or BPIV vector genome or antigenome for constructing bovine-human chimeric PIV of the invention may inco ⁇ orate heterologous antigenic determinant(s) selected from the measles virus HA and F proteins, or antigenic domains, fragments and epitopes thereof.
  • a transcription unit comprising an open reading frame (ORF) of a measles virus HA gene is added to or inco ⁇ orated within a BPIV or HPIV3 vector genome or antigenome.
  • bovine-human chimeric PIV of the invention may be used as vectors to inco ⁇ orate heterologous antigenic determinant(s) from respiratory syncytial virus (RSV), for example by inco ⁇ orating one or more genes or genome segments that encode(s) RSV F and/or G glycoprotein or immunogenic domain(s) or epitope(s) thereof.
  • RSV respiratory syncytial virus
  • the cloning of RSV cPNA and other disclosure useful within the invention is provided in U.S. Patent Application No. 08/720,132, filed September 27, 1996 (corresponding to priority U.S. Provisional Patent Application No. 60/007,083, filed September 27, 1995); U.S. Patent Application No. 09/444,067, filed November 19, 1999 (corresponding to priority U.S.
  • human-bovine chimeric PIV which inco ⁇ orate at least one antigenic determinant from a heterologous PIV or non-PIV pathogen.
  • one or more individual gene(s) or genome segment(s) of HPIV3 may be replaced with counte ⁇ art gene(s) or genome segment(s) from human RSV, or an RSV gene or genome segment can be inserted or added as an supernumerary gene.
  • a selected, heterologous genome segment e.g.
  • encoding a cytoplasmic tail, transmembrane domain or ectodomain of an RSV glycoprotein is substituted for a counte ⁇ art genome segment in, e.g., the same gene in HPIV3 or within a different gene in HPIV3, or added within a non-coding sequence of the HPIV3 genome or antigenome to yield a chimeric PIV-RS V glycoprotein.
  • a genome segment from an F gene of human RSV is substituted for a counte ⁇ art HPIV3 genome segment to yield constructs encoding chimeric proteins, e.g.
  • fusion proteins having a cytoplasmic tail and/or transmembrane domain of PIV fused to an ectodomain of RSV to yield a novel attenuated virus, and/or a multivalent vaccine immunogenic against both PIV and RSV.
  • these documents describe methods and procedures for mutagenizing, isolating and characterizing PIV to obtain attenuated mutant strains (e.g., temperature sensitive (ts), cold passaged (cp) cold-adapted (ca), small plaque (sp) and host-range restricted (hr) mutant strains) and for identifying the genetic changes that specify the attenuated phenotype.
  • mutant strains e.g., temperature sensitive (ts), cold passaged (cp) cold-adapted (ca), small plaque (sp) and host-range restricted (hr) mutant strains
  • ts temperature sensitive
  • cp cold passaged
  • sp small plaque
  • hr host-range restricted
  • Also disclosed in the above-inco ⁇ orated references are methods for constructing and evaluating infectious recombinant PIV that are modified to inco ⁇ orate phenotype-specific mutations identified in biologically-derived PIV and non-PIV mutants, e.g., cold passaged (cp), cold adapted (ca), host range restricted (hr), small plaque (sp), and/or temperature sensitive (ts) mutants, for example the JS HPIV3 cp45 mutant strain. Other mutations may be attenuating without an auxiliary marker phenotype. Mutations identified in these mutants can be readily adopted in human- bovine chimeric PIV.
  • one or more attenuating mutations occur in the polymerase L protein, e.g., at a position corresponding to Tyr 9 2 , Leu 992 , or Thr 1558 of JS wild type HPIV3.
  • these mutations are inco ⁇ orated in human- bovine chimeric PIV of the invention by an identical, or conservative, amino acid substitution as identified in the biological mutant.
  • PIV recombinants may inco ⁇ orate a mutation wherein Tyr 942 is replaced by His, Leu 992 is replaced by Phe, and/or Thr ⁇ ss is replaced by He. Substitutions that are conservative to these replacement amino acids are also useful to achieve a desired mutant phenotype.
  • exemplary mutations adopted from a biologically derived PIV mutant include one or more mutations in the N protein, including specific mutations at a position corresponding to residues Val 96 or Ser 389 of JS. In more detailed aspects, these mutations are represented as Val 96 to Ala or Ser 389 to Ala or substitutions that are conservative thereto. Also useful within recombinant PIV of the invention are amino acid substitution in the C protein, e.g., a mutation at a position corresponding to Ile 96 of JS, preferably represented by an identical or conservative substitution of Ile 96 to Thr.
  • Further exemplary mutations adopted from biologically derived PIV mutants include one or more mutations in the F protein, including mutations adopted from JS cp45 at a position corresponding to residues Ile 0 or Ala ⁇ o of JS, preferably represented by acid substitutions Ile 20 to Val or Ala ⁇ o to Thr or substitutions conservative thereto.
  • Other human-bovine chimeric PIV within the invention adopt one or more amino acid substitutions in the HN protein, as exemplified herein below by a recombinant PIV adopting a mutation at a position corresponding to residue Val 84 of JS, preferably represented by the substitution Val 38 to Ala.
  • Yet additional examples within this aspect of the invention include human- bovine chimeric PIV which inco ⁇ orate one or more mutations in noncoding portions of the PIV genome or antigenome, for example in a 3 ' leader sequence.
  • Exemplary mutations in this context may be engineered at a position in the 3 ' leader of a recombinant virus at a position corresponding to nucleotide 23, 24, 28, or 45 of JS.
  • Yet additional exemplary mutations may be engineered in the N gene start sequence, for example by changing one or more nucleotides in the N gene start sequence, e.g., at a position corresponding to nucleotide 62 of JS.
  • human-bovine chimeric PIV inco ⁇ orate a T to C change at nucleotide 23, a C to T change at nucleotide 24, a G to T change at nucleotide 28, and/or a T to A change at nucleotide 45. Additional mutations in extragenic sequences are exemplified by a A to T change in N gene start sequence at a position corresponding to nucleotide 62 of JS.
  • a large "menu" of attenuating mutations is provided, each of which can be combined with any other mutation(s) for adjusting the level of attenuation, immunogenicity and genetic stability in a recombinant PTV bearing C, D, and/or V deletion or knock out mutation(s).
  • many recombinant PIVs of the invention will include one or more, and preferably two or more, mutations from biologically derived PIV mutants, e.g., any one or combination of mutations identified in JS cp45.
  • Preferred PIV recombinants within the invention will inco ⁇ orate a plurality and up to a full complement of the mutations present in JS cp45 or other biologically derived mutant PIV strains.
  • these mutations are stabilized against reversion in human-bovine chimeric PIV by multiple nucleotide substitutions in a codon specifying each mutation.
  • Additional mutations that may be inco ⁇ orated in human-bovine chimeric PIV of the invention are mutations, e.g., attenuating mutations, identified in heterologous PIV or more distantly related nonsegmented negative stranded RNA viruses.
  • attenuating and other desired mutations identified in one negative stranded RNA virus may be "transferred", e.g., introduced by mutagenesis in a corresponding position, within the genome or antigenome of the human-bovine chimeric PIV.
  • desired mutations in one heterologous negative stranded RNA virus are transferred to the PIV recipient (e.g., bovine or human PIV, respectively).
  • 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 function of the wild- type residue).
  • Negative stranded RNA viruses from which exemplary mutations are identified and transferred into human-bovine chimeric PIV of the invention include other PIVs (e.g., HPIVl, HPIV2, HPTV3, HPTV4A, HPIV4B and BPIV), RSV, Sendai virus (SeV), Newcastle disease virus (NDV), simian virus 5 (SV5), measles virus (MeV), rinde ⁇ est virus, canine distemper virus (CDV), rabies virus (RaV) and vesicular stomatitis virus (VSV), among others.
  • PIVs e.g., HPIVl, HPIV2, HPTV3, HPTV4A, HPIV4B and BPIV
  • RSV Sendai virus
  • NDV Newcastle disease virus
  • SV5 Sendai virus
  • NDV Newcastle disease virus
  • SV5 simian virus 5
  • Measles virus Measles virus
  • rinde ⁇ est virus canine distemper
  • a variety of exemplary mutations for use within the invention are disclosed in the above-inco ⁇ orated reference, including but not limited to an amino acid substitution of phenylalanine at position 521 of the RSV L protein corresponding to and therefore transferable to a substitution of phenylalanine (or a conservatively related amino acid) at position 456 of the HPIV3 L protein.
  • mutations marked by deletions or insertions these can be introduced as corresponding deletions or insertions into the recombinant virus, either within the background genome or antigenome or within the heterologous gene or genome segment inco ⁇ orated therein.
  • the particular size and amino acid sequence of the deleted or inserted protein fragment can vary.
  • Yet additional human-bovine PIV vaccine candidates within the invention can be achieved by modifying the chimeric PTV genome or antigenome to encode an analogous mutation to an attenuating mutation identified in Sendai virus (SeV).
  • the attenuating mutation comprises an amino acid substitution of phenylalanine at position 170 of the C protein of SeV.
  • the PIV genome or antigenome is modified to encode an alteration of a conserved residue that corresponds conservatively to the alteration marking the attenuating mutation in the heterologous, SeV mutant.
  • the mutation is inco ⁇ orated within a recombinant HPIV3 protein and comprises an amino acid substitution of phenylalanine at position 164 of the C protein of HPIV3.
  • target proteins are amenable to introduction of attenuating mutations from one negative stranded RNA virus at a corresponding site within chimeric human-bovine PIV of the invention.
  • five target proteins are strictly conserved and show moderate to high degrees of sequence identity for specific regions or domains.
  • all known members of the order share a homologous constellation of five proteins: a nucleocapsid protein (N), a nucleocapsid phosphoprotein (P), a nonglycosylated matrix (M) protein, at least one surface glycoprotein (HN, F, H, or G) and a large polymerase (L) protein.
  • proteins all represent useful targets for inco ⁇ orating attenuating mutations by altering one or more conserved residues in a protein of the recombinant virus at a site corresponding to the site of an attenuating mutation identified in the heterologous, mutant virus.
  • the methods for transferring heterologous mutations into chimeric human-bovine PIV of the invention are based on identification of an attenuating mutation in a first negative stranded RNA virus.
  • the mutation identified in terms of mutant versus wild-type sequence at the subject amino acid position(s) marking the site of the mutation, provides an index for sequence comparison against a homologous protein in the chimeric virus (either in the background genome or antigenome or in the heterologous gene or gene segment added or substituted therein) that is the target for recombinant attenuation.
  • the attenuating mutation may be previously known or may be identified by mutagenic and reverse genetics techniques applied to generate and characterize biologically-derived mutant virus.
  • attenuating mutations of interest may be generated and characterized de novo, e.g., by site directed mutagenesis and conventional screening methods .
  • Each attenuating mutation identified in a negative stranded RNA virus provides an index for sequence comparison against a homologous protein in one or more heterologous negative stranded virus(es).
  • existing sequence alignments may be analyzed, or conventional sequence alignment methods may be employed to yield sequence comparisons for analysis, to identify corresponding protein regions and amino acid positions between the protein bearing the attenuating mutation and a homologous protein of a different virus that is the target recombinant virus for attenuation.
  • the genome or antigenome of the target virus is recombinantly modified to encode an amino acid deletion, substitution, or insertion to alter the conserved residue(s) in the target virus protein and thereby confer an analogous, attenuated phenotype on the recombinant virus.
  • the wild-type identity of residue(s) at amino acid positions marking an attenuating mutation in one negative stranded RNA virus may be conserved strictly, or by conservative substitution, at the corresponding amino acid position(s) in the target, human-bovine chimeric virus protein.
  • the corresponding residue(s) in the target virus protein may be identical, or may be conservatively related in terms of amino acid side-group structure and function, to the wild- type residue(s) found to be altered by the attenuating mutation in the heterologous, mutant virus.
