EP1179054A2 - Use of recombinant parainfluenza viruses (pivs) as vectors to protect against infection and disease caused by piv and other human pathogens - Google Patents

Use of recombinant parainfluenza viruses (pivs) as vectors to protect against infection and disease caused by piv and other human pathogens

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Publication number
EP1179054A2
EP1179054A2 EP00984052A EP00984052A EP1179054A2 EP 1179054 A2 EP1179054 A2 EP 1179054A2 EP 00984052 A EP00984052 A EP 00984052A EP 00984052 A EP00984052 A EP 00984052A EP 1179054 A2 EP1179054 A2 EP 1179054A2
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EP
European Patent Office
Prior art keywords
piv
genome
antigenome
chimeric
gene
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EP00984052A
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German (de)
English (en)
French (fr)
Inventor
Brian R. Murphy
Peter L. Collins
Alexander C. Schmidt
Anna P. Durbin
Mario H. Skiadopoulos
Tao Tao
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US Department of Health and Human Services
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US Department of Health and Human Services
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Priority claimed from US09/458,813 external-priority patent/US7314631B1/en
Priority claimed from US09/459,062 external-priority patent/US7250171B1/en
Application filed by US Department of Health and Human Services filed Critical US Department of Health and Human Services
Publication of EP1179054A2 publication Critical patent/EP1179054A2/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • 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
    • C12N2760/18622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18611—Respirovirus, e.g. Bovine, human parainfluenza 1,3
    • C12N2760/18641—Use of virus, viral particle or viral elements as a vector
    • C12N2760/18643—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • HPIV3 Human parainfluenza virus type 3
  • RSV respiratory syncytial virus
  • HPIV1 and HPIV2 are the principal etiologic agents of laryngotracheobronchitis (croup) and also can cause severe pneumonia and bronchiolitis (Collins et al., 3rd ed. In "Fields Virology.” B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B.
  • HPIV1, 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. JJ_:1-15, 1988).
  • HPIV1, HPIV2, and HPIV3 are distinct serotypes which do not elicit significant cross- protective immunity.
  • BPIV3 bovine PIV
  • a second PIV3 vaccine candidate, JS cp45 is a cold-adapted mutant of the JS wildtype (wt) strain of HPIV3 (Karron et al., J. Inf. Pis. 172:1445-1450, 1995b; Belshe et al., J. Med. Virol. 10:235-42. 1982).
  • This live, attenuated, cold-passaged (cp) PIV3 vaccine candidate exhibits temperature-sensitive (ts), cold-adaptation (ca), and attenuation (atf) phenotypes which are stable after viral replication in vivo.
  • 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 (Hall et al., Virus Res. 22:173-184. 1992: Karron et al.. J. Inf. Pis. 172:1445-1450. 1995b).
  • the most promising prospects to date are live attenuated vaccine viruses since these have been shown to be efficacious in non-human primates even in the presence of passively transferred antibodies, an experimental situation that simulates that present in the very young infant who possesses maternally acquired antibodies (Crowe et al., Vaccine 13:847-855, 1995; Purbin et al., J. Infect. Pis.
  • the live attenuated PlV3cp45 vaccine candidate was derived from the JS strain of HPIV3 via serial passage in cell culture at low temperature and has been found to be protective against HPIV3 challenge in experimental animals and to be satisfactorily attenuated, genetically stable, and immunogenic in seronegative human infants and children (Belshe et al, J. Med. Virol. 10:235-242, 1982; Belshe et al., Infect. Immun. 37:160-5, 1982; Clements et al., J. Clin. Microbiol. 29:1175- 82, 1991; Crookshanks et al.. J. Med. Virol. 13:243-9. 1984; Hall et al., Virus Res.
  • recombinant PNA technology has recently made it possible to recover infectious negative-stranded RNA viruses from cPNA (for reviews, see Conzelmann, J. Gen. Virol. 77:381-89, 1996; Palese et al., Proc. Natl. Acad. Sci. U.S.A. 93: 11354-58, 1996).
  • recombinant rescue has been reported for infectious respiratory syncytial virus (RSV), rabies virus (RaV), simian virus 5 (SV5), rinderpest virus, Newcastle disease virus (NPV), vesicular stomatitis virus
  • VSV VSV
  • MeV measles virus
  • SeV Sendai virus
  • these disclosures allow for genetic manipulation of viral cPNA cones 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. Additionally, 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
  • infectious wild type recombinant PIV3, (r)PIV3, as well as a number of ts derivatives have now been recovered from cPNA, 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 PlV3c/?45.
  • a chimeric PIVl vaccine candidate has been generated using the PIV3 cPNA rescue system by replacing the PIV3 HN and F open reading frames (ORFs) with those of PIVl in a PIV3 full-length cPNA that contains the three attenuating mutations in L.
  • the recombinant chimeric virus derived from this cPNA is designated rP_V3-l.cp45L (Skiadopoulos et al., J. Virol. 72:1762-8, 1998; Tao et al., I 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 PIVl.
  • This recombinant vaccine candidate is highly attenuated in the upper and lower respiratory tract of hamsters and induces a high level of protection against HPIV1 infection (Skiadopoulos et al., Vaccine 18:503-510, 1999).
  • Examples in this context include vesicular stomatitis virus, an ungulate pathogen with no history of administration to humans except for a few laboratory accidents; Sendai virus, a mouse pathogen with no history of administration to humans; simian virus 5, a canine pathogen with no history of administration to humans; and an attenuated strain of measles virus which must be administered systemically and would be neutralized by measles-specific antibodies present in nearly all humans due to maternal antibodies and widespread use of a licensed vaccine.
  • some of these prior vector candidates have adverse effects, such as immunosupression, which are directly inconsistent with their use as vectors.
  • one must identify vectors whose growth characteristics, tropisms, and other biological properties make them appropriate as vectors for human use. It is further necessary to develop a viable vaccination strategy, including an immunogenic and efficacious route of administration.
  • measles virus has been considered for use a vector for the protective antigen of hepatitis B virus (Singh et al., J. Virol. 21:4823-8, 1999).
  • this combined measles virus- hepatitis B virus vaccine could only be given, like the licensed measles virus vaccine, after nine months of age, whereas the current hepatitis B virus vaccine is recommended for use in early infancy.
  • measles virus vaccine is administered parenterally and is very sensitive to neutralization and immunosuppression by maternal antibodies, and therefore is not effective if administered before 9-15 months of age. Thus, it could not be used to vector antigens that cause disease in early infancy and therefore would not useful for viruses such as RSV and the HPIVs.
  • virus-mediated immunosuppression Another well known, characteristic effect of measles virus infection is virus-mediated immunosuppression, which can last several months. Immunosuppression would not be a desirable feature for a vector.
  • the attenuated measles virus vaccine was associated with altered immune responses and excess mortality when administered at increased dose, which might be due at least in part to virus-induced immunosuppression and indicates that even an attenuated measles virus might not be appropriate as a vector.
  • the use of measles virus as a vector would be inconsistent with the global effort to eradicate this pathogen. Indeed, for these reasons it would be desirable to end the use of live measles virus and replace the present measles virus vaccine with a PIV vector that expresses measles virus protective antigens, as described herein.
  • Rabies virus a rare cause of infection of humans, has been considered for use as a vector (Mebatsion et al., Proc. Natl. Acad. Sci. USA 93:7310-4, 1996), but it is unlikely that a vector that is 100% fatal for humans would be developed for use as a live attenuated virus vector, especially since immunity to the rabies virus, which is not a ubiquitous human pathogen, is not needed for the general population. While mumps and measles viruses are less pathogenic, infection by either virus can involve undesirable features. Mumps virus infects the parotid gland and can spread to the testes, sometimes resulting in sterility.
  • Measles virus establishes a viremia, and the widespread nature of its infection is exemplified by the associated widespread rash. Mild encephalitis during mumps and measles infection is not uncommon. Measles virus also is associated with a rare progressive fatal neurological disease called subacute sclerosing encephalitis. In contrast, PIV infection and disease in normal individuals is limited to the respiratory tract, a site that is much more advantageous for immunization than the parental route. Viremia and spread to second sites can occur in severely immunocompromised experimental animals and humans, but this is not a characteristic of the typical PIV infection. Acute respiratory tract disease is the only disease associated with PIVs. Thus, use of PIVs as vectors will, on the basis of their biological characteristics, avoid complications such as interaction of virus with peripheral lymphocytes, leading to immunosuppression, or infection of secondary organs such as the testes or central nervous system, leading to other complications.
  • measles virus a host of human pathogens for which a vector-based vaccine approach may be desirable.
  • a live attenuated vaccine has been available for more than three decades and has been largely successful in eradicating measles disease in the United States.
  • the World Health Organization estimates that more than 45 million cases of measles still occur annually, particularly in developing countries, and the virus contributes to approximately one million deaths per year
  • Measles virus is a member of the Morbillivirus genus of the Paramyxoviridae family (Griffin et al., In “Fields Virology”. B. N. Fields, P. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267- 1312. Lippincott-Raven Publishers, Philadelphia, 1996). It is one of the most contagious infectious agents known to man and is transmitted from person to person via the respiratory route (Griffin et al., In "Fields Virology”. B. N. Fields, P. M. Knipe, P.
  • the measles virus has a complex pathogenesis, involving replication in both the respiratory tract and various systemic sites (Griffin et al., In "Fields Virology". B. N. Fields, P. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267- 1312. Lippincott-Raven Publishers, Philadelphia, 1996).
  • both the wild type measles virus and the vaccine virus are very readily neutralized by antibodies, and the measles virus vaccine is rendered non-infectious by even very low levels of maternally- acquired measles virus-specific neutralizing antibodies (Halsey et al., N. Engl. J. Med. 313:544-9, 1985; Osterhaus et al., Vaccine 16:1479-81, 1998).
  • the vaccine virus is not given until the passively-acquired maternal antibodies have decreased to undetectable levels.
  • measles virus vaccine is not given until 12 to 15 months of age, a time when almost all children are readily infected with the measles virus vaccine.
  • measles virus continues to have a high mortality rate, especially in children within the latter half of the first year of life (Gellin et al., J. Infect. Pis. 170:S3-14, 1994; Taylor et al., Am. J. Epidemiol. 122:788-94, 1988). This occurs because the measles virus, which is highly prevalent in these regions, is able to infect that subset of infants in whom maternally-acquired measles virus-specific antibody levels have decreased to a non- protective level.
  • the first strategy for developing an early measles vaccine involved administration of the licensed live attenuated measles virus vaccine to infants about six months of age by one of the following two methods (Cutts et al., Biologicals 25:323-38, 1997).
  • the live attenuated measles virus was administered intranasally by drops (Black et al., New Eng. J. Med. 263 ⁇ l65-169; 1960; Kok et al., Trans. R. Soc. Trop. Med. Hvg. 77:171-6, 1983; Simasathien et al., Vaccine 15:329-34, 1997) or into the lower respiratory tract by aerosol (Sabin et al., J.
  • the live attenuated poliovirus vaccine viruses are able to infect the mucosal surface of the gastrointestinal tract or the respiratory tract of young infants, including those with maternal antibodies, resulting in the induction of a protective immune response.
  • Replication-competent vaccinia recombinants expressing the protective antigens of RSV have also been shown to be ineffective in inducing a protective immune response when they are administered parenterally in the presence of passive antibody (Murphy et al., J. Virol. 62:3907-10, 1988a), but they readily protected such hosts when administered intranasally.
  • replication-competent vaccinia virus recombinants are not sufficiently attenuated for use in immunocompromised hosts such as persons with human immunodeficiency virus (HIV) infection (Fenner et al., World Health Organization, Geneva, 1988; Redfield et al., N. Engl. J. Med. 116:673-676, 1987), and their administration by the intranasal route even to immunocompetent individuals would be problematic. Therefore they are not being pursued as vectors for use in human infants, some of whom could be infected with HIV.
  • HIV human immunodeficiency virus
  • MVA The MVA vector, which was derived by more than 500 passages in chick embryo cells (Marchr et al., Infection 1:6-14, 1975; Meyer et al., J. Gen. Virol. 22:1031-1038, 1991), has also been evaluated as a potential vaccine vector for the protective antigens of several paramyxoviruses (Ourbin et al., J. Infect. Pis. 129:1345-51, 1999a; Wyatt et al., Vaccine 14:1451-1458, 1996). MVA is a highly attenuated host range mutant that replicates well in avian cells but not in most mammalian cells, including those obtained from monkeys and humans (Blanchard et al., J. Gen.
  • Avipox vaccine vectors which have a host range restriction similar to that of MVA, also have been constructed that express measles virus protective antigens (Taylor et al., Virology 187:321-8, 1992).
  • MVA is non-pathogenic in immunocompromised hosts and has been administered to large numbers of humans without incident (Mayr et al., Monbl. Bakteriol. [B] 167:375-90. 1978; Stickle et al., Ptsch. Med. Schuschr. 99:2386-92, 1974; Werner et al, Archives of Virology 64:247- 256, 1980).
  • both the immunogenicity and efficacy of MVA expressing a paramyxovirus protective antigen were abrogated in passively-immunized rhesus monkeys whether delivered by a parenteral or a topical route (Purbin et al., Virology 235:323-332. 1999).
  • MVA recombinants expressing parainfluenza virus antigens unlike replication-competent vaccinia virus recombinants, lacked protective efficacy when given by a mucosal route to animals with passively-acquired antibodies, and it is unlikely that they, or the similar avipox vectors, can be used in infants with maternally-acquired measles virus antibodies.
  • VSV vesicular stomatitis virus
  • VSV is an animal virus that can cause disease in humans
  • development of this recombinant for use in humans will require that a VSV backbone that is satisfactorily attenuated in human infants be first identified (Roberts et al., J. Virol. 73:3723- 32, 1999), but such clinical studies have not been initiated.
  • the present invention provides chimeric parainfluenza viruses (PIVs) that are infectious in humans and other mammals and are useful in various compositions to generate desired immune responses against one or more PIVs, or against a PIV and one or more additional pathogens in a host susceptible to infection therefrom.
  • PIVs parainfluenza viruses
  • the invention provides novel methods for designing and producing attenuated, chimeric PIVs that are useful as vaccine agents for preventing and/or treating infection and related disease symptoms attributable to PIV and one or more additional pathogens.
  • novel, isolated polynucleotide molecules and vectors incorporating such molecules that comprise a chimeric PIV genome or antigenome including a partial or complete PIV vector genome or antigenome combined or integrated with one or more heterologous genes or genome segments that encode single or multiple antigenic determinants of a heterologous pathogen or of multiple heterologous pathogens.
  • methods and compositions incorporating a chimeric PIV for prophylaxis and treatment of infection by both a selected PIV and one or more heterologous pathogens, e.g., a heterologous PIV or a non-PIV pathogen such as a measles virus e.g., a heterologous PIV or a non-PIV pathogen such as a measles virus.
  • the invention thus involves methods and compositions for developing live vaccine candidates based on chimeras that employ a parainfluenza virus or subviral particle that is recombinantly modified to incorporate one or more antigenic determinants of a heterologous pathogen(s).
  • Chimeric PIVs of the invention are constructed through a cONA- based virus recovery system. Recombinant chimeric PIVs made from cPNA replicate independently and are propagated in a similar manner as biologically-derived viruses.
  • the recombinant viruses are engineered to inco ⁇ orate nucleotide sequences from both a vector (i.e., a "recipient” or “background") PIV genome or antigenome, and one or more heterologous "donor” sequences encoding one or more antigenic determinants of a different PIV or heterologous pathogen — to produce an infectious, chimeric virus or subviral particle.
  • candidate vaccine viruses are recombinantly engineered to elicit an immune response against one or more PIVs or a polyspecific response against a selected PIV and a non-PIV pathogen in a mammalian host susceptible to infection therefrom.
  • the PIV and or non-PIV pathogen(s) from which the heterologous sequences encoding the antigenic determinant(s) are human pathogens and the host is a human host.
  • the vector PIV is a human PIV, although non-human PIVs, for example a bovine PIV (BPIV), can be employed as a vector to inco ⁇ orate antigenic determinants of human PIVs and other human pathogens.
  • Chimeric PIVs according to the invention may elicit an immune response against a specific PIV, e.g., HPIVl, HPIV2, HPIV3, or a polyspecific immune response against multiple PIVs, e.g., HPIVl and HPIV2.
  • chimeric PIVs of the invention may elicit a polyspecific immune response against one or more PIVs and a non-PIV pathogen such as measles virus.
  • Exemplary chimeric PIV of the invention inco ⁇ orate a chimeric PIV genome or antigenome as described above, 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 PIV of the invention include a partial or complete "vector" PIV genome or antigenome derived from or patterned after a human PIV or non-human PIV combined with one or more heterologous gene(s) or genome segment(s) of a different PIV or other pathogen to form the chimeric PIV genome or antigenome.
  • chimeric PIV inco ⁇ orate a partial or complete human PIV vector genome or antigenome combined with one or more heterologous gene(s) or genome segment(s) from a second human PIV or a non-PIV pathogen such as measles virus.
  • the PIV "vector" genome or antigenome typically acts as a recipient or carrier to which are added or inco ⁇ orated one or more "donor” genes or genome segments of a heterologous pathogen.
  • donor genes or genome segments of a heterologous pathogen typically, polynucleotides encoding one or more antigenic determinants of the heterologous pathogen are added to or substituted within the vector genome or antigenome to yield a chimeric PIV that thus acquires the ability to elicit an immune response in a selected host against the heterologous pathogen.
  • the chimeric virus may exhibit other novel phenotypic characteristics compared to one or both of the vector PIV and heterologous pathogens.
  • chimeric PIVs are attenuated for greater efficacy as a vaccine candidate by inco ⁇ oration of large polynucleotide inserts which specify the level of attenuation in the resulting chimeric virus dependent upon the size of the insert.
  • Preferred chimeric PIV vaccine candidates of the invention bear one or more major antigenic determinants of a human PIV, e.g., of HPIV 1 , HPIV2 or HPIV3, and thus elicit an effective immune response against the selected PIV in human hosts.
  • the antigenic determinant which is specific for a selected human PIV may be encoded by the vector genome or antigenome, or may be inserted within or joined to the PIV vector genome or antigenome as a heterologous polynucleotide sequence from a different PIV.
  • the major protective antigens of human PIVs are their HN and F glycoproteins, although other proteins can also contribute to a protective or therapeutic immune response.
  • polynucleotides encoding antigenic determinants that may be present in the vector genome or antigenome, or integrated therewith as a heterologous gene or genome segment, may encode one or more PIV N, P, C, P, V, M, F, HN and/or L protein(s) or selected immunogenic fragment(s) or epitope(s) thereof from any human PIV.
  • the chimeric PIV includes a vector genome or antigenome that is a partial or complete human PIV (HPIV) genome or antigenome, for example of HPIV3, and further includes one or more heterologous gene(s) or genome segment(s) encoding antigenic determinant(s) of at least one heterologous PIV, for example HPIVl and/or HPIV2.
  • HPIV human PIV
  • 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).
  • one or more genes or genome segments encoding one or more antigenic determinants of HPIVl may be added to or substituted within the partial or complete HPIV3 genome or antigenome.
  • the antigenic determinant(s) of HPIVl is/are selected from HPIVl HN and F glycoproteins or comprise one or more antigenic domains, fragments or epitopes of the HN and/or F glycoproteins.
  • both of the HPIVl genes encoding the HN and F glycoproteins are substituted for counte ⁇ art HPIV3 HN and F genes in the HPIV3 vector genome or antigenome. These constructs yield chimeric PIVs that elicit a mono- or poly-specific immune response in humans to HPIV3 and/or HPIVl .
  • one or more genes or genome segments encoding one or more antigenic determinants of HPIV2 is/are added to, or inco ⁇ orated within, a partial or complete HPIV3 genome or antigenome, yielding a new or additional immunospecificity of the resultant chimera against HPIV2 alone, or against HPIV3 and HPIV2.
  • one or more HPIV2 genes or genome segments encoding one or more HN and/or F glycoproteins or antigenic domains, fragments or epitopes thereof is/are added to or inco ⁇ orated within the partial or complete HPIV3 vector genome or antigenome.
  • multiple heterologous genes or genome segments encoding antigenic determinants of multiple heterologous PIVs are added to or inco ⁇ orated within a partial or complete PIV vector genome or antigenome, preferably an HPIV vector genome or antigenome.
  • heterologous genes or genome segments encoding antigenic determinants from both HPIVl and HPIV2 are added to or inco ⁇ orated within a partial or complete HPIV3 vector genome or antigenome.
  • one or more HPIVl genes or genome segments encoding one or more HN and/or F glycoproteins (or antigenic domains, fragments or epitopes thereof) and one or more HPIV2 genes or genome segments encoding HN and/or F glycoproteins, antigenic domains, fragments or epitopes, is/are added to or inco ⁇ orated within the 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, which is further modified by addition or inco ⁇ oration of one or more genes or gene segments encoding single or multiple antigenic determinants of HPIV2.
  • This is readily achieved within the invention, for example, by adding or substituting a transcription unit comprising an open reading frame (ORF) of an HPIV2 HN within the chimeric HPIV3-1 vector genome or antigenome.
  • chimeric PIVs of the invention are based on a human PIV vector genome or antigenome which is employed as a recipient for inco ⁇ oration of major antigenic determinants from a non-PIV pathogen.
  • Pathogens from which one or more antigenic determinants may be adopted into the chimeric PIV vaccine candidate include, but are not limited to, 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.
  • a human PIV genome or antigenome can be employed as a vector for inco ⁇ oration of one or more major antigenic determinants from a wide range of non-PIV pathogens.
  • Representative major antigens that can be inco ⁇ orated within chimeric PIVs of the invention include, but are not limited to the measles virus HA and F proteins; the F, G, SH and M2 proteins of subgroup A and subgroup B respiratory syncytial virus, mumps virus HN and F proteins, human papilloma virus LI protein, type 1 or type 2 human immunodeficiency virus gpl60 protein, he ⁇ es simplex virus and cytomegalovirus gB, gC, gO, gE, gG, gH, gl, gJ, gK, gL, and gM proteins, rabies virus G Protein, Epstein Barr Virus gp350 protein; filovirus G protein, bunyavirus G
  • Various human PIV vectors can be employed to carry heterologous antigenic determinants of non-PIV pathogens to elicit one or more specific humoral or cell mediated immune responses against the antigenic determinant(s) carried by the chimeric vaccine virus and hence elicit an effective immune response against the wild-type "donor" pathogen in susceptible hosts.
  • one or more heterologous genes or genome segments from the donor pathogen is joined to or inserted within a partial or complete HPIV3 genome or antigenome.
  • the heterologous gene or genome segment may be inco ⁇ orated within a chimeric HPIV vector genome or antigenome, for example a partial or complete HPIV3 genome or antigenome bearing one or more genes or genome segments of a heterologous PIV.
  • the gene(s) or genome segment(s) encoding the antigenic determinant(s) of a non-PIV pathogen may be combined with a partial or complete chimeric HPIV3-1 vector genome or antigenome, e.g., as described above having one or both HPIVl genes encoding HN and F glycoproteins substituted for counte ⁇ art HPIV3 HN and F genes.
  • the gene(s) or genome segment(s) encoding the antigenic determinant(s) of a non-PIV pathogen may be combined with a partial or complete chimeric genome or antigenome that inco ⁇ orates single or multiple antigenic determinants of HPIV2, e.g., an HPIV2 HN gene, within an HPIVl or HPIV3 vector genome or antigenome, or a chimeric HPIV3-1 vector genome or antigemome as described above.
  • the heterologous gene(s) or genome segment(s) encoding one or more measles antigenic determinant(s) may be combined with any of the PIV vectors or chimeric PIV vectors disclosed herein.
  • the vector genome or antigenome is a partial or complete HPIV3 genome or antigenome, or a chimeric HPIV genome or antigenome comprising a partial or complete HPIV3 genome or antigenome having one or more genes or genome segments encoding antigenic determinant(s) of a heterologous HPIV added or inco ⁇ orated therein.
  • a transcription unit comprising an open reading frame (ORF) of a measles virus HA gene is added to a HPI V3 vector genome or antigenome at various positions, yielding exemplary chimeric PIV/measles vaccine candidates rPIV3(HA HN-L), rPIV3(HA N-P), rcp45L(HA N-P), rPIV3(HA P-M), or r ⁇ p45L(HA P-M).
  • ORF open reading frame
  • the PIV vector genome or antigenome is a chimeric HPIV genome or antigenome comprising a partial or complete HPIV3 genome or antigenome having one or more gene(s) or genome segment(s) encoding one or more antigenic determinant(s) of HPIVl added or inco ⁇ orated therein.
  • This construct may be used as a vector, e.g., for measles virus, wherein the heterologous antigenic determinant(s) is/are selected from the measles virus HA and F proteins and 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 HPIV3-1 vector genome or antigenome having both the HPIV3 HN and F ORFs substituted by the HN and F ORFs of HPIVl.
  • ORF open reading frame
  • rPIV3-l HAP-M or rPIV3-l HAP-M cp45L.
  • the partial or complete PIV vector genome or antigenome is combined with one or more "supernumerary" (i.e., additional to a full complement of genes, whether present in a wild-type vector or in a mutant, e.g., chimeric vector backbone) heterologous gene(s) or genome segment(s) to form the chimeric PIV genome or antigenome.
  • "supernumerary" i.e., additional to a full complement of genes, whether present in a wild-type vector or in a mutant, e.g., chimeric vector backbone
  • the vector genome or antigenome is often a complete HPIV3 or HPIV3-1 chimeric genome or antigenome, and the supernumerary heterologous gene(s) or genome segment(s) are selected from HPIVl HN, HPIVl F, HPIV2 HN, HPIV2 F, measles HA, and/or a translationally silent synthetic gene unit.
  • HPIVl HN and/or HPIV2 HN ORF(s) is/are inserted within the HPIV3 vector genome or antigenome, respectively.
  • the HPIVl HN, HPIV2 HN, and measles virus HA ORFs are inserted between the N/P, P/M, and HN/L genes, respectively.
  • the HPIVl HN and HPIV2 HN genes may be inserted between the N/P and P/M genes, respectively and a 3918-nt GU insert is added between the HN and L genes.
  • vaccine candidates identified herein below as rHPI V3 1 HNN-P, rHPIV3 1 HNP-M, rHPI V3 2HNN- P, rHPIV3 2HNP-M, rHPIV3 1 HNN-P 2HNP-M, rHPIV3 1 HNN-P 2HNP-M HAHN-L, and rHPIV3 1HNN-P 2HNP-M 3918GUHN-L.
  • chimeric PIV of the invention may contain protective antigens from one, two, three, four or more different pathogens.
  • vaccine candidates are provided which contain protective antigens from one to four pathogens selected from HPIV3, HPIVl, HPIV2, and measles virus.
  • one or more supernumerary heterologous gene(s) or genome segment(s) can be added which may add a total length of supernumerary foreign sequence to the recombinant genome or antigenome of 30% to 50% or greater (e.g., compared to the wild-type HPIV3 genome length of 15,462 nt).
