WO2013116770A1 - Replication-defective flavivirus vaccines and vaccine vectors - Google Patents

Replication-defective flavivirus vaccines and vaccine vectors Download PDF

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WO2013116770A1
WO2013116770A1 PCT/US2013/024495 US2013024495W WO2013116770A1 WO 2013116770 A1 WO2013116770 A1 WO 2013116770A1 US 2013024495 W US2013024495 W US 2013024495W WO 2013116770 A1 WO2013116770 A1 WO 2013116770A1
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virus
flavivirus
protein
replication
sequences
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PCT/US2013/024495
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French (fr)
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Konstantin V. Pugachev
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Sanofi Pasteur Biologics, Llc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20111—Lyssavirus, e.g. rabies virus
    • C12N2760/20134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2770/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This invention relates to replication-defective fiavivirus vaccines and vaccine vectors, and corresponding compositions and methods.
  • Flaviviruses are distributed worldwide and represent a global public health problem.
  • Fiavivirus pathogens include yellow fever (YF), dengue types 1-4 (DEN1-4), Japanese encephalitis (JE), West Nile (WN), tick- borne encephalitis (TBE), and other viruses from the TBE serocomplex, such as Kyasanur Forest disease (KFD) and Omsk hemorrhagic fever (OHF) viruses.
  • YF yellow fever
  • DEN1-4 dengue types 1-4
  • JE Japanese encephalitis
  • WN West Nile
  • TBE tick- borne encephalitis
  • JE [inactivated vaccines (INV) and LAV] are available. No licensed human vaccines are currently available against DEN and WN.
  • Veterinary vaccines have been in use including, for example, vaccines against WN in horses (INV, recombinant and live chimeric vaccines), JE (INV and LAV) to prevent encephalitis in horses and stillbirth in pigs in Asia, louping ill fiavivirus (INV) to prevent neurologic disease in sheep in the UK, and TBE (INV) used in farm animals in Czech Republic (INV) (Monath and Heinz, Flaviviruses, in Fields et al. Eds., Fields Virology, 3rd Edition, Philadelphia, New York, Lippincott-Raven Publishers, 1996, pp. 961-1034).
  • Tick-borne encephalitis is the most important tick-borne viral disease of humans. It is endemic in parts of Europe and Northern Asia, causing more than 10,000 hospitalizations annually, with a case-fatality rate 0.5-1.5% in Europe and 6-40% in Siberia and the Far East. A significant proportion of patients suffer from long-lasting neuropsychiatric sequelae. Inactivated vaccines produced in chick embryo cell cultures have proven effective in preventing the disease. For example, an 86% vaccination coverage of the Austrian population (the highest among European countries) has resulted in an approximately 90% reduction of hospitalized cases (Heinz and Kunz, Arch. Virol. Suppl. 18:201-205, 2004).
  • the inactivated vaccines are expensive and require three inoculations for primary immunization. Periodic boosters (every 2-5 years) are required to maintain immunity. Therefore, a less costly TBE vaccine, which is effective after one-two doses and provides durable, such as life-long immunity (similar to that achieved by YF 17D immunization) is needed, and indeed has been identified by the WHO as a major priority.
  • TBE LAV candidates in the past several decades by means of empirical or rational attenuation of TBE virus parent per se or chimerization of TBE or Langat (LGT, a naturally attenuated flavi virus that is closely related (serologically) to TBE) viruses with dengue 4 virus has faced difficulties due to problems with residual virulence of candidates and/or low immunogenicity/overattenuation (Wright et al., Vaccine 26:882-890, 2008; Maximova et al, J. Virol. 82:5255-5268, 2008; Rumyantsev et al., Vaccine 24:133-143, 2006; Kofler et al., Arch. Virol. Suppl. 18:191-200, 2004; and references therein).
  • LGT Langat
  • Flaviviruses are small, enveloped, plus-strand RNA viruses transmitted primarily by arthropod vectors (mosquitoes or ticks) to natural hosts, which are primarily vertebrate animals, such as various mammals, including humans, and birds.