  • analogous attenuation in the recombinant virus may be achieved according to the methods of the invention by modifying the recombinant genome or antigenome of the target virus to encode the amino acid deletion, substitution, or insertion to alter the conserved residue(s).
  • the genome or antigenome it is preferable to modify the genome or antigenome to encode an alteration of the conserved residue(s) that corresponds conservatively to the alteration marking the attenuating mutation in the heterologous, mutant virus.
  • a substitution should be engineered at the corresponding residue(s) in the recombinant virus.
  • the substitution will be identical or conservative 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 function of the wild-type residue).
  • mutations marked by deletions or insertions these can transferred as corresponding deletions or insertions into the recombinant virus, however the particular size and amino acid sequence of the deleted or inserted protein fragment can vary.
  • mutations thus transferred from heterologous mutant negative stranded viruses may confer a variety of phenotypes within human-bovine chimeric PIV of the invention, in addition to or associated with the desired, an attenuated phenotype.
  • exemplary mutations inco ⁇ orated within recombinant proteins of the virus may confer temperature sensitive (ts), cold-adapted (ca), small plaque (sp), or host range restricted (hr) phenotypes, or a change in growth or immunogenicity, in addition to or associated with the attenuated phenotype.
  • Attenuating mutations in biologically derived PIV and other nonsegmented negative stranded RNA viruses for inco ⁇ oration within human-bovine chimeric PIV may occur naturally or may be introduced into wild-type PIV strains by well known mutagenesis procedures.
  • incompletely attenuated parental PIV 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 described in the above inco ⁇ orated references.
  • biologically derived PTV any PIV not produced by recombinant means.
  • biologically derived PIV include all naturally occurring PIV, including, e.g., naturally occurring PIV having a wild-type genomic sequence and PIV having allelic or mutant genomic variations from a reference wild-type RSV sequence, e.g., PIV having a mutation specifying an attenuated phenotype.
  • biologically derived PIV include PIV mutants derived from a parental PIV by, inter alia, artificial mutagenesis and selection procedures.
  • a sufficiently attenuated biologically derived PIV mutant can be accomplished by several known methods.
  • One such procedure involves subjecting a partially attenuated virus to passage in cell culture at progressively lower, attenuating temperatures.
  • partially attenuated mutants are produced by passage in cell cultures at suboptimal temperatures.
  • a cp mutant or other partially attenuated PIV strain is adapted to efficient growth at a lower temperature by passage in culture. This selection of mutant PIV during cold-passage substantially reduces any residual virulence in the derivative strains as compared to the partially attenuated parent.
  • specific mutations can be introduced into biologically derived PIV 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 of the attenuated derivative.
  • Means for the introduction of ts mutations into PIV include replication of the virus in the presence of a mutagen such as 5-fluorouridine according to generally known procedures. Other chemical mutagens can also be used. Attenuation can result from a ts mutation in almost any PIV gene, although a particularly amenable target for this pu ⁇ ose has been found to be the polymerase (L) gene.
  • the level of temperature sensitivity of replication in exemplary attenuated PIV 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 PIV correlate with the mutant's shutoff temperature.
  • the JS cp45 HPJV3 mutant has been found to be relatively stable genetically, highly immunogenic, and satisfactorily attenuated.
  • the above-inco ⁇ orated references also disclose how to routinely distinguish between silent incidental mutations and those responsible for phenotype differences by introducing the mutations, separately and in various combinations, into the genome or antigenome of infectious PIV 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.
  • Mutations thus identified are compiled into a "menu” and are then introduced as desired, singly or in combination, to adjust a human-bovine chimeric PIV to an appropriate level of attenuation, immunogenicity, genetic resistance to reversion from an attenuated phenotype, etc., as desired.
  • the ability to produce infectious PIV from cDNA permits introduction of specific engineered changes within human-bovine chimeric PIV.
  • infectious, recombinant PIVs are employed for identification of specific mutation(s) in biologically derived, attenuated PIV strains, for example mutations which specify ts, ca, att and other phenotypes.
  • Desired mutations are thus identified and introduced into recombinant, human-bovine chimeric PIV vaccine strains.
  • the capability of producing virus from cDNA allows for routine inco ⁇ oration 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 can 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 PIV are single nucleotide changes, other "site specific" mutations can also be inco ⁇ orated by recombinant techniques into biologically derived or recombinant PIV.
  • 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 PIV sequence, from a sequence of a selected mutant PIV strain, or from a parent recombinant PIV clone subjected to mutagenesis).
  • Such site-specific mutations may be inco ⁇ orated at, or within the region of, a selected, biologically derived point mutation.
  • the mutations can be introduced in various other contexts within a PIV 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 PIV mutants typically retain a desired attenuating phenotype, but may additionally exhibit altered phenotypic characteristics unrelated to attenuation, e.g., enhanced or broadened immunogenicity, and/or improved growth.
  • site-specific mutants include recombinant PIV designed to inco ⁇ orate 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 PIV clone, yielding a biologically derived or recombinant PIV 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 the human-bovine chimeric PIV disclosed herein include deletions, insertions, substitutions or rearrangements of one or more gene(s) or genome segment(s). Particularly useful are deletions involving one or more gene(s) or genome segment(s), which deletions have been shown to yield additional desired phenotypic effects for adjusting the characteristics of human-bovine chimeric PIV within the invention.
  • U.S. Patent Application Serial No. 09/350,821 filed by Durbin et al.
  • these modifications may specify one or more desired phenotypic changes including (i) altered growth properties in cell culture, (ii) attenuation in the upper and/or lower respiratory tract of mammals, (iii) a change in viral plaque size, (iv) a change in cytopathic effect, and (v) a change in immunogenicity.
  • One such exemplary "knock out" mutant lacking C ORF expression, designated rC-KO was able to induce a protective immune response against wild type HPIV3 challenge in a non-human primate model despite its beneficial attenuation phenotype.
  • human-bovine chimeric PIV inco ⁇ orate deletion or knock out mutations in a C, D, and/or V ORF(s) which alters or ablates expression of the selected gene(s) or genome segment(s).
  • This can be achieved, e.g., by introducing a frame shift mutation or termination codon within a selected coding sequence, altering translational start sites, changing the position of a gene or introducing an upstream start codon to alter its rate of expression, changing GS and/or GE transcription signals to alter phenotype, or modifying an RNA editing site (e.g., growth, temperature restrictions on transcription, etc.).
  • human-bovine chimeric PIVs are provided in which expression of one or more gene(s), e.g., a C, D, and/or V ORF(s), is ablated at the translational or transcriptional level without deletion of the gene or of a segment thereof, by, e.g., introducing multiple translational termination codons into a translational open reading frame (ORF), altering an initiation codon, or modifying an editing site.
  • ORF translational open reading frame
  • knock-out virus will often exhibit reduced growth rates and small plaque sizes in tissue culture.
  • these methods 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.
  • knock-out virus phenotypes produced without deletion of a gene or genome segment can be alternatively produced by deletion mutagenesis, as described, to effectively preclude correcting mutations that may restore synthesis of a target protein.
  • C, D, and/or V ORF(s) deletion and knock out mutants can be made using alternate designs and methods that are well known in the art (as described, for example, in (Kretschmer et al, Virology 216:309-316, 1996; Radecke et al, Virology 217:418-421, 1996; and Kato et al, 1987a, supra; and Schneider et al, 1997, supra; each inco ⁇ orated herein by reference).
  • nucleotide modifications in human-bovine chimeric PIV may alter small numbers of 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), or nearly complete or complete genes (e.g., 1,000-1,500 nucleotides, 1,500-2,500 nucleotides, 2,500-5,000, nucleotides, 5,000-6,5000 nucleotides or more) 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 genome segment, whereas large block(s) of bases are involved when genes or large genome 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
  • the invention provides for supplementation of mutations adopted into a recombinant PIV clone from biologically derived PIV, e.g., cp and ts mutations, with additional types of mutations involving the same or different genes in a further modified PIV clone.
  • Each of the PIV genes can be selectively altered in terms of expression levels, or can be added, deleted, substituted or rearranged, in whole or in part, alone or in combination with other desired modifications, to yield a human-bovine chimeric PIV exhibiting novel vaccine characteristics.
  • the present invention also provides a range of additional methods for attenuating or otherwise modifying the phenotype of human-bovine chimeric PIV based on recombinant engineering of infectious PIV clones.
  • a variety of alterations can be produced in an isolated polynucleotide sequence encoding a targeted gene or genome segment, including a donor or recipient gene or genome segment in a chimeric PIV genome or antigenome for inco ⁇ oration 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 genome or antigenome, as well as mutations which delete, substitute, introduce or rearrange whole gene(s) or genome segment(s), within a human-bovine chimeric PTV clone.
  • modifications in the human-bovine chimeric PIV which simply alter or ablate expression of a selected gene, e.g., by introducing a termination codon within a selected PIV coding sequence or altering its translational start site or RNA editing site, changing the position of a PTV 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 RNA(s).
  • modifications in the human-bovine chimeric PIV which simply alter or ablate expression of a selected gene, e.g., by introducing a termination codon within a selected PIV coding sequence or altering its
  • any PIV gene or genome segment which is not essential for growth can be ablated or otherwise modified in a recombinant PIV to yield desired effects on virulence, pathogenesis, immunogenicity and other phenotypic characters.
  • coding sequences noncoding, leader, trailer and intergenic regions can be similarly deleted, substituted or modified and their phenotypic effects readily analyzed, e.g., by the use of minireplicons and recombinant PIV.
  • a variety of other genetic alterations can be produced in a PIV genome or antigenome for inco ⁇ oration into human-bovine chimeric PIV, alone or together with one or more attenuating mutations adopted from a biologically derived mutant PIV, e.g., to adjust growth, attenuation, immunogenicity, genetic stability or provide other advantageous structural and/or phenotypic effects.
  • additional types of mutations are also disclosed in the foregoing inco ⁇ orated references and can be readily engineered into human-bovine chimeric PIV of the invention.
  • restriction site markers are routinely introduced within the human-bovine chimeric PIV antigenome or genome to facilitate cDNA construction and manipulation.
  • genes in a human-bovine chimeric PIV can be changed, a PIV genome promoter replaced with its antigenome counte ⁇ art, portions of genes removed or substituted, and even entire genes deleted.
  • 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.
  • mutations for inco ⁇ oration into human-bovine chimeric PIV of the invention include mutations directed toward cis-acting signals, which can be identified, e.g., by mutational analysis of PIV minigenomes. For example, insertional and deletional analysis of the leader and trailer and flanking sequences identifies viral promoters and transcription signals and provides 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 affect RNA replication or transcription.
  • any of these mutations can be inserted into a human-bovine chimeric PIV antigenome or genome as described herein. Evaluation and manipulation of trans-acting proteins and cis-acting RNA sequences using the complete antigenome cDNA is assisted by the use of PIV minigenomes as described in the above-inco ⁇ orated references.
  • Additional mutations within the human-bovine chimeric PIV involve replacement of the 3' end of genome with its counte ⁇ art from antigenome, which is associated with changes in RNA replication and transcription.
  • the level of expression of specific PIV 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 (Hans et al, Current Biol. 6:315-324, 1996, inco ⁇ orated herein by reference). Optimization by recombinant methods of the codon usage of the mRNAs encoding the HN and F proteins of PIV, which are the major protective antigens, will provide improved expression for these genes.
  • a sequence surrounding a translational start site (preferably including a nucleotide in the -3 position) of a selected PIV gene is modified, alone or in combination with introduction of an upstream start codon, to modulate PIV gene expression by specifying up- or down-regulation of translation (Kozak et al., J. Mol. Biol. 196:947-950, 1987, inco ⁇ orated herein by reference).
  • gene expression of a human-bovine chimeric PIV can be modulated by altering a transcriptional GS or GE signal of any selected gene(s) of the virus.
  • levels of gene expression in the human-bovine chimeric PIV are modified at the level of transcription.