  • the addition of one or more supernumerary heterologous gene(s) or genome segment(s) in this context often specifies an attenuation phenotype of the chimeric PIV, which exhibits at least a 10-to 100-fold, often 100- to 1, 000-fold, and up to a 1,000- to 10,000-fold or greater decrease in replication in the upper and/or lower respiratory tract.
  • a heterologous gene or genome segment of a donor PIV or non-PIV pathogen may be added or substituted at any operable position in the vector genome or antigenome.
  • the position of a gene or gene segment substitution will correspond to a wild-type gene order position of a counte ⁇ art gene or genome segment within the partial or complete PIV vector 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 counte ⁇ art gene or genome segment within the background genome or antigenome, to enhance or reduce expression, respectively, of the heterologous gene or genome segment.
  • a heterologous genome segment for example a genome segment encoding an immunogenic ectodomain of a heterologous PIV or non-PIV pathogen, can be substituted for a corresponding genome segment in a counte ⁇ art gene in the PIV vector genome or antigenome to yield constructs encoding chimeric proteins, e.g. fusion proteins having a cytoplasmic tail and/or transmembrane domain of one PIV fused to an ectodomain of another PIV or non-PIV pathogen.
  • chimeric proteins e.g. fusion proteins having a cytoplasmic tail and/or transmembrane domain of one PIV fused to an ectodomain of another PIV or non-PIV pathogen.
  • a chimeric PIV genome or antigenome may be engineered to encode a polyspecific chimeric glycoprotein in the recombinant virus or subviral particle having immunogenic glycoprotein domains or epitopes from two different pathogens.
  • heterologous genes or genome segments from one PIV or non-PIV pathogen can be added (i.e., without substitution) within a PIV vector genome or antigenome to create novel immunogenic properties within the resultant clone.
  • the heterologous gene or genome segment may be added as a supernumerary gene or genome segment, optionally for the additional piupose of attenuating the resultant chimeric virus, in combination with a complete PIV vector genome or antigenome.
  • heterologous gene or genome segment may be added in conjunction with deletion of a selected gene or genome segment in the vector genome or antigenome.
  • the heterologous gene or genome segment is added at an intergenic position within the partial or complete PIV vector genome or antigenome.
  • the gene or genome segment can be inserted within other noncoding regions of the genome, for example, within 5' or 3' noncoding regions or in other positions where noncoding nucleotides occur within the vector genome or antigenome.
  • regions of the vector genome or antigenome represent target sites for disruption or modification of regulatory functions associated with introduction of the heterologous gene or genome segment.
  • Attenuating mutations may be generated de novo and tested for attenuating effects according to well known rational design mutagenesis strategies.
  • the attenuating mutations may be identified in existing biologically derived mutant PIV or other viruses and thereafter inco ⁇ orated into a chimeric PIV of the invention.
  • Attenuating mutations in the latter context are readily identified and inco ⁇ orated into a chimeric PIV, either by inserting the mutation within the vector genome or antigenome by cloning or mutagenizing the vector genome or antigenome to contain the attenuating mutation.
  • attenuating mutations are engineered within the vector genome or antigenome and are imported or copied from biologically derived, attenuated PIV mutants. These are recognized to include, for example, cold passaged (cp), cold adapted (ca), host range restricted (hr), small plaque (sp), and/or temperature sensitive (ts) PIV mutants.
  • one or more attenuating mutations present in the well characterized JS HPIV3 cp45 mutant strain are inco ⁇ orated within chimeric PIV of the invention, preferably including one or more mutations identified in the polymerase L protein, e.g., at a position corresponding to Tyr 42 , Leu 92 , or Thr 1 58 of JS.
  • attenuating mutations present in the JS HPIV3 cp 45 mutant strain are introduced in the N protein of chimeric PIV clones, for example which encode amino acid substitution(s) at a position corresponding to residues Val 6 or Ser 38 of JS.
  • Yet additional useful attenuating mutations encode amino acid substitution(s) in the C protein, e.g., at a position corresponding to Ile 96 of JS and in the M protein, e.g., at a position corresponding to Pro 19 (for example a Proj 9 to Thr mutation).
  • Other mutations identified in PIV3 JS cp45 that can be adopted to adjust attenuation of a chimeric PIV of the invention are found in the F protein, e.g., at a position corresponding to Ile 420 or Ala ⁇ o of JS, and in the HN protein, e.g., at a position corresponding to residue Val 38 of JS.
  • Attenuating mutations from biologically derived PIV mutants for inco ⁇ oration into chimeric PIV of the invention also include 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 cp45.
  • 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 cp45.
  • chimeric PIVs are constructed which include one or more, and preferably two or more, mutations of HPIV3 JS cp45.
  • chimeric PIVs of the invention selected for vaccine use often have two and sometimes three or more attenuating mutations from biologically derived PIV mutants or like model sources to achieve a satisfactory level of attenuation for broad clinical use.
  • these attenuating mutations inco ⁇ orated within recombinant chimeric PIVs of the invention are stabilized by multiple nucleotide substitutions in a codon specifying the mutation.
  • Introduction of attenuating and other desired phenotype-specifying mutations into a selected PIV vector may be achieved by transferring a heterologous gene or genome segment containing the mutation, e.g., a gene encoding a mutant L protein, or portion thereof, into the PIV vector genome or antigenome.
  • the mutation may be present in the selected vector genome or antigenome, and the introduced heterologous gene or genome segment may bear no mutations, or may bear one or more additional different mutations.
  • the vector genome or antigenome is modified at one or more sites corresponding to a site of mutation in a heterologous "donor" 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 a mutation identified in the donor virus (see, PCT/US00/09695 filed April 12, 2000 and its priority U.S. Provisional Patent Application Serial No. 60/129,006, filed April 13, 1999, inco ⁇ orated herein by reference).
  • a heterologous "donor” virus e.g., a heterologous bovine or human PIV or a non-PIV negative stranded RNA virus
  • a conservatively related, mutation e.g., a conservative amino acid substitution
  • a PIV vector genome or antigenome is modified at one or more sites corresponding to a site of mutation in HPIV3 JS cp45, as enumerated above, 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 inco ⁇ orating attenuating mutations into PIV vectors 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.
  • Attenuating mutations from biologically derived PIV mutants for inco ⁇ oration into human-bovine chimeric PIV of the invention also include 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 cp45.
  • 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 cp45.
  • Additional mutations which can be adopted or transferred to PIV vectors of the invention may be identified in non-PIV nonsegmented negative stranded RNA viruses and inco ⁇ orated 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, inco ⁇ orated herein by reference.
  • additional attenuating mutations can be readily adopted or engineered within chimeric PIVs of the invention that are identified in other viruses, particularly other nonsegmented negative stranded RNA viruses.
  • chimeric PIVs with or without attenuating mutations modeled after biologically derived attenuated mutant viruses, are constructed to have additional nucleotide modification(s) to yield a desired phenotypic, structural, or functional change.
  • the selected nucleotide modification will be made within the partial or complete PIV vector genome, but such modifications can be made as well within any heterologous gene or genome segment that contributes to the chimeric clone.
  • These modifications preferably specify a desired 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 cONAs for ease of manipulation and identification.
  • nucleotide changes within the genome or antigenome of a 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.
  • Target genes for mutation in this context include any of the PIV genes, including the nucleocapsid protein N, phosphoprotein P, large polymerase subunit L, matrix protein M, hemagglutinin- neuraminidase protein HN, fusion protein F, and the products of the C, P and V open reading frames (ORFs).
  • each of these proteins can be selectively deleted, substituted or rearranged, in whole or in part, alone or in combination with other desired modifications, to achieve novel deletion or knock out mutants.
  • one or more of the C, O, 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 16:578-587, 1997 and Schneider et al., Virology 222:314-322, 1997, inco ⁇ orated herein by reference).
  • one or more of the C, O, 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 Ourbin et al. on July 9, 1999, inco ⁇ orated herein by reference).
  • Alternative nucleotide modifications in 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.
  • a cis-acting regulatory sequence of one PIV gene is changed to correspond to a heterologous regulatory sequence, which may be a counte ⁇ art 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.
  • a variety of other genetic alterations can be produced in a chimeric PIV genome or antigenome, alone or together with one or more attenuating mutations adopted from a biologically derived mutant PIV.
  • genes or genome segments from non-PIV sources may be inserted in whole or in part.
  • the invention provides methods for attenuating chimeric PIV vaccine candidates based on host range effects due to the introduction of one or more gene(s) or genome segment(s) from, e.g., a non-human PIV into a human PIV vector-based chimeric virus.
  • host range attenuation can be conferred on a HPIV-vector based chimeric construct by introduction of nucleotide sequences from a bovine PIV (BPIV) (see, e.g., as disclosed in U.S. Application Serial No. 09/586,479, filed June 1, 2000, corresponding to U.S. Provisional Application Serial No. 60/143,134 filed on July 9, 1999, inco ⁇ orated herein by reference).
  • BPIV bovine PIV
  • the percent amino acid identity for each of the N proteins is 86%, for P is 65%, M 93%, F 83%, HN 77%, and L 91%. All of these proteins are therefore candidates for introduction into a HPIV vector to yield an attenuated chimeric virus which cannot readily be altered by reversion.
  • the vector genome or antigenome is an HPIV3 genome or antigenome and the heterologous gene or genome segment is a N ORF derived from a selected BPIV3 strain.
  • chimeric PIV are provided within the invention based on a vector genome or antigenome which is a human-bovine chimeric PIV genome or antigenome.
  • the human-bovine chimeric vector genome or antigenome is combined with one or more heterologous gene(s) or genome segment(s) encoding one or more antigenic determinant(s) of a heterologous pathogen 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.
  • a heterologous pathogen selected from measles virus, subgroup A and subgroup B respiratory syncytial viruses, mumps virus, human papilloma viruses, type 1 and type 2
  • a human-bovine chimeric vector genome or antigenome comprises a partial or complete HPIV genome or antigenome combined with one or more heterologous gene(s) or genome segment(s) from a BPIV.
  • a transcription unit comprising an open reading frame (ORF) of a BPIV3 N ORF is substituted in the vector genome or antigenome for a corresponding N ORF of a HPIV3 vector genome.
  • the vector genome or antigenome is combined with a measles virus HA gene, or a selected antigenic determinant of another pathogen, as a supernumerary gene insert, as exemplified by the vaccine candidate identified below as rHPIV3-NB HAP-M.
  • the human-bovine chimeric vector genome or antigenome comprises a partial or complete HPIV genome or antigenome combined with one or more heterologous gene(s) or genome segment(s) from a BPIV.
  • one or more HPIV gene(s) or genome segment(s) encoding HN and/or F glycoproteins or one or more immunogenic domain(s), fragment(s) or epitope(s) thereof may be added to or inco ⁇ orated within a partial or complete bovine genome or antigenome to form the vector genome or antigenome.
  • both HPIV3 genes encoding HN and F glycoproteins are substituted for corresponding BPIV3 HN and F genes to form the vector genome or antigenome.
  • the vector genome or antigenome is combined with a RSV F and or G gene, or a selected antigenic determinant of another pathogen, as a supernumerary gene insert, as exemplified by the vaccine candidates identified below as rBHPIV3-Gl or rB/HPIV3-Fl .
  • a chimeric human-bovine vector inco ⁇ orates one or more HPIVl HN and/or F gene(s) or genome segment(s) encoding one or more immunogenic domain(s), fragment(s) or epitope(s) thereof, and the vector is further modified by inco ⁇ oration of one or more HPIV2 HN and/or F gene(s) or genome segment(s) encoding one or more immunogenic domain(s), fragment(s) or epitope(s) thereof to form the chimeric genome or antigenome which expresses protective antigen(s) from both HPIVl and HPIV2.
  • This category of chimeric PIV is exemplified by various vaccine candidates identified below as rB/HPIV3.1-2F; rB/HPIV3.1-2HN; or rB/HPIV3.1-2F,2HN.
  • the order of genes can be changed to cause attenuation or reduce or enhance expression of a particular gene.
  • a PIV genome promoter can be replaced with its antigenome counte ⁇ art to yield additional desired phenotypic changes.
  • Oifferent 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 regions or elsewhere.
  • Nontranslated gene sequences can be removed to increase capacity for inserting foreign sequences.
  • polynucleotide molecules or vectors encoding the 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 or fungus) capable of eliciting a protective immune response in an intended host.
  • a microbial pathogen e.g., virus, bacterium or fungus
  • chimeric PIVs are constructed that inco ⁇ orate a gene encoding a cytokine to yield novel phenotypic and immunogenic effects in the resulting chimera.
  • the invention provides related cPNA clones and vectors which inco ⁇ orate a PIV vector genome or antigenome and heterologous polynucleotide(s) encoding one or more heterologous antigenic determinants, wherein the clones and vectors optionally inco ⁇ orate mutations and related modifications specifying one or more attenuating mutations or other phenotypic changes as described above.
  • Heterologous sequences encoding antigenic determinants and/or specifying desired phenotypic changes are introduced in selected combinations, e.g., into an isolated polynucleotide which is a recombinant cPNA vector genome or antigenome, to produce a suitably attenuated, infectious virus or subviral particle in accordance with the methods described herein.
  • These methods coupled with routine phenotypic evaluation, provide a large assemblage of chimeric PIVs having such desired characteristics as attenuation, temperature sensitivity, altered immunogenicity, cold-adaptation, small plaque size, host range restriction, genetic stability, etc.
  • Preferred vaccine viruses among these candidates are attenuated and yet sufficiently immunogenic to elicit a protective immune response in the vaccinated mammalian host.
  • compositions e.g., isolated polynucleotides and vectors inco ⁇ orating a chimeric PIV-encoding cPNA
  • methods for producing an isolated infectious chimeric PIV.
  • novel, isolated polynucleotide molecules and vectors inco ⁇ orating such molecules that comprise a 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 can alternatively be expressed directly from the genome or antigenome cPNA.
  • the vector(s) is/are preferably expressed or coexpressed in a cell or cell-free lysate, thereby producing an infectious chimeric parainfluenza virus particle or subviral particle.
  • infectious viral or subviral particles or derivatives thereof.
  • An infectious virus is comparable to the authentic PIV 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 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 cPNA.
  • the genome or antigenome and N, P and L proteins combine to produce an infectious chimeric parainfluenza virus or subviral particle.
  • an expression vector comprising an isolated polynucleotide molecule encoding a chimeric PIV, and an expression vector comprising one or more isolated polynucleotide molecules encoding N, P, and L proteins of a PIV.
  • the genome or antigenome and N, P, and L proteins combine to produce an infectious PIV particle, such as a viral or subviral particle.
  • the chimeric PIVs of the invention are useful in various compositions to generate a desired immune response against one or more PIVs, or against PIV and a non-PIV pathogen, in a host susceptible to infection therefrom.
  • Chimeric PIV recombinants are capable of eliciting a mono- or poly-specific protective immune response in an infected mammalian host, yet are sufficiently attenuated so as to not cause unacceptable symptoms of disease in the immunized host.
  • 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 chimeric parainfluenza virus or subviral particle as described above.
  • the vaccine is comprised of a chimeric PIV having at least one, and preferably two or more additional mutations or other nucleotide modifications that specify a suitable balance of attenuation and immunogenicity.
  • the vaccine can be formulated in a dose of 10 to 10 PFU of attenuated virus.
  • the vaccine may comprise attenuated chimeric PIV that elicits an immune response against a single PIV strain or against multiple PIV strains or groups.
  • 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 one or more PIVs, or against PIV and a non-PIV pathogen, in a mammalian subject.
  • the method comprises administering a formulation of an immunologically sufficient amount a chimeric PIV in a physiologically acceptable carrier and/or adjuvant.
  • the immunogenic composition is a vaccine comprised of a chimeric PIV having at least one, and preferably two or more attenuating mutations or other nucleotide modifications specifying a desired phenotype and/or level of attenuation 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 an attenuated chimeric PIV 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 measles or RSV.
  • chimeric PIVs 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.
  • 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 measles or RSV.
  • the immunogenic compositions of the invention are administered to the upper respiratory tract, e.g., by spray, droplet or aerosol.
  • the immunogenic composition is administered to the upper respiratory tract, e.g., by spray, droplet or aerosol.
  • the invention also provides novel combinatorial vaccines and coordinate vaccination protocols for multiple pathogenic agents, including multiple PIVs and/or PIV and a non-PIV pathogen.
  • selected targets for early vaccination according to these compositions include RSV and PIV3, which each cause significant amount of illness within the first four months of life, whereas most of the illness caused by PIVl and PIV2 occurs after six months of age (Collins et al., In Fields Virology. Vol. 1, pp. 1205-1243, Lippincott-Raven Publishers, Philadelphia, 1996; Reed et al., J. Infect. Pis. 175:807-13. 1997).
  • a preferred immunization sequence employing live attenuated RSV and PIV vaccines is to administer RSV and PIV3 as early as one month of age (e.g., at one and two months of age) followed by a bivalent PIVl and PIV2 vaccine at four and six months of age. It is thus desirable to employ the methods of the invention to administer multiple PIV vaccines, including one or more chimeric PIV vaccines, coordinately, e.g., simultaneously in a mixture or separately in a defined temporal sequence (e.g., in a daily or weekly sequence), wherein each vaccine virus preferably expresses a different heterologous protective antigen.
  • Such a coordinate/sequential immunization strategy which is able to induce secondary antibody responses to multiple viral respiratory pathogens, provides a highly powerful and extremely flexible immunization regimen that is driven by the need to immunize against each of the three PIV viruses and other pathogens in early infancy.
  • the presence of multiple PIV serotypes and their unique epidemiology with PIV3 disease occurring at an earlier age than that of PIVl and PIV2 makes it desirable to sequentially immunize an infant with different PIV vectors each expressing the same heterologous antigenic determinant such as the measles virus HA.
  • This sequential immunization permits the induction of the high titer of antibody to the heterologous protein that is characteristic of the secondary antibody response.
  • early infants e.g. 2-4 month old infants
  • an attenuated chimeric virus of the invention for example a chimeric HPIV3 expressing the measles virus
  • HA protein and also adapted to elicit an immune response against HPIV3, such as rcp45L(HA P-M).
  • an immune response against HPIV3, such as rcp45L(HA P-M) e.g., rcp45L(HA P-M).
  • the infant is again immunized but with a different, secondary vector construct, such as the rPIV3-l cp45L virus expressing the measles virus HA gene and the HPIVl antigenic determinants as the functional, obligate glycoproteins of the vector.
  • the vaccinee will elicit a primary antibody response to both the PIV3 HN and F proteins and to the measles virus HA protein, but not to the PIVl HN and F protein.
  • the vaccinee Upon secondary immunization with the rPIV3-l cp45L expressing the measles virus HA, the vaccinee will be readily infected with the vaccine because of the absence of antibody to the PIVl HN and F proteins and will develop both a primary antibody response to the PIVl HN and F protective antigens and a high titered secondary antibody response to the heterologous measles virus HA protein.
  • a similar sequential immunization schedule can be developed where immunity is sequentially elicited against HPIV3 and then HPIV2 by one or more of the chimeric vaccine viruses disclosed herein, simultaneous with stimulation of an initial and then secondary, high titer protective response against measles or another non-PIV pathogen.
  • Figures 1A and IB illustrate insertion of the HA gene of measles virus into the HPIV3 genome (Note: all of the figures presented herein and related descriptions refer to the positive-sense antigenome of HPIV3, 5' to 3').
  • Figure 1A provides a diagram (top; not to scale) of the 1926 nt insert containing the complete open reading frame of the hemagglutinin (HA) gene of the Edmonston wildtype strain of measles virus engineered to express the measles virus HA from an extra transcriptional unit.
  • HA hemagglutinin
  • the insert contains, in 5' to 3' order: an AfUl site; nts 3699-3731 from the HPIV3 antigenome which contains the P/M gene junction, including downstream noncoding sequence for the P gene, its gene-end signal, the intergenic region, and the M gene-start signal; three additional nts (GCG); the complete measles virus HA ORF; HPIV3 nt 3594-3623 from the downstream noncoding region of the P gene; and a second AfH ⁇ site.
  • FIG. 1A Panel 1 illustrates the complete antigenome of the JS wildtype strain of HPIV3 (rPIV3) with the introduced AfTII site in the 3 '-noncoding region of the N gene before (top) and after (bottom) insertion of the measles HA ORF.
  • Panel 2 illustrates the complete antigenome of the JS wildtype strain of HPIV3 (rPIV3) with the introduced Aflll site in the 3 '-noncoding region of the P gene before (top) and after (bottom) insertion of the measles HA ORF.
  • SEQ IP NO. 1 and SEQ IP NO. 2 are shown in Fig. 1 A.
  • Figure IB provides a diagram (top; not to scale) of the 2028 nt insert containing the compete ORF of the HA gene of measles virus.
  • the insert contains, in 5' to 3' order: a Stul site; nts 8602 to 8620 from the HPIV3 antigenome, which consist of downstream noncoding sequence from the HN gene and its gene-end signal; the conserved HPIV3 intergenic trinucleotide; nts 6733 to 6805 from the HPIV3 antigenome, which contains the HN gene-start and upstream noncoding region; the measles virus HA ORF; HPIV3 nts 8525-8597, which are downstream noncoding sequences from the HN gene; and a second Stwl site.
  • the construction is designed to, upon insertion, regenerate the HPIV3 HN gene containing the St ⁇ l site, and place the measles virus ORF directly after it flanked by the transcription signals and noncoding region of the HPIV3 HN gene.
  • the complete antigenome of HPIV3 JS wildtype (rPIV3) with the introduced Stwl site at nt position 8600 in the 3 '-noncoding region of the HN gene is illustrated in the next (middle) diagram. Below is the antigenome of HPIV3 expressing the measles HA protein inserted into the Stul site.
  • the HA cPNA used for this insertion came from an existing plasmid, rather than from the Edmonston wild type measles virus, which was used for the insertions in the N/P and P/M regions.
  • This cPNA had two amino acid differences from the HA protein inserted in Fig 1 A, and their location in the HA gene of measles virus is indicated by the asterisks in Figure IB. SEQ IP NO. 3 and 4 are shown in Fig. IB.
  • Figure 2 illustrates expression of the HA protein of measles virus by rHPIV3- measles virus-HA chimeric viruses in LLC-MK2 cells.
  • the figure presents a radioimmunoprecipitation assay (RIP A) demonstrating that the measles HA protein is expressed by the recombinant chimeric viruses rcp45L(HA P-M) and rcp45L(HA N-P), and by the Edmonston wild type strain of measles virus (Measles), but not by the rJS wild type HPIV3 (rJS).
  • RIP A radioimmunoprecipitation assay
  • Lanes A S-labeled infected cell lysates were immunoprecipitated by a mixture of three monoclonal antibodies specific to the HPIV3 HN protein).
  • the 64kO band corresponding to the HN protein (open arrow) is present in each of the three HPIV3 infected cell lysates (lanes 3, 5, and 7), but not in the measles virus infected cell lysates (lane 9), confirming that the ⁇ cp45L(HA P-M) and rcp45L(HA N-P) chimeras are indeed HPIV3 and express similar levels of HN proteins.
  • Lanes (b) ⁇ 35 S-labeled infected cell lysates were immunoprecipitated by a mixture of monoclonal antibodies which recognizes the HA glycoprotein of measles virus (79-XV-V17, 80-III-B2, 81-1-366) (Hummel et al., J. Virol. 69:1913-6, 1995; Sheshberanies et al., Arch. Virol. 81:251-68, 1985, each inco ⁇ orated herein by reference).
  • the 76kP band corresponding to the HA protein (closed arrow) is present in lysates from cells infected with the rcp45L(HA) chimeric viruses (lanes 6, 8) and the measles virus (lane 10), but not in the lysates from rJS infected cells (lane 4), a HPIV3 wild type virus which does not encode a measles virus HA gene.
  • Figure 3 illustrates insertion of the HPIV2 HN gene as an extra transcription/translation uni+t into the antigenomic cPNA encoding rPIV3-l or rPIV3-lcp45 chimeric virus (Note: rPIV3-l is a rPIV3 in which the HN and F genes were replaced by those of HPIVl, and rPIV3-lcp45 is a version which contains, in addition, 12 mutations from the cp45 attenuated virus).
  • the HPIV2 HN gene was amplified from vRNA of HPIV2 using RT-PCR with HPIV2 HN gene specific primers (Panel A).
  • the amplified cPNA carrying a primer-introduced Ncol site at its 5 '-end and a Hind ⁇ ll site at its 3 '-end, was digested with Nco ⁇ -Hindl ⁇ l and ligated into pLit.PI V31 H ⁇ hc, that had been digested with Ncol-Hindlll, to generate pLit.PIV32H ⁇ hc (Panel B).
  • the pLit.PIV32HNhc plasmid was used as a template to produce a modified PIV2 HN cassette (Panel C), which has a PpuMl site at its 5 '-end and an introduced PpuMl site at its 3 '-end.
  • This cassette contained, from left to right: the PpuMl site at the 5'-end, a partial 5 '-untranslated region (UTR) of PIV3 HN, the PIV2 HN ORF, a 3 ' -UTR of PI V3 HN, the gene-end, intergenic, gene-start sequence that exists at the PIV3 HN and L gene junction, a portion of the 5 '-untranslated region of PIV3 L, and the introduced PpuMl site at the 3 '-end.
  • UTR 5 '-untranslated region
  • This cPNA cassette was digested with PpuMl and then ligated to p38' ⁇ PIV3 lhc, that had been digested with PpuMl, to generate p38' ⁇ PIV31hc.2HN (Panel P).
  • the 8.5 Kb BspEl-Sphl fragment was assembled into the BspEl-Sphl window of pFLC.2G+.hc or pFLCcp45 to generate the final full-length antigenomic cPNA, pFLC.3-lhc.2HN (Panel E) or pFLC.3-lhc.c/?45.2HN (Panel F), respectively.
  • pFLC.2G+.hc and pFLCc 45 are full-length antigenomic clones encoding wild type rPIV3-l and rPIV3 45, respectively, that have been described previously (Skiadopoulos et al., J. Virol. 21:1374-81, 1999a; Tao et al., J. Virol. 22:2955-2961, 1998, inco ⁇ orated herein by reference).
  • Figure 4 details and verifies construction of the rPIV3-1.2HN chimeric virus carrying the PIV2 HN ORF insert between the PIVl F and HN genes.
  • Panel A depicts the differences in the structures of rPIV3-l and rPIV3-1.2HN, which contains the PIV2 HN ORF insert between the PIVl F and HN ORFs of rPIV3-l.
  • the arrows indicate the approximate locations of the RT-PCR primers used to amplify fragments analyzed in Panels B-O.
  • Panels B and C depict the expected sizes of the restriction enzyme digestion fragments generated from the RT-PCR products amplified from rPIV3-l and rPIV3-1.2HN using either the PpuMl or Ncol restriction endonucleases, with the fragment sizes in base pairs (bp) indicated, and the results presented in panel P.
  • vRNA extracted from virus harvested from rPIV3-1.2HN or from rPIV3-l infected LLC-MK2 cells was used as a template in the presence and absence of reverse transcriptase (RT) to amplify cPNA fragments by PCR using primers indicated in panel A.