  • the flavivirus genomic RNA molecule is about 11,000 nucleotides (nt) in length and encompasses a long open reading frame (ORF) flanked by 5' and 3' untranslated terminal regions (UTRs) of about 120 and 500 nucleotides in length,
  • the ORF encodes a polyprotein precursor that is cleaved co- and post-translationally to generate individual viral proteins.
  • the proteins are encoded in the order: C-prM/M-E-NSl- NS2A/2B-NS3-NS4A/4B-NS5, where C (core/capsid), prM/M (pre-membrane/membrane), and E (envelope) are the structural proteins, i.e., the components of viral particles, and the NS proteins are non-structural proteins, which are involved in intracellular virus replication. Flavivirus replication occurs in the cytoplasm.
  • processing of the polyprotein starts with translocation of the prM portion of the polyprotein into the lumen of endoplasmic reticulum (ER) of infected cells, followed by translocation of E and NS1 portions, as directed by the hydrophobic signals for the prM, E, and NS 1 proteins.
  • Amino-termini of prM, E, and NS1 proteins are generated by cleavage with cellular signalase, which is located on the luminal side of the ER membrane, and the resulting individual proteins remain carboxy-terminally anchored in the membrane. Most of the remaining cleavages, in the nonstructural region, are carried out by the viral NS2B/NS3 serine protease.
  • the viral protease is also responsible for generating the C- terminus of the mature C protein found in progeny virions.
  • Newly synthesized genomic RNA molecules and the C protein form a dense spherical nucleocapsid, which becomes surrounded by cellular membrane in which the E and prM proteins are embedded.
  • the mature M protein is produced by cleavage of prM shortly prior to virus release by cellular furin or a similar protease.
  • E the major protein of the envelope, is the principal target for neutralizing antibodies, the main correlate of immunity against flavivirus infection.
  • Virus-specific cytotoxic T-lymphocyte (CTL) response is the other key attribute of immunity.
  • Multiple CD8+ and CD4+ CTL epitopes have been characterized in various flavivirus structural and non-structural proteins.
  • innate immune responses contribute to both virus clearance and regulating the development of adaptive immune responses and immunologic memory.
  • PAV pseudoinfectious virus
  • PIVs are replication-defective viruses attenuated by a deletion(s). Unlike live flavivirus vaccines, they undergo a single round replication in vivo (or optionally limited rounds, for two-component constructs; see below), which may provide benefits with respect to safety. PIVs also do not induce viremia and systemic infection. Further, unlike inactivated vaccines, PIVs mimic whole virus infection, which can result in increased efficacy due to the induction of robust B- and T-cell responses, higher durability of immunity, and decreased dose requirements. Similar to whole viruses, PIV vaccines target antigen-presenting cells, such as dendritic cells, stimulate toll-like receptors (TLRs), and induce balanced Thl/Th2 immunity.
  • TLRs toll-like receptors
  • s- PIV pseudoinfectious virus
  • the deletion does not remove the first -20 codons of the C protein, which contain an RNA cyclization sequence, and a similar number of codons at the end of C, which encode a viral protease cleavage site and the signal peptide for prM.
  • the s-PrV can be propagated, e.g., during manufacture, in substrate (helper) cell cultures in which the C protein is supplied in trans, e.g., in stably transfected cells producing the C protein (or a larger helper cassette including C protein), or in cells containing an alphavirus replicon [e.g., a Venezuelan equine encephalitis virus (VEE) replicon] expressing the C protein or another intracellular expression vector expressing the C protein.
  • VEE Venezuelan equine encephalitis virus
  • VLPs empty virus-like particles
  • a T-cell response should also be induced via MHCI presentation of viral epitopes. This approach has been applied to YF 17D virus and WN viruses and WN/JE and WN/DEN2 chimeric viruses (Mason et al., Virology 351 :432-443, 2006; Suzuki et al., J. Virol