  • the position of a selected gene in the PIV 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 (see, U.S. Provisional Patent Application entitled RESPIRATORY SYNCYTIAL VIRUS VACCINES EXPRESSING PROTECTIVE ANTIGENS FROM PROMOTER- PROXIMAL GENES, filed by Krempl et al. on June 23, 2000 and identified by Attorney Docket No. 15280-424000US, inco ⁇ orated herein by reference).
  • modulation of expression for specific genes can be achieved yielding reductions or increases of gene expression from two-fold, more typically four- fold, up to ten- fold or more compared to wild-type levels often attended by a commensurate decrease in expression levels for reciprocally, positionally substituted genes.
  • transpositioning changes yield novel human-bovine chimeric PIV having attenuated phenotypes, for example due to decreased expression of selected viral proteins involved in RNA replication, or having other desirable properties such as increased antigen expression.
  • one or more of the PIV N, P, M, HN and/or F gene(s), or genome segment(s), is/are shifted to a more promoter-proximal or promoter-distal location in the chimeric genome or antigenome (e.g., by inserting, deleting or rearranging one or more polynucleotides in the background genome or antigenome, which may or may not involve the heterologous gene or genome segment as a "displacement polynucleotide" compared to the wild type position(s) of the subject gene(s) or genome segment(s) within the recombinant or background genome or antigenome.
  • a more promoter-proximal or promoter-distal location in the chimeric genome or antigenome e.g., by inserting, deleting or rearranging one or more polynucleotides in the background genome or antigenome, which may or may not involve the heterologous gene or genome segment as a "displacement polynucleo
  • Infectious human-bovine chimeric PIV 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 PIV or a parent PIV.
  • an immunogenic epitope from a heterologous PIV strain or type, or from a non-PIV source such as RSV can be added to a recombinant clone by appropriate nucleotide changes in the polynucleotide sequence encoding the genome or antigenome.
  • mutant PIV of the invention can be engineered to add or ablate (e.g., by amino acid insertion, substitution or deletion) immunogenic proteins, protein domains, or forms of specific proteins associated with desirable or undesirable immunological reactions.
  • additional genes or genome segments may be inserted into or proximate to the human-bovine chimeric PIV 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 PIV genes identified above, as well as non-PIV genes.
  • Non-PIV genes of interest include those encoding cytokines (e.g., IL-2 through IL-18, especially IL-2, IL-6 and IL- 12, IL-18, etc.).
  • Gamma-interferon, and proteins rich in T helper cell epitopes are additional proteins. These additional proteins can be expressed either as a separate protein, or as a supernumerary copy of an existing PIV proteins, such as HN or F. This provides the ability to modify and improve the immune responses against PIV both quantitatively and qualitatively.
  • compositions e.g., isolated polynucleotides and vectors inco ⁇ orating human-bovine chimeric PIV-encoding cDNA
  • infectious PIV are generated from a PIV genome or antigenome, a nucleocapsid (N) protein, a nucleocapsid phosphoprotein (P), and a large (L) polymerase protein.
  • compositions and methods are provided for introducing the aforementioned structural and phenotypic changes into a recombinant PIV to yield infectious, attenuated vaccine viruses.
  • infectious, human- bovine chimeric PJV clone can be achieved by a variety of well known methods.
  • infectious clone with regard to DNA 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.
  • a variety of other mutagenesis techniques are known and available for use in producing the mutations of interest in the PIV antigenome or genome cDNA. Mutations can vary from single nucleotide changes to replacement of large cDNA pieces containing one or more genes or genome regions.
  • mutations are introduced by using the Muta-gene phagemid in vitro mutagenesis kit available from Bio-Rad.
  • cDNA encoding a portion of a PTV genome or antigenome is cloned into the plasmid pTZ 18U, 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.
  • Infectious PTV of the invention are produced by intracellular or cell-free coexpression of one or more isolated polynucleotide molecules that encode a PIV genome or antigenome RNA, together with one or more polynucleotides encoding viral proteins necessary to generate a transcribing, replicating nucleocapsid.
  • viral proteins useful for coexpression to yield infectious PIV are the major nucleocapsid protein (N) protein, nucleocapsid phosphoprotein (P), large (L) polymerase protein, fusion protein (F), hemagglutinin-neuraminidase glycoprotein (HN), and matrix (M) protein.
  • N nucleocapsid protein
  • P nucleocapsid phosphoprotein
  • L large
  • F fusion protein
  • HN hemagglutinin-neuraminidase glycoprotein
  • M matrix
  • products of the C, D and V ORFs of PIV are also useful in this context.
  • cDNAs encoding a PIV genome or antigenome are constructed for intracellular or in vitro coexpression with the necessary viral proteins to form infectious PIV.
  • PIV antigenome is meant an isolated positive-sense polynucleotide molecule which serves as a template for synthesis of progeny PIV genome.
  • a cD A is constructed which is a positive-sense version of the PIV genome corresponding to the replicative intermediate RNA, or antigenome, so as to minimize the possibility of hybridizing with positive-sense transcripts of complementing sequences encoding proteins necessary to generate a transcribing, replicating nucleocapsid.
  • the genome or antigenome of a recombinant PIV need only contain those genes or portions thereof necessary to render the viral or subviral particles encoded thereby infectious.
  • 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.
  • the PIV genome or antigenome encodes all functions necessary for viral growth, replication, and infection without the participation of a helper virus or viral function provided by a plasmid or helper cell line.
  • recombinant PIV is meant a PIV or PIV-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 PIV gene expression, for example, a promoter, a structural or coding sequence which is transcribed into PIV RNA, and appropriate transcription initiation and termination sequences.
  • the genome or antigenome is coexpressed with those PIV N, P and L proteins necessary to (i) produce a nucleocapsid capable of RNA replication, and (ii) render progeny nucleocapsids competent for both RNA replication and transcription.
  • PIV proteins Transcription by the genome nucleocapsid provides the other PIV proteins and initiates a productive infection. Alternatively, additional PIV proteins needed for a productive infection can be supplied by coexpression.
  • Synthesis of PIV antigenome or genome together with the above- mentioned viral proteins can also be achieved in vitro (cell-free), e.g., using a combined transcription-translation reaction, followed by transfection into cells.
  • antigenome or genome RNA can be synthesized in vitro and transfected into cells expressing PIV proteins.
  • complementing sequences encoding proteins necessary to generate a transcribing, replicating PIV 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 PIV cDNA.
  • monoclonal antibodies which react immunologically with the helper virus but not the virus encoded by the PIV 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 PIV cPNA to provide antigenic diversity from the helper virus, such as in the HN or F glycoprotein genes.
  • the N, P, L and other desired PIV proteins are encoded by one or more non- viral expression vectors, which can be the same or separate from that which encodes the genome or antigenome. Additional proteins may be included as desired, each encoded by its own vector or by a vector encoding one or more of the N, P, L and other desired PIV proteins, or the complete genome or antigenome.
  • Expression of the genome or antigenome and proteins from transfected plasmids can be achieved, for example, by 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, inco ⁇ orated herein by reference in its entirety).
  • the viral proteins, and/or T7 RNA polymerase can also be provided by transformed mammalian cells or by transfection of preformed mRNA or protein.
  • a PIV antigenome may be constructed for use in the present invention by, e.g., assembling cloned cPNA segments, representing in aggregate the complete antigenome, by polymerase chain reaction or the like (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 Piego, 1990; each inco ⁇ orated herein by reference in its entirety) of reverse-transcribed copies of PIV mRNA or genome RNA.
  • PCR polymerase chain reaction
  • a first construct is generated which comprises cPNAs containing the left hand end of the antigenome, spanning from an appropriate promoter (e.g., T7 RNA polymerase promoter) and assembled in an appropriate expression vector, such as a plasmid, cosmid, phage, or PNA virus vector.
  • an appropriate promoter e.g., T7 RNA polymerase promoter
  • the vector may be modified by mutagenesis and/or insertion of synthetic polylinker containing unique restriction sites designed to facilitate assembly.
  • synthetic polylinker containing unique restriction sites designed to facilitate assembly.
  • the N, P, L and other desired PIV proteins can be assembled in one or more separate vectors.
  • the right hand end 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 be any of the other suitable ribozymes such as that of hepatitis delta virus (Perrotta et al, Nature 350:434-436. 1991), inco ⁇ orated herein by reference in its entirety) which would yield a 3' end free of non- PIV nucleotides.
  • the left- and right-hand ends are then joined via a common restriction site.
  • nucleotide insertions, deletions and rearrangements can be made in the PIV genome or antigenome during or after construction of the cPNA.
  • specific desired nucleotide sequences can be synthesized and inserted at appropriate regions in the cPNA using convenient restriction enzyme sites.
  • Such techniques as site-specific mutagenesis, alanine scanning, PCR mutagenesis, or other such techniques well known in the art can be used to introduce mutations into the cPNA.
  • cPNA encoding the genome or antigenome
  • reverse transcription-PCR using improved PCR conditions (e.g., as described in Cheng et al, Proc. Natl. Acad. Sci. USA 91:5695-5699, 1994, inco ⁇ orated herein by reference) to reduce the number of subunit cPNA 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.
  • Pifferent DNA vectors e.g., cosmids
  • Isolated polynucleotides encoding the genome or antigenome may be inserted into appropriate host cells by transfection, electroporation, mechanical insertion, transduction or the like, into cells which are capable of supporting a productive PIV infection, e.g., HEp-2, FRhL-DBS2, LLC-MK2, MRC-5, and Vero cells.
  • Transfection of isolated polynucleotide sequences may be introduced into cultured cells by, for example, calcium phosphate-mediated transfection (Wigler et al, Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virolo g v 52:456.
  • the N, P, L and other desired PIV proteins are encoded by one or more helper viruses which is phenotypically distinguishable from that which encodes the genome or antigenome.
  • the N, P, L and other desired PIV proteins can also be 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 one or more of the N, P, L and other desired PIV proteins, or the complete genome or antigenome.
  • infectious clones of PIV the invention permits a wide range of alterations to be recombinantly produced within the PIV genome (or antigenome), yielding defined mutations which specify desired phenotypic changes.
  • infectious clone cDNA or its product, synthetic or otherwise, RNA capable of being directly inco ⁇ orated into infectious virions which can be transcribed into genomic or antigenomic RNA capable of serving as a template to produce the genome of infectious viral or subviral particles.
  • defined mutations can be introduced by a variety of conventional techniques (e.g., site-directed mutagenesis) into a cDNA copy of the genome or antigenome.
  • genomic or antigenomic cDNA sub fragments to assemble a complete genome or antigenome cDNA as described herein has the advantage that each region can be manipulated separately, where small cDNA subjects provide for better ease of manipulation than large cDNA subjects, and then readily assembled into a complete cDNA.
  • the complete antigenome or genome cDNA, or a selected subfragment thereof can be used as a template for oligonucleotide-directed mutagenesis.
  • a mutated subfragment can then be assembled into the complete antigenome or genome cDNA.
  • a variety of other mutagenesis techniques are known and can be routinely adapted for use in producing the mutations of interest in a PIV antigenome or genome cDNA of the invention.
  • mutations are introduced by using the MUTA-gene® phagemid in vitro mutagenesis kit available from Bio-Rad Laboratories.
  • cDNA encoding an PIV 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 is then amplified.
  • Genome segments can correspond to structural and/or functional domains, e.g., cytoplasmic, transmembrane or ectodomains of proteins, active sites such as sites that mediate binding or other biochemical interactions with different proteins, epitopic sites, e.g., sites that stimulate antibody binding and/or humoral or cell mediated immune responses, etc.
  • Useful genome segments in this regard range from about 15-35 nucleotides in the case of genome 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.
  • the ability to introduce defined mutations into infectious PIV has many applications, including the manipulation of PIV pathogenic and immunogenic mechanisms.
  • PIV proteins including the N, P, M, F, HN, and L proteins and C, D and V ORF products
  • the functions of PIV proteins can be manipulated by introducing mutations which ablate or reduce the level of protein expression, or which yield mutant protein.