  • RT reverse transcriptase
  • PCR fragments were absent in RT-PCR reactions lacking RT indicating that the template employed for amplification of the PNA fragments was RNA and not contaminating cPNA (Lanes A and C of panel P).
  • rPIV3-1.2HN vRNA (Lane B) yielded a fragment that was approximately 2kb larger than that of its rPIV3-l parent (Lane P) indicating the presence of an insert of 2kb.
  • the PpuMl digestion of the RT-PCR product from rPIV3- 1.2HN (Lane 1 ) produced three bands of the expected sizes indicating the presence of two PpuMl sites and PpuMl digestion of the RT- PCR product from rPIV3-l produced two bands of the expected sizes for rPIV3-l (Lane 2) indicating the presence of just one PpuMl site.
  • the Ncol digestion of the RT-PCR product from rPIV3-1.2H ⁇ (Lane 5) produced 4 bands including the 0.5 kb fragment indicative of the HPIV2 HN gene and the Ncol digestion of the RT-PCR product from rPIV3-l (Lane 6) produced the expected two fragments.
  • M identifies the lane containing the 1 kb PNA ladder used as nucleotide (nt) size markers (Life Technology). Similar results confirmed the presence of the HPIV2 HN insert in rPIV3-lc/?45.2HN.
  • Figure 5 demonstrates that rPIV3-1.2HN expresses the HPIV2 HN protein. LLC-MK2 monolayers were infected with rPIV3-l , rPIV3-l .2HN, or the PIV2/V94 wild type virus at a MOI of 5. Infected monolayers were incubated at 32°C and labeled with S- met and 35 S-cys mixture from 18-36 hours post-infection.
  • Figure 6 depicts the location and construction of gene unit (GU) insertions or HN gene 3 '-noncoding region (NCR) extensions.
  • the nucleotide sequences and unique restriction enzyme cloning sites of the GU and NCR insertion sites are shown in panels A and B, respectively.
  • Cis-acting transcriptional signal sequences i.e., gene-end (GE), intergenic (IG), and gene-start (GS) signal sequences, are indicated.
  • Panel A an oligonucleotide duplex specifying the HN GE, IG and GS signal sequences as well as the unique restriction enzyme recognition sequences are shown inserted into the introduced Stwl restriction site (underlined nucleotides) (see Figure IB and Example I for the location of the introduced Stwl site).
  • a restriction fragment from an RSV antigenome plasmid was cloned into the Hpal site.
  • a short oligonucleotide duplex was inserted into the Mlul site of the multiple cloning site, so that the total length of the insert would conform to the rule of six.
  • Figure 7 illustrates open reading frames (ORFs) in the 3079 bp RSV insert.
  • the six possible reading frames in the 3079 bp RSV fragment are shown (three in each orientation; 3, 2, 1, -1, -2, -3).
  • Short bars represent translation start codons.
  • Long bars represent translation stop codons.
  • the 3079 bp fragment was inserted into the HN 3' NCR (NCR ins) or between the HN and L genes as a gene unit (GU ins) in such an orientation that the reading frames encountered by the PIV3 translation machinery correspond to -3, -2 and -1 in the figure.
  • NCR ins HN 3' NCR
  • GU ins gene unit
  • Figure 8 demonstrates that rPIV3 insertion and extension mutants contain inserts of the appropriate size.
  • RT-PCR was performed using a PI V3 -specific primer pair flanking the insertion site, and RT-PCR products were separated by agarose gel electrophoresis.
  • the expected size of the RT-PCR fragment for rPIV3wt (also referred to as rJS) is 3497 bp and that for each of the other rPIV3s GU or NCR mutants is increased in length depending on the size of the insertion.
  • Panel A depicts GU insertion (ins) mutants: 1. rPIV3 wt; 2. rl68 nt GU ins; 2. r678 nt GU ins; 3.
  • M Hindl ⁇ l restriction enzyme digestion products of lamda phage PNA. Sizes of relevant size markers are indicated.
  • Panel B depicts NCR insertion mutants: 1. rPIV3 wt; 2. r258 nt NCR ins; 3. r972 nt NCR ins; 4. rl404 nt NCR ins; 5. r3126 nt NCR ins; 6. r3894 nt NCR ins.
  • M Hindlll restriction enzyme digestion products of lambda phage PNA. Sizes of relevant size markers are indicated.
  • Figures 9A-9C present multi-step growth curves of GU and NCR insertion mutations compared with rHPIV3 wt and rcp45L- LLC-MK2 monolayers in 6-well plates were infected with each HPIV3 in triplicate at a multiplicity of infection (m.o.i.) of 0.01 and were washed 4 times after removal of the virus supernatant. At 0 hr and at 24 hrs intervals for 6 days post-infection, 0.5 ml virus medium from each well was harvested and 0.5 ml fresh medium was added to each well. Harvested samples were stored at -80°C.
  • Virus present in the samples was quantified by titration on LLC-MK2 monolayers in 96-well plates incubated at 32°C. The titers of viruses are expressed as TCIP 50 /ml. The average of three independent infections from one experiment is shown. The lower limit of detection is 0.7 log 10 TCIP 5 o/ml.
  • Figure 10 illustrates the strategy for placing a supernumerary gene insert between the P and M genes of rHPI V3.
  • the downstream (3') NCR of the rHPIV3 P gene was modified to contain an Aflll restriction site at antigenomic sequence positions 3693- 3698 (Purbin, J. Virol. 74:6821-31, 2000, inco ⁇ orated herein by reference).
  • This site was then used to insert an oligonucleotide duplex (shown at the top) that contains HPIV3 cis- acting transcriptional signal sequences, i.e., gene-end (GE), intergenic (IG), and gene-start (GS) motifs.
  • the duplex also contains a series of restriction enzyme recognition sequences available for insertion of foreign ORFs.
  • the cloning sites were Ncol and Hzndlll. Insertion of a foreign ORF into the multiple cloning sites places it under the control of a set of ⁇ PIV3 transcription signals, so that in the final recombinant virus the gene is transcribed into a separate mR ⁇ A by the HPIV3 polymerase. As necessary, a short oligonucleotide duplex was inserted into the Mlul site of the multiple cloning site to adjust the final length of the genome to be an even multiple of six, which has been shown to be a requirement for efficient R ⁇ A replication (Calain et al., J. Virol.
  • Figure 11 is a diagram (not to scale) of the genomes of a series of chimeric rHPIV3s that contain one, two or three supernumerary gene inserts, each of which encodes a protective antigen of PIVl, PIV2, or measles virus.
  • Schematic representation of rHPIV3s (not to scale) showing the relative position of the added insert(s) encoding the HN (hemagglutinin-neuraminidase) glycoprotein of HPIVl ( TM) or HPIV2 ( ⁇ ) or the HA (hemagglutinin) glycoprotein of measles virus (t ⁇ ) inserted into the rHPIV3 backbone (I — I).
  • the rHPIV3 construct that is diagrammed at the bottom contains a 3918-nt insert (GU) that does not encode a protein ( ⁇ ) (Skiadopoulos et al., Virology 222:225-34, 2000, inco ⁇ orated herein by reference). Each foreign insert is under the control of a set of HPIV3 gene start and gene end transcription signals and is expressed as a separate mRNA.
  • a. LLC- MK2 monolayers on 6 well plates (Costar) were separately infected in triplicate at an m.o.i. of 0.01 with each of the indicated viruses.
  • Supematants were harvested on days 5, 6 and 7 and virus was quantified as described previously (Skiadopoulos et al., Virology 272:225-34, 2000). The mean peak titer obtained for each virus is shown as log 10 TCIOso/ml.
  • b Mean of two experiments. Serially-diluted viruses were incubated at 32°C and 39°C on LLC-MK2 monolayer cultures for 7 days, and the presence of virus was determined by hemadsorbtion with guinea pig erythrocytes. The mean reduction in titer at 39°C compared to that of 32°C is shown.
  • Figure 12 provides a diagram (not to scale) illustrating insertion of a supernumerary gene insert into an rHPIV3 backbone, rHPIV3-N ⁇ , in which the HPIV3 N ORF has been replaced by its BPIV3 counte ⁇ art, conferring an attenuation phenotype due to host range restriction (Bailly et al., J. Virol. 74:3188-3195, 2000a, inco ⁇ orated herein by reference). Schematic representations are shown of rHPIV3 (top) and biologically derived BPIV3 (bottom).
  • the relative position of the N ORF sequence derived from the Kansas strain of BPIV3 ( )and the measles virus hemagglutinin gene( ⁇ - ⁇ 5) in the PIV3 backbone are shown.
  • the foreign sequence is under the control of a set of HPIV3 transcription signals.
  • a portion of the plasmid vector containing the NgoMIV site is shown
  • FIG. 13 illustrates insertion of RSV G or F as an additional, supernumerary gene in a promoter-proximal position into the genome of rB/HPIV3.
  • rB/HPIV3 is a recombinant version of BPIV3 in which the BPIV3 F and HN genes have been replaced by their HPIV3 counte ⁇ arts (FH and HN H respectively).
  • a Blpl site was created in the B/HPIV3 backbone immediately upstream of the ATG start codon of the N ORF.
  • RSV G or F open reading frames were inserted into this Blpl site.
  • the downstream end of either RSV insert was designed to contain a PIV3 gene end (GE) and gene start (GS) sequences (AAGTAAGAAAAA (SEQ IP NO. 8) and AGGATTAAAG, respectively, in positive sense) separated by the intergenic sequence CTT.
  • GE PIV3 gene end
  • GS gene start
  • AGGATTAAAG AGGATTAAAG
  • AGGATTAAAGAACTTTACCGAAAGGTAAGGGGAAAGAAATCCTAAGAGCTTAG CGATG SEQ IP NO. 9
  • GCTTAGCGATG SEQ IP NO. 10
  • AAGCTAGCGCTTAGC SEQ IP NO. 11
  • GCTTAGCAAAAAGCTAGCACAATG SEQ IP NO. 12
  • Figure 14 illustrates multicycle replication of rB/HPIV3-Gl, rB/HPIV3-Fl and their recombinant and biological parent viruses in simian LLC-MK2 cells.
  • Triplicate monolayer cultures were infected at an input MOI of 0.01 TCIO 50 per cell with rB/HPIV3- Gl, rB/HPIV3-Fl, or the following control viruses: rBPIV3 Ka, which is the recombinant version of BPIV3 strain Ka; rB/HPIV3, with is the version of rBPIV3 in which the BPIV3 F and HN glycoprotein genes were replaced with their HPIV3 counte ⁇ arts; HPIV3 JS, which is biologically-derived HPIV3 strain JS; and BPIV3 Ka, which is the biologically-derived version of BPIV3 strain Ka.
  • the virus titers are shown as mean log ⁇ 0 TCIPso/ml of triplicate samples. The lower limit of detection of this assay
  • Figure 15 is a diagram (not to scale) of the genomes of rBPIV3 (#1) and a series of chimeric rB/HPIV3s (#2-6) that contain substitutions of BPIV3 F and HN genes by those of HPIV 3 (#2) or HPIVl (#3-6), and one or two supernumerary gene inserts encoding the F and or HN ORF of HPIV2 (#4-6).
  • Schematic representation of the rB/HPIV3.1 chimeric viruses (not to scale) showing the relative position of the supernumerary gene encoding the F or HN glycoprotein of HPIV2 (F2 and HN2, respectively).
  • Each foreign insert is under the control of a set of HPIV3 gene start and gene end transcription signals and is designed to be expressed as a separate mRNA.
  • Figure 16 provides a diagram (not to scale) illustrating the insertion of a the measles virus HA coding sequence into several different rPIV3 backbones. Three backbones are illustrated: wild type rHPIV3 (top construct); wild type rHPIV3-l (second construct from top) (Tao et al. J. Virol.
  • Figure 17 illustrates construction of the PIV3-PIV2 chimeric antigenomic cPNA pFLC.PIV32hc encoding the full-length PIV2 HN and F proteins.
  • the cPNA fragment containing the full-length PIV2 F ORF flanked by the indicated restriction sites (Al) was amplified from PIV2/V94 vRNA using RT-PCR and a PIV2 F specific primer pair (1, 2 in Table 22). This fragment was digested with Ncol plus BamHI (Cl) and ligated to the Ncol-BamHI windown of pLit.PIV31.fhc (B 1 ) to generate pLit.PIV32Fhc (01 ).
  • the cPNA fragment containing the full-length PIV2 HJN ORF flanked by the indicated restrction sites (A2) was amplified from PIV2/V94 vRNA using RT-PCR and a PIV2 HN specific primer pair (3, 4 in Table 22).
  • This fragment was digested with Ncol plus Hindlll (C2) and ligated to the Ncol-Hindlll window of pLit.PIV31.HNhc (B2) to generate pLit.PIV32HNhc (02).
  • pLit.PIV32Fhc and pLit.PIV32HNhc were digested with PpuMl and Spel and assembled together to generate pLit.PIV32hc (E).
  • pLit.PIV32hc was further digested with BspEI and Spel and introduced into the BspEl-Spel window of p38' ⁇ PIV31hc (F) to generate p38' ⁇ PIC32hc (G).
  • the chimeric PIV3-PIV2 construct was introduced into the BspEl-Sphl window of pFLC.2G+hc to generate pFLC.PIC32hc (H).
  • Figure 18 depicts construction of full-length PIV3-PIV2 chimeric antigenomic cPNA pFLC.PIV32TM and pFLC.PIV32TMcp45, which encode F and HN proteins containing PIV2-derived ectodomains and PI V3 -derived transmembrane and cytoplasmic domains.
  • the region of the PIV3 F ORF, in pLit.PIV3.F3a (Al) encoding the ectodomain was deleted (Cl) by PCR using a PIV3 F specific primer pair (9, 10 in Table 22.
  • the region of the PIV2 F ORF encoding the ectodomain was amplified from pLit.PI V32Fhc (Bl) using PCR and PIV2 F specific primer pair (5, 6 in Table 22). The two resulting fragments (Cl and Ol) were ligated to generate pLit.PIV32FTM (El).
  • the region of the PIV3 HN ORF, in pLit.PI V3.HN4 (A2), encoding the ectodomain was deleted (C2) by PCR using a PIV3 HN specific primer pair (11, 12 in Table 22).
  • the region of the PIV2 HN ORF encoding the ectodomain was amplified from pLit.PIV32HNhc (B2) by PCR and a PIV2 HN specific primer pair (8, 9 in Table 22). Those two PNA fragments (C2 and P2) were ligated together to generate pLit.PIV32HNTM (E2). pLit.PIV32FTM and pLit.PIV32HNTM were digested with PpuMl and Spel and assembled to generate pLit.PIV32TM (F).
  • the BspEI-Spel fragment from pLit.PIV32TM was ligated to the BspEI-Spel window of p38'_PIV3 lhc (G) to generate p38'_PIV32TM (H).
  • the insert containing chimeric PIV3-PIV2 F and HN was introduced as a 6.5 kb BspEl-Sphl fragment into the BspEl-Sphl window of pFLC.2G+.hc and pFLCcp45 to generate pFLC.PIV32TM and pFLC.PIV32TM 45 (I), respectively.
  • Figure 19 shows construction of full-length PIV3-PIV2 chimeric antigenomic cPNA pFLC.PIV32CT and pFLC.PIV32Ctcp45 which encode F and HN proteins containing a PIV2-derived ectodomain, a PIV2-derived transmembrane domain, and a PIV3 -derived cytoplasmic domain.
  • the region of the PIV3 F ORF in pLit.PI V3.F3 a (Al) encoding the ectodomain and the transmembrane domain was deleted (Cl) by PCR using a PIV3 F specific primer pair (17, 18 in Table 22).
  • the region of the PIV2 F ORF encoding the ectodomain plus the transmembrane domain was amplified from pLit.PIV32Fhc (Bl) using PCR and a PIV2 F specific primer pair (13, 14 in Table 22). The two resulting fragments (Cl and Ol) were ligated to generate pLit.PIV32FCT (El).
  • the region of the PIV3 HN ORF in pLit.PI V3.HN4 (A2), encoding the ectodomain and transmembrane domain was deleted (C2) by PCR using a PIV3 HN specific primer pair (19, 20 in Table 22).
  • the region of the PIV2 HN ORF encoding the ectodomain plus the transmembrane domain was amplified from pLit.PIV32HNhc (B2) by PCR using a PIV2 HN specific primer pair (15, 16 in Table 22). Those two DNA fragments (C2 and D2) were ligated to generate pLit.PIV32HNCT (E2). pLit.PIV32FCT and pLit.PI V32HNCT were digested with PpuMl and Spel and assembled to generate pLit.PIV32CT (F).
  • the BspEI-Spel fragment from pLit.PIV32CT was ligated to the BspEI-Spel window of p38'_PIV3 lhc (G) to generate p38'_PIV32CT (H).
  • the insert containing chimeric PIV3-PIV2 F and HN was introduced as a 6.5 kb BspEl-Sphl fragment into the BspEl-Sphl window of pFLC.2G+.hc and pFLC.cp45 to generate pFLC.PIV32CT and pFLC.PIV32CTcp45 (I), respectively.
  • Figure 20 details genetic structures of the PIV3-PIV2 chimeric viruses and the gene junction sequences for rPIV3-2CT and rPIV3-2TM.
  • Panel A illustrates the genetic structures of rPIV3-2 chimeric viruses (middle three diagrams) are compared with that of rPIV3 (top diagram) and rPIV3-l (bottom diagram) viruses.
  • the cp45 derivatives are shown marked with arrows depicting the relative positions of cp45 mutations.
  • For the cp45 derivatives only the F and HN genes are different while the remaining genes remained identical, all from PIV3. From top to bottom, the three chimeric PIV3-PIV2 viruses carry decreasing amount of PIV3 glycoprotein genes.
  • Panel B provides the nucleotide sequences of the junctions of the chimeric F and FIN glycoprotein genes for rPIV3-2TM are given along with the protein translation. The shaded portions represent sequences from PIV2. The amino acids are numbered with respect to their positions in the corresponding wild type glycoproteins. Three extra nucleotides were inserted in PIV3-PIV2 HN TM as indicated to make the construct conform to rule of six.
  • Panel C shows the nucleotide sequences of the junctions of the chimeric F and HN glycoprotein genes for rPIV3-2CT, given along with the protein translation. The shaded portions represent sequences from PIV2.
  • amino acids are numbered with respect to their positions in the corresponding wild type glycoproteins.
  • Figure 21 documents multicycle replication of rPIV3-2 chimeric viruses compared with that of rPIV3/JS and PIV2/V94 wild type parent viruses.
  • Panel A the rPIV3-2TM and rPIV3-2TMcp45 viruses, along with the rPIV3/JS and PIV2/V94 wt parent viruses, were used to infect LLC-MK2 cells in 6 well plates, each in triplicate, at an MOI of 0.01. All cultures were incubated at 32°C. After a 1 hour adso ⁇ tion period, the inocula were removed, and the cells were washed three times with serum-free OptiMEM. The cultures were overlayed with 2 ml per well of the same medium.
  • rPIV3-2TM and rPIV3-2TMcp45 infected plates 0.5 mg/ml of p-trypsin was added to each well. Aliquots of 0.5 ml were taken from each well at 24 hour intervals for 6 days, flash frozen on dry ice, and stored at -80°C. Each aliquot was replaced with 0.5 ml of fresh medium with or without p- trypsin as indicated above. The virus present in the aliquots was titered on LLC-MK2 plates with liquid overlay at 32°C for 7 days, and the endpoints were identified with hemadso ⁇ tion.
  • Panel B The rPIV3-2CT and rPIV3-2CTcp45, along with the rPIV3/JS and PIV2/V94 wt parent viruses, were used to infect LLC-MK2 in 6 well plates, each in triplicate, as described in Panel A. Aliquots were taken and processed in the same manner as described in Panel A. Virus titers are expressed as logl0TCID50/ml ⁇ standard errors for both experiments presented in Panel A and B.
  • the instant invention provides methods and compositions for the production and use of novel, chimeric parainfluenza viruses (PIVs) and associated vaccines.
  • the chimeric viruses of the invention are infectious and immunogenic in humans and other mammals and are useful for generating immune responses against one or more PIVs, for example against one or more human PIVs (HPIVs).
  • HPIVs human PIVs
  • chimeric PIVs are provided that elicit an immune response against a selected PIV and one or more additional pathogens, for example against both a HPIV and measles virus.
  • the immune response elicited can involve either or both humoral and/or cell mediated responses.
  • chimeric PIVs of the invention are attenuated to yield a desired balance of attenuation and immunogenicity for vaccine use.
  • the invention thus provides novel methods for designing and producing attenuated, chimeric PIVs that are useful as vaccine agents for preventing and/or treating infection and related disease symptoms attributable to PIV and other pathogens.
  • chimeric parainfluenza viruses or subviral particles are constructed using a PIV "vector" genome or antigenome that is recombinantly modified to inco ⁇ orate one or more antigenic determinants of a heterologous pathogen.
  • the vector genome or antigenome is comprised of a partial or complete PIV genome or antigenome, which may itself inco ⁇ orate nucleotide modifications such as attenuating mutations.
  • the vector genome or antigenome is modified to form a chimeric structure through inco ⁇ oration of a heterologous gene or genome segment. More specifically, chimeric PIVs of the invention are constructed through a cDNA-based virus recovery system that yields recombinant viruses that inco ⁇ orate a partial or complete vector or "background" PIV genome or antigenome combined with one or more "donor" nucleotide sequences encoding the heterologous antigenic determinant(s).
  • the PIV vector comprises a HPIV genome or antigenome, although non-human PIVs, for example a bovine PIV (BPIV), can be employed as a vector to inco ⁇ orate antigenic determinants of human PIVs and other human pathogens.
  • BPIV bovine PIV
  • a human PIV3 (HPIV3) vector genome or antigenome is modified to inco ⁇ orate one or more genes or genome segments that encode antigenic determinant(s) of one or more heterologous PIVs (e.g., HPIVl and or HPIV2), and/or a non-PIV pathogen (e.g., measles virus).
  • heterologous PIVs e.g., HPIVl and or HPIV2
  • a non-PIV pathogen e.g., measles virus.
  • chimeric PIVs of the invention may elicit an immune response against a specific PIV, e.g., HPIVl, HPIV2, and/or HPIV3, or against a non-PIV pathogen.
  • compositions and methods are provided for eliciting a polyspecific immune response against multiple PIVs, e.g., HPIVl and HPIV3, or against one or more HPIVs and a non-PIV pathogen such as measles virus.
  • Exemplary chimeric PIV of the invention inco ⁇ orate a chimeric PIV genome or antigenome as described above, 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.
  • 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 PIV inco ⁇ orate a partial or complete human PIV vector genome or antigenome combined with one or more heterologous gene(s) or genome segment(s) from a second human PIV or a non-PIV pathogen such as measles virus.
  • the PIV "vector" genome or antigenome typically acts as a recipient or carrier to which are added or inco ⁇ orated one or more "donor" genes or genome segments of a heterologous pathogen.
  • polynucleotides encoding one or more antigenic determinants of the heterologous pathogen are added to or substituted within the vector genome or antigenome to yield a chimeric PIV that thus acquires the ability to elicit an immune response in a selected host against the heterologous pathogen.
  • the chimeric virus may exhibit other novel phenotypic characteristics compared to one or both of the vector PIV and heterologous pathogens.
  • the partial or complete vector genome or antigenome generally acts as a backbone into which heterologous genes or genome segments of a different pathogen are inco ⁇ orated.
  • the heterologous pathogen is a different PIV from which one or more gene(s) or genome segment(s) is/are of are combined with, or substituted within, the vector genome or antigenome.
  • the addition or substitution of heterologous genes or genome segments within the vector PIV strain may confer an increase or decrease in attenuation, growth changes, or other desired phenotypic changes as compared with the corresponding phenotype(s) of the unmodified vector and donor viruses.
  • Heterologous genes and genome segments from other PIVs that may be selected as inserts or additions within chimeric PIV of the invention include genes or genome segments encoding the PIV N, P, C, D, V, M, F, HN and or L protein(s) or one or more antigenic determinant(s) thereof.
  • Heterologous genes or genome segments of one PIV may be added as a supernumerary genomic element to a partial or complete genome or antigenome of a different PIV.
  • one or more heterologous gene(s) or genome segment(s) of one PIV may be substituted at a position corresponding to a wild-type gene order position of a counte ⁇ art gene(s) or genome segment(s) that is deleted within the PIV vector 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 the counte ⁇ art gene or genome segment within the vector genome or antigenome to enhance or reduce, respectively, expression of the heterologous gene or genome segment.
  • heterologous immunogenic proteins, protein domains and immunogenic epitopes to produce 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 pathogen within a recipient PIV vector genome or antigenome can generate an immune response directed against the donor pathogen, the PIV vector, or against both the donor pathogen and vector.
  • chimeric PIV may be constructed that express a chimeric protein, for example an immunogenic glycoprotein having a cytoplasmic tail and/or transmembrane domain specific to a vector fused to a heterologous ectodomain of a different PIV or non-PIV pathogen to provide a fusion protein that elicits an immune response against the heterologous pathogen.
  • a chimeric protein for example an immunogenic glycoprotein having a cytoplasmic tail and/or transmembrane domain specific to a vector fused to a heterologous ectodomain of a different PIV or non-PIV pathogen to provide a fusion protein that elicits an immune response against the heterologous pathogen.
  • a heterologous genome segment encoding a glycoprotein ectodomain from a human PIVl HN or F glycoprotein may be joined with a genome segment encoding the corresponding HPIV3 HN or F glycoprotein cytoplasmic and transmembrane domains to form a HPIV3-1 chimeric glycoprotein that elicits an immune response against HPIVl.
  • PIV of the invention expressing a chimeric glycoprotein comprise a major nucleocapsid (N) protein, a nucleocapsid phosphoprotein (P), a large polymerase protein (L), and a HPIV vector genome or antigenome that is modified to encode a chimeric glycoprotein.
  • the chimeric glycoprotein inco ⁇ orates one or more heterologous antigenic domains, fragments, or epitopes of a second, antigenically distinct HPIV.
  • this is achieved by substitution within the HPIV vector genome or antigenome of one or more heterologous genome segments of the second HPIV that encode one or more antigenic domains, fragments, or epitopes, whereby the genome or antigenome encodes the chimeric glycoprotein that is antigenically distinct from the parent, vector virus.
  • the heterologous genome segment or segments preferably encode a glycoprotein ectodomain or immunogenic portion or epitope thereof, and optionally include other portions of the heterologous or "donor" glycoprotein, for example both an ectodomain and transmembrane region that are substituted for counte ⁇ art glycoprotein ecto- and transmembrane domains in the vector genome or antigenome.
  • Preferred chimeric glycoproteins in this context may be selected from HPIV HN and/or F glycoproteins, and the vector genome or antigenome may be modified to encode multiple chimeric glycoproteins.
  • the HPIV vector genome or antigenome is a partial HPIV3 genome or antigenome and the second, antigenically distinct HPIV is either HPIVl or HPIV2.
  • both glycoprotein ectodomain(s) of HPIV2 HN and F glycoproteins are substituted for corresponding HN and F glycoprotein ectodomains in the HPIV3 vector genome or antigenome.