  • Various genome RNA structural features such as promoters, intergenic regions, and transcription signals, can also be routinely manipulated within the methods and compositions of the invention.
  • the effects of trans-acting proteins and cis-acting RNA sequences can be readily determined, for example, using a complete antigenome cDNA in parallel assays employing PTV minigenomes (Dimock et al, J. Virol. 67:2772-2778, 1993, inco ⁇ orated herein by reference in its entirety), whose rescue-dependent status is useful in characterizing those mutants that may be too inhibitory to be recovered in replication-independent infectious virus.
  • substitutions, insertions, deletions or rearrangements of genes or genome segments within recombinant PIV of the invention are made in structural or functional relation to an existing, "counte ⁇ art" gene or genome segment from the same or different PTV or other source.
  • Such modifications yield novel recombinants having desired phenotypic changes compared to wild-type or parental PIV or other viral strains.
  • recombinants of this type may express a chimeric protein having a cytoplasmic tail and/or transmembrane domain of one PIV fused to an ectodomain of another PIV.
  • Other exemplary recombinants of this type express duplicate protein regions, such as duplicate immunogenic regions.
  • counte ⁇ art genes, genome segments, proteins or protein regions are typically from heterologous sources (e.g., from different PIV genes, or representing the same (i.e., homologous or allelic) gene or genome segment in different PTV types or strains).
  • Typical counte ⁇ arts selected in this context share gross structural features, e.g., each counte ⁇ art may encode a comparable protein or protein structural domain, such as a cytoplasmic domain, transmembrane domain, ectodomain, binding site or region, epitopic site or region, etc.
  • Counte ⁇ art domains and their encoding genome segments embrace an assemblage of species having a range of size and sequence variations defined by a common biological activity among the domain or genome segment variants.
  • Counte ⁇ art genes and genome segments, as well as other polynucleotides disclosed herein for producing recombinant PIV within the invention often share substantial sequence identity with a selected polynucleotide "reference sequence,” e.g., with another selected counte ⁇ art sequence.
  • a "reference sequence” is a defined sequence used as a basis for sequence comparison, for example, a segment of a full-length cDNA or gene, or a complete cDNA or gene sequence. Generally, a reference sequence 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; inco ⁇ orated herein by reference), by the homology alignment algorithm of Needleman & Wunsch, (J. Mol. Biol. 48:443, 1970; inco ⁇ orated herein by reference), by the search for similarity method of Pearson & Lipman, (Proc. Natl. Acad. Sci. USA
  • 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.
  • the identical nucleic acid base e.g., A, T, C, G, U, or I
  • 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 recombinant PIV 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. Preferably, 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 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.
  • Abbreviations for the twenty naturally occurring amino acids used herein follow conventional usage (Immunology - A Synthesis, 2nd ed., E.S. Golub & D.R. Gren, eds., Sinauer Associates, Sunderland, MA, 1991, inco ⁇ orated herein by reference).
  • Stereoisomers e.g., D-amino acids of the twenty conventional 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, ⁇ -N,N,N-trimethyllysine, ⁇ -N-acetyllysine, O- phosphoserine, N-acetylserine, N-formylmethionine, 3 -methylhistidine, 5 -hydroxy lysine, ⁇ -N-methylarginine, and other similar amino acids and imino acids (e.g., 4- hydroxyproline).
  • amino acids may be modified by glycosylation, phosphorylation and the like.
  • virus 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.
  • the recombinant PIV of the invention are not only viable and more appropriately attenuated than previous vaccine candidates, but are more stable genetically in vitro-- 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.
  • Recombinant PIV of the invention can be tested in various well known and generally accepted in vitro and in vitro 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 PIV
  • in vitro assays the modified virus (e.g., a multiply attenuated, biologically derived or recombinant PIV) is tested, e.g., for temperature sensitivity of virus replication, i.e. ts phenotype, and for the small plaque or other desired phenotype.
  • Modified viruses are further tested in animal models of PIV infection. A variety of animal models have been described and are summarized in various references inco ⁇ orated herein.
  • PIV model systems including rodents and non-human primates, for evaluating attenuation and immunogenic activity of PIV vaccine candidates are widely accepted in the art, and the data obtained therefrom correlate well with
  • the invention also provides isolated, infectious recombinant PTV viral compositions for vaccine use.
  • the attenuated virus which is a component of a vaccine is in an isolated and typically purified form.
  • isolated is meant to refer to PIV 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 where it can be propagated and characterized in a controlled setting.
  • attenuated PIV of the invention may be produced by an infected cell culture, separated from the cell culture and added to a stabilizer.
  • recombinant PIV produced according to the present invention can be used directly in vaccine formulations, or lyophilized, as desired, using lyophilization protocols well known to the artisan. Lyophilized virus will typically be maintained at about 4°C. 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.
  • PTV vaccines of the invention contain as an active ingredient an immunogenically effective amount of PIV produced as described herein.
  • the modified virus may be introduced into a host with a physiologically acceptable carrier and/or adjuvant.
  • a physiologically acceptable carrier and/or adjuvant 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, MPLTM (3-o-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamilton, MT) and IL-12 (Genetics Institute, Cambridge MA), among many other suitable adjuvants well known in the art.
  • the immune system of the host responds to the vaccine by producing antibodies specific for PIV virus proteins, e.g., F and HN glycoproteins.
  • PIV virus proteins e.g., F and HN glycoproteins.
  • the host becomes at least partially or completely immune to PTV infection, or resistant to developing moderate or severe PIV infection, particularly of the lower respiratory tract.
  • the host to which the vaccines are administered can be any mammal which is susceptible to infection by PTV or a closely related virus and which host is capable of generating a protective immune response to the antigens of the vaccinizing strain. Accordingly, the invention provides methods for creating vaccines for a variety of human and veterinary uses.
  • the vaccine compositions containing the PIV of the invention are administered to a host susceptible to or otherwise at risk for PIV infection to enhance the host's own immune response capabilities. Such an amount is defined to be a "immunogenically effective dose.”
  • the precise amount of PIV to be administered within an effective dose will depend on the host's state of health and weight, the mode of administration, the nature of the formulation, etc., but will generally range from about 10 to about 10 plaque forming units (PFU) or more of virus per host, more commonly from about 10 4 to 10 6 PFU virus per host.
  • the vaccine formulations should provide a quantity of modified PIV of the invention sufficient to effectively protect the host patient against serious or life-threatening PIV infection.
  • the PIV produced in accordance with the present invention can be combined with viruses of other PTV serotypes or strains to achieve protection against multiple PTV serotypes or strains.
  • protection against multiple PIV serotypes or strains can be achieved by combining protective epitopes of multiple serotypes or strains engineered into one virus, as described herein.
  • viruses typically when different viruses are administered they will be in admixture and administered simultaneously, but they may also be administered separately. Immunization with one strain may protect against different strains of the same or different serotype.
  • the PIV vaccines of the invention can be employed as a vector for protective antigens of other pathogens, such as respiratory syncytial virus (RSV) or measles virus, by inco ⁇ orating the sequences encoding those protective antigens into the PIV genome or antigenome which is used to produce infectious PIV, as described herein (see, e.g., U.S. Provisional Patent Application Serial No. 60/170,195, filed December 10, 1999 by Mu ⁇ hy et al, inco ⁇ orated herein by reference).
  • RSV respiratory syncytial virus
  • the precise amount of recombinant PIV 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 10 to about 10 plaque forming units (PFU) or more of virus per patient, more commonly from about 10 4 to 10 6 PFU virus per patient.
  • the vaccine formulations should provide a quantity of attenuated PIV sufficient to effectively stimulate or induce an anti-PIV 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 PTV.
  • 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) PIV infection.
  • PTV vaccines produced in accordance with the present invention can be combined with viruses expressing antigens of another subgroup or strain of PIV to achieve protection against multiple PIV subgroups or strains.
  • the vaccine virus may inco ⁇ orate protective epitopes of multiple PTV strains or subgroups engineered into one PTV clone, as described herein.
  • the PIV 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 PIV. 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 vitro and in vitro. In many instances the host antibodies will also neutralize wild-type virus of a different, non- vaccine subgroup.
  • Preferred PIV vaccine candidates of the invention exhibit a very substantial diminution of virulence when compared to wild-type virus that is circulating naturally in humans.
  • the 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. However, its virulence is sufficiently abrogated such that severe lower respiratory tract infections in the vaccinated or incidental host do not occur.
  • the level of attenuation of PIV vaccine candidates 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 PIV or other attenuated PIV which have been evaluated as candidate vaccine strains.
  • the attenuated 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 or rhesus monkey, compared to the levels of replication of wild-type virus, e.g., 10- to 1000-fold less.
  • 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 PTV in the nasopharynx of an infected host are well known in the literature.
  • Levels of induced immunity provided by the vaccines of the invention can also be monitored by measuring amounts of neutralizing secretory and serum antibodies. Based on these measurements, vaccine dosages can be adjusted or vaccinations repeated as necessary to maintain desired levels of protection. Further, different vaccine viruses may be advantageous for different recipient groups. For example, an engineered PIV strain expressing an additional protein rich in T cell epitopes may be particularly advantageous for adults rather than for infants.
  • the PIV is employed as a vector for transient gene therapy of the respiratory tract.
  • the recombinant PIV genome or antigenome inco ⁇ orates 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 PIV expression.
  • PTV produced by coexpressing the recombinant PIV genome or antigenome with the N, P, L and other desired PTV proteins, and containing a sequence encoding the gene product of interest is administered to a patient.
  • Administration is typically by aerosol, nebulizer, or other topical application to the respiratory tract of the patient being treated.
  • Recombinant PIV 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 may be administered within this method are preferably suitable for transient expression, including, for example, 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.
  • interleukin-2 interleukin-4
  • gamma-interferon GM-CSF
  • G-CSF G-CSF
  • erythropoietin erythropoietin
  • cytokines glucocerebrosidase
  • phenylalanine hydroxylase phenylalanine hydroxylase
  • a chimeric recombinant virus was constructed in which the N ORF of the JS strain of HPIV3 was replaced by that of either the Ka or SF strain of BPIV3. These chimeric viruses possess the HN and F glycoproteins of the HPIV3 parent and will induce a high level of immunity to HPIV3 in primates. Both chimeric viruses were successfully recovered. Both grew to high titer in cell culture and both were found to be attenuated in rhesus monkeys. Thus, the N protein was identified as an exemplary protein that contributes to the host range phenotype of BPIV3.
  • the complete consensus nucleotide sequence for each of the Ka or SF BPTV3 strains was determined from RT-PCR products generated from virion RNA. These sequences are set forth in Figures 1A-1G, and Figures 2A-2G, respectively.
  • the full length cDNA encoding a complete 15456 nucleotide (nt) antigenomic RNA of BPIV3 Ka is set forth in Figures 1A-1G herein (see also GenBank accession #AF178654). It was noted during construction of one exemplary cDNA used within the present examples that the nucleotide sequence departed from the published GenBank sequence by two, non- material nucleotide differences — that may be attributed to sequencing error or other factors.
  • the former cDNA contains a sequence beginning at nucleotide 18, ACTGGTT, (SEQ ID NO: 1) whereas the corresponding published sequence (GenBank accession #AF178654; Figures 1A-1G) reads ACTTGCT (SEQ ID NO: 2) (differing nucleotides at positions 21 and 23 are underscored).
  • PCR was carried out on the first strand product using the Advantage cDNA PCR kit (Clontech Laboratories, Palo Alto, CA). Ka and SF genomes were each amplified by PCR in 3 or 4 overlapping fragments using primers homologous to regions of RNA conserved among previously-published paramyxovirus sequences. Each primer pair was constructed to include matching restriction enzyme sites (not represented in the sequence targeted for amplification).
  • a separate random library was generated for each amplicon by digesting a set of PCR products with the appropriate restriction enzyme, followed by gel-purification, ligation of the products into tandem arrays and sonication.
  • a random library was generated from this pool of sheared cDNA sequences by cloning a subset (approx. 500 bp fragments) into Ml 3.
  • the nucleotide sequences of cDNA inserts were determined by automated DNA sequencing using the Taq DYE Deoxy Terminator cycle sequencing kit (ABI, Foster City, CA).
  • a continuous sequence (contig) was assembled for each of the original large RT-PCR fragments with sufficient redundancy that each nucleotide position was confirmed by a minimum 3 independent Ml 3 clones.
  • the 5' and 3' terminal genomic sequences of Ka and SF were converted to cDNA using the system for Rapid Amplification of cDNA Ends (Life Technologies, Gaithersburg, MD) and sequenced by automated sequencing.
  • the N ORF of the Ka or SF virus was initially selected for replacement of the corresponding gene in the HPIV3 virus because the N gene represents a gene with an intermediate level of sequence divergence among the six HPIV3 and BPTV3 proteins.
  • Human-bovine chimeric full-length PIV3 genomes were constructed by introducing the BPIV3 Ka or SF N coding region as a replacement for its HPIV3 counte ⁇ art into the rJS cDNA p3/7(131)2G which encodes a complete copy of HPIV3 positive-sense antigenomic RNA (see, e.g., Durbin et al., 1991 a, supra; Hoffman et al, 1991, supra; Skiadopoulos et al, 1998, supra; U.S. Patent Application Serial No. 09/083,793, filed May 22, 1998, (corresponding to International Publication No. WO
  • BPIV3 and HPIV3 N coding regions with flanking sequences were first subcloned and further modified to permit an exchange of just the N ORF.
  • pUCl 19JSN bearing the HPTV3 N gene and the plasmids with a BPIV3 N Ka or SF gene (pBSKaN and PBSSFN) were subjected to mutagenesis using the method of Kunkel (Proc. Natl. Acad. Sci. USA 82:488-492, 1985, inco ⁇ orated herein by reference) to introduce Ncol and Afffl restriction enzyme recognition sites at translational start and stop sites, respectively (Figure 3, panel A).
  • the BPIV3 N coding region was introduced as an Ncol/Afllll fragment into pUCl 19 JSN-Ncol/Aflll as a replacement for the HPIV3 N coding region ( Figure 3, panel B).
  • the chimeric N genes which contain the HPTV3 3' and 5', noncoding sequences and the BPIV3 ORF, were modified by site-directed mutagenesis to restore the original HPIV3 noncoding sequence and BPTV3 coding sequence.
  • the resulting chimeric PIV3 plasmids comprising a human PIV 3 background genome or antigenome inco ⁇ orating a BPIV3 N protein, designated pB/HPIV3NKa or pB/HPrV3NSF, contained the full-length rJS antigenome in which the N ORF encoded the BPTV3 Ka or SF N protein.
  • Chimeric antigenomic HPIV3/BPIV3 cDNAs were transfected individually into HEp-2 cells grown to near-confluence in 6-well plates along with two previously-described support plasmids, pTM(P) and pTM(L), Lipofectace (Life Technologies, Gaithersburg, MD), and a modified vaccinia virus recombinant that expresses bacteriophage T7 RNA polymerase (MVA-T7) as previously described (Durbin et al, Virology 234:74-83, 1997b).
  • An N support plasmid used in previous work was omitted because the antigenomic plasmid expressed sufficient levels of the N protein.
  • the cultures were maintained for 3.5 days at 32°C after which supernatants were harvested, passaged in LLC-MK2 cells and plaque-purified 3 times in LLC-MK2 cells.
  • the identities of the chimeric viruses inco ⁇ orating a human PIV 3 background genome or antigenome and a BPTV3 N protein (designated as rHPIV3-N ⁇ chimeric recombinants or, more specifically, as "cKa” and "cSF” chimeric viruses recovered from the transfections were confirmed by sequencing RT-PCR products containing the regions of start and stop codons from virion RNA isolated , after amplification of triply plaque- purified virus (Figure 5).
  • This amplified product and the corresponding amplified HPIV3 rJS and BPTV3 Ka or SF sequences were also subjected to Taql digestion to confirm the chimeric identity of cKa and cSF viruses (Figure 6).
  • Taql digestion profiles were distinct for the 3 parental and 2 chimeric viruses and each parental profile included Taql fragments of unique size, allowing the contribution of sequence of rJS, Ka and SF parents to the chimeric viruses to be verified.
  • the recovered cKa and cSF chimeric recombinants each contained the expected sequences as designed.
  • Efficient replication of live attenuated virus vaccines in tissue culture cells is a feature of human-bovine chimeric PIV of the invention that permits efficient manufacture of the recombinant vaccine materials.
  • the multicycle replication of rJS parent, cKa, Ka parent, cSF, and SF parent in a bovine cell line (MDBK) and in a simian cell line (LLC-MK2) was determined by infecting cells with virus at a multiplicity of infection of 0.01 and harvesting samples (in triplicate) over a five day period of time (Figure 7) as previously described (Tao et al, 1998, supra, inco ⁇ orated herein by reference).
  • the chimeric viruses replicated efficiently in both cell lines like their human or bovine parent viruses without significant delay in replication or a significant reduction in the titer of virus achieved.
  • the chimeric viruses replicated to over 10 7 0 TCID 5 o/ml which is well above the 10 40 or 10 5 0 dose of live attenuated human or bovine PIV vaccines currently being used in human clinical trials (Karron et al, 1996, supra; Karron et al, 1995a, supra; and Karron et al, 1995b, supra).
  • Both the SF and Ka BPIV3s are attenuated for the upper and the lower respiratory tract of the rhesus monkey (van Wyke Coelingh et al, 1988, supra). This attenuation phenotype correlates with attenuation in humans (Karron et al, 1995a, supra) as indicated by the fact that Ka is highly restricted in replication in the upper respiratory tract of fully susceptible seronegative infants and children. The absence of cough, croup, bronchiolitis, or pneumonia in the BPIV3 -infected vaccinees suggests that the Ka BPIV3 virus is attenuated for the lower respiratory tract as well. Therefore, the rhesus monkey is widely accepted as a reasonably correlative model to evaluate attenuation of candidate PIV vaccine viruses and their efficacy against challenge with wild type PIV.
  • the rJS, cKa, Ka parent, cSF, and SF parent were administered intranasally and intratracheally at a dose of 10 5 0 TCID 50 per site to rhesus monkeys. Replication was monitored using previously described procedures for obtaining samples from the upper (nasopharyngeal swab specimens) and lower (tracheal lavage specimens) respiratory tract and for titering the virus in LLC-MK2 cells (Hall et al, 1992, supra).
  • the cKa and cSF recombinants were significantly attenuated for the upper respiratory tract (Table 1) exhibiting, respectively, a 63-fold or a 32-fold reduction in mean peak virus titer compared to that of the rJS HPIV3 parent. Both cKa and cSF were also attenuated for the lower respiratory tract, but this difference was only statistically significant for cSF.
  • the low level of replication of rJS in the lower respiratory tract made it difficult to demonstrate in a statistically-significant fashion further restriction of replication due to an attenuation phenotype at this site.
  • each chimeric virus cKa and cSF
  • the level of replication of each chimeric virus, cKa and cSF was not significantly different from its bovine parent in the upper or the lower respiratory tract, although the chimeric viruses each replicated somewhat better than their BPIV3 parents in the upper respiratory tract.
  • the acquisition of the N gene of either the Ka or SF BPIV3 by rJS HPIV3 attenuated the human virus for rhesus monkeys to a level approximately equivalent to that of the BPIV parent.
  • the HPIV3/BPIV3 chimeric recombinants replicated efficiently in tissue culture cells in vitro, it is clear that the phenotype of host range restricted replication manifested by the two bovine parental viruses was transferred to HPIV3 by the N ORF.
  • the observation that the level of replication of cKa and cSF is slightly greater than that of their BPIV parents in the upper respiratory tract suggests that additional bovine genes will contribute to the host range attenuation phenotype at this site.
  • the titer of virus present at each site was determined for each monkey on LLC-MK2 cell monolayers, and the titers presented are mean peak titers (Hall et al, 1992, supra).
  • Previous infection with either chimeric virus induced a high level of resistance to replication of the rJS challenge virus in both the upper and lower respiratory tract.
  • Monkeys previously infected with cKa manifested a 300-fold reduction of replication of wild type HPIV3 (rJS) in the upper respiratory tract and a 1000-fold reduction in the lower tract compared to uninoculated control monkeys.
  • Monkeys previously infected with cSF manifested a 2000-fold reduction of replication of rJS in the upper respiratory tract and a 1000-fold reduction in the lower tract compared to uninoculated control monkeys.
  • HPIV3 -specific HAI-responses induced by the chimeric viruses were statistically indistinguishable from that induced by immunization with rJS.
  • An additional unexpected result demonstrated herein is that, following challenge of the monkeys with HPIV3, the level of HAI antibody in monkeys initially immunized with cKa-N or cSF-N was significantly greater than levels observed in animals immunized with rJS, Ka or SF.
  • certain aspects of the invention are directed to chimeric viruses that are attenuated by one or more host-range attenuating genetic elements of BPTV3, wherein the chimeric virus retains the immunogenicity of a human PIV, for example HPIVl, HPIV2 or HPIV3.
  • the major neutralization and protective antigens of HPTVs are the F and HN glycoproteins.
  • certain human-bovine chimeric constructs for developing live-attenuated HPIV vaccine viruses will contain one or more of the HPIV3 F and HN glycoprotein genes, or one or more genome segments encoding an immunogenic domain, region or epitope of HN and/or F, in a backbone in which one or more genes or genome segments (e.g., from N, P, C, V, D, M, and/or L) are derived from BPIV3.
  • the subject genes or genome segments may encode proteins or selected protein domains or immunogenic fragments.
  • additional BPIV genetic elements including cis-acting promoter and transcription signals in the leader, trailer, gene-start, gene-end, editing, and intergenic regions, can be used to confer an attenuation phenotype on the resulting chimeric vaccine candidate.
  • two chimeric viruses (rHPIV3-P ⁇ ) and (rHPIV3- M B ) are constructed bearing the P or M ORF, respectively, of the wild type Kansas/15626/84 strain of BPIV3 (see Figures 8-10).
  • the ORFs for the C, D, and V proteins, as well as the sequence for the RNA editing site are contained within the sequence containing the P ORF. Therefore transfer of the P ORF also transfers these genetic elements.
  • rHPIV3 cDNA plasmids bearing the P or M ORF of BPIV3 were assembled by first introducing unique restriction sites near the start and stop codons of the human P or M ORF and its bovine counte ⁇ art using site-directed mutagenesis. The P or M ORF of HPIV3 was then exchanged with that of its BPIV3 counte ⁇ art using molecular cloning techniques as described herein and in Bailly et al, J. Virol. 74:3188-3195, 2000: Durbin et al, Virologv 235:323-332. 1997a; Durbin et al, Virologv 234:74-83. 1997b; Durbin et al.
  • pUC(M-B) The P ORF of HPTV3 in pUC(M-B) (Skiadopoulos et al, Virol. 73:1374-1381, 1999) was first modified.
  • pUC(M-B) is a subclone of the full-length HPIV3 cDNA plasmid p3/7(131)2G. This plasmid contains nt 1-3903 of the complete HPIV3 antigenomic RNA sequence (HPIV3 JS strain; GenBank accession #Z11575).
  • pUC(M-B) was modified by site-directed mutagenesis using the Clontech site-directed mutagenesis kit as described previously (Skiadopoulos et al, Virol.
  • bovine PIV3 P ORF in ⁇ UC(DF) was engineered at the analogous P ORF translation start and stop positions to introduce Ncol (TCATGG (SEQ ID NO: 7) to CCATGG (SEQ ID NO: 4); BPIV3 nts 1782-1787; BPIV3 Kansas strain; GenBank accession #AF178654) and Mel (GCCAAC (SEQ ID NO: 8) to GCTAGC (SEQ TD NO: 9); BPIV3 nts 3578-3583) restriction sites, respectively.