  • PIV2 ectodomain and transmembrane regions of one or both HN and/or F glycoproteins are fused to one or more corresponding PIV3 cytoplasmic tail region(s) to form the chimeric glycoprotein.
  • a heterologous gene or genome segment of the donor pathogen may be added or substituted at any operable position in the vector genome or antigenome.
  • heterologous genes or genome segments from a non-PIV pathogen can be added (i.e., without substitution) within a PIV vector genome or antigenome to create novel immunogenic properties within the resultant clone.
  • the heterologous gene or genome segment may be added as a supernumerary gene or genome segment, optionally for the additional pu ⁇ ose of attenuating the resultant chimeric virus, in combination with a complete PIV vector genome or antigenome.
  • the heterologous gene or genome segment may be added in conjunction with deletion of a selected gene or genome segment in the vector genome or antigenome.
  • the heterologous gene or genome segment is added at an intergenic position within the partial or complete PIV vector genome or antigenome.
  • the gene or genome segment can be inserted within other noncoding regions of the genome, for example, within 5' or 3' noncoding regions or in other positions where noncoding nucleotides occur within the vector genome or antigenome.
  • the heterologous gene or genome segment is inserted at a non-coding site overlapping a cis-acting regulatory sequence within the vector genome or antigenome, e.g., within a sequence required for efficient replication, transcription, and/or translation. These regions of the vector genome or antigenome represent target sites for disruption or modification of regulatory functions associated with introduction of the heterologous gene or genome segment.
  • the term “gene” generally refers to a portion of a subject genome, e.g., a 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 gene is also interchangeable with the term “translational open reading frame", or ORF, particularly in the case where a protein, such as the PIV C protein, is expressed from an additional ORF rather than from a unique mRNA.
  • the genome is a single strand of negative-sense RNA 15462 nucleotides (nt) in length (Galinski et al., Virology 165: 499-510, 1988; Stokes et al., Virus Res. 25:91-103, 1992).
  • At least eight proteins are encoded by the HPIV3 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., 3rd ed. In "Fields Virology," B.
  • the viral genome of all PIVs 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.
  • 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 genome 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 Kingsbury, D.W. (Ed.), The Paramyxoviruses. 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).
  • one or more PIV gene(s) or genome segment(s) may be deleted, inserted or substituted in whole or in part. This means that partial or complete 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 genome segment encodes at least one immunogenic epitope capable of eliciting a humoral or cell mediated immune response in a mammalian host.
  • the genome segment may also encode an immunogenic fragment or protein domain.
  • the donor genome segment may encode multiple immunogenic domains or epitopes, including recombinantly synthesized sequences that comprise multiple, repeating or different, immunogenic domains or epitopes.
  • Alternative chimeric PIV of the invention will contain protective antigenic determinants of HPIVl, HPIV2 and/or HPIV3. This is preferably achieved by expression of one or more HN and/or F genes or genome segments by the vector PIV, or as extra or substitute genes from the heterologous donor pathogen.
  • a HPIV3-1 or HPIV3-2 chimeric virus may be constructed for use as a vaccine or vector strain, in which the HPIVl or HPIV2 HN and/or F genes replace their PIV3 counte ⁇ art(s) (Skiadopoulos et al. Vaccine 18:503-510, 1999; Tao et al.. Vaccine 17:1100-1108. 1999; U.S. Patent Application Serial No.
  • a chimeric PIVl 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 PIVl in a PIV3 full-length cDNA that contains the three attenuating mutations in L.
  • ORFs open reading frames
  • rPIV3-l.c/?45L The recombinant chimeric virus derived from this cDNA is designated rPIV3-l.c/?45L (Skiadopoulos et al.. J. Virol. 72:1762-8. 1998; Tao et al.. J. Virol. 72:2955-2961. 1998: Tao et al.. Vaccine 17:1100-1108. 1999, inco ⁇ orated herein by reference). rPIV3-l.cp45L is attenuated in hamsters and induced a high level of resistance to challenge with PIVl .
  • a recombinant chimeric virus designated ⁇ PTV3-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., Vaccine 18:503-510, 1999, inco ⁇ orated herein by reference).
  • the chimeric PIV bear one or more major antigenic determinants of a human PIV, or against multiple human PIVs, including HPIVl, HPIV2 or HPIV3. These preferred vaccine candidates elicit an effective immune response in humans against one or more selected HPIVs.
  • the antigenic determinant(s) that elicit(s) an immune response against HPIV may be encoded by the vector genome or antigenome, or may be inserted within or joined to the PIV vector genome or antigenome as a heterologous gene or gene segment.
  • the major protective antigens of human PIVs are their HN and F glycoproteins.
  • all PIV genes are candidates for encoding antigenic determinants of interest, including internal protein genes which may encode such determinants as, for example, CTL epitopes.
  • Preferred chimeric PIV vaccine viruses of the invention bear one or more major antigenic determinants from each of a plurality of HPIVs or from a HPIV and a non- PIV pathogen.
  • Chimeric PIV thus constructed include a partial or complete HPIV genome or antigenome, for example of HPIV3, and one or more heterologous gene(s) or genome segment(s) encoding antigenic determinant(s) of a heterologous PIV, for example HPIVl or HPIV2.
  • one or more genes or genome segments encoding one or more antigenic determinants of HPIVl or HPIV2 may be added to or substituted within a partial or complete HPIV3 genome or antigenome.
  • both HPIVl genes encoding the HN and F glycoproteins are substituted for counte ⁇ art HPIV3 HN and F genes in a chimeric PIV vaccine candidate.
  • These and other constructs yield chimeric PIVs that elicit either a mono- or poly-specific immune response in humans to one or more HPIVs. Further detailed aspects of the invention are provided in United States Patent Application entitled CONSTRUCTION AND USE OF
  • RECOMBINANT PARAINFLUENZA VIRUSES EXPRESSING A CHIMERIC GLYCOPROTEIN filed on December 10, 1999 by Tao et al. and identified by Attorney Docket No. 17634-000340
  • heterologous genes or genome segments encoding antigenic determinants from both HPIVl and HPIV2 are added to or inco ⁇ orated within a partial or complete HPIV3 vector genome or antigenome.
  • one or more HPIVl genes or genome segments encoding HN and/or F glycoproteins, or antigenic determinant(s) thereof, and one or more HPIV2 genes or genome segments encoding HN and/or F glycoproteins or antigenic determinants can be added to or inco ⁇ orated 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.
  • This vector construct can be further modified by addition or inco ⁇ oration of one or more genes or gene segments encoding antigenic determinant(s) of HPIV2.
  • specific constructs exemplifying the invention are provided which yield chimeric PIVs having antigenic determinants of both HPIVl and HPIV2, as exemplified by the vaccine candidates rPIV3-1.2HN and rPIV3-lcp45.2HN described herein below.
  • chimeric PIV inco ⁇ orate a HPIV vector genome or antigenome modified to express one or more major antigenic determinants of non-PIV pathogen, for example measles virus.
  • the methods of the invention are generally adaptable for inco ⁇ oration of antigenic determinants from a wide range of additional pathogens within chimeric PIV vaccine candidates.
  • the invention also provides for development of vaccine candidates against subgroup A and subgroup B respiratory syncytial viruses (RSV), 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, among other pathogens.
  • pathogens that may be targeted for vaccine development according to the methods of the invention include viral and bacterial pathogens, as well as protozoans and multicellular pathogens.
  • exemplary pathogens including the measles virus HA and F proteins; the F, G, SH and M2 proteins of RSV, mumps virus HN and F proteins, human papilloma virus LI protein, type 1 or type 2 human immunodeficiency virus gpl60 protein, he ⁇ es simplex virus and cytomegalovirus gB, gC, gD, gE, gG, gH, gl, gj, gK, gL, and gM proteins, rabies virus G protein, Epstein Barr Virus gp350 protein; filo virus G protein, bunyavirus G protein, flavivirus E and NS1 proteins, and alphavirus E.
  • antigens as well as other antigens known in the art for the enumerated pathogens and others, are well characterized to the extent that many of their antigenic determinants, including the full length proteins and their constituent antigenic domains, fragments and epitopes, are identified, mapped and characterized for their respective immunogenic activities.
  • mapping studies that identify and characterize major antigens of diverse pathogens for use within the invention are epitope mapping studies directed to the hemagglutinin-neuraminidase (HN) gene of HPIV3 (van
  • the HN epitopes were located in predicted coil regions. Epitopes recognized by MAbs which inhibit neuramimdase activity of the virus were located in a region which appears to be structurally conserved among several paramyxovirus HN proteins and which may represent the sialic acid-binding site of the HN molecule. This exemplary work, employing conventional antigenic mapping methods, identified single amino acids which are important for the integrity of HN epitopes. Most of these epitopes are located in the C-terminal half of the molecule, as expected for a protein anchored at its N terminus (Elango et al., J. Virol. 52:481-489, 1986).
  • RSV respiratory syncytial virus
  • MAbs monoclonal antibodies
  • MAb-resistant mutants Thirteen MAb-resistant mutants (MARMs) were selected, and the neutralization patterns of the MAbs with either MARMs or RSV clinical strains identified a minimum of 16 epitopes. MARMs selected with antibodies to six of the site A and AB epitopes displayed a small-plaque phenotype, which is consistent with an alteration in a biologically active region of the F molecule. Analysis of MARMs also indicated that these neutralization epitopes occupy topographically distinct but conformationally interdependent regions with unique biological and immunological properties. Antigenic variation in F epitopes was then examined by using 23 clinical isolates (18 subgroup A and 5 subgroup B) in cross-neutralization assays with the 18 anti-F MAbs.
  • the invention provides numerous human and non-human PIV vectors, including bovine PIV (BPIV) vectors. These vectors are readily modified according the recombinant methods described herein to carry heterologous antigenic determinants and elicit one or more specific humoral or cell mediated immune responses against the heterologous pathogen and vector PIV.
  • BPIV bovine PIV
  • one or more heterologous genes or genome segments from a donor pathogen is combined with a HPIV3 vector genome or antigenome.
  • the heterologous gene or genome segment is inco ⁇ orated within a chimeric HPIV vector genome or antigenome, for example a chimeric HPIV3-1 vector genome or antigenome having one or both HPIVl genes encoding the HN and F glycoproteins substituted for their counte ⁇ art HPIV3 HN and or F gene(s).
  • a transcription unit comprising an open reading frame (ORF) of the measles virus HA gene is added to a HPIV3 vector genome or antigenome at various positions, yielding exemplary chimeric PIV/measles vaccine candidates rPIV3(HA HN-L), rPIV3(HA N-P), rcp45L(HA N-P), rPIV3(HA P-M), or rc/?45L(HA P-M).
  • chimeric PIV for vaccine use may inco ⁇ orate one or more antigenic determinants of HPIV2, for example an HPIV2 HN gene, within a chimeric HPIV3-1 vector genome or antigemome.
  • chimeric PIVs are engineered that inco ⁇ orate heterologous nucleotide sequences encoding protective antigens from respiratory syncytial virus (RSV) to produce infectious, attenuated vaccine candidates.
  • RSV respiratory syncytial virus
  • the cloning of RSV cPNA and other disclosure is provided in U.S. Provisional Patent Application No. 60/007,083, filed September 27, 1995; U.S. Patent Application No. 08/720,132, filed September 27, 1996; U.S. Provisional Patent Application No. 60/021,773, filed July 15, 1996; U.S. Provisional Patent Application No. 60/046,141, filed May 9, 1997; U.S. Provisional Patent Application No. 60/047,634, filed May 23, 1997; U.S. Patent
  • PIV chimeras inco ⁇ orating one or more RSV antigenic determinants preferably comprise a human PIV (e.g., HPIVl, HPIV2, HPIV3) vector genome or antigenome with a heterologous gene or genome segment encoding an antigenic RSV glycoprotein, protein domain (e.g., a glycoprotein ectodomain) or one or more immunogenic epitopes.
  • a human PIV e.g., HPIVl, HPIV2, HPIV3
  • a heterologous gene or genome segment encoding an antigenic RSV glycoprotein, protein domain (e.g., a glycoprotein ectodomain) or one or more immunogenic epitopes.
  • one or more genes or genome segments from RSV F and/or G genes is/are combined with the vector genome or antigenome to form the chimeric PIV vaccine candidate.
  • chimeric proteins for example fusion proteins having a cytoplasmic tail and/or transmembrane domain of PIV fused to an ectodomain of RSV to yield a novel attenuated virus that elicits a multivalent immune response against both PIV and RSV
  • 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 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 cp 45 mutant strain. Mutations identified in these mutants can be readily inco ⁇ orated into chimeric PIV of the instant invention.
  • cp cold passaged
  • ca cold adapted
  • hr host range restricted
  • sp small plaque
  • ts temperature sensitive mutants
  • one or more attenuating mutations occur in the polymerase L protein, e.g., at a position corresponding to Tyr 942 , Leu 9 2 , or Thri 558 of JS cp45.
  • these mutations are inco ⁇ orated in chimeric PIV of the invention by an identical, or conservative, amino acid substitution as identified in the biological mutant.
  • chimeric PIV for vaccine use inco ⁇ orate one or more mutation wherein Tyr 42 is replaced by His, Leu 2 is replaced by Phe, and/or Thr ⁇ s is replaced by He. Substitutions that are conservative to these replacement amino acids are also useful to achieve desired attenuation in chimeric vaccine candidates.
  • the HPIV3 JS cp45 strain has been deposited under the terms of the Budapest Treaty with the American Type Culture
  • exemplary mutations that can be adopted in chimeric PIVs from biologically derived PIV mutants include one or more mutations in the N protein, including specific mutations at a position corresponding to residues Val 9 or Ser 389 of JS cp45. 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 chimeric PIV of the invention are amino acid substitution in the C protein, e.g., a mutation at a position corresponding to Ile 6 of JS cp45, preferably represented by an identical or conservative substitution of Ile 96 to Thr.
  • exemplary mutations that can be 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 420 or Ala ⁇ o of JS cp45, preferably represented by acid substitutions Ile 420 to Val or Ala ⁇ o to Thr or substitutions conservative thereto.
  • chimeric PIV of the invention can adopt one or more amino acid substitutions in the HN protein, as exemplified by a mutation at a position corresponding to residue Val 38 of JS cp45, preferably represented by the substitution Val 384 to Ala.
  • Yet additional embodiments of the invention include 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, that specify desired phenotypic changes such as attenuation.
  • Exemplary mutations in this context may be engineered at a position in the 3' leader of the chimeric virus at a position corresponding to nucleotide 23, 24, 28, or 45 of JS cp45.
  • 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 cp45.
  • 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 an A to T change in the N gene start sequence at a position corresponding to nucleotide 62 of JS.
  • chimeric PIV recombinants e.g., having the HN and F genes of HPIVl substituted into a partial HPIV3 background genome or antigenome, which is further modified to bear one or more of the attenuating mutations identified in HPIV3 JS cp45.
  • One such chimeric recombinant inco ⁇ orates all of the attenuating mutations identified in the L gene of cp45. It has since been shown that all of the cp45 mutations outside of the heterologous (HPIVl) HN and F genes can be inco ⁇ orated in a HPIV3-1 recombinant to yield an attenuated, chimeric vaccine candidate.
  • chimeric PIVs 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 chimeric PIVs 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 chimeric PIV by multiple nucleotide substitutions in a codon specifying each mutation.
  • mutations that may be inco ⁇ orated in chimeric PIV of the invention are mutations, e.g., attenuating mutations, identified in heterologous PIV or other nonsegmented negative stranded RNA viruses.
  • attenuating and other desired mutations identified in one negative stranded RNA virus may be "transferred", e.g., copied, to a corresponding position within the genome or antigenome of a chimeric PIV.
  • desired mutations in one heterologous negative stranded RNA virus are transferred to the chimeric PIV recipient (either in the vector genome or antigenome or in the heterologous donor gene or genome segment).
  • an amino acid substitution marks a site of mutation in the mutant virus compared to the corresponding wild-type sequence
  • a similar substitution can be engineered at the corresponding residue(s) in the recombinant virus.
  • the substitution will specify an identical or conservative amino acid to the substitute residue present in the mutant viral protein.
  • it is also possible to 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 a recombinant PIV of the invention include other PIVs (e.g., HPIVl, HPIV2, HPIV3, BPIV and MPIV), RSV, Sendai virus (SeV), Newcastle disease virus (NOV), simian virus 5 (SV5), measles virus (MeV), rinde ⁇ est virus, canine distemper virus (CPV), rabies virus (RaV) and vesicular stomatitis virus (VSV), among others.
  • PIVs e.g., HPIVl, HPIV2, HPIV3, BPIV and MPIV
  • RSV Sendai virus
  • NOV Newcastle disease virus
  • SV5 Sendai virus
  • NOV Newcastle disease virus
  • SV5 Sendai virus
  • MeV Newcastle disease virus
  • SV5 simian virus 5
  • Measles virus Measles virus
  • rinde ⁇ est virus canine dis
  • a variety of exemplary mutations are disclosed, 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, however the particular size and amino acid sequence of the deleted or inserted protein fragment can vary.
  • Attenuating mutations in biologically derived PIV and other nonsegmented negative stranded RNA viruses for inco ⁇ oration within chimeric PIV of the invention 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 PIV 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 PIV sequence, e.g., PIV having a mutation specifying an attenuated phenotype.
  • biologically derived PIV include PIV mutants derived from a parental PIV by, ter 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 piupose 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. In experimental animals and humans, both the replication and virulence of PIV correlate with the mutant's shutoff temperature.
  • the JS cp45 HPIV3 mutant has been found to be relatively stable genetically, highly immunogenic, and satisfactorily attenuated. Nucleotide sequence analysis of this biologically derived virus, and of recombinant viruses inco ⁇ orating various individual and combined mutations found therein, indicates that each level of increased attenuation is associated with specific nucleotide and amino acid substitutions.
  • 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 viruses 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 chimeric PIV of the invention 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 cPNA permits introduction of specific engineered changes within 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. Pesired mutations are thus identified and introduced into chimeric PIV vaccine strains.
  • cPNA virus from cPNA allows for routine inco ⁇ oration of these mutations, individually or in various selected combinations, into a full-length cPNA clone, whereafter the phenotypes of rescued recombinant viruses containing the introduced mutations to be readily determined.
  • desired phenotypes e.g., a cp or ts phenotype
  • the invention provides for other, site-specific modifications at, or within close proximity to, the identified mutation.
  • 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).
  • 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 PIV with greater 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 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.
  • PIV genome or antigenome to inco ⁇ orate a mutation that alters the coding assignment of an initiation codon or mutation(s) that introduce one or one or more stop codon(s).
  • one or more of the C, O, and or V ORFs can be deleted in whole or in part to render the corresponding protein(s) partially or entirely non-functional or to disrupt protein expression altogether.
  • 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 exemplary "knock out" mutant PIV 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.
  • chimeric PIV inco ⁇ orate deletion or knock out mutations in a C, O, and or V ORF(s) or other non- essential gene 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.).
  • chimeric PIVs are provided in which expression of one or more gene(s), e.g., a C, O, 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, O, 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 212:418-421, 1996; Kato et al., EMBO J. 16:578-587, 1987; and Schneider et al., Virology 277:314-322. 1996, each inco ⁇ orated herein by reference).
  • Nucleotide modifications that may be introduced into chimeric PIV constructs of the invention 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, 5,00-6,5000 nucleotides or more) in the vector genome or antigenome or heterologous, donor gene or genome segment, 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
  • the invention provides for supplementation of mutations adopted into a chimeric 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 chimeric PIV exhibiting novel vaccine characteristics.
  • the present invention also provides a range of additional methods for attenuating or otherwise modifying the phenotype of a 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 nucleotide sequence 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 chimeric PIV clone.
  • modifications in chimeric PIV of the invention 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 PIV gene relative to an operably linked promoter, introducing an upstream start codon to alter rates of expression, modifying (e.g., by changing position, altering an existing sequence, or substituting an existing sequence with a heterologous sequence) GS and/or GE transcription signals to alter phenotype (e.g., growth, temperature restrictions on transcription, etc.), and various other deletions, substitutions, additions and rearrangements that specify quantitative or qualitative changes in viral replication, transcription of selected gene(s), or translation of selected protein(s).
  • 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 a 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 chimeric PIV of the invention.
  • restriction site markers are routinely introduced within chimeric PIVs to facilitate cPNA construction and manipulation.
  • chimeric PIV construct can be changed, a PIV genome promoter replaced with its antigenome counte ⁇ art, portions of genes removed or substituted, and even entire genes deleted.
  • Pifferent 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 chimeric PIV constructs of the invention include mutations directed toward cis-acting signals, which can be readily 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 chimeric PIV antigenome or genome as described herein. Evaluation and manipulation of trans-acting proteins and cis-acting RNA sequences using the complete antigenome cPNA is assisted by the use of PIV minigenomes as described in the above-inco ⁇ orated references.
  • Additional mutations within chimeric PIVs of the invention may also include 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 (Haas 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 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.
  • gene expression of a 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 a chimeric PIV vaccine candidate 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.
  • 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.
  • These and other transpositioning changes yield novel chimeric PIV vector virus 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.
  • chimeric PIVs useful in vaccine formulations can be conveniently modified to accommodate antigenic drift in circulating virus.
  • the modification will be in the HN and/or F proteins.
  • 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 cPNA 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 PIV BPIV
  • MIV murine PIV
  • 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.
  • the invention provides methods for attenuating chimeric PIV vaccine candidates based on the further construction of chimeras between HPIV and a non-human PIV, for example HPIV3 and BPIV3 (e.g., as disclosed in U.S. Patent Application Serial No. 09/586,479, filed June 1, 2000 by Schmidt et al.; Schmidt et al., J. Virol. 24:8922-9, 2000, each inco ⁇ orated herein by reference).
  • This method of attenuation is based on host range effects due to the introduction of one or more gene(s) or genome segment(s) of the non-human PIV into a human PIV vector-based chimeric virus.
  • HPIV3 and BPIV3 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., J. Infect. Pis. 152:655-662, 1988, inco ⁇ orated herein by reference).
  • HPIV3 and BPIV3 Although the basis of the host range differences between HPIV3 and BPIV3 remains 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 vector 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 chimeric PIV clone.
  • Replacement of the HPIV3 N ORF of HPIV3 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.
  • Ka and SF HPIV3/BPIV3 chimeric recombinants are viable and replicate 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. This property of efficient replication in vitro is important since it permits efficient manufacture of this biological.
  • Ka and the SF HPIV3/BPIV3 chimeric recombinants (termed cKa and cSF), bearing only one bovine gene, are nearly equivalent to their BPIV3 parents in the degree of host range restriction in the respiratory tract of the rhesus monkey.
  • the cKa and cSF viruses exhibit approximately a 60-fold or 30-fold reduction, respectively, in replication in the upper respiratory tract of rhesus monkeys compared to replication of HPIV3.
  • BPIV3 genes will also confer desired levels of host range restriction within chimeric PIV of the invention.
  • a list of attenuating determinants will be readily identified in heterologous genes and genome segments of BPIV and other non-human PIVs that will confer, in appropriate combination, a desired level of host range restriction and immunogenicity on chimeric PIV selected for vaccine use.
  • Chimeric human-bovine PIV for use as vectors within the present 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 PIV genome or antigenome.
  • chimeric PIV inco ⁇ orate 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 counte ⁇ art, human or bovine PIV.
  • Heterologous genes or genome segments from the counte ⁇ art, 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 (U.S. Patent Application Serial No. 09/586,479, filed June 1, 2000 by Schmidt et al.; Schmidt et al., J. Virol. 24:8922-9, 2000, each inco ⁇ orated herein by reference).
  • Genes and genome segments that may be selected for use as heterologous substitutions or additions within human-bovine chimeric PIV vectors include genes or genome segments encoding a PIV N, P, C, O, V, M, F, SH (where appropriate), HN and/or L protein(s) or portion(s) thereof.
  • genes and genome segments encoding non-PIV proteins for example, an SH protein as found in mumps and SV5 viruses, may be inco ⁇ orated 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.
  • Certain human-bovine chimeric PIV vectors for use within 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 BPIV 3 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.
  • Any 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 PIV will include one or more of the HPIV protective antigens such as the HN or F glycoproteins.
  • human-bovine chimeric PIV bearing one or more bovine gene(s) or genome segment(s) exhibits 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.
  • a human PIV is attenuated by the addition or substitution of one or more bovine gene(s) or genome segment(s) to a partial or complete human, e.g., HPIV3, PIV background genome or antigenome.
  • the HPIV3 N gene is substituted by the BPIV3 N gene to yield a novel human-bovine chimeric PIV vector, and within this vector the measles HA gene is substituted to yield a multivalent, HPIV/measles vaccine candidate, as exemplified by the recombinant rHPIV3-N ⁇ HA P . M described below.
  • the degree of host range restriction exhibited by human-bovine chimeric PIV vectors for developing 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 vectors 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 vector for developing vaccines against PIV and other pathogens, based on host range effects.
  • human-bovine chimeric PIV vectors comprise a BPIV recipient or backbone virus that inco ⁇ orates one or more heterologous gene(s) that encode an HPIV HN and/or F glycoprotein(s).
  • the chimeric PIV may inco ⁇ orate 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).
  • These immunogenic proteins, domains and epitopes are particularly useful within human-bovine chimeric PIV because they generate novel immune responses in an immunized host.
  • 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 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.
  • the RSV A glycoprotein genes G and F were successfully inserted as additional heterologous ORF to yield multivalent, HPIV/RSV vaccine candidates exemplified by the recombinant viruses rB/HPIV3-Gl and rB/HPIV3-Fl described below.
  • human-bovine chimeric PIV vectors additionally inco ⁇ orate 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.
  • Substitution or addition of 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 vector 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 FIN 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.
  • the human PIV HN or F glycoprotein or parts thereof may be joined with a genome segment encoding an HN or F glycoprotein or parts thereof from another PIV strain or serotype.
  • Attenuated, human-bovine chimeric PIV vectors 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. These can include mutations in RNA regulatory sequences or in encoded proteins. These attenuating mutations may be generated de novo and tested for attenuating effects according to a rational design mutagenesis strategy. Alternatively, the attenuating mutations may be identified in existing biologically derived mutant PIV and thereafter inco ⁇ orated into a human-bovine chimeric PIV of the invention.
  • 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.
  • Infectious chimeric PIV vector 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, parental (i.e., vector or heterologous donor) PIV or non-PIV pathogen .
  • a wild-type, parental (i.e., vector or heterologous donor) PIV or non-PIV pathogen i.e., vector or heterologous donor
  • one or more supplemental immunogenic epitope(s), protein domains, or proteins from a heterologous PIV strain or type, or from a non-PIV pathogen such as measles or RSV can be added to a chimeric PIV by appropriate nucleotide changes in the chimeric genome or antigenome.