  • Ncol TCATGG (SEQ ID NO: 7) to CCATGG (SEQ ID NO: 4); BPIV3 nts 1782-1787; BPIV3 Kansas strain; GenBank accession #AF178654) and Mel (GCCAAC (SEQ ID NO: 8) to GCTAGC (SEQ TD NO: 9); BPIV3 nts 3578-3583) restriction sites, respectively.
  • the specific sequence at the P ORF translation start and stop is given in Table 3.
  • This table provides a nucleotide sequence comparison of the genomic region of rHPIV3-P B flanking the translation initiation codon (ATG, in bold type) and the translation termination codon (TAG or TAA, in bold type) of the P ORF.
  • the nucleotide sequencing of the 2156 bp RT-PCR fragment generated from vRNA of rHPIV3-P ⁇ around the junctions of the BPIV3 P ORF and the HPTV3 flanking sequences was determined using a Perkin Elmer ABI 310 sequencer, and the engineered sequences for the rHPIV3-P ⁇ were confirmed to be present.
  • the determined sequence of rHPIV3-P ⁇ is indicated in comparison to that of its two parents.
  • the introduced i zel restriction enzyme recognition sequence following the P ORF stop codon in the chimera is italicized and the BPTV3 sequences are underlined.
  • the human PTV3 P ORF present in the Ncol to Nhel fragment in the modified pUC(M-B) was exchanged with that of the modified BPIV3 P ORF in pUC(DF).
  • the sequence immediately preceding the translation initiation site was changed by site-directed mutagenesis to restore the original HPIV3 sequence (CCATGG (SEQ ID NO: 4) to TGATGG (SEQ ID NO:3); HPIV3 nts 1782-1787).
  • CCATGG SEQ ID NO: 4
  • TGATGG SEQ ID NO:3
  • HPIV3 nts 1782-1787 HPIV3 nts 1782-1787
  • pUC(M-B) and pUC(B-BII) are subclones of p3/7(131)2G that contain HPIV3 nts 1-3903 and 3903-5261, respectively.
  • pUC(M-B) was modified by site directed mutagenesis to introduce an Sphl restriction site near the M ORF translation initiation codon (AAATGA (SEQ ID NO: 16) to GCATGC (SEQ ID NO: 17); HPIV3 nts 3751-3756).
  • pUC(B-BII) was modified to contain an Mel restriction site after the translation termination codon (AATCTC (SEQ TD NO: 18) to GCTAGC (SEQ ID NO: 9); HPIV3 nts 4816-4821).
  • the bovine PIV3 M ORF in pUC(Fl) was mutagenized near the analogous M ORF translation start and stop positions to introduce Sphl (CAATGA (SEQ ID NO: 19) to GCATGC (SEQ ID NO: 17); BPIV3 nts 3733-3738) and el (ATCAAC (SEQ ID NO: 20) to GCTAGC (SEQ TD NO: 9); BPTV3 nts 4792-4797) restriction sites, respectively.
  • HPIV3 M ORF The two portions of the modified HPIV3 M ORF were cloned into the p(Left+2G) plasmid (Durbin et al, Virologv 235:323-332, 1997a) (HPIV3 nts 1-7437). Standard molecular cloning techniques were then employed to exchange the human PIV3 M ORF for that of the BPTV3 in the Sphl to Nhel fragment as referenced above.
  • the specific sequence at the M ORF translation start and stop is given in Table 4.
  • This table provides a nucleotide sequence comparison of the genomic region of rHPTV3-M B flanking the translation initiation codon (ATG, in bold type) and the translation termination codon (TAG or TAA, in bold type) of the M ORF.
  • the nucleotide sequencing of the 3445 bp RT-PCR fragment generated from vRNA of rHPIV3-M B around the junctions of the BPIV3 M ORF and the HPIV3 flanking sequences was determined using a Perkin Elmer ABI 310 sequencer, and the engineered sequences for the rHPTV3-M B were confirmed to be present.
  • the determined sequence of rHPIV3-M ⁇ is indicated in comparison to that of its two parents.
  • the introduced Nhel restriction enzyme recognition sequence following the M ORF stop codon in the chimera is italicized and the BPIV3 specific sequences are underlined.
  • the M ORF of BPIV3 is 6 nt shorter than that of HPIV3, and thus the rule of six was maintained.
  • Chimeric virus was recovered by transfection of HEp-2 cells with each individual chimeric full-length cDNA, the pTM(N), ⁇ TM(PnoC) and pTM(L) support plasmids, and with MVA-T7 infection as described previously (Durbin et al, Virology 235:323-332, 1997a; Durbin et al, Virologv 234:74-83, 1997b; Durbin et al, Virologv 261:319-330, 1999; Skiadopoulos et al, Virol. 73:1374-1381, 1999).
  • rHPIV3-P B and rHPIV3-M B replicated efficiently in tissue culture.
  • rHPIV3-P B replicated to an average titer of 10 7 8 TCID 5 o/ml
  • rHPIV3-M B replicated to an average titer of 10 8 0 TCID 50 /ml, compared to 10 8 2 TCID 50 /ml, for rHPIV3 wt.
  • both rHPIV3-P B and rHPIV3-M ⁇ were viable and exhibited efficient replication in vitro.
  • rHPIV3-P B and ⁇ HPIV3-M B were confirmed by isolation of the viral RNA (vRNA) and generation of specific RT-PCR fragments containing the BPTV3 ORF flanked by HPIV3 sequence using previously described techniques (Skiadopoulos et al, J. Virol. 72:1762-1768, 1998; Skiadopoulos et al, Virol 73: 1374- 1381, 1999), with PCR primers flanking the BPIV3 P or BPIV3 M ORF ( Figure 8, panels A and B, Figure 9, panels A-C, and Figure 10, panels A-C).
  • Sense (HPIV3 nts 1629- 1661) and antisense primers (HPIV3 nts 3802-3763) were used to generate the 2156 bp fragment containing the BPTV3 P ORF in rHPIV3-P B ( Figure 8).
  • Sense (HPIV3 nts 1629-1661) and antisense primers (HPTV3 nts 5079-5041) were used to generate the 3445 bp fragment containing the BPIV3 M ORF in rHPIV3-M B ( Figure 10).
  • a chimeric virus was generated in which the N open reading frame (ORF) of rHPIV3 was replaced with that of BPIV3, and the resulting chimeric virus, rHPTV3-NB, replicated efficiently in vitro but was attenuated for the respiratory tract of rhesus monkeys.
  • ORF N open reading frame
  • the ⁇ HPIV3-N B chimera induced high titers of antibody in rhesus monkeys to HPIV3 and protected them against wild type HPIV3 challenge.
  • the unrestricted nature of the replication phenotype of rHPIV3-N B in vitro is a highly desirable, because it makes it possible to efficiently produce recombinant virus for vaccine use.
  • the BPIV3 N protein has been demonstrated herein to be sufficiently compatible with human PIV3 internal proteins to permit efficient growth in vitro, while at the same time it is shown that replacement of this single ORF confers a host range restriction phenotype in vitro.
  • Each virus was administered intranasally and intratracheally at a dose of 10 5 0 TCID 50 per site.
  • the mean peak titer at each site was quantified for each virus by plaque titration in LLC-MK2 cells (e.g., as described by Bailly et al, J. Virol. 74:3188-3195, 2000; Hall et al, Virus Res. 22:173-184, 1992; and Schmidt et al, J. Virol. 74:8922-9, 2000, each inco ⁇ orated herein by reference), and the results are presented in Table 5.
  • the rHPIV3 NB, rHPIV3 PB, and rHPIV3 MB chimeric viruses were each restricted in replication in the upper respiratory tract compared to the level of replication of rHPIV3 (Table 5), demonstrating that each bovine ORF can specify an attenuation phenotype when transferred to rHPIV3.
  • Table 5 the level of replication of rHPIV3
  • rHPIV3 NB and rHPIV3 P B were attenuated and replicated to levels slightly lower than those of the highly attenuated BPIV3 Ka virus.
  • Monkeys were inoculated intranasally and intratracheally with 10 TCID 50 of virus in a 1 ml inoculum at each site.
  • Groups 1 and 5 contain animals from previous rhesus studies (Bailley et al., J Virol. 74:3188-3195, 2000, and Schmidt et al, J Virol 74:8922-9, 2000).
  • Group 5 contains 2 new animals from the present study.
  • Group 1 contains 4 animals from the present study.
  • c. Mean of the peak virus titers for each animal in its group irrespective of sampling day. S.E. - standard error. d.
  • Virus titrations were performed on LLC-MK2 cells at 32°C. The limit of detection of virus titer was 10 TCID 5 o/ml.
  • e Nasopharyngeal swab samples were collected on days 1 to 10 post-infection.
  • rHPIV3 N ⁇ , rHPIV3 P B , and rHPIV3 M B induce a high level of resistance of rhesus monkeys to wild type HPIV3 challenge.
  • each chimeric virus induced a hemagglutination-inhibiting (HAI) antibody response to HPTV3 that was 8-fold or greater in magnitude than that induced by immunization with the BPIV3 parent and was almost as immunogenic as rHPIV3.
  • HAI hemagglutination-inhibiting
  • rhesus monkeys previously immunized with the parent viruses or with rHPTV3 NB, rHPIV3 P B , or rHPIV3 M B were challenged intranasally and intratracheally with 10 6 TCTD 50 of the biologically derived JS strain of HPTV3 virus on day 28 or 31 post- immunization.
  • the animals immunized with the recombinant or biologically derived PIV3s were protected against challenge with HPTV3 (Table 6) as indicated by a 100-fold or greater reduction in replication of challenge virus in both the upper and the lower respiratory tract of the animals immunized with rHPIV3 N B , rHPTV3 P B , or rHPIV3 M B compared to that of control animals.
  • Serum HAI tier is expressed as the mean reciprocal log 2 ⁇ standard error.
  • Virus titrations were performed on LLC-MK2 cells at 32°C. The limit of detection of virus titer was 10TCID 50 /ml. Nasopharyngeal swab and Trachael lavage samples were collected on days 2, 4,6, 8 and 10 post-challenge. The tiers on day 0 were ⁇ 10 TCID50/ml.
  • groups 2 and 4 the data presented includes historical data from studies reported previously (Bailley et al, J Virol.
  • the control group consisted of animals that were not immunized, or that had received 10 5 pfu of respiratory syncytial virus (2 new animals in the present study).
  • backbone bearing the HPIV3 F and HN genes substituted for the counte ⁇ art BPIV3 glycoprotein genes, were generated to assess the effect of glycoprotein substitution on replication of HPIV3 and BPIV3 in the upper and lower respiratory tract of rhesus monkeys.
  • the F and HN genes of HPIV 3 were replaced with their BPTV3 counte ⁇ arts, resulting in a chimeric recombinant designated rHPIV3-
  • the reciprocal chimeric recombinant PIV3 ( ⁇ BPIV3-FHHN H ) was constructed by replacing the F and HN genes of a recombinant BPIV3 (rBPTV3) with their HPIV3 counte ⁇ arts.
  • rBPTV3 recombinant BPIV3
  • the introduction of the HPTV3 F and HN ORFs into the BPIV3 backbone combines the antigenic determinants of HPIV3 with the backbone of BPIV3 and thus provides and improved vaccine candidate compared with parental BPIV3.
  • HEp-2 and simian LLC-MK2 monolayer cell cultures were maintained in MEM medium (Life Technologies, Gaithersburg, MD) supplemented with 5% fetal bovine serum (Summit Biotechnology, Ft. Collins, CO), 50ug/ml gentamicin sulfate, and 4mM glutamine (Life Technologies, Gaithersburg, MD).