  • chimeric PIVs 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
  • genes of interest in this context include genes encoding cytokines, for example, an interleukin (e.g., interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 5 (IL- 5), interleukin 6 (IL6), interleukin 18 (IL-18)), tumor necrosis factor alpha (TNF ⁇ ), interferon gamma (IFN ⁇ ), or granulocyte-macrophage colony stimulating factor (GM-CSF), as well as IL-2 through IL-18, especially IL-2, IL-6 and IL-12, and IL-18, gamma-interferon (see, e.g., U.S. Application No. 09/614
  • Oeletions, insertions, substitutions and other mutations involving changes of whole viral genes or genome segments within chimeric PIV of the invention yield highly stable vaccine candidates, which are particularly important in the case of immunosuppressed individuals. Many of these changes will result in attenuation of resultant vaccine strains, whereas others will specify different types of desired phenotypic changes.
  • accessory (i.e., not essential for in vitro growth) genes are excellent candidates to encode proteins that specifically interfere with host immunity (see, e.g., Kato et al., EMBO. J. 16:578-87, 1997, inco ⁇ orated herein by reference). Ablation of such genes in vaccine viruses is expected to reduce virulence and pathogenesis and/or improve immunogenicity.
  • infectious, chimeric PIV clone can be achieved by a variety of well known methods.
  • infectious clone with regard to PNA is meant cPNA 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 cPNA copy of the genome or antigenome.
  • antigenome or genome cPNA subfragments to assemble a complete antigenome or genome cPNA as described herein has the advantage that each region can be manipulated separately (smaller cPNAs are easier to manipulate than large ones) and then readily assembled into a complete cPNA.
  • the complete antigenome or genome cPNA, or any subfragment thereof can be used as template for oligonucleotide-directed mutagenesis.
  • mutations are introduced by using the Muta-gene phagemid in vitro mutagenesis kit available from Bio-Rad.
  • cPNA encoding a portion of a PIV genome or antigenome is cloned into the plasmid pTZ18U, and used to transform CJ236 cells (Life Technologies, Gaithersburg, MP).
  • 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.
  • the invention also provides methods for producing infectious chimeric PIV from one or more isolated polynucleotides, e.g., one or more cPNAs.
  • cPNA encoding a PIV genome or antigenome is 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 the template for the synthesis of progeny PIV genome.
  • a cPNA 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 the complementing sequences that encode proteins necessary to generate a transcribing, replicating nucleocapsid, i.e., sequences that encode N, P, and L proteins.
  • the genome or antigenome of the recombinant PIV of the invention 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, or can be expressed directly from the genome or antigenome cPNA.
  • 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 proteins necessary to (i) produce a nucleocapsid capable of RNA replication, and (ii) render progeny nucleocapsids competent for both RNA replication and transcription. Transcription by the genome nucleocapsid provides the other PIV proteins and initiates a productive infection. Alternatively, additional PIV proteins needed for a productive infection can be supplied by coexpression.
  • Infectious PIV 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, P 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 an isolated positive-sense polynucleotide molecule which serves as a template for synthesis of progeny PIV genome.
  • a cDNA 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. 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 cDNA 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 cDNA 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 Diego, 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 cDNAs 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 DNA 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 cDNA.
  • specific desired nucleotide sequences can be synthesized and inserted at appropriate regions in the cDNA 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 cDNA.
  • cDNA encoding the genome or antigenome include reverse transcription-PCR using improved PCR conditions (e.g., as described in Cheng et al., Proc. Natl. Acad. Sci. U.S.A. 91:5695-5699, 1994), inco ⁇ orated herein by reference) to reduce the number of subunit cDNA components to as few as one or two pieces.
  • different promoters can be used (e.g., T3, SP6) or different ribozymes (e.g., that of hepatitis delta virus.
  • Different DNA vectors e.g., cosmids
  • cosmids can be used for propagation to better accommodate the larger size genome or antigenome.
  • 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.
  • 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 2:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et al., ed., Current Protocols in Molecular Biology.
  • 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 subfragments 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
  • 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 PIV minigenomes (Dimock et al., J. Virol. 67: 2772-8, 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 PIV 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 PIV 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), by the homology alignment algorithm of Needleman & Wunsch, .
  • sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
  • the reference sequence may be a subset of a larger sequence.
  • proteins and protein regions encoded by 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.
  • 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
  • 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 vivo-- 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 vivo models to confirm adequate attenuation, resistance to phenotypic reversion, and immunogenicity for vaccine use.
  • the modified virus e.g., a multiply attenuated, biologically derived or recombinant 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
  • the invention also provides isolated, infectious recombinant PIV 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 "1-1" and HEPES, with or without adjuvant, as further described below.
  • a stabilizing solution e.g., saline or comprising SPG, Mg "1-1" and HEPES, with or without adjuvant, as further described below.
  • PIV 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 proteins, e.g., F and HN glycoproteins.
  • PIV proteins e.g., F and HN glycoproteins.
  • the host becomes at least partially or completely immune to PIV 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 PIV or a closely related virus and which host is capable of generating a protective immune response to the antigens of the vaccinizing strain.
  • 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
  • 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 3 to about 10 7 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 PIV serotypes or strains to achieve protection against multiple PIV 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.
  • 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.
  • 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 PIV.
  • 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.
  • adults who are particularly susceptible to repeated or serious PIV infection such as, for example, health care workers, day care workers, family members of young children, the elderly, individuals with compromised cardiopulmonary function, may require multiple immunizations to establish and/or maintain protective immune responses.
  • Levels of induced immunity can be monitored by measuring amounts of neutralizing secretory and serum antibodies, and dosages adjusted or vaccinations repeated as necessary to maintain desired levels of protection.
  • different vaccine viruses may be indicated for administration to different recipient groups.
  • an engineered PIV strain expressing a cytokine or an additional protein rich in T cell epitopes may be particularly advantageous for adults rather than for infants.
  • PIV 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 PIV strains or subgroups engineered into one PIV 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.
  • 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, 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 PIV 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.
  • the infectious PIV produced by coexpressing the recombinant PIV genome or antigenome with the N, P, L and other desired PIV 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.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the HA gene of the measles virus is inserted as an extra gene into one of three gene junctions of a JS wild type or attenuated strain of HPIV3, namely, the N/P, P/M, or HN/L junction, and recombinant chimeric viruses were recovered. Insertion of the measles HA gene at three different positions in the HPIV3 genome illustrates the range of useful constructs for transferring antigenic determinants from foreign pathogens into PIV vectors.
  • the chimeric rHPIV bearing the measles virus HA insertion in a wild type rHPIV3 background replicated efficiently in vitro but was restricted in replication in hamsters compared to that of the rHPIV3 virus from which it was derived.
  • the recombinant chimeric HPIV3 bearing the measles virus HA insertion in an attenuated rHPIV3 background replicated in vitro and in hamsters to a level that was also slightly less than that of the attenuated rHPIV3 ⁇ p45L mutant virus from which it was derived.
  • the amount of HA protein expressed by cells infected with the attenuated rHPI V3 -measles virus HA recombinants with the HA gene in the N/P or P/M junction was very high and even exceeded that seen in cells infected with native measles virus.
  • the level of replication of the rHPIV3cp45L with a measles virus HA insert in the N/P or P/M junction was 10-fold lower in the upper respiratory tract of the hamster than that of the rHPIV3-cp45L parent virus indicating that gene insertions can unexpectedly contribute to the attenuation of an HPIV3 vector.
  • Attenuated chimeric recombinant HPIV vectors combining a backbone of HPIV3 and one or more antigenic determinants of HPIVl, can also be used as vectors to express additional foreign antigens (e.g., of HPIV2 or a non-PIV virus).
  • additional foreign antigens e.g., of HPIV2 or a non-PIV virus.
  • rPIV3-l a non-attenuated recombinant bearing major antigens of HPIVl
  • rPIV3-lcp45 an attenuated recombinant bearing HPIVl major antigens
  • Each virus exhibited a level of temperature sensitivity of replication in vitro similar to that of its rPIV3-l or rPIV3-lcp45 parent virus.
  • the insertion of the PIV2 HN attenuated both the rPIV3-l and rPIV3-c/?45 viruses in hamsters, a finding similar to that observed with the insertion of the measles viruses HA into rJS and into rPIV3cp45.
  • Infection of hamsters with these antigenic rPIV3-l recombinants bearing the PIV2 HN gene insert induced serum antibody responses reactive against both HPIVl and HPIV2.
  • an attenuated rHPIV3 or rHPIV3-l vaccine candidate as a vector to infect the respiratory tract of susceptible hosts and thereby induce a vigorous antibody response to foreign protective antigens expressed from an extra gene unit, as well as against the HPIV vector itself.
  • the presence of three antigenic serotypes of HPIV which do not provide significant cross-protection, allows for more effective, sequential immunization of human infants with antigenically distinct variants of HPIV each bearing the same or different heterologous antigenic determinant(s), e.g., a protective antigen, antigenic domain or epitope of measles virus or of one or more different viral or microbial pathogens.
  • Sequential immunization permits development of a primary immune response to the foreign protein, which is boosted during subsequent infections with a secondary, antigenically- distinct HPIV bearing one or more heterologous antigenic determinants, e.g., a protective antigen, antigenic domain or epitope of measles virus or of one or more different viral or microbial pathogens.
  • a secondary, antigenically- distinct HPIV bearing one or more heterologous antigenic determinants e.g., a protective antigen, antigenic domain or epitope of measles virus or of one or more different viral or microbial pathogens.
  • the immunity induced to one HPIV vector can be circumvented by boosting with an antigenically distinct HPIV vector.
  • successful immunization of animals that are immune to PIV3 has been achieved with attenuated PIV3-1 vaccine candidates, confirming the feasibility of sequential immunization with serotypically distinct PIV viruses even if these PIVs share proteins other than HN and F.
  • vector based vaccine constructs of the invention are useful to boost immune responses by a second, third or fourth administration of the same HPIV vector or by sequential use of different vectors.
  • the invention In preferred sequential vaccination methods of the invention, it is desirable to sequentially immunize an infant with different PIV vectors each expressing the same heterologous antigenic determinant such as the measles virus HA.
  • This sequential immunization permits the induction of the high titer of antibody to the heterologous protein that is characteristic of the secondary antibody response.
  • early infants e.g. 2-4 month old infants
  • an attenuated chimeric HPIV3 expressing a heterologous antigenic determinant, for example the measles virus HA protein, and also adapted to elicit an immune response against HPIV3.
  • a vaccine candidate useful in this context is the rcp45L(HA P-M) recombinant.
  • An exemplary vaccine candidate in this context is the rPIV3-l cp45L virus expressing the measles virus HA gene and HPIVl antigenic determinants as functional, obligate glycoproteins of the vector.
  • the vaccinee will elicit a primary antibody response to both the PIV3 HN and F proteins and to the measles virus HA protein, but not to the PIVl HN and F protein.
  • the vaccinee Upon secondary immunization with the rPIV3-l cp45L expressing the measles virus HA, the vaccinee will be readily infected with the vaccine because of the absence of antibody to the PIVl HN and F proteins and will develop both a primary antibody response to the PIVl HN and F protective antigens and a high titered secondary antibody response to the heterologous measles virus HA protein.
  • a similar sequential immunization schedule can be developed where immunity is sequentially elicited against HPIV3 and then HPIV2 by one or more of the chimeric vaccine viruses disclosed herein, simultaneous with stimulation of an initial and then secondary, high titer protective response against measles or another non-PIV pathogen.
  • This sequential immunization strategy preferably employing different serotypes of PIV as primary and secondary vectors, effectively circumvents immunity that is induced to the primary vector, a factor ultimately limiting the usefulness of vectors with only one serotype.
  • exemplary coordinate vaccination protocols may inco ⁇ orate two, three, four and up to six or more separate chimeric HPIV vaccine viruses administered simultaneously (e.g., in a polyspecific vaccine mixture) in a primary vaccination step, e.g., at one, two or four months of age.
  • two or more and up to a full panel of HPIV-based vaccine viruses can be administered that separately express one or more antigenic determinants (i.e., whole antigens, immunogenic domains, or epitopes) selected from the G protein of RSV subgroup A, the F protein of RSV subgroup A, the G protein of RSV subgroup B, the F protein of RSV subgroup B, the HA protein of measles virus, and/or the F protein of measles virus.
  • antigenic determinants i.e., whole antigens, immunogenic domains, or epitopes
  • Coordinate booster administration of these same PIV3-based vaccine constructs can be repeated at two months of age.
  • a separate panel of 2-6 or more antigenically distinct (referring to vector antigenic specificity) live attenuated HPIV-based vaccine viruses can be administered in a secondary vaccination step.
  • secondary vaccination may involve concurrent administration of a mixture or multiple formulations that contain(s) multiple HPIV3-1 vaccine constructs that collectively express RSV G from subgroup A, RSV F from subgroup A, RSV F from subgroup B, RSV G from subgroup B, measles virus HA, and/or measles virus F, or antigenic determinants from any combination of these proteins.
  • This secondary immunization provides a boost in immunity to each of the heterologous RSV and measles virus proteins or antigenic determinant(s) thereof.
  • a tertiary vaccination step involving administration of one-six or more separate live attenuated PIV3-2 vector-based vaccine recombinants can be coordinately administered that separately or collectively express RSV G from subgroup A, RSV F from subgroup A, RSV G from subgroup B, RSV F from subgroup B, measles virus HA, and/or measles virus F, or antigenic determinant(s) thereof.
  • rPIV3 and rPIV3-l vaccines may be administered in booster formulations.
  • nucleotide sequences into a rHPIV that both direct the expression of a foreign protein and that attenuate the virus in an animal host, or to use nucleotide insertions separately to attenuate candidate vaccine viruses.
  • gene units of varying lengths were inserted into a wild type HPIV3 backbone and the effects of gene unit length on attenuation were examined. These novel gene unit insertions were engineered to not contain a significant ORF which permitted an evaluation of the effect of gene unit length independently of an effect of the expressed protein of that gene.
  • heterologous sequences were inserted as an extra gene unit of sizes between 168 nt and 3918 nt between the HN and L genes.
  • control cPNA constructions and viruses were made in which insertions of similar sizes were placed in the 3 '-noncoding region of the HN gene and hence did not involve the addition of an extra gene. These viruses were made to assess the effect of an increase in the overall genome length and in gene number on attenuation. The insertion of an extra gene unit is expected to decrease the transcription of genes downstream of the insertion site which will affect both the overall abundance and ratios of the expressed proteins.
  • gene insertions or extensions larger than about 3000 nts in length attenuated the wild type virus for the upper and lower respiratory tract of hamsters.
  • Gene insertions of about 2000 nts in length further attenuated the rHPIV3cp45L vaccine candidate for the upper respiratory tract.
  • gene insertions can have the dual effect of both attenuating a candidate vaccine virus and inducing a protective effect against a second virus.
  • Gene extensions in the 3'- noncoding region of a gene, which cannot express additional proteins, can also be attenuating in and of themselves.
  • gene insertion length is a determinant of attenuation.
  • GU and NCR insertions within recombinant PIV of the invention produce an attenuation phenotype characterized by efficient replication in vitro and decreased replication in vivo, a phenotype not previously described for other paramyxovirus insertions.
  • the mechanism of attenuation resulting from a GU insertion may result from one or more of the following factors acting predominantly in vivo.
  • the addition of an extra gene unit may decrease the level of transcription of downstream genes since there is a transcriptional gradient in which more promoter-proximal genes are transcribed at a higher rate than the more promoter-distal genes.
  • the decreased expression of the downstream gene products resulting from the decreased abundance of their mRNAs could result in attenuation if their gene product is limiting or if a specific ratio of gene products that is required for efficient replication is altered. It is thought that the transcription gradient is a consequence of the transcriptase complex falling off the template during transcription as well as during the transfer across gene junctions.
  • the increase in the overall length of the genome and the extra mRNAs transcribed may increase the level of viral double stranded RNA made which in turn may induce a higher level of the antiviral activity of the interferon system.
  • the overall level of genome replication may be reduced due to the increase in length of the genome and the antigenome. This may result from a disengagement of replicase complexes from the template during replication of the genomic RNA or antigenomic RNA.
  • the decreased amount of genome available for packaging into virions may result in a decrease in virus yield which results in attenuation.
  • the mechanism of attenuation resulting from a NCR insertion may result from one or more of the following factors.
  • the extra length of the 3 '-end of HN mRNA resulting from the NCR insertion may contribute to the instability of the mRNA and lead to a decrease in the expression of the HN protein.
  • the increase in the overall length of the genome and the extra length of the HN mRNA may increase the level of viral double stranded RNA made that can induce a higher level of the antiviral activity of the interferon system.
  • the overall level of genome replication may be reduced due to the increase in length of the genome and the antigenome.
  • the in vitro and in vivo growth properties of the GU and NCR insertions into PIV3 are distinct from previous findings with other single-stranded, negative-sense RNA viruses, cited above.
  • Previously tested insertions examined expressed proteins, whereby the independent effect of the length of insertions on viral growth in vivo cannot be determined.
  • the present findings demonstrate that the GU and NCR insertions greater than 3 kb specify an attenuation phenotype that is independent of expressed protein. Shorter insertions, e.g., greater than about 2 kb, specify further attenuation in a partially attenuated recipient.
  • the GU and NCR insertions specify restricted replication in vivo in the absence of restricted replication in vitro.
  • the attenuation phenotype in vivo is seen when the insertion is either in the form of a GU or a NCR insertion - other documented insertions are in the form of GU only.
  • the attenuation of replication in vivo specified by a GU or NCR insertion that does not encode a protein represents a unique way to attenuate members of the Mononegavirales in vivo.
  • the Pmtl to Ba HI fragment of p3/7(131)2G+ (nt 1215-3903 of the PIV3 antigenome ⁇ was subcloned into the plasmid pUCl 19 ⁇ pUCl 19(Pm/I-_3 ⁇ wHI) ⁇ which had been modified to include a PmR site in the multiple cloning region.
  • Two independent single- stranded mutagenesis reactions were performed on pUCl l9(Pmll -BamHI) using Kunkel's method (Kunkel et al., Methods Enzymol.
  • the first reaction introduced an Aflll site in the 3' (downstream)-noncoding region of the N gene by mutating the CTAAAT sequence at nts 1677-1682 of the antigenome to
  • CTTAAG (pAffll N-P)
  • the second, separate, reaction introduced an Aflll site in the in the 3 '-noncoding region of the P gene by mutating the TCAATC sequence at nts 3693-3698 of the antigenome to CTTAAG (pAflll P-M).
  • the HA ORF of measles virus Edmonston strain was amplified from Edmonston wild type virus by reverse transcription polymerase chain reaction (RT-PCR).
  • the nt sequence of the Edmonston wild type HA open reading frame (ORF) is in GenBank Accession # U03669, inco ⁇ orated herein by reference (note that this sequence is the ORF only without the upstream 3 nts or the stop codon).
  • Measles virus RNA was purified from clarified medium using TRIzol-LS (Life Technologies, Gaithersburg, MO) following the manufacturer's recommended procedure.
  • RT-PCR was performed with the Advantage RT- for-PCR and Advantage-HF PCR kits (Clontech, Palo Alto, CA) following the recommended protocols.
  • Primers were used to generate a PCR fragment spanning the entire ORF of the measles virus HA gene flanked by PIV3 non-coding sequence and Aflll restriction sites.
  • the forward primer 5'- TTAATC7T .4GAATATACAAATAAGAAAAACTTAGGATTAAAGAGCGATGTCAC CACAACGAGACCGGATAAATGCCTTCTAC-3 ' (SEQ IP NO.
  • Aflll site (italicized) downstream (in the positive-sense complement) of PIV3 noncoding sequence derived from the P gene, nt 3594- 3623 (underlined), and the end of the measles HA ORF (bolded).
  • the resultant PCR fragment was then digested with Aflll and cloned into p(AfHl N-P) and p(Aflll P-M) to create pUC 119(HA N-P) and pUC 119(HA P-M) respectively.
  • pUC 119(HA N-P) and pUC 119(HA P-M) were sequenced over the entire Aflll insert using dRhodamine Terminator Cycle Sequencing Ready Reaction (ABI prism, PE Applied Biosystems, Foster city, CA), and the sequence was confirmed to be correct.
  • pFLCcp45L encodes the three amino acid changes in the L gene of PIV3 cp45 (aa position 942, 992, and 1558) which confer most of the temperature-sensitivity and attenuation of the cp45 vaccine candidate virus (Skiadopoulos et al., J. Virol. 72:1762-8. 1998, inco ⁇ orated herein by reference), and the transfer of the XhoI-NgoMl fragment transferred those mutations.
  • a HPIV3 chimeric cPNA was constructed by PCR to include a heterologous polynucleotide sequence, exemplified by the measles virus HA gene, encoding a heterologous antigenic determinant of the measles virus, flanked by the transcription signals and the noncoding regions of the HPIV3 HN gene.
  • This cPNA was designed to be combined with an rPIV3 vector as an extra gene following the HN gene.
  • Kunkel mutagenesis Kunkel mutagenesis (Kunkel et al., Methods Enzymol. 154:367-382.
  • CC-3' contains a Stwl site (italicized) followed by HPIV3 sequence (underlined) which includes the downstream end of the HN gene (HPIV3 nts 8602-8620), an intergenic region, and the gene-start signal and sequence from the upstream end of the HN gene (HPIV3 nt 6733-6753).
  • Mlul site 16 contains an Mlul site (italicized) downstream of the start of the measles HA ORF (bolded) followed by the complement to HPIV3 nts 6744-6805 (underlined), which are part of the upstream HN noncoding region.
  • the Mlul site present in the introduced measles virus ORF was created by changing nt 27 from T (in the wild type Edmonston HA gene) to C and nt 30 from C to G. Both of these changes are noncoding in the measles virus ORF.
  • the PCR was performed using p3/7(131)2G-Stu as template.
  • PCR fragment 1 The resulting product, termed PCR fragment 1 , is flanked by a Stwl site at the 5 '- end and an Mlul site at the 3 '-end and contains the first 36 nt of the measles HA ORF downstream of noncoding sequence from the HPIV3 HN gene.
  • the second PCR reaction synthesized the right-hand end of the HN gene.
  • CTATTGGGTCCTTCC-3 ' contains the Xmal (italics) and the end of the measles HA ORF (bold), followed by HPIV3 nts 8525-8566 (underlined) representing part of the downstream nontranslated region of the HN gene.
  • the template for the PCR was p3/7(131)2G-Stu.
  • PCR fragment 2 which resulted from this reaction contains the last 35 nt of the measles HA ORF and approximately 2800 nt of the L ORF of PIV3 and is flanked by an Xmal site and an Sphl site (which occurs naturally at HPIV3 position 11317).
  • the third PCR reaction amplified the largest, central portion of the measles HA ORF from the template cPNA pTM-7, a plasmid which contains the HA ORF of the Edmonston strain of measles virus supplied by the ATCC.
  • the measles virus HA ORF contained in PTM-7 contains 2 amino acid differences from pTM-7 of the Edmonston wild type HA sequence used for insertion into the N-P and M-P junction, and these were at amino acid positions 46 (F to S) and at position 481 (Y to N).
  • the forward primer 5'- CGGATAA CGCG7TCTACAAAGATAACC-3' (SEQ IP NO. 18) (MWI site italicized) and reverse primer 5'-CGGATAA CGCG7TCTACAAAGATAACC-3' (SEQ IP NO. 18) (Xmal site italicized) amplified PCR fragment 3 which contained nts 19-1838 of the measles HA ORF.
  • PCR fragment 1 was digested with Stwl and MwI while PCR fragment 3 was digested with Mlul and Xmal. These two digested fragments were then cloned by triple ligation into the Stul-Xmal window of pUCl 18 which had been modified to include a Stwl site in its multiple cloning region.
  • the resultant plasmid, pUCl 18(HA 1+3) was digested with Stwl and Xmal while PCR fragment 2 was digested with Xmal and Sphl.
  • the two digested products were then cloned into the Stul-Sphl window of p3/7(131)2G-Stu, resulting in the plasmid pFLC(HA HN-L).
  • the Stul-Sphl fragment including the entire measles HA ORF, was then sequenced using dRhodamine Terminator Cycle Sequencing Ready Reaction (ABI prism, PE Applied Biosystems, Foster city, CA). The chimeric construct sequence was confirmed.
  • the measles virus HA ORF flanked by HPIV3 transcription signals was inserted as an extra gene into the N/P, P/M, or HN/L junction of an antigenomic cPNA vector comprising a wild type HPIV3 or into the N/P or P/M junction of an antigenomic cPNA vector comprising an attenuated HPIV3.
  • the five full-length vector cPNAs bearing the measles HA ORF as a separate gene were transfected separately into HEp-2 cells on six-well plates (Costar, Cambridge, MA) together with the support plasmids ⁇ pTM(N), pTM(P no C), and pTM(L) ⁇ , and
  • pTM(P no C) is a derivative of pTM(P) (Purbin et al., Virology 261:319-330, 1999) in which the C ORF expression has been silenced by mutation of the C start codon.
  • the transfection harvest was passaged onto a fresh monolayer of Vero cells in a T25 flask and incubated for 5 days at 32°C (referred to as passage 1).
  • the presence of HPIV3 in the passage 1 harvest was determined by plaque titration on LLC-MK2 monolayer cultures with plaques visualized by immunoperoxidase staining with HPIV3 HN- specific and measles HA-specific monoclonal antibodies as previously described (Purbin et al., Virology 235:323-332, 1997, inco ⁇ orated herein by reference).
  • rPIV3(HA HN-L) virus present in the supernatant of the appropriate passage 1 harvest was biologically-cloned by plaque purification three times on LLC-MK2 cells as previously described (Hall et al., Virus Res. 22:173-184, 1992, inco ⁇ orated herein by reference).
  • rPIV3(HA N-P), rc/?45L(HA N-P), rPIV3(HA P-M), and rcp45L(HA P-M) were biologically-cloned from their respective passage 1 harvests by terminal dilution using serial 2-fold dilutions on 96- well plates (12 wells per dilution) of Vero cell monolayers.
  • the biologically-cloned recombinant viruses from the third round of plaque purification or from the second or third round of terminal dilution were then amplified twice in LLC-MK2 cells ⁇ rPIV3(HA HN-L ⁇ or Vero cells ⁇ rPIV3(HA N-P), rcp45L(HA N-P), rPIV3(HA P-M), rcp45L(HA P-M) ⁇ at 32°C to produce virus for further characterization.
  • each passage 1 harvest was analyzed by RT-PCR using three different primer pairs; one pair for each location of the HA ORF insert.
  • the first primer pair amplified a fragment of PIV 3 spanning nucleotides 1596-1968 of the full-length HPIV3 genome, which includes the N/P insertion site. This fragment size increased to 2298 nucleotides with the measles HA ORF inserted between the N and P genes.
  • the second primer pair amplified a fragment of PIV3 spanning nucleotides 3438-3866 of the full-length HPIV3 genome, which includes the P/M insertion site.
  • Monolayers of LLC-MK2 cells in T25 flasks were infected at a multiplicity of infection (MOI) of 5 with either rc/?45L(HA N-P), rcp45L(HA P-M), rJS or were mock infected.
  • MOI multiplicity of infection
  • Monolayers of Vero cells in T25 flasks were infected with the Edmonston wild type strain of measles virus at an MOI of 5.
  • Vero cell monolayers were chosen for the measles Edmonston virus infection because measles virus does not grow well in LLC-MK2 cells. At 24 hours post-infection, the monolayer was washed with methionine-minus PMEM (Life Technologies).
  • 35 S methionine was added to PMEM-minus media at a concentration of lOuCi/ml and 1 ml was added to each flask which was then incubated at 32°C for 6 hours.
  • the cells were harvested and washed 3 times in PBS.
  • the cell pellets were resuspended in 1 ml RIP A buffer ⁇ 1% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100 (Sigma), 0.2% (w/v) SPS, 150mM NaCl, 50mM Tris-HCl, pH 7.4 ⁇ , freeze-thawed and clarified by centrifugation at 6500 X G for 5 minutes.
  • the cell extract was transferred to a fresh eppendorf tube and a mixture of monoclonal antibodies which recognizes the HA glycoprotein of measles virus (79-XV-V17, 80-III-B2, 81-1-366) (Hummel et al., J. Virol. 69:1913-6, 1995; Sheshberanig et al., Arch. Virol.
  • rcp45L(HA P-M) and rcp45L(HA N-P) expressed the measles virus HA protein to a greater extent than did the Edmonston wild type strain of measles virus indicating that these constructs efficiently expressed the measles virus HA from the N/P and P/M junctions of the attenuated strain rcp45L.
  • rcp45L(HA N-P) and rcp45L(HA P-M) were confirmed to be HPIV3-based by their reactivity with the PIV3 anti-HN monoclonal antibodies.
  • the level of temperature sensitivity of replication of the chimeric rPIV3s bearing the measles virus HA insertion was evaluated to assess whether acquisition of the HA insert modified the level of replication in the chimeric virus compared to the parental, vector virus at various temperatures (Table 1).
  • chimeric derivatives of both wild type vector viruses bearing the measles virus HA gene were slightly restricted in replication at 40°C (Table 1).
  • the two attenuated rPIV3s bearing the measles virus HA gene, rcp45L(N-P) and rcp45L(HA P- M) possessed a level of temperature sensitivity similar to that of the rcp45L parental, vector virus with rcp45L(HA P-M) being slightly more ts than its parent.
  • the viruses bearing the inserts replicated in tissue culture similarly to the parental vector rPIV3 from which they were derived, with only a slight increase in temperature sensitivity.
  • rPIV3 can readily serve as a vector to accommodate the HA insert at different sites without major alteration in replication in vitro, and that rPIV3(HA) chimeric viruses can readily accommodate the further addition of one or more attenuating mutations.
  • Table 1 Replication at permissive and elevated temperatures of recombinant HPIV3s expressing the HA protein of measles virus as an extra gene in the N-P, P-M, or HN-L junctions.
  • Underlined titer represents the lowest restrictive temperature at which a 100-fold or greater reduction in titer from that at 32°C is seen and defines the shut-off temperature of the virus.
  • the levels of replication of chimeric rPIV3s bearing an antigenic determinant of the measles virus was compared with that of their parent rPIV3s to determine if the acquisition of the determinant, exemplified by an HA insert, significantly modified their ability to replicate and to induce an immune response in vivo.
  • the nasal turbinates and lungs were homogenized in 10% or 20% w/v suspension of L-15 (Quality Biologicals, Gaithersburg, MO) respectively, and the samples were rapidly frozen. Virus present in the samples was titered on 96 well plates of LLC-MK2 cell monolayers and incubated at 32°C for 7 days. Virus was detected by hemadso ⁇ tion, and the mean log 10 TCID 50 /g was calculated for each group of hamsters. Insertion of the HA gene into wild type rJS (Table 2) restricted its replication 4 to 20-fold in the upper respiratory tract and up to five-fold in the lower respiratory tract indicating only a slight effect of the acquisition of the HA gene on replication of wild type rJS virus in hamsters.
  • each of the two rcp45(HA) antigenic chimeras was 10-fold less in the upper respiratory tract of hamsters (Table 3)-than that of ⁇ cp45L, the recombinant parent virus bearing the three attenuating ts mutations in the L protein, but was the same as the rcp45L parent in the lower respiratory tract.
  • Table 3 The replication of each of the two rcp45(HA) antigenic chimeras was 10-fold less in the upper respiratory tract of hamsters (Table 3)-than that of ⁇ cp45L, the recombinant parent virus bearing the three attenuating ts mutations in the L protein, but was the same as the rcp45L parent in the lower respiratory tract.
  • the serum antibody response to HPIV3 was evaluated by hemagglutination-inhibition (HAI) assay as previously described (van Wyke Coelingh et al., Virology 143:569-582, 1985, inco ⁇ orated herein by reference), and the serum antibody response to measles virus was evaluated by 60% plaque-reduction assay as previously described (Coates et al., Am. J. Epidemiol. 81:299-313, 1966, inco ⁇ orated herein by reference). These results were compared with that from an additional control group of cotton rats that received 10 5 ° of the live-attenuated measles virus (Moraten strain) administered intramuscularly on day 0.
  • HAI hemagglutination-inhibition
  • the level of measles virus-neutralizing serum antibodies induced by the rPIV3(HA) recombinants were on average 5 -fold greater than that achieved by the intramuscular immunization with the live attenuated measles virus vaccine.
  • the serum antibody response to HPIV3 produced by all the chimeric viruses was also robust and comparable to that produced by infection with wild type rJS.
  • Serum antibody titer to measles virus (60% Serum antibody
  • rcp45L 3 18 ⁇ 3.3 ⁇ 0 ⁇ 3.3 ⁇ 0 ⁇ 2.0 ⁇ 0 10.7 ⁇ 0.2 rcp45L(HA P-M) 4 24 ⁇ 3.3 ⁇ 0 12.8 ⁇ 0.1 ⁇ 2.0 ⁇ 0 9.2 ⁇ 0.2 rcp45L(HA N-P) 5 6 ⁇ 3.3 ⁇ 0 13.4 ⁇ 0.4 ⁇ 2.0 ⁇ 0 10.8 ⁇ 0.3 rPIV3(HA P-M) 6 6 ⁇ 3.3 ⁇ 0 13.3 ⁇ 0.3 ⁇ 2.0 ⁇ 0 10.3 ⁇ 0.2
  • Measles virus 4 ⁇ 3.3 ⁇ 0 10.8 ⁇ 0.2 ⁇ 2.0 ⁇ 0 ⁇ 2.0 ⁇ 0 (Moraten) 7 rJS* ⁇ 3.3 ⁇ 0 ⁇ 3.3 ⁇ 0 ⁇ 2.0 ⁇ 0 10.7 ⁇ 0.2
  • Virus was administered at a dose of 10 ⁇ - ⁇ PFU in a 0.1 ml inoculum intranasally on day 0 to all animals with the exception of those in the measles virus group which received virus by intramuscular injection.
  • rPIV3-l is a recombinant chimeric HPIV3 in which the HN and F genes have been replaced by those of HPIVl (see, e.g., Skiadopoulos et al., Vaccine 18:503-510, 1999; Tao et al., Vaccine 17:1100-1108, 1999; U.S. Patent Application Serial No. 09/083,793, filed May 22, 1998; U.S. Patent Application Serial No. 09/458,813, filed Pecember 10, 1999; U.S. Patent Application Serial No. 09/459,062, filed Pecember 10, 1999, each inco ⁇ orated herein by reference).
  • the HN gene of HPIV2 was inserted into the rPIV3-l chimeric virus that served as a vector to produce a chimeric derivative virus, bearing an introduced heterologous antigenic determinant from HPIV2, able to protect against both HPIVl and HPIV2.
  • the HPIV2 HN gene also was inserted into an attenuated derivative of rPIV3-l, designated rPIV3-lc/?45, which contains 12 of the 15 cp45 mutations, i.e., those mutations on genes other than HN and F, inserted into the rPIV3 backbone
  • HPIV2 wild type virus The source of the HPIV2 wild type virus was the wild type strain V9412-6 (designated PIV2/V94) (Tao et al., Vaccine T.1100-1108, 1999), which was isolated in Vero cells from a nasal wash that was obtained in 1994 from a child with a natural HPIV2 infection.
  • PIV2/V94 was plaque purified 3 times on Vero cells before being amplified twice on Vero cells using OptiMEM tissue culture medium without FBS.
  • a cPNA clone of the HN gene of PIV2/V94 was generated from virion RNA by reverse transcription (RT) using random hexamers and Superscript Preamplification System (Life Technologies) followed by PCR using Advantage cPNA Synthesis kit (Clontech, Palo Alto, CA) and synthetic primers which introduced Ncol-Hindl ⁇ l sites flanking the HN cPNA ( Figure 3 A).
  • the sequences of these primers were: (with HPIV specific sequences in upper case, restriction sites underlined, nts which are non-HPIV or which are altered from wt in lower case, and start and stop codons in bold), upstream HPIV2 HN 5'- gggccATGGAAGATTAC AGC AAT-3 ' (SEQ IP NO. 19); downstream HPIV2 HN 5'- caataagcTT AAAGC ATT AGTTCCC-3 ' (SEQ IP NO. 20).
  • the HN PCR fragment was digested with Ncol-Hindlll and cloned into pLit.PIV3 lHNhc to generate pLit.32HNhc ( Figure 3 B).
  • HPIV2 HN heterologous gene insert in pLit.32HNhc was completely sequenced using the ThermoSequenase Kit and P-labeled terminators (Pharmacia Amersham, Piscataway, NJ) and was confirmed to contain the authentic sequence of the PIV2/94 HN coding region.
  • HPIV2 HN gene in pLit.32HNhc was further modified by PCR and Peep Vent thermostable PNA polymerase (New England Biolab, Beverly, MA) to introduce P wMI sites for cloning into the unique EpwMI site in p38' ⁇ PIV3 lhc, Figure 3C
  • the modified cPNA bearing the HPIV2 HN ORF consists of (from left to right) a partial 5 '-untranslated region (5'-UTR) of HPIV3 HN including the PpwMI site at the 5 '-end, the HPIV2 HN ORF, the 3'- UTR of HPIV3 HN, a complete set of HPIV3 transcription signals (i.e.
  • pFLC.2G+.hc and pFLCcp45 are full-length antigenomic clones encoding wt rPIV3-l and rPIV3cp45, respectively, as described previously (Skiadopoulos et al., J. Virol. 21:1374-81, 1999; Tao et al., J. Virol. 72:2955-2961, 1998, each inco ⁇ orated herein by reference).
  • Confluent HEp-2 cells were transfected with pFLC.31hc.2HN or pFLC.3- lhc.cp45.2HN plus the pTM(N), pTM(P no C), and pTM(L) support plasmids in the presence of MVA-T7 as previously described (Purbin et al., Virology 235:323-332, 1997, inco ⁇ orated herein by reference).
  • the recombinant chimeric viruses recovered from transfection were activated by addition of TPCK trypsin (Catalog No.
  • RNA from each recovered recombinant chimeric virus was amplified on LLC-MK2 cells and concentrated by polyethylene glycol (PEG) precipitation (Mbiguino et al, J. Virol. Methods 11:161-170, 1991, inco ⁇ orated herein by reference).
  • Virion RNA (vRNA) was extracted with Trizol (Life Technologies) and used as template to synthesize first strand cPNA using Superscript Preamplification system (Life Technologies, Gaithersburg, MP) and random hexamer primers as described above.
  • the synthesized cPNA was amplified by PCR with the Advantage cPNA Synthesis kit (Clontech, Palo Alto, CA) with primers specific for HPIVl F and HPIVl HN coding region (for HPIVl F 5'-AGTGGCTAATTGCATTGCATCCACAT-3' (SEQ IP NO. 23) and for HPIVl HN 5'-GCCGTCTGCATGGTGAATAGCAAT-3') (SEQ IP NO. 24).
  • the relative locations of the PIVl F and HN primers are indicated by arrows in Figures 3 and 4. Amplified PNA fragments were digested and analyzed on agarose gels ( Figure 4).
  • Pata for rPIV3-lcp45.2HN is not shown, but was comparable and confirmed in structure.
  • rPIV3- 1.2HN and rPIV3-lcp45.2HN each contained the insert of the expected size, and the digestion patterns with a number of restriction enzymes confirmed the identity and authenticity of the inserts. The presence of the cp45 mutations in rPIV3-lcp45.2HN was also confirmed.
  • Cells were then fed with 1 ml of methionine and cysteine deficient PMEM supplemented with 120 ⁇ Ci of ProMix 35S-methionine and 35S-cysteine mixture (Pharmacia Amersham, Piscataway, NJ) and incubated for 18 hours at 32°C. Cells were scraped into medium, pelleted by brief centrifugation in a microfuge, and washed three times with cold PBS.
  • methionine and cysteine deficient PMEM supplemented with 120 ⁇ Ci of ProMix 35S-methionine and 35S-cysteine mixture (Pharmacia Amersham, Piscataway, NJ) and incubated for 18 hours at 32°C.
  • Cells were scraped into medium, pelleted by brief centrifugation in a microfuge, and washed three times with cold PBS.
  • Each cell pellet was resuspended in 1 ml RIPA buffer (1% sodium deoxycholate, 1% Triton X-100, 0.2% SPS, 150 mM NaCl, and 50 mM Tris-HCl, pH7.4) containing 250 units/ml of Benzonase (Sigma), freeze/thawed once, and clarified by centrifugation at 12,000 X g for 5 min in a microfuge. Clarified supematants were transferred to a clean microfuge tube, mixed with 50 ⁇ l of anti- HPIV2 HN monoclonal antibody (mAb) 150S1 (Tsurudome et al., Virology 171 :38-48.
  • mAb anti- HPIV2 HN monoclonal antibody
  • the level of temperature sensitivity of replication of rPIV3-1.2HN and rPIV3-l.cp45.2HN in LLC-MK2 cells was evaluated to determine if the acquisition of the HN ORF of HPIV2 by rPIV3-l wild type or attenuated viruses employed as vectors altered the level of temperature sensitivity of replication in the resultant chimeric derivatives bearing the heterologous antigenic determinant of HPIV2 compared to the parental, vector viruses (Table 6).
  • rPIV3-1.2HN and rPIV3-lcp45.2HN were serially diluted l :10 in lX L15 supplemented with 0.5 ⁇ g/ml TPCK trypsin and used to infect LLC- MK2 monolayers in 96 well plates in quadruplicate. Infected plates were placed at various temperatures for 7 days before the virus titers were determined by hemadso ⁇ tion using 0.2% guinea pig erythrocytes (in IX PBS). The virus titers are presented as log t oTCIDso ⁇ standard error (S.E.).
  • rPIV3-1.2HN and rPIV3-lcp45.2HN exhibited a level of temperature sensitivity similar to that of their parental, vector viruses, i.e. rPIV3-l and rPIV3-lcp45, respectively, each of which lacks the HPIV2 HN insert. This indicated that the introduction of one extra transcription translation unit in rPIV3-1.2HN and rPIV3- lcp45.2HN, does not significantly alter their level of temperature sensitivity of replication in vitro.
  • the rPIV3-l viruses carrying the PIV2 HN insertion have a temperature sensitive phenotype similar to that of their parental virus.
  • rPIV3-l expressing the PIV2 HN gene termed rPIV2-1.2HN
  • rPIV2-1.2HN is more restricted in replication than its rPIV3-l parent as indicated by a 30-fold reduction in virus titer in both the upper and lower respiratory tracts of hamsters.
  • the insertion of a transcription translation unit expressing the PIV2 HN protein into rPIV3-l attenuates the virus for hamsters.
  • the attenuating effect of insertion of a transcription/translation unit containing PIV2 HN ORF into rPIV3-l was slightly more than that observed for the insertion of a similar unit containing the measles HA ORF into the recombinant JS strain of wild type PIV3.
  • the rPIV3-lcp45.2HN virus was 1,000-fold more restricted in replication than the rPIV3-lcp45 parent indicating that the attenuating effect of the PIV2 HN insertion and the cp45 mutations are additive. It should be possible to adjust the level of attenuation as needed by adding fewer cp45 mutations than the 12 that are present in rPIV3-l.cp45.2HN.
  • Immunizing virus Serum neutralizing antibody titer against indicated Titer of challenge virus in indicated tissues virus (reciprocal mean log 2 ⁇ SE) b (log 10 TCID 50 /g ⁇ SE) c
  • a Hamsters in groups of 12 were immunized with 10 5 3 TCID 50 of indicated virus intranasally on day 0.
  • the serum neutralizing antibody titer was determined on LLC-MK2, and t titers are expressed as reciprocal mean log 2 ⁇ standard error (SE).
  • c Half of the hamsters from each immunized group were challenged with 10 6 TCID 50 PIV2 on day 29, and the remaining half were challenged with 10 6 TCID 50 PIVl on d 32.
  • Tissue samples were harvested 4 days after challenge, and challenge virus titers are expressed as log ⁇ 0 TCID 5 o/gram of tissue ⁇ SE.
  • NT nasal turbinates.
  • PIV3 provided no resistance against either PIVl or PIV2 (Tao, Vaccine 17: 1100-1108, 1999), while previous infection with PIV2 wild type virus and rPIV3-l induced complete resistance to replication of PIV2 and PIVl challenge viruses, respectively.
  • rPIV3-l .2HN induced antibody to both PIVl and PIV2 and included strong resistance to both PIVl and PIV2 as indicated by the 1,000- to 10,000-fold reduction in replication of each virus in the upper and lower respiratory tract of rPIV3-l .2HN immunized hamsters.
  • rPIV3-1.2HN carrying the cp45 mutations failed to induce significant resistance to replication of wild type PIVl or PIV2 challenge virus indicating that this particular recombinant chimeric virus is over-attenuated in hamsters.
  • Introduction of one or several selected cp45 mutations, rather than the complete set of 12 mutations, into rPIV3-1.2HN can be done to adjust the level of attenuation of rPIV3-1.2HN to an appropriate level.
  • the gene insert was relatively large (approximately 1900 nts). Further examples are provided herein that indicate the size of the insert specifies a selectable level of attenuation of the resulting recombinant virus.
  • Insertion mutations were constructed in a pUC based plasmid, pUCl 18-Stu, containing theXhol to Sphl fragment (HPIV3 nts 7437-11317) of the full length HPIV3 clone p3/7(131)2G-Stu.
  • Two separate plasmids were constructed as acceptor plasmids for insertion of GUs and HN gene 3 '-NCR extensions ( Figure 6). In each, a synthetic oligonucleotide duplex containing multiple cloning sites was inserted into the unique Stu I site.
  • the inserted sequence for the GU insertion plasmid contained a HN gene-end (GE) signal sequence, the conserved intergenic (IG) trinucleotide sequence, and a L gene-start (GS) signal sequence, cis-acting sequences that direct termination of the HN gene transcription and initiation of transcription of the inserted sequence, respectively ( Figure 6). Additional unique restriction endonuclease sites were included in the multiple cloning region to facilitate subsequent screening and subcloning.
  • the 3 '-NCR extension acceptor plasmid was similarly designed and constructed, but it lacked the cis-acting GE, IG, and GS sequences at its 5 '-end ( Figure 6B, Table 9).
  • the RSV antigenomic plasmid d53RSV sites or subgenomic plasmid pUCl 18FM2 were digested with the appropriate restriction enzymes, and fragments of the desired sizes were isolated by electrophoresis on agarose gels and ligated individually into the unique Hpal site of the GU or the HN gene 3 '-NCR extension acceptor plasmid ( Figure 6; Table 9). Clones were screened to identify ones in which the RSV restriction fragments were inserted in the reverse orientation, an orientation in which all reading frames contained multiple stop codons ( Figure 7).
  • the specific RSV sequences and size of the short synthetic oligonucleotides added are summarized in Table 9. Plasmid clones were sequenced through all restriction enzyme sites used for subcloning, and Xhol-Sphl fragments containing insertion mutations conforming to the rule of six, either as GUs or HN gene NCR extensions, were cloned into the full-length PIV3 cDNA plasmid p3/ 7(131)2G+.
  • Restriction Restriction sites ICR multiple cloning site (58 nt) + GU insertion NCR insertion (total fragment size and nt position in the RSV site (32 nt) + rule 6 rule of 6 (total nts inserted) nts inserted)
  • Source of RSV sequence is pUCl 18FM2, a plasmid containing a subgenomic cDNA fragment of RSV subgroup A as described previously (Juhasz, K. et al, J Virol., 71:5814-5819, 1997.).
  • Source of RSV sequence is D53sites, a plasmid containing the entire RSV subgroup A cDNA sequence with several introduced point mutations as described previously. The previously described D53sites plasmid was used to derive the rAsites virus descried in Whitehead, S. et al. J. Virol. 72:4467-4471, 1998.
  • D53sites a plasmid containing the entire RSV subgroup A cDNA sequence with several introduced point mutations as described previously. The previously described D53sites plasmid was used to derive the rAsites virus descried in Whitehead, S. et al. J. Virol. 72:4467-4471, 1998.
  • the gel purified 1356 nt fragment contained a 1 nt deletion compared to the predicted 1357 nt restriction endonuclease cleavage product, d.
  • the 1850 nt fragment is a product of two 3' to 3' adjoined 925 nt restriction fragments, e.
  • the following oligonucleotides were inserted into the Mlul restriction site to conform all the inserted foreign sequences to the rule of six: 13mer: CGCGGCAGGCCTG (SEQ ID NO. 25); 14mer: CGCGGCGAGGCCTG (SEQ ID NO. 26); 15mer: CGCGAGGCCTCCGCG (SEQ ID NO. 27); 16mer: CGCGCCGCGGAGGCCT (SEQ ID NO. 28); 17mer: CGCGCCCGCGGAGGCCT (SEQ ID NO. 29). nd, not done.
  • LipofectACE (Life Technologies, MD), and the monolayers were infected with MVA-T7 as described previously (Durbin et al., Virology 215:323-332, 1997; Skiadopoulos et al., J. Virol. 72:1762-8, 1998, each inco ⁇ orated herein by reference). After incubation at 32°C for 4 days, the transfection harvest was passaged onto LLC-MK2 cells in T-25 flasks which were incubated at 32°C for four to eight days.
  • the clarified medium supernatant was subjected to plaque purification on LLC-MK2 cells as described previously (Durbin et al., Virology 215:323-332, 1997; Hall et al., Virus Res. 22:173-184, 1992; Skiadopoulos et al., J. Virol. 72:1762-8, 1998, each inco ⁇ orated herein by reference).
  • Each biologically-cloned recombinant virus was amplified twice in LLC-MK2 cells at 32°C to produce virus for further characterization.
  • Virus was concentrated from clarified medium by polyethylene glycol precipitation (Mbiguino et al.. J. Virol.
  • RNA was extracted with Trizol Reagent (Life Technologies). Reverse transcription was performed on vRNA using the Superscript II Preamplification System (Life Technologies) with random hexamer primers.
  • the Advantage cDNA PCR kit (Clontech, C A) and sense (PIV3 nt 7108-7137) and antisense primers (PI V3 nt 10605-10576) were used to amplify fragments for restriction endonuclease digestion or sequence analysis.
  • the PCR fragments were analyzed by agarose gel electrophoresis ( Figure 8) and sequencing. Each of the recovered rPIV3 insertion mutants contained insertions of the indicated sizes and they were next evaluated for their biological properties.
  • the growth properties of the rPIV3 GU and NCR insertion mutants were compared to rPIV3 wt and rcp45L in vitro. As shown in Figure 9, the rate of replication and the peak virus titer of each of the rPIV3s containing either the GU or NCR insertions was indistinguishable from that of rPIV3 wt indicating that insertion of sequences of at least 3918 nts in length does not affect virus replication in vitro.
  • the insertion of the HA gene of measles virus into the rJS wildtype and the attenuated cp45L virus further attenuated each virus for hamsters. Since the HA gene of measles virus is 1936 nt in length, we examined the effect of a similar size gene insertion (1908 nt) on replication of rcp45L. The 1908 gene insertion differs from the measles virus HA gene insertion in that it cannot synthesize a large polypeptide.
  • GU insertions can have dual roles in the design of recombinant vaccines. The first role is to encode a protective antigen of a pathogen, and the second role is to confer an attenuation phenotype.
  • HN gene NCR insertions ranging in size from 258 nt up to 1404 nt did not significantly reduce viral replication in the respiratory tract of hamsters (Table 3).
  • viruses bearing HN gene NCR insertions of 1404 nts or greater yielded viruses that were slightly ts at 40°C with a gradient of temperature sensitivity proportional to the size of the insertion.
  • Addition of the 1908 nt GU insertion to the cp 5 ⁇ backbone yielded a virus that was almost 100-fold more ts at 38°C compared to rcp45 ⁇ _, demonstrating that the ts phenotype specified by the 1908 nt GU insertion and by the L gene ts mutations is additive.
  • Table 12 Efficiency of plaque formation of rPIV3 GU and NCR insertion mutants at permissive and non-permissive temperatures
  • Plaques were enumerated by immunoperoxidase staining after incubation for 6 days at the indicated temperature. Values which are underlined and in bold type represent the lowest restrictive temperature at which there was at least a 100-fold reduction of plaquing efficiency compared to the titer at 32°C, which is defined as the shut-off temperature of plaque formation.
  • rHPIV3 provides an effective vector for foreign viral protective antigens expressed as additional, supernumerary genes, as exemplified by the measles virus hemagglutinin (HA) glycoprotein gene.
  • HA hemagglutinin
  • the rHPIV3-l antigenic chimeric virus, a recombinant HPIV3 in which the PIV3 F and HN genes were replaced by their HPIVl counte ⁇ arts provides an effective vector the HPIV2 hemagglutinin-neuraminidase (HN) glycoprotein.
  • the foreign coding sequence was designed and constructed to be under the control of a set of HPIV3 gene start and gene end transcription signals, inserted into the vector genome as an additional, supernumerary gene, and expressed as a separate mRNA by the HPIV3 polymerase.
  • Expression of the measles virus HA or the HPIV2 HN glycoprotein from a supernumerary gene insert by the rHPIV3 or rHPIV3-l vector was determined to be stable over multiple rounds of replication.
  • Hamsters infected with the rHPIV3 vector expressing the measles virus HA or the rHPIV3-l vector expressing the HPIV2 HN glycoprotein induced a protective immune response to HPIV3 and measles virus, or to HPIVl and HPIV2, respectively.
  • a single rHPI V3 vector expressing the protective antigen of measles virus induced a protective immune response against two human pathogens, namely, HPIV3 via an immune response to the glycoproteins present in the vector backbone and measles virus via the HA protective antigen expressed from the extra gene inserted into rHPIV3.
  • the measles virus glycoprotein was not inco ⁇ orated into the infectious HPIV3 vector virus, and hence its expression would not be expected to alter the tropism of the vector nor render it susceptible to neutralization with measles virus-specific antibodies.
  • rHPIV3-l vector expressing the protective HN antigen of HPIV2 induced a protective immune response against two human pathogens, namely, HPIVl via an immune response to the glycoproteins present in the vector backbone and HPIV2 via the HN protective antigen expressed from the extra gene inserted into rHPIV3-l .
  • Modification of a single recombinant vaccine virus to induce immunity against multiple pathogens has several advantages. It is much more feasible and expeditious to develop a single attenuated backbone expressing antigens against multiple pathogens than it is to develop a separate attenuated vaccine against each pathogen.