  • the wild type BPTV3 strain Kansas/15626/84 (Clone 5-2-4, Lot BPI3-1) (BPTV3 Ka), the HPIV3 JS wild type, its recombinant version (rHPIV3), and the rHPIV3 virus containing the BPTV3 Ka N ORF in place of the HPIV3-N ORF (rHPIV3-N B ) are each described above (see also, Clements et al, 1991, supra; Karron et al, 1995a, supra; Bailly et al, 2000, supra; and Durbin et al, 1997, supra).
  • PIVs were propagated at 32°C in LLC-MK2 cells (ATCC CCL-7), as previously described (Hall et al, 1992, supra).
  • the modified vaccinia strain Ankara (MV A) recombinant virus that expresses bacteriophage T7 RNA polymerase is described by Wyatt et al (1995, supra).
  • a full length cDNA was constructed to encode the complete 15456 nucleotide (nt) antigenomic RNA of BPIV3 Ka, as described above.
  • the cDNA was assembled from 4 subclones derived from reverse transcription (RT) of viral RNA using the Superscript II Pre-amplification System (Life Technologies, Gaithersburg, MD) and polymerase chain reaction (PCR) amplification with a High Fidelity PCR kit (Clontech Laboratories, Palo Alto, CA).
  • RT-PCR products were cloned into modified pUC19 plasmids (New England Biolabs, Beverly, MA) using the following naturally occurring internal restriction enzyme recognition sites: Sma I (BPIV3 Ka sequence position ntl86), Pst I (nt 2896), Mlu I (nt 6192), Sac II (nt 10452) and Bsp LU11 (nt 15412).
  • Sma I BPIV3 Ka sequence position ntl86
  • Pst I nt 2896
  • Mlu I nt 6192
  • Sac II nt 10452
  • Bsp LU11 nt 15412
  • Multiple subclones of the antigenomic cDNA were sequenced using a Perkin Elmer ABI 310 sequencer with dRhodamine Terminator Cycle Sequencing (Perkin Elmer Applied Biosystems, Warrington, UK), and only those matching the consensus sequence of BPIV 3 Ka were used for assembly of the full length clone.
  • BPIV3 Ka The 3' and 5' ends of BPIV3 Ka were cloned and the assembly of the full length cDNA took place in the previously described p(Right) vector (Durbin et al, 1997, supra), which we modified to contain a new polylinker with restriction enzyme recognition sites for Xho I, Sma I, Mlu I, Sac II, Eco RI, Hind III and Rsrll.
  • the full length cDNA clone ⁇ BPIV3(l 84) contained the following elements in 3' to 5' order: a T7 promoter followed by 2 non- viral guanosine residues, the complete antigenomic sequence of BPIV3 Ka, a hepatitis delta virus ribozyme and a T7 polymerase transcription terminator (Bailly et al, 2000, supra; and Durbin et al., 1997a, supra).
  • the nucleotide number given for the position of restriction enzyme recognition sites indicates the nucleotide after which the enzyme cuts, not the first nucleotide of the restriction enzyme recognition site.
  • the sequence was changed from TCCAACATTGCA (SEQ. ID. NO. 27) to TCCACCGGTGCA (SEQ. ID. NO. 28) in rBPIV3 and from CGGACGTATCTA (SEQ. ID. NO. 29) to CGCACCGGTGTA (SEQ. ID. NO. 30) in rHPIV3 (recognition sites underlined).
  • BsfWl restriction sites were introduced in the downstream non-coding region of the HN gene at nt 8595 of the rBPIV3 sequence and at nt 8601 of the rHPIV3 JS sequence.
  • the sequence was changed from GATATAAAGA (SEQ. ID. NO. 31) to GACGTACGGA (SEQ. ID. NO. 32) in rBPTV3 to give pBPIVs(l 07) and from GACAAAAGGG (SEQ. ID. NO. 33) to GACGTACGGG (SEQ. ID. NO. 34) in rHPIV3 to give pHPIVs(106).
  • the F and HN genes were exchanged between pBPIVs(107) and pHPIV3s(l 06) by digestion of each with SgrAI and BsiWl, gel purification of the fragments, and assembly of the appropriate fragments into the two full length cDNAs.
  • BPIV3 backbone bearing the HPIV3 F and HN genes designated pBPIV(215), encoded 15438 nts of viral sequence, of which nts 4811 to 8577 came from HPIV3, and it was used to derive rBPIV3-F H HN H ( Figures 11 A-l 1C).
  • BPIV3 support plasmids for recovery of virus from cDNA.
  • Support plasmids encoding the BPIV3 Ka N, P and L genes were assembled in modified pUC19 vectors and then cloned into the previously described pTM vector (Durbin et al, 1997a, supra).
  • an Nco I site was introduced at the start codon of the N, P and L open reading frames (ORFs) using site-directed mutagenesis.
  • the Nco I restriction site and a naturally occurring restriction site downstream of each ORF was used for cloning into pTM.
  • HEp-2 cells (approximately 1.5 x 10 6 cells per well of a six-well plate) were grown to 90% confluence and transfected with 0.2 ⁇ g each of the BPIV3 support plasmids ⁇ TM(N) and ⁇ TM(P), and 0.1 ⁇ g of pTM(L), along with 5 ⁇ g of the full length antigenomic cDNA and 12 ⁇ l LipofectACE (Life Technologies, Gaithersburg, MD). Each transfection mixture also contained 1.5 x 10 7 plaque forming units (PFU) of MVA-T7, as previously described (Durbin et al, 1997, supra).
  • PFU plaque forming units
  • the cultures were incubated at 32°C for 12 hrs before the medium was replaced with MEM (Life Technologies, Gaithersburg, MD) containing 10% fetal bovine serum.
  • MEM Life Technologies, Gaithersburg, MD
  • the supernatants were harvested after incubation at 32°C for an additional three days, and were passaged onto LLC-MK2 cell monolayers in 25cm 2 flasks and incubated for 5 days at 32°C.
  • Virus present in the supernatant was plaque-purified three times prior to amplification and characterization.
  • heterologous F and HN genes in the bovine or human PTV3 backbone was confirmed in plaque-purified recombinant viruses by RT-PCR of viral RNA isolated from infected cells or supernatant, which was performed using a primer pair that recognizes conserved sequences in rBPIV3 and rHPIV3.
  • the multicycle growth kinetics of BPIV3 Ka, rHPIV3-F B HN B , rBPIV3- F H HN H , rHPTV3-N B and rHPIV3 in LLC-MK2 cells were determined by infecting cells in triplicate at a multiplicity of infection (MOI) of 0.01 and harvesting samples at 24 hr intervals over a six day period, as previously described (Tao et al, 1998, supra). Samples were flash- frozen and titered in a single assay on LLC-MK2 cell monolayers in 96 well plates at 32°C, as described (Durbin et al. Virology 261:319-330, 1999b, inco ⁇ orated herein by reference) .
  • MOI multiplicity of infection
  • Rhesus monkeys seronegative for PIV3 as determined by hemagglutination-inhibition (HAI) assay were inoculated intranasally and intratracheally in groups of 2 or 4 animals with 10 5 tissue culture infectious dose 50 (TCID 50 ) per ml of BPTV3 Ka, rHPIV3-F B HN B , rBPIV3-F H HN H; rHPTV3-N B or rHPTV3.
  • TCID 50 tissue culture infectious dose 50
  • Nasopharyngeal swabs were collected daily on days 1 to 11 and on day 13. Tracheal lavage samples were collected on days 2, 4, 6, 8, and 10 post- infection.
  • Nasopharyngeal swab samples were collected on days 3, 4, 5, 6, 7 and 8, and tracheal lavage samples on days 4, 6 and 8 post challenge. Samples were titered in a single assay as described above. Serum was collected on day 28 post challenge.
  • a complete BPIV3 antigenomic cDNA designated pBPIV( 184), was constructed to encode the consensus sequence of BPIV3 Ka.
  • This BPIV3 antigenomic cDNA was further modified by the introduction of unique SgrAI and BsiWl sites into the downstream noncoding region of the M and HN genes, respectively.
  • the same restriction sites were introduced into the downstream noncoding region of the M and HN genes of a previously described complete HPIV3 antigenomic cDNA, p3/7(131)2G (Durbin et al, 1997a, supra).
  • the F and HN glycoprotein genes of HPIV3 and BPIV3 were swapped by exchanging this SgrAl-Bsi ⁇ l restriction fragment.
  • HPIV3 antigenomic cDNA bearing the BPIV3 F and HN genes was designated pHPIV(215)
  • BPIV3 antigenomic cDNA bearing the HPTV3 F and HN genes was designated pBPIV(215).
  • the antigenomic cDNAs pBPTV(184), pHPIV(215), pBPIV(215) and p3/7(131)2G were separately transfected into HEp-2 cells along with the three BPIV3 support plasmids pTM(N), pTM(P) and pTM(L), and the cells were simultaneously infected with recombinant MVA expressing the T7 RNA polymerase.
  • RNA or RNA from supernatant from each cloned virus was analyzed by RT-PCR using a primer pair that recognized identical sequences in HPTV3 JS and BPTV3 Ka.
  • the primer pair amplified a 4.8 kb fragment of DNA corresponding to the downstream end of the M gene, the F and HN genes, and the upstream end of the L gene (nts 4206-9035 in rBPTV3, nts 4224-9041 in rHPIV3, nts 4206-9017 in rBPIV3-F H HN H , and nts 4224-9059 in rHPIV3-F B HN B ).
  • the generation of each PCR product was dependent upon the inclusion of reverse transcriptase, indicating that each was derived from viral RNA and not from contaminating cDNA.
  • PCR products were then digested with Eco RI, which would be predicted to yield a different, unique restriction enzyme digest pattern for each of the four viruses ( Figure 12). In each case, the predicted pattern was observed, confirming the identity of the backbone and the inserted F and HN genes.
  • nucleotide sequencing was performed on the RT- PCR products to confirm the presence of the introduced restriction sites and flanking sequences.
  • CPE cytopathic effect
  • BPIV3/HPIV3 chimeric viruses replicate efficiently in cell culture.
  • the F and HN genes of the BPIV3/HPIV3 chimeric viruses are determinants of the host range restriction of replication of BPIV3 Ka in the respiratory tract of rhesus monkeys.
  • rHPIV3-F B HN B and ⁇ BPIV3-F H HN H were evaluated for their ability to replicate in the upper and lower respiratory tract of rhesus monkeys.
  • the effects of introduction of the BPIV3 F and HN genes into HPIV3 on attenuation of replication in rhesus monkeys was demonstrated, as described above for the BPIV3 N protein (see also, Bailly et al, 2000, supra).
  • Each chimeric virus was administered intranasally and intratracheally to rhesus monkeys at a dose of 10 5 TCID 50 per site.
  • the level of replication of the chimeric viruses was compared to that of the rHPIV3 and BPIV3 parental viruses and to that of ⁇ HPIV3-N B (Table 7). Since the rHPIV3 parental virus replicated to a low to moderate level in the lower respiratory tract, meaningful comparisons between groups could only be made for replication in the upper respiratory tract.
  • the level of replication of rHPIV3- FBHN B was similar to that of its BPIV3 parent and substantially lower than that of its HPTV3 parent (Table 3; Figure 14, panel A).
  • BPIV3 glycoprotein genes contained one or more major determinants of the host range attenuation phenotype of BPTV3 for rhesus monkeys.
  • the magnitude and pattern of replication of rHPIV3- F B HN B and ⁇ HPIV3-N B were very similar, indicating that each of the two bovine genetic elements, namely the N gene versus the F and HN genes, attenuate HPIV3 to a similar extent.
  • the groups with 6 animals contain 4 animals each from a previous rhesus study (Bailly et al., 2000, supra).
  • ⁇ BPIV3-F H HN H chimeric virus replicated significantly less well than rHPTV3 (Table 5), and it grouped with BPIV3 in a Duncan multiple range test.
  • panel B suggested that rBPIV3- F H HNH replicated to a level intermediate between that of its HPIV3 and BPIV3 parents.