  • Each pathogen offers different challenges for manipulation, attenuation and demonstration of safety and efficacy, and it would be a daunting task to attempt to develop an attenuated version of each of a series of pathogens. It is also simpler and easier to prepare, handle, and administer a single vaccine virus than to undertake these activities with several different attenuated viruses. Reducing the number of vaccine viruses also will help simplify the crowded schedule of pediatric immunizations.
  • viruses can be administered as a mixture, but this complicates vaccine development, since each component must be shown to be safe separately, and then shown to be safe and efficacious as a mixture.
  • One particular problem with the administration of mixtures of viruses is the common phenomenon of viral interference, in which one or more of the viruses in the mixture interferes with the replication of one or more of the other components. This may result in reduced replication and immunogenicity for one or more components. This common problem is obviated by the use of a single vector backbone.
  • viruses such as measles virus have particular safety concerns, it would be safer to use a single, comparatively benign virus such as PIV as a vector bearing multiple supernumerary antigens, as opposed to a mixture of separately-attenuated viruses, each of which must be developed and validated separately.
  • HPIVs are constructed and shown to serve as vectors for more than one supernumerary gene with satisfactory characteristics of replication and immunogenicity for development of vaccine viruses.
  • this example describes the design, construction, recovery, and characterization of rHPIV3s expressing one, two or three supernumerary genes from the following list: (i) the hemagglutinin-neuraminidase (HN) of HPIVl (Washington 20993/1964 strain); (ii) the HN of HPIV2 (V9412 strain); (iii) the hemagglutinin (HA) of the wild type Edmonston strain of measles virus; and (iv) a 3918-nt translationally-silent synthetic gene called gene unit (GU) (see above).
  • HN hemagglutinin-neuraminidase
  • HN hemagglutin-neuraminidase
  • V9412 strain the HN of HPIV2
  • HA hemagglutinin
  • GU 39
  • the added genes were inserted into rHPIV3 between the nucleoprotein (N) and phosphoprotein (P) genes, between the P and membrane protein (M) genes, or between the HN and large polymerase (L) genes.
  • N nucleoprotein
  • P phosphoprotein
  • M P and membrane protein
  • L large polymerase
  • HPIVl HN and HPIV2 HN genes between the N/P and P/M genes were performed as follows: Plasmid pUCl 19(4 711 N-P), a subclone of the HPIV3 antigenomic cDNA (Durbin, J. Virol. 74:6821-31, 2000, inco ⁇ orated herein by reference), was modified by site directed mutagenesis to insert a unique Aflll site into (i) the downstream noncoding region of the HPIV3 N gene (CTAAAT to CTTAAG. HPIV3 nts 1677-1682), or (ii) the downstream noncoding region of the HPIV3 P gene (TCAATC to CTTAAG. HPIV3 nts 3693-3698).
  • Each Aflll site was then modified by the insertion of an oligonucleotide duplex, creating the intermediate plasmids PUC(GE/GS-N-H) N - P and pUC(GE/GS-N-H)p.M, respectively.
  • the inserted duplex contained an HPIV3 gene-end (GE) sequence, the conserved intergenic (IG) trinucleotide sequence, and an HPIV3 gene-start (GS) sequence, which are cis-acting signals that direct transcriptional termination and initiation, respectively (Figure 10).
  • Additional unique restriction endonuclease sites were included in the multiple cloning region to facilitate subsequent subcloning and screening, including Ncol and Hwdlll sites for addition of the ⁇ PIV1 and ⁇ PIV2 H ⁇ ORFs.
  • a foreign ORF inserted into the multiple cloning site would be under the control of a set of HPIV3 transcription signals and expressed as a separate mR ⁇ A by the HPIV3 polymerase.
  • the multiple cloning site also contained an Mlu I site for inserting oligonucleotides of varying lengths as necessary to make the entire inserted sequence conform to the rule of six (Calain et al., J. Virol. 67:4822-30, 1993; Durbin et al., Virology 234:74-83. 1997b; 1999a Skiadopoulos et al., Virology 272:225-34. 2000).
  • HPIVl H ⁇ ORF available as an Ncol to Hwdlll restriction fragment of p38' ⁇ 31hc #6 (Tao et al., J. Virol. 22:2955-2961, 1998), was inserted into the Ncol to H dIII sites of P UC(GE/GS- ⁇ - ⁇ ) ⁇ - P and pUC(GE/GS-N- ⁇ ) P-M to generate pUC lHN N- p and pUC IHN P - M , respectively. Short oligonucleotide duplexes were inserted in the unique Mlul restriction site to adjust the sequence to conform to the rule of six.
  • HPIV2 HN ORF available within an Ncol to H dIII restriction fragment of p32 ⁇ nhc#3 31hc (Tao et al, J. Virol. 22:2955-2961, 1998, inco ⁇ orated herein by reference), was inserted into the Ncol to Hwdlll sites of pUC(GE/GS- ⁇ - ⁇ ) ⁇ - p and pUC(GE/GS-H-N)p -M to generate pUC 2HN N- p and pUC 2HN P-M , respectively. Short oligonucleotide duplexes were inserted in the unique Mlul restriction site to adjust the sequence to conform to the rule of six.
  • the inserted oligonucleotide was one nucleotide shorter that that required to specify that the genome of the recovered virus would conform to the rule of six. Therefore, all cDNAs bearing the HIV2 HN gene insertion did not conform to the rule of six. Nonetheless, virus was recovered from each of these cDNAs.
  • These chimeric subgenomic cDNAs were cloned into the full-length PIV3 antigenomic cDNA pFLC HPIV3 wt to yield pFLC PIV3 2HN ⁇ . P) and pFLC PIV3 2HN (P . M) , respectively ( Figure 11, plasmids from which the fourth and fifth recombinant viruses from the top were isolated).
  • HPIV3 antigenomic cDNAs were assembled that contained up to three supernumerary foreign genes in various combinations and locations in the HPIV3 backbone ( Figure 11). These antigenomic cDNAs were assembled from the subgenomic cDNAs described above in which the HN of HPIVl or HPIV2 was inserted between the N and P genes or the P and M genes. Other subclones used for assembly contained the measles virus HA gene between the P/M genes or HN/L genes as described above. Another subclone used in assembly contained the 3918-nt GU between the HN and L genes, as described above.
  • 1HN N - P 2HN P- MGUHN-L ( Figure 11, bottom), was approximately 23 kb in length. This is 50% longer than wild-type HPIV3, and longer than any previously described biologically derived or recombinant paramyxovirus.
  • HPIV3 antigenomic cDNAs bearing single or multiple supernumerary genes of heterologous paramyxovirus protective antigens were separately transfected into HEp-2 monolayer cultures on six-well plates (Costar, Cambridge, MA) together with the support plasmids pTM(N), pTM(P no C), and pTM(L) and LipofectACE (Life Technologies, Gaithersburg, MD) and the cells were simultaneously infected with
  • MVA-T7 a replication-defective vaccinia virus recombinant encoding the bacteriophage T7 polymerase protein using techniques previously described (Durbin et al., Virology 235:323- 332, 1997a; Skiadopoulos et al., Virology 272:225-34, 2000, each inco ⁇ orated herein by reference). After incubation at 32°C for up to four days, the transfection harvest was passaged onto LLC-MK2 monolayer cultures in a 25 cm 2 flask and the cells were incubated for 5 days at 32°C. The virus recovered from the cell supernatant was further passaged on LLC-MK2 cells at 32°C to amplify the virus.
  • rHPIV3s bearing single or multiple foreign gene inserts were biologically-cloned by plaque purification on LLC-MK2 cells as previously described (Skiadopoulos et al., J. Virol. 21:1374-81, 1999a, inco ⁇ orated herein by reference). Viral suspensions derived from biologically cloned virus were amplified on LLC-MK2 cells and yielded final titers of 10 and 10 TCID 0 /ml, similar to the range of titers typically obtained for wt rHPIV3. Recombinant viruses were assayed for their ability to grow at 39°C.
  • rHPIV3 1HN N-P rHPIV3 1HN N- P2HNP -M HAHN-L
  • rHPIV3 1HN N-P 2HN P-M 3918 GU HN - L were 100 to 1000-fold restricted for replication at 39°C compared to the replication at the permissive temperature.
  • Viral RNA (vRNA) was isolated from biologically cloned recombinant chimeric viruses as described above (see also, Skiadopoulos et al., J. Virol. 73:1374-81. 1999a, inco ⁇ orated herein by reference).
  • RT-PCR reverse transcription and polymerase chain reaction
  • rHPIV3 or rHPIV3-l viruses expressing one supernumerary viral protective antigen gene replicated efficiently in vitro and in vivo and induced protective immune responses against both the vector virus and the virus represented by the supernumerary antigen gene.
  • a rHPIV could accommodate two or more supernumerary genes and retain the ability to replicate efficiently in vitro and in vivo and to induce protective immune responses against both the vector and the expressed supernumerary antigens. The present example indicates that this is indeed possible.
  • HPIV2 HN gene between the HPIV3 N and P genes or between the P and M genes also exhibited a modest reduction (about 10 to 20-fold) in replication in the respiratory tract of hamsters (Table 13 groups 3 and 4).
  • insertion of the HPIVl HN gene between the P and M genes or between the N and P resulted in over approximately 100-fold reduction of replication in the upper and lower respiratory tract of hamsters (Table 13, groups 1 and 2). Since the HPIVl HN, HPIV2 HN, and measles virus HA gene insertions are all of approximately the same size (1794 nt, 1781 nt, and 1926 nt, respectively), this was unlikely to be due to insert length.
  • the greater level of attenuation conferred by the introduction the HPIVl HN gene likely is due to an additional attenuating effect that is specific to the expression of the HPIVl HN protein on replication of the HPIV3 vector.
  • a supernumerary antigen can attenuate rHPIV3 for hamsters above and beyond the modest attenuation due to inserting an additional gene.
  • the rHPIV3 chimeric recombinant viruses exhibited a gradient of attenuation that was a function of the number of supernumerary gene inserts.
  • the viruses bearing three added genes exhibited the greatest effect, and were reduced approximately 10,000 - 108,000 fold in replication in the upper and lower respiratory tract of the infected hamsters (Table 13, groups 9 and 10).
  • the rHPIV3 chimeric recombinant virus bearing two gene inserts exhibited an intermediate level of attenuation, and was reduced approximately 12-80 fold (Table 13, group 8).
  • rHPIV3 chimeric recombinant viruses bearing one supernumerary gene except those bearing the HPIVl HN gene) were reduced only approximately 10-25 fold (groups 3-7 in Table 13).
  • rHPIV3 chimeric recombinant viruses bearing one, two, or three supernumerary gene inserts replicated in all animals.
  • All rHPIV3 viruses elicited a strong immune response to the HPIV3 backbone with the exception of the viruses bearing the three supernumerary gene insertions.
  • the reduced or absent immune response in hamsters infected with either the rHPIV3 IHN N - P 2HN N - P HA HN - L or rHPIV3 IHN N - P 2HN N - P 3918GU HN - L was likely a result of these viruses being overly attenuated for replication in hamsters.
  • the immune response to the vectored antigens in the viruses bearing three foreign genes was also low or undetectable.
  • viruses bearing single or double foreign gene insertions induced an immune response against each of the additional antigens, demonstrating that the vectored foreign genes are immunogenic in hamsters, and as in the example of rHPI V3 IHN N - P 2HN N - P (Table 14; group 11) can be used to induce a strong immune response to three different viruses: HPIVl, HPIV2 and HPIV3.
  • Table 14
  • Mean antibody response in groups of hamsters (n 6) inoculated intranasally with 10° TCID 50 rHPIV3s expressing the hemagglutinin-neuraminidase protein of HPIVl (IHN), HPIV2 (2HN) or measles virus hemagglutination (HA) inserted between the N and P genes (N-P), the P and M genes (P-M) or the HN and L genes (HN-L) of rHPIV3.
  • c. Mean hemagglutination inhibiting antibody (HAI) titer to HPIV3.
  • d. Mean neutralizing antibody titer to HPIVl .
  • e. Mean HAI antibody titer to HPIV2.
  • f. Mean neutralizing antibody titer to measles virus (60% plaque reduction neutral
  • HPIV3 human parainfluenza virus type 3
  • ORF nucleoprotein
  • ⁇ HPIV3-N B cKa-N (Bailly et al., J. Virol. 74:3188-3195, 2000a, inco ⁇ orated herein by reference) and is referred to here as ⁇ HPIV3-N B .
  • rHPIV3-N ⁇ grew to a titer comparable to that of the rHPIV3 and BPIV3 parent viruses in LLC-MK2 monkey kidney and Madin Darby bovine kidney cells (Bailly et al., J. Virol. 74:3188-3195, 2000a).
  • the heterologous nature of the N protein did not impede replication of rHPIV3-N ⁇ in vitro.
  • ⁇ HPIV3-N B was restricted in replication in rhesus monkeys to a similar extent as its BPIV3 parent virus (Bailly et al., J. Virol. 74:3188- 3195, 2000a). This identified the BPIV3 N protein as a determinant of the host range restriction of replication of BPIV3 in primates.
  • the rHPIV3-N ⁇ chimeric virus thus combines the antigenic determinants of HPIV3 with the host range restriction and attenuation phenotype of BPIV3.
  • the host range restriction is anticipated to be based on numerous amino acid differences, it is anticipated that the attenuation phenotype of rHPIV3-N ⁇ will be stable genetically even following prolonged replication in vivo.
  • ⁇ HPIV3-N B Despite its restricted replication in rhesus monkeys, ⁇ HPIV3-N B induced a high level of resistance to challenge of the monkeys with wild type (wt) HPIV3, and this level of resistance was indistinguishable from that conferred by immunization with wt rHPI V3.
  • the infectivity, attenuation, and immunogenicity of ⁇ HPIV3-N B suggest that this novel chimeric virus is an excellent candidate as a HPIV3 vaccine (Bailly et al., J. Virol. 74:3188-3195, 2000a).
  • chimeric viruses are excellent candidates to serve as an attenuated vector for the expression of supernumerary protective antigens, such as the HA of measles virus.
  • supernumerary protective antigens such as the HA of measles virus.
  • the vector component of the resulting chimeric virus induces an immune response against HPIV3, and the added supernumerary genes induce immune responses against their respective heterologous pathogens.
  • a bivalent attenuated vaccine virus is made that simultaneously induces immune response to HPIV3 and measles virus.
  • rHPIV3 can be used as a vector for expression of the measles virus hemagglutinin (HA) protein.
  • HA hemagglutinin
  • ⁇ C P45 L HA(N-P) and rcp45 HA(HN-L) attenuated vectors expressing the measles virus HA gene possessed three attenuating amino acid point mutations in the vector backbone.
  • the rHPIV3-N ⁇ vector of the present invention will likely be even more stable than vectors having an attenuation phenotype based on three amino acid point mutations.
  • Inserts that affect replication in vitro or in vivo can be problematic for development of specific vaccines such as rHPIV3-N ⁇ .
  • a candidate virus that is highly restricted in replication in vitro would be difficult to manufacture, and one that is highly restricted in replication in vivo could be overattenuated and not useful as a vaccine.
  • rHPIV3-Ne chimeric virus expressing the measles virus HA glycoprotein designated rHPIV3-N ⁇ HA (P - M) , would be satisfactorily immunogenic in primates against both HPIV3 and measles virus since all previous studies with HPIV3 expressing HA were conducted in a rodent model.
  • the full length antigenomic cPNA plasmid pFLC HPIV3-N B HA( P - M ) ( Figure 12) was constructed in two steps. First, the previously-constructed pLeft-N ⁇ plasmid contains the 3' half of the HPIV3 antigenomic cPNA (HPIV3 nts 1-7437, the latter position being an Xhol site within the HN gene) with the HPIV3 N ORF replaced by that of BPIV3 (Bailly et al., J. Virol. 74:3188-3195, 2000a, inco ⁇ orated herein by reference). The PshAl- NgoMlY fragment was excised from this plasmid.
  • the PshAl site is at position 2147 in the HPIV3 antigenome sequence (see Figure 12) and the NgoMTV site occurs in the vector sequence, and so this removes all of the HPIV3 sequence downstream of the PshAl site.
  • This fragment was replaced by the PshAl-NgoMlW fragment from the previously- constructed plasmid pLeft HA (P - M ), which contains the measles virus HA ORF under the control of HPIV3 transcription signals and inserted between the HPIV3 N and P genes (Purbin, J. Virol. 24:6821-31, 2000, inco ⁇ orated herein by reference). This yielded pLeft- N ⁇ HAp -M .
  • rHPIV3-N ⁇ HAp -M was recovered from HEp-2 cells transfected with pFLC HPIV3-N B HA P - M .
  • pFLC HPIV3-N B HA P-M was transfected into HEp-2 cells on six-well plates (Costar, Cambridge, MA) together with the support plasmids pTM(N), pTM(P no C), and pTM(L) and LipofectACE (Life Technologies, Gaithersburg, MD), and the cells were simultaneously infected with MVA-T7, a replication-defective vaccinia virus recombinant encoding the bacteriophage T7 polymerase protein, as described above.
  • RT- PCR was performed using specific oligonucleotide primer pairs spanning the BPIV3 N ORF or the measles virus HA gene, and the amplified cDNAs were analyzed by restriction endonuclease digestion and partial DNA sequencing as described above. This confirmed the presence of the BPIV3 N ORF substitution and the measles virus HA supernumerary gene insert.
  • measles virus HA protein was initially confirmed by immunostaining plaques formed on LLC-MK2 monolayer cultures infected with rHPIV3- N B HA P - M using mouse monoclonal antibodies specific to the measles virus HA protein and goat anti-mouse peroxidase-conjugated antibodies, as described previously (Durbin, J. Virol. 24:6821-31, 2000, inco ⁇ orated herein by reference).
  • ⁇ HP ⁇ V3-N B HA P - M replicates to the same level as rHPIV3-N ⁇ in the respiratory tract of rhesus monkeys. It was next determined whether the acquisition of the measles virus HA insert significantly decreased the replication of rHPIV3-N ⁇ in the upper and lower respiratory tract, as was observed when a supernumerary gene was inserted into an attenuated HPIV3 backbone lacking a bovine chimeric component. It was also determined whether rHPIV3-N ⁇ HA P - M replicated sufficiently to induce an immune response against both HPIV3 and measles virus in vivo.
  • rHPIV3-N ⁇ HA P _ M The replication of rHPIV3-N ⁇ HA P _ M in the upper and lower respiratory tract of rhesus monkeys was compared to that of its rHPIV3-N ⁇ parent as well as wt HPIV3 and wt BPIV3 (Table 15).
  • Rhesus monkeys that were seronegative for both HPIV3 and measles virus were inoculated simultaneously by the intranasal (IN) and intratracheal (IT) routes with one milliliter per site of LI 5 medium containing 10 5 TCID 50 of virus suspension, as described previously (Bailly et al., J. Virol. 74:3188-3195, 2000a).
  • NP Nasopharyngeal
  • TL tracheal lavage
  • a chimeric human/bovine PIV3 containing the measles virus hemagglutinin gene is satisfactorily attenuated for replication in the upper and lower respiratory tract of rhesus monkeys, induces antibodies to both HPIV3 and measles virus, and protects against HPIV3 wild type virus challenge
  • NP swab Tracheal reciprocal log 2 mean reciprocal NP swab Tracheal SE) for HPIV3 log 2 ⁇ SE) (day 87 post lavage ⁇ S.E.) for log 2 ⁇ SE) lavage on day 56/59 h first immunization/
  • the present study included 4 monkeys that received rHP_V3-N B HA (P . M) and two monkeys in each of the groups that received rHPIV3 wt, rHPIV3-N B, orBPIV3 Ka. With the exception of the group that received rHPIV3-N B HA ( p. M) , the data presented includes historical data from studies reported in Bailey et al., J. Virol. 74:3188-3195, 2000, and Schmidt et al., J. Virol. 74:8922-8929, 2000. b.
  • the limit of detection of virus titer was 10 TCID 5 o/ml. d. In the present study sera were collected from monkeys on day 31 post immunization and animals were then challenged with HPIV3. In the two previous studies, monkeys were sampled and challenged on day 28 post immunization. e. Sera collected for the present study and from the two previous studies were assayed at the same time. Serum HAI titer is expressed as the mean reciprocal log 2 ⁇ standar error, SE.
  • ⁇ P* samples were collected on days 0, 2, 4, 6 and 8 post challenge.
  • the titers obtained for NP and TL samples on day 0 were ⁇ 2.0 log 10 TCID 50 ml.
  • rHPIV3-N ⁇ HA P - M and rHPIV3-N ⁇ conferred a comparable, high level of protection against challenge with wt HPIV3 as indicated by a 100 to 1000-fold reduction in wt HPIV3 replication in the respiratory tract of immunized monkeys.
  • rHPIV3-N B HA P - M Immunogenicity of rHPIV3-N B HA P - M was then compared with that of the licensed Moraten strain of live attenuated measles virus vaccine in rhesus monkeys, a species in which both PIV3 and measles virus replicate efficiently.
  • Rhesus monkeys previously infected with a rHPIV3 virus or with rHPIV3-N ⁇ HA P - M were immunized parenterally (IM) with 10 6 pfu of the Moraten strain of live-attenuated measles virus vaccine on day 59, and serum samples were taken on day 87 and analyzed for neutralizing antibodies against measles virus (Table 15).
  • rHPIV3-N ⁇ HAp. M as a vaccine for measles virus over the Moraten vaccine is that the PIV vector can be admimstered by the intranasal route, whereas live-attenuated measles virus vaccines are not consistently infectious by this route, probably as a consequence of their attenuation and adaptation to cell culture. This makes it possible to immunize with rHPIV3-N ⁇ HAp. M in early infancy, an age group that cannot be immunized with a current live attenuated measles virus vaccine such as the Moraten strain because of the neutralizing and immimosuppressive effects of maternal antibodies (Durbin, J. Virol.
  • the rHPIV3-N ⁇ HA (P - M) candidate vaccine offers a unique opportunity to immunize against two major causes of severe pediatric disease, namely, HPIV3 and measles virus.
  • HPIV3 and measles virus Unlike the currently licensed measles virus vaccines, we expect that chimeric rHPIV3-N ⁇ HA (P - M) and other human-bovine chimeric vector constructs, expressing the major antigenic determinant of measles virus or other heterologous pathogens, can be used to induce a strong immune response to, e.g., measles virus, in infants and children younger than six months of age (Durbin, J.
  • a recombinant chimeric human-bovine PIV was constructed in which the BPIV3 F and HN genes were replaced with those of HPIV3.
  • This recombinant chimeric bovine-human virus rB/HPIV3 was shown to be fully competent for replication in cell culture, whereas in rhesus monkeys it displayed the host range-restricted, attenuated phenotype characteristic of BPIV3 and was highly immunogenic and protective (U.S. Patent Application Serial No. 09/586,479, filed June 1, 2000 by Schmidt et al.; Schmidt et al., J. Virol. 74:8922-9, 2000, each inco ⁇ orated herein by reference).
  • restriction enzyme recognition sites were introduced to facilitate the insertion of foreign, supernumerary genes into the genome of the chimeric B/HPIV3 virus genome.
  • the sites were designed so that they did not disrupt any of the BPIV3 replication and transcription cis-acting elements. This specific example will describe insertion into the Blpl site ( Figure 13).
  • RSV subgroup A glycoprotein genes G and F were modified for insertion into the promoter-proximal Blpl site of B/HPIV3 ( Figure 13).
  • the strategy was to express each heterologous ORF as an additional, separate mR ⁇ A, and hence it was important that it be introduced into the rB/HPIV3 genome so that it was preceded by a BPIV3 gene start signal and followed by a BPIV3 gene end signal.
  • the Blpl insertion site followed the gene start signal of the ⁇ gene ( Figure 13).
  • the RSV ORF needed to be modified by insertion of a Blpl site at its upstream end and addition of a BPIV3 gene end signal, intergenic region, gene start signal, and Blpl site at its downstream end.
  • the forward PCR primer used was (5 ' to 3 ')
  • the reverse primer was (5' to 3') AAAAAGCTAAGCGCTAGCCTTTAATCCTAAGTTTTTCTTACTTTTTTTACTACTG GC GTGGTGTGTTGGGTGGAGATGAAGGTTGTGATGGG (SEQ ID NO. 3 ⁇ )(Blp I site underlined, ORF translational initiation and termination triplets in bold).
  • the forward PCR primer used was (5' to 3')
  • AAAGGCCTGCTTAGCAAAAAGCTAGCACAATGGAGTTGCTAATCC TCAAAGCAAAT GCAATTACC (SEQ ID NO. 32), and the reverse primer was (5' to 3') AAAAGCTAAGCGCTAGCTTCTTTAATCCTAAGTTTTTCTTACTTTTATTAGTTACT AAATGCAATATTATTTATACCACTCAGTTGATC (SEQ ID NO. 33)(Blp I site underlined, ORF translational initiation and termination triplets in bold).
  • the PCR products were digested with Blpl and cloned into the modified full length cDNA clone using standard molecular cloning techniques.
  • the resulting full length cDNA containing the RSV A G ORF was designated pB/HPIV3-G A l and the plasmid containing the F ORF was designated PB/HPIV3-F A 1.
  • the nucleotide sequence of each inserted gene was confirmed by restriction enzyme digestion and automated sequencing. All constructs were designed so that the final genome nucleotide length was a multiple of six, which has been shown to be a requirement for efficient RNA replication (Calain et al., J. Virol. 67:4822-30, 1993, inco ⁇ orated herein by reference).
  • rB/HPIV3-Gl and rB/HPIV3-Fl viruses were recovered from the cDNAs PB/HPIV3-G A 1 and pB/HPIV3-F A l, respectively. This was accomplished by the previously-described method in which HEp-2 cells were transfected with the respective antigenomic cDNA together with BPIV3 N, P and L support plasmids. The cells were simultaneously infected with a recombinant vaccinia virus, strain MVA, expressing the T7 RNA polymerase gene. The recovered recombinant viruses were cloned biologically by sequential terminal dilution in Vero cells.
  • rB/HPIV3-Gl and rB HPIV3-Fl viruses replicate efficiently in cell culture.
  • the multicycle growth kinetics of rB/HPIV3-Gl and rB/HPIV3-Fl in LLC- MK2 cells were determined by infecting LLC-MK2 cell monolayers in triplicate at a multiplicity of infection (MOI) of 0.01 and harvesting samples at 24-hour intervals over a seven day period, as previously described (Bailly et al., J. Virol. 74:3188-3195, 2000a, inco ⁇ orated herein by reference). These two viruses were compared with BPIV3 Ka,
  • HPIV3 JS, rBIV3 Ka, and rB/HPIV3 ( Figure 14).
  • the final titer achieved for each of the six viruses were similar with one exception: rB/HPIV3-Fl was approximately 8-fold reduced in its replicative capacity compared to the other viruses ( Figure 14). This might be an effect of having this large gene in a promoter-proximal position, or might be an effect of the expression of a second fusogenic protein, or both.
  • rB/HPIV3-Fl induced massive syncytia, comparable to what is observed with wild type RSV infection and greater than that observed with rB/HPIV3 or the other parental viruses.