  • the interpretation that ⁇ BPIV3-F H HNH replicates to a level intermediate between that of its parents is supported by Friedman's test of consistency of ranks (Sprent, P., "A Generalization Of The Sign Test," Applied Nonparametric Statistical Methods, pp.
  • the chimeric BPIV3 bearing HPIV3 glycoprotein genes induces serum HAI antibody to HPIV3 and a high level of resistance to wt HPIV3 challenge.
  • ⁇ BPIV3-F H HNH has important features that make it a candidate live attenuated virus vaccine against HPIV3, including attenuating genes from BPIV3 and the antigenic specificity of HPIV3, i.e. the F and HN glycoproteins, which are the major protective antigens. Therefore, its immunogenicity and protective efficacy against challenge with HPTV3 were documented.
  • Rhesus monkeys were immunized by infection with BPIV3 Ka, rHPIV3-F B HN B , rBPIV3-F H HN H , rHPIV3-N B , or rHPIV3. They were challenged 28 days later with HPIV3 JS wild type virus. Serum samples were taken prior to the initial infection on day 0 and prior to the challenge.
  • BPIV3 and ⁇ HPIV3-F B HN B induced serum HAI antibodies that reacted more efficiently with BPIV3 than HPIV3, whereas the converse was the case for HPIV3 and ⁇ BPIV3-F H HN H .
  • the origin of the glycoprotein genes in each virus determined whether the HAI antibody response was directed predominantly against HPIV3 or against BPIV3.
  • the replication of challenge HPTV3 virus was significantly reduced in the upper and lower respiratory tract of previously immunized monkeys (Table 8). Although the level of protective efficacy against HPIV3 was not significantly different among the different viruses, viruses bearing HPTV3 F and HN were consistently more protective in the upper respiratory tract than were viruses bearing BPIV3 F and HN. This is in accordance with the higher level of HPIV3 -specific serum HAI antibodies induced by viruses bearing HPIV3 F and HN.
  • the groups with 6 animals contain 4 animals each from a previous rhesus study (Bailly et al., 2000, supra). Mean of the peak virus titers for each animal in its group irrespective of sampling day.
  • Virus titrations were performed on LLC-MK2 cells. The limit of detectability of virus titer was 10TCID 5 o/ml. Mean viral titers were compared using a Duncan Multiple
  • Range test ( 0.05). Within each column, mean titers with different letters are statistically different. Titers indicated with two letters are not significantly different from those indicated with either letter. The group of unimmunized animals were not included in the Duncan analysis at the day of challenge. Nasopharyngeal swab samples were collected on days 3 to 8 post challenge.
  • the invention provides for importation of BPIV genes into a virulent HPIV backbone and visa versa to yield novel, human- bovine chimeric PIV vaccine candidates.
  • exemplary chimeric recombinants disclosed in the present example ⁇ BPIV3-F H HN H and its ⁇ HPIV3-F B HNB counterpart, replicated in vitro as well as the respective parental viruses. It was also confirmed that the F and HN exchange between the BPIV3 and HPIV3 is compatible since the considerably more divergent HPIVl F and HN proteins were highly functional in a HPIV3 background (Tao et al, J. Virol.
  • BPIV3-F H HN H replicated in the upper respiratory tract of rhesus monkeys to a level intermediate between that of its HPIV3 and BPIV3 parents indicating that the BPIV3 F and HN genes make an independent contribution to the overall attenuation of BPIV3 for primates.
  • the overall attenuation of BPTV3 virus thus is the sum of two or more genetic elements, one of which is the set of F and HN genes and others are indicated to be N, M, and P.
  • ⁇ BPIV3-F H HN H represents such a viras, and, in the present example, immunization of rhesus monkeys with ⁇ BPIV3-F H HN H induced a higher level of antibody to HPTV3 than did immunization with BPIV3.
  • rBPIV3 -FHHN H conferred a level of protection against replication of HPIV3 challenge in the upper and lower respiratory tract that was statistically indistinguishable from that conferred by a previous infection with rHPIV3.
  • rHPIV3-NB which is attenuated by the BPIV3 N protein but possesses HPIV3 protective antigens, also induced a high level of resistance to HPIV3 challenge.
  • rHPTV3-N B induced higher levels of antibodies to HPIV3 than rBPIV3-F H HN H .
  • ⁇ BPIV3-FHHNH replicates to higher levels in rhesus monkeys than BPIV3, although it is significantly attenuated compared to HPIV3. Since the level of replication of BPIV3 in humans is low (Karron et al, J. Infect. Pis. 171:1107-1114, 1995), this increase is expected to be well tolerated among vaccinees.
  • additional methods to attenuate human-bovine chimeric viruses of the invention are disclosed herein to ensure that the vaccine viruses replicate only to moderate levels, for example in human infants, to prevent unacceptable respiratory tract illness among vaccinees.
  • the slight increase in replication of rBPIV3 -FHHNH in primates offers an opportunity to use rBPIV3 -F H HNH as a vector for heterologous viral antigens such as glycoproteins of other PTVs (e.g., HPIVl and HPIV2), the RSV F and G glycoproteins, and the measles HA glycoprotein, which can be incorporated as added or substituted gene(s) or genome segment(s) into the attenuated HPIV3 vaccine candidate.
  • heterologous viral antigens such as glycoproteins of other PTVs (e.g., HPIVl and HPIV2), the RSV F and G glycoproteins, and the measles HA glycoprotein, which can be incorporated as added or substituted gene(s) or genome segment(s) into the attenuated HPIV3 vaccine candidate.
  • the slight increase in replication of rBPTV3-F H HN H in monkeys over that of BPTV3 can be offset by the addition of foreign viral protective antigens, e.g., RSV glycoproteins, whose addition provides a selected level of attenuation.
  • the data presented here further defined the basis for the host range restriction of BPTV3 for primates and identify rBPTV3 -F H HN H as a potential vaccine candidate against HPTV3 and as a vector for heterologous viral antigens.
  • BPIV3 bovine parainfluenza type 3
  • HPTV3 infection The Kansas strain of bovine parainfluenza type 3 (BPIV3) is restricted for replication in the respiratory tract of humans and non-human primates and is currently being evaluated as a live-attenuated vaccine candidate to protect human infants from the severe lower respiratory tract disease caused by HPTV3 infection (Karron et al, 1996; Karron et al, 1995).
  • N, P and M ORFs of BPIV3 were shown to confer attenuation of replication in the respiratory tract of rhesus monkeys when substituted for the corresponding human ORF in rHPIV3.
  • rHPIV3 N B , rHPIV3 P B , or rHPIV3 M B chimeras which contained the human PIV3 F and HN glycoproteins, were immunogenic and protective against HPIV3 challenge.
  • a chimeric HPIV3 was constrcuted bearing the L ORF of BPIV3 in place of the human PIV3 L ORF ( Figure 15).
  • HPIV3 cDNA plasmid bearing the L ORF of BPTV3 (BPTV3 nts 8640-15341; GenBank accession #AF178654) in place of the human PIV3 L ORF (HPIV3 nts 8646-15347; GenBank accession #Z11575) was assembled by introducing unique restriction sites near the start and stop codons of the human L ORF and its bovine counterpart using site directed mutagenesis.
  • the L ORF of HPIV3 was then exchanged with that of its bovine counterpart using standard molecular cloning techniques. The sequence immediately before and after the translation start codon of the exchanged ORF was subsequently mutagenized back to human or bovine PIV3 sequence, respectively.
  • Chimeric virus was recovered by transfection of HEp-2 cells with the chimeric full-length cDNA, with the ⁇ TM(N), ⁇ TM(PnoC) and ⁇ TM(L) support plasmids, and with the recombinant vaccinia virus MVA-T7, as described previously (see, e.g., Durbin et al, Virologv 235:323-332, 1997; Skiadopoulos et al, Virologv 260:125- 135, 1999b, each incorporated herein by reference).
  • rHPIV3 L B replicated efficiently in tissue culture, growing to a mean titer of 10 • (TC ⁇ D 50 /ml). The identity of rHPIV3 L B was confirmed by isolation of the viral RNA (vRNA) which was used to amplify specific fragments by RT-PCR (see, e.g., Skiadopoulos et al.

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Abstract

L'invention porte sur des chimères de virus de la parainfluenza de l'homme et du bovin (HBPIV) et d'autres mammifères, infectieuses et atténuées, qui, seules ou en combinaison dans des préparations de vaccins, peuvent servir à susciter une réponse immunitaire anti-PIV. L'invention porte également sur des molécules et vecteurs isolés comportant un génome ou antigénome de chimère de PIV incluant un génome ou antigénome partiel ou complet de PIV 'd'environnement' humain ou bovin combiné ou intégré à un ou plusieurs segments de gènes ou génomes d'un PIV différent. Les chimères de HBPIV de l'invention comportent un génome ou antigénome partiel ou complet de PIV 'd'environnement' humain ou bovin dérivant du ou modelé sur le HBPIV, et combiné à un ou plusieurs segments de gènes ou génomes d'un PIV différent de manière à former un génome ou antigénome de PIV humain et bovin (HBPIV). Selon certains aspects de l'invention, les chimères de PIV comportent un génome ou antigénome partiel ou complet de PIV 'd'environnement' humain combiné à un ou plusieurs segments de gènes ou génomes hétérologues d'un PIV bovin (BPIV) le virus chimère résultant étant atténué en raison de la restriction due à la réactivité inter-espèces. Selon d'autres aspects de l'invention les chimères de HBPIV comportent un génome ou antigénome partiel ou complet de PIV 'd'environnement' bovin combiné à un ou plusieurs segments de gènes ou génomes hétérologues d'un gène de PIV humain codant pour une protéine immunogène, un domaine de protéine ou un épitope de PIV humain, par exemple codé par un PIV HN et/ou segments de gènes ou génomes de glycoprotéine F. La chimère de HBPIV de l'invention s'avère également utile comme vecteur de mise au point de vaccins contre d'autres pathogènes. La HBPIV de l'invention présente diverses mutations additionnelles et modifications nucléotidiques permettant d'obtenir certains effets phénotypiques et structurels recherchés
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EP1572947A2 (fr) * 2002-09-18 2005-09-14 The United States Of America 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
US7678376B2 (en) 2000-03-21 2010-03-16 Medimmune, Llc Recombinant parainfluenza virus expression systems and vaccines
CN113322241A (zh) * 2021-05-14 2021-08-31 金宇保灵生物药品有限公司 一种牛副流感病毒3型病毒株bpiv3-nm及其应用

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7678376B2 (en) 2000-03-21 2010-03-16 Medimmune, Llc Recombinant parainfluenza virus expression systems and vaccines
US8084037B2 (en) 2000-03-21 2011-12-27 Medimmune, Llc Recombinant parainfluenza virus expression systems and vaccines
EP1572947A2 (fr) * 2002-09-18 2005-09-14 The United States Of America 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
EP1572947A4 (fr) * 2002-09-18 2008-09-03 Us Health 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
US7919301B2 (en) 2002-09-18 2011-04-05 The United States of America as represented by the Secretary, Department of Health of Human Services Recovery of recombinant human parainfluenza virus type 2 (HPIV2) from CDNA and use of recombinant HPIV2 in immunogenic compositions and as vectors to elicit immune responses against PIV and other human pathogens
US8367074B2 (en) 2002-09-18 2013-02-05 The United States Of America As Represented By The Secretary Of The Department Of Health & Human Services Recovery of recombinant human parainfluenza virus type 2 (HYPIV2) from cDNA and use of recombinant HPIV2 in immunogenic compositions and as vectors to elicit immune responses against PIV and other human pathogens
CN113322241A (zh) * 2021-05-14 2021-08-31 金宇保灵生物药品有限公司 一种牛副流感病毒3型病毒株bpiv3-nm及其应用
CN113322241B (zh) * 2021-05-14 2023-06-13 金宇保灵生物药品有限公司 一种牛副流感病毒3型病毒株bpiv3-nm及其应用

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