  • rB/HPIV3-Gl induced less cytopathic effect and few syncytia in LLC-MK2 cells, comparable to rB/HPIV3.
  • rB/HPIV3-Fl and rB/HPIV3-Gl grew to a final titer of at least 10 7 TCID 50 /ml in LLC-MK2 cells and in Vero cells. This indicates that each virus is fully-permissive for growth which will allow cost-efficient vaccine manufacture.
  • the rB HPIV3-Gl and rB/HPIV3-Fl viruses replicate efficiently in the respiratory tract of hamsters
  • rB/HPI V3 -G 1 and rB/HPI V3 -F 1 were evaluated for their ability to replicate in the upper and lower respiratory tract of hamsters.
  • Each virus was administered intranasally at a dose of 10 6 TCID 50 , and one group received both rB/HPIV3-Gl and rB/HPIV3-Fl . Animals from each group were sacrificed on days 4 and 5 post infection, and the virus titer in the nasal turbinates and lungs were determined by serial dilution.
  • the titer of virus from the mixed infection of rB/HPIV3-Gl and rB/HPIV3-Fl appeared to be somewhat reduced in the lower respiratory tract on day 4, but this was not statistically significant.
  • Replication of one of the control viruses, BPIV3 Ka was somewhat reduced in the lower respiratory tract on day 5: this also was not statistically significant, and indicates that these small differences likely are not important.
  • the rB/HPIV3-Gl and rB/HPIV3-Fl viruses appeared to be fully competent for replication in vivo, despite the presence of the 0.9 kb G or 1.8 kb F supernumerary gene next to the promoter.
  • Table 16 rB/HPIV3 bearing the RSV G or F ORF as a supernumerary gene in the promoter proximal position replicates efficiently in the respiratory tract of hamsters.
  • HPIV3 JS wild type 6 6.6 ⁇ 0.1 (A) 6.5 ⁇ 0.1 (A) 6.0 ⁇ 0.2 (AB) 6.0 ⁇ 0.4 (A)
  • the limit of detectability of virus was 10 245 TCID 50 /g tissue.
  • S.E. standard error.
  • c Mean virus titers were assigned to similar groups (A, B, C, D) by the Tukey-Kramer test. Within each column, mean titers with different letters are statistically different (p ⁇ 0.05). Titers indicated with two letters are not significantly different from those indicated with either letter.
  • the rB/HPIV3-Gl and rB/HPIV3-Fl viruses induce serum antibodies to both HPIV3 and RSV
  • each of the viruses induced a titer of PIV3-specific antibody that was indistinguishable from that of their parent virus rB/HPIV3 (Table 18).
  • the rB/HPIV3 vector bearing the F or G gene of RSV induced strong immune responses against both the RSV insert and the PIV vector.
  • Immunization of hamsters with rB/HPIV3 expressing the RSV G or F ORF as a supernumerary gene induces an antibody response against the RSV G or F protein.
  • Titers in the pre serum specimen represent non-specific background levels of antibody in this sensitive ELISA.
  • Immunization of hamsters with rB/HPIV3s expressing the RSV G or F ORF induces neutralizing antibodies against RSV as well as hemagglutination-inhibiting (HAI) antibodies against HPIV3.
  • HAI hemagglutination-inhibiting
  • the rB/HPIV3-Gl and rB/HPIV3-Fl viruses induce resistance to replication of HPIV3 and RSV challenge virus.
  • rB/HPIV3-Gl Animals that received rB/HPIV3-Gl, or rB/HPIV3-Fl, or both viruses, exhibited a high level of resistance to replication of the RSV challenge virus.
  • the level of protective efficacy of the rB/HPIV3-Fl virus against the RSV challenge appeared to be marginally less than that of the rB/HPIV3-Gl virus or of the RSV control. However, this difference was not significantly different.
  • the rB/HPIV3 vector bearing either the F or G gene of RSV induced a level of protective efficacy that was comparable to that of complete infectious RSV.
  • Groups of 6 hamsters were inoculated intranasally with 10 6 TCID 50 of the indicated PIV3 or 10 6 PFU of RSV in a 0.1 ml inoculum.
  • HPIV3 titrations were performed on LLC-MK2 cells. The limit of detectability of virus was 10 1 7 TCID 50 /g tissue.
  • Quantitation of RSV was determined by plaque numeration on HEp-2 cells. The limit of detectability of virus was 10 l 7 PFU/g tissue.
  • d Mean virus titers were assigned to similar groups (A, B, C) by the Tukey-Kramer test. Within each column, mean titers with different letters are statistically different
  • the chimeric rHPIV3-l virus which has a HPIV3 backbone in which the HPIV3 HN and F genes have been replaced by their HPIVl counte ⁇ arts, serves as a useful vector for the HPIV2 HN protein as a supernumerary gene.
  • This chimeric vector, rHPIV3- 1.2HN is demonstrated herein to induce resistance to replication of both HPIVl and HPIV2 in hamsters.
  • the rHPIV3-1.2HN recombinant virus contains elements from each of the three serotypes of HPIV that cause significant disease: the internal genes of serotype 3 combined with the HN and F glycoprotein genes of serotype 1 , and the HN protective antigen of serotype 2 as a supernumerary gene.
  • the present example provides yet another approach to deriving a PIV-based vector vaccine to protect against both PIVl and PIV2.
  • the rB/HPIV3 was modified by the substitution of the human PIV3 HN and F proteins by those of HPIVl .
  • This virus designated rB/HPI V3.1, contains the PIVl HN and F glycoproteins as part of the vector backbone, intended to induce neutralizing antibodies and immunity to HPIVl .
  • This virus was used in the present example as a vector to express the HN and F proteins of HPIV2 singly or together as supernumerary gene(s).
  • Three viruses were recovered and shown to be fully viable: rB/HPIV3.1 -2F; rB/HPIV3.1 -2HN; or rB/HPIV3.1 -2F,2HN, and each expressed the PIV2 F and/or HN gene as a supernumerary gene or genes.
  • rB/HPIV3.1-2F,2HN which expresses both the PIV2 F and/or HN proteins from two supernumerary genes and the PIVl F and HN genes from the vector backbone, thus expresses both major protective antigens, i.e., the F and HN of glycoproteins, of PIVl and PIV2 from a single virus.
  • This approach optimizes the vaccine's protective efficacy and minimizes manufacturing costs since it accomplishes this increased immunogenicity using only one virus. It also likely will be simpler, safer and more effective to immunize infants and children with a single multivalent virus compared to a mixture of several viruses.
  • a full length cDNA of the BPIV3 Kansas strain in which the F and HN glycoprotein genes of the bovine .virus had been replaced with the corresponding genes of the HPIV3 JS strain (rB/HPIV3) was constructed as previously described (Schmidt et al., L Virol. 74:8922-9, 2000, inco ⁇ orated herein by reference).
  • This cDNA was modified to contain three additional unique restriction enzyme recognition sites (Figure 15). Specifically, a Blpl site was introduced preceding the N ORF (nucleotide (nt) 103-109), an Ascl site (nt 1676-83) was introduced preceding the N gene end sequence and a Notl site (nt 3674-3681) was introduced preceding the P gene end sequence.
  • HPIV3 transcription signals was modified by PCR mutagenesis to create a SgrAl restriction enzyme recognition site preceding the F gene and a BsiWl site preceding the HN gene end sequence, analogous to the position of the SgrAl and BsiWl sites that had been introduced previously into rB/HPIV3 (Schmidt et al., J. Virol. 74:8922-9, 2000).
  • the mutagenic forward primer used to create the SgrAl site was (5' to 3')
  • TGC (SEQ ID NO. 35) (BsiWl site underlined).
  • the SgrAl and BsiWl sites were used to replace, as a single DNA fragment, the HPIV3 F and HN genes in rB/HPIV3 with the HPIVl F and HN genes from the modified 3.1hcR6 plasmid. This yielded the full length antigenomic cDNA pB/HPIV3.1, consisting of HPIVl F and HN open reading frames under the control of HPIV3 transcription signals in a background that is derived from BPIV3.
  • the HPIV2 F ORF needed to be modified by insertion of a Notl site and addition of a BPIV3 gene end signal, intergenic region and gene start signal at its upstream end, and a Notl site at its downstream end.
  • the forward PCR primer used was (5' to 3') AAAATATAGCGGCCGCAAGTAAGAAAAACTTAGGATTAAAGGCGGATGGATCA CCTGCATCCAATGATAGTATGCATTTTTGTTATGTACACTGG (SEQ ID NO.
  • the reverse primer was (5' to 3') AAAATATAGCGGCCGCTTTTACTAAGATATCCCATATATGTTTCCATGATTGTTC TTGGAAAAGACGGCAGG (SEQ ID NO. 37) (Notl site underlined, ORF translational initiation and termination triplets in bold).
  • the reverse primer was (5' to 3') GGAAAGGCGCGCCAAAGTAAGAAAAACTTAGGATTAAAGGCGGA
  • PCR products were digested with Notl (HPIV2 F insert) or Ascl (HPI V2
  • the genome nucleotide length of the recovered chimeric viruses is as follows: pB/HPIV3.1 : 15492; pB/HPIV3.1 -2HN: 17250; pB/HPIV3.1-2F: 17190; pB/HPIV3.1-2HN,2F: 18948.
  • rB/HPIV3.1 , rB/HPIV3.1 -2F, rB/HPIV3.1 -2HN, and rB/HPIV3.1 -2F,2HN chimeric viruses were recovered from the cDNAs pB/HPIV3.1, pB/HPIV3.1-2F, pB/HPIV3.1-2HN, and pB/HPIV3.1-2F,2HN, respectively. This was accomplished by the previously-described method in which HEp-2 cells were transfected with the respective antigenomic cDNA together with BPIV3 N, P and L support plasmids. The cells were simultaneously infected with a recombinant vaccinia virus, strain MVA, expressing the T7 RNA polymerase gene.
  • Porcine trypsin was added to the cell culture medium to activate the HPIVl F protein, as previously described (Tao et al., J. Virol. 72:2955-2961, 1998).
  • the recovered recombinant viruses were cloned biologically by sequential terminal dilution in Vero cells. All of the recombinant viruses replicated efficiently, induced CPE in Vero cells within 5 days and rendered the cell monolayer positive for hemadso ⁇ tion.
  • the presence of the inserted HPIV2 F and HN gene in the backbone of each recovered recombinant virus was confirmed by RT-PCR of viral RNA isolated from infected cells followed by restriction enzyme digestion and DNA sequencing. The sequence of the inserted gene and flanking regions in the recovered recombinant viruses was identical to that of the starting antigenomic cDNA.
  • the chimeric rHPIV3-l virus which has a HPIV3 backbone in which the HPIV3 HN and F genes have been replaced by their HPIVl counte ⁇ arts, was shown above to serve as a useful vector for the HPIV2 HN protein as a supernumerary gene.
  • This chimeric vector, rHPIV3-l .2HN was able to induce resistance to replication of both HPIVl and HPIV2 in hamsters. This finding illustrates the siuprising flexibility of the PIV expression system.
  • this particular virus contained elements from each of the three serotypes of HPIV: the internal genes of serotype 3 combined with the HN and F glycoprotein genes of serotype 1, and the HN protective antigen of serotype 2 as a supernumerary gene.
  • a further derivative, rHPIV3-1.2HNcp45 L was also made that contained attenuating mutations from the cp45 HPIV3 vaccine candidate.
  • a PIV vector can be represented as comprising three components: the internal vector backbone genes, which can contain attenuating mutations as desired; the vector glycoprotein genes, which can be of the same or of a heterologous serotype; and one or more supernumerary genes encoding protective antigens for additional pathogens. In most cases, these supernumerary antigens are not inco ⁇ orated into the virion and hence do not change the neutralization or tropism characteristics of the virus.
  • each PIV vector is a bivalent or multivalent vaccine in which the vector itself induces immunity against an important human pathogen and each supernumerary antigen induces immunity against an additional pathogen.
  • the flexibility of the PIV vector system is further demonstrated by using the rHPIV3-l virus, as well as its attenuated rHPIV3-lcp45 L derivative, as vectors to express measles virus HA as a supernumerary gene.
  • This provides a new bivalent vaccine candidate for HPIVl and measles virus.
  • measles virus HA can be vectored by rHPIV3 and attenuated derivatives thereof, bearing the serotype 3 antigenic determinants, or by rHPIV3-l and attenuated derivatives thereof, bearing the serotype 1 antigenic determinants.
  • the present example details the use of the techniques of reverse genetics to develop a live-attenuated HPIVl candidate vaccine, rPIV3-l HAp. M cp45 L , expressing as a supernumerary gene the major measles virus protective antigen, the HA glycoprotein (Durbin, J. Virol. 74:6821-31, 2000, inco ⁇ orated herein by reference), for use in infants and young children to induce an immune response against both measles virus and HPIVl .
  • rHPIV3 HA P - M CP45 candidate vaccine (bearing the serotype 3 antigenic determinants) was followed by the rHPIV3-l HAp. M cp45L candidate vaccine (bearing the serotype 1 antigenic determinants).
  • Hamsters immunized with these viruses developed antibodies to the HPIV3 and HPIVl antigens present in the backbone of the vectors and also maintained high titers of antibodies to the vectored antigen, the measles virus HA expressed as a supernumerary antigen from both the HPIV3 and HPIVl candidate vaccine viruses. Construction of rHPIV3-l HA(p. ) and rHPIV3-l HA(p.M) C/>45L. wild type and attenuated versions of rHPIV3-l expressing measles virus HA as a supernumerary gene.
  • pFLC HPIV3-1 HA was constructed using the above-described pFLC HPIV3 HA (P _ M) in which the wild type measles virus Edmonston strain HA gene ORF was inserted as a supernumerary gene between the P and M genes of rHPI V3.
  • pFLC HPIV3 HA was digested with BspEl to Sphl and the cDNA fragment lacking the 6487 bp BspEl to Sphl sequence was isolated.
  • pFLC 2G+.hc a full-length antigenomic cDNA plasmid bearing the F and HN ORFs of PIVl in place of those of HPIV3 (Tao et al., J. Virol. 72:2955-2961, 1998) was digested with BspEl and Sphl, and the 6541 bp fragment (plasmid nts 4830-11371) containing the HPIVl glycoprotein genes in the HPIV3 backbone was inserted into the BspEl to Sphl window of pFLC HPIV3 HA P-M to give pFLC HPIV3-1 HA P . M ( Figure 15).
  • the cp45 L mutations present in the L gene ORF are the major ts and att determinants of the HPIV3 cp45 candidate vaccine (Skiadopoulos et al., J. Virol. 72:1762-8, 1998) and were previously shown to confer attenuation of replication to the rHPIV3-l cp45y ⁇ in the respiratory tract of hamsters (Tao et al., Vaccine 17:1100-8, 1999).
  • the pFLC HPIV3-1 HA P-M was then modified to encode these three ts mutations to yield pFLC HPIV3-1 HA P - M cp45_.
  • Figure 16 This was accomplished by inserting the Sphl to NgoMIV restriction endonuclease fragment of pFLC HPIV3 cp45 (plasmid nts 11317 -15929) (Skiadopoulos et al., J. Virol. 72:1762-8, 1998) into the Sphl to NgoMIV window of pFLC HPIV3-1 HA P-M .
  • pFLC HPIV3-1 HA (P-M ) or pFLC HPIV3-1 HA (P . M) cp45 L was transfected separately into HEp-2 cells on six- well plates (Costar, Cambridge, MA) together with the support plasmids pTM( ⁇ ), pTM(P no C), and pTM(L) and LipofectACE (Life Technologies, Gaithersburg, MD) and the cells were simultaneously infected with MVA-T7, a replication- defective vaccinia virus recombinant encoding the bacteriophage T7 polymerase protein as previously described (Skiadopoulos et al., Vaccine 18:503-10, 1999b, inco ⁇ orated herein by reference).
  • the transfection harvest was passaged onto LLC-MK2 cells in a 25 cm flask, and the cells were incubated for 5 days at 32°C.
  • the virus recovered from the cell supernatant was further passaged on LLC-MK2 monolayer cultures with trypsin at 32°C to amplify the virus.
  • rPIV3-l HA P - M and rPIV3-l HA P-M cp4 ⁇ were biologically cloned by terminal dilution on LLC-MK2 monolayer cultures at 32°C as previously described (Skiadopoulos et al., Vaccine 18:503-10, 1999b).
  • Viral suspensions derived from biologically cloned virus were amplified on LLC-MK2 monolayer cultures.
  • Viral RNA was isolated from biologically cloned recombinant chimeric viruses as described above.
  • RT-PCR was performed using rHPIV3-l HAp. M or rHPIV3-l HAp.M C/?45 L vRNA as template and specific oligonucleotide primers that spanned the HA gene insert or the cp45 mutations in the L gene.
  • the RT-PCR products were analyzed by restriction endonuclease digestion and partial DNA sequencing of the PCR products as described above. This confirmed the presence of the measles virus HA gene inserted between the P and M genes of rHPIV3-l and the presence of the ⁇ p45 L gene mutations in its attenuated derivative.
  • TCIDjo of rHPIV3-l, rHPIV3-l HA P-M , rHPIV3-l cp45 L , or rHPIV3-l HA P-M cp45 L Four days after inoculation the lungs and nasal turbinates were harvested and titers of virus were determined as described previously (Skiadopoulos et al., Vaccine 18:503-10, 1999b). The titers are expressed as mean log 10 TCID 50 /gram tissue (Table 20). The recombinant rHPIV3- 1 HAp.
  • a sequential immunization schedule employing immunization with the attenuated rHPIV3 HA P -MC/J45L chimeric vaccine candidate followed by the attenuated rHPIV3-l HAp.M ⁇ p45 L vaccine candidate induces antibodies to the HPIV3 and HPIVl antigens of the vector backbones and induces and maintains high titers of antibodies to the shared vectored antigen, the measles virus HA.
  • M cp45 _ vaccine was clearly immunogenic in animals previously immune to HPIV3 as indicated by the response of hamsters in Group 4. These animals, which were immunized with rHPIV3 cp45 ⁇ _ on day 0, developed a moderately high titer of neutralizing antibodies to measles virus on day 94, 35 days following immunization with rHPIV3-l HAp.M cp45_. on day 59. Significantly, hamsters that were first immunized with rHPIV3 HAp. M c/?45_.
  • HPIV3 vaccine such as rHPIV3 HAp.
  • M cp45 ⁇ will be given within the first four months of life followed two months later by an HPIVl vaccine such as rHPIV3-l HA P .
  • M cp45 L Sudopoulos et al., Vaccine 18:503-10, 1999b, inco ⁇ orated herein by reference.
  • human infants characteristically develop low titers of antibodies to viral glycoprotein antigens administered within the first six months of life, due to immunologic immaturity, immunosuppression by maternal antibodies, and other factors (Karron et al., Pediatr. Infect. Pis. J.
  • the present example indicates that it is possible to sequentially immunize animals with two serologically distinct live attenuated PIV vaccines, each of which expresses the measles virus HA, to develop antibodies to the HPIV3 and HPIVl antigens of the vector backbone, and to maintain high titers of antibodies to the vectored antigen, the measles virus HA.
  • Mean serum neutralizing antibody titer to HPIVl is expressed as the reciprocal mean log 2 ⁇ S.E.
  • Mean serum neutralizing antibody titer to wild type measles virus is expressed as the reciprocal mean log 2 ⁇ standard error, PRN, plaque reduction neutralization.
  • the present example details development of a live attenuated PIV2 candidate vaccine virus for use in infants and young children using reverse genetic techniques.
  • the recovered viruses designated rPIV3-2CT in which the PIV2 ectodomain and transmembrane domain was fused to the PIV3 cytoplasmic domain and rPIV3-2TM in which the PIV2 ectodomain was fused to the PIV3 transmembrane and cytoplasmic tail domain, possessed similar, although not identical, in vitro and in vivo phenotypes. Thus, it appears that only the cytoplasmic tail of the HN or F glycoprotein of PIV3 is required for successful recovery of PIV2-PIV3 chimeric viruses.
  • the rPIV3-2 recombinant chimeric viruses exhibit a strong host range phenotype, i.e. they replicate efficiently in vitro but are strongly restricted in replication in vivo. This attenuation in vivo occurs in the absence of any added mutations from cp45.
  • rPIV3-2CT and rPIV3-2TM replicated efficiently in vitro, they were highly attenuated in both the upper and the lower respiratory tract of hamsters and African green monkeys (AGMs), indicating that chimerization of the HN and F proteins of PIV2 and PIV3 itself specified an attenuation phenotype in vivo.
  • a phenotype including efficient replication in vitro and highly restricted groth in vivo is greatly desired for vaccine candidates.
  • rPIV3-2CT and rPIV3-2TM were further modified by the introduction of the 12 PIV3 cp45 mutations located outside of the HN and F coding sequences to derive rPIV3-2CTcp45 and rPIV3-2TMcp45. These derivatives replicated efficiently in vitro but were even further attenuated in hamsters and AGMs indicating that the attenuation specified by the glycoprotein chimerization and by the cp45 mutations was additive.
  • the wild type PIVl strain used in this study PIVl /Washington/20993/ 1964 (PIVl/Wash64) (Mu ⁇ hy et al., Infect. Immun. 12:62-68, 1975, inco ⁇ orated herein by reference), was propagated in LLC-MK2 cells (ATCC CCL 7.1) as previously described (Tao et al., J. Virol. 72:2955-2961, 1998, inco ⁇ orated herein by reference).
  • the PIV wild type virus, strain V9412-6, designated PIV2/V94 was isolated in qualified Vero cells from a nasal wash of a sick child in 1994 .
  • PIV2/V94 was plaque purified three times on Vero cells before being amplified twoce on Vero cells using OptiMEM without FBS.
  • the wild type cDNA-derived recombinant PIV3/JS strain (rPIV3/JS) was propagated as previously described (Durbin et al., Virology 235:323-332, 1997, inco ⁇ orated herein by reference).
  • the modified vaccinia Ankara virus (MVA) recombinant that expresses the bacteriophage T7 RNA polymerase was generously provided by Drs. L. Wyatt and B. Moss (Wyatt et al., Virology 210:202-205, 1995, inco ⁇ orated herein by reference).
  • HEp-2 cells (ATCC CCL 23) were maintained in MEM (Life Technologies, Gaithersburg, MD) with 10% fetal bovine serum, 50 ⁇ g/ml gentamicin sulfate, and 2 mM glutamine. Vero cells below passage 150 were maintained in serum-free medium VP-SFM (Formula No. 96-0353 S A, Life Technologies) with 50 ⁇ g/ml gentamicin sulfate and 2 mM glutamine.
  • MEM Life Technologies, Gaithersburg, MD
  • Vero cells below passage 150 were maintained in serum-free medium VP-SFM (Formula No. 96-0353 S A, Life Technologies) with 50 ⁇ g/ml gentamicin sulfate and 2 mM glutamine.
  • viruses were amplified on cultured cells and concentrated by polyethylene glycol precipitation as previously described (Mbiguino et al., J. Virol. Methods 31 : 161-170, 1991, inco ⁇ orated herein by reference).
  • Virion RNA was extracted from the virus pellet using Trizol reagent (Life Technologies) and used as template for reverse transcription (RT) with the Superscript Preamplification system (Life Technologies).
  • the cDNA was further PCR amplified using the Advantage cDNA kit (Clontech, Palo Alto, CA).
  • the RT-PCR amplified DNA was purified from agarose gels using NA45 DEAE membrane as suggested by the manufacturer (Schleicher & Schuell, Keene, NH). Sequencing was performed with the dRhodamine dye terminator cycling squencing kit (Perkin Elmer, Forster City, CA) and an ABI 310 Gene Analyzer (Perkin Elmer, Forster City, CA). Construction of the chimeric PIV3-PI V2 antigenomic cDNAs encoding the complete PIV2 F and HN proteins or chimeric F and HN proteins containing a PIV2-derived ectodomain and PIV3-derived cytoplasmic tail domain
  • a DNA encoding a full-length PIV3 antigenomic RNA was constructed in which the PIV3 F and HN ORFs were replaced by their PIV2 counte ⁇ arts following the strategy described previously (Tao et al., J. Virol. 72:2955-2961, 1998) for PIV3-PIV1. Details of this construction are presented in Figure 17.
  • PIV2/V94 propagated in Vero cells was concentrated and virion RNA (vRNA) was extracted from the virus pellet using Trizol reagent.
  • the F and HN ORFs of PIV2/V94 were reverse transcribed from vRNA using random hexamer primers and the Superscript Preamplification System before being amplified by PCR using the cDNA Advantage kit and primer pairs specific to PIV2 F and HN genes, respectively (1, 2 and 3, 4; Table 22).
  • the amplified cDNA fragment of PIV2 F ORF was digested with Ncol plus BamHI and ligated into the Ncol -BamHI window of pLit.PIV31.Fhc (Tao et al., J. Virol. 72:2955-2961, 1998, inco ⁇ orated herein by reference) to generate pLit.PIV32Fhc.
  • the BspEI site in the PIV3 full-length cDNA is unique and we planned to use it to exchange segments between cDNAs (see Figures 17-19). Therefore, a BspEI site that was found in the PIV2 F ORF was removed by site-directed mutagenesis without affecting the amino acid sequence.
  • the cDNA fragment of PIV2 HN ORF was digested with Ncol plus Hindlll and ligated into the Ncol-Hindlll window of pLit.PIV31.HNhc (Tao et al., J. Virol. 72:2955-2961 , 1998) to generate pLit.PIV32HNhc.
  • the PIV2 ORFs in pLit.PIV32Fhc and pLit.PIV32HNhc were sequenced, and the sequence was found to be as designed.
  • the nucleotide sequences for the PIV2 F and HN ORFs are submitted in the GenBank.
  • pLit.PIV32Fhc and pLit.PIV32HNhc were each digested with PpuMl plus Spel and assembled to generate pLit.PIV32hc.
  • the 4 kb BspEI-Spel fragment of pLit.PIV32hc was introduced into the BspEI-Spel window of p38' ⁇ PIV3 lhc
  • PIV2 F sense 5069" 5088" pFLC.PIV32CT ATGCATCACCTGCATCCAAT (SEQ ID NO. 50)
  • PIV3 F sense 6620" 6642 d pFLC.PIV32CT AAGTATTACAGAATTCAAAAGAG (SEQ ID NO. 54)
  • PIV2 F Sense 6608c,d 6630 ,'c,d Chimera confirmation ACCGCAGCTGTAGCAATAGT (SEQ ID NO. 56)
  • PIV3 M sense 4759 .
  • d 4780 Chimera confirmation GATACTATCCTAATATTATTGC (SEQ ID NO. 58)
  • the numbers are the nt positions in the full-length antigenomic cDNA construct pFLC.PIV32hc.
  • the numbers are the nt positions in the full-length antigenomic cDNA construct pFLC.PIV32TM and pFLC.PIV32TMcp45.
  • the numbers are the nt positions in the full-length antigenomic cDNA construct ⁇ FLC.PIV32CT and pFLC.PIV32CTcp45.

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CN104093422B (zh) * 2012-01-24 2018-02-23 乔治亚大学研究基金公司 基于副流感病毒5的疫苗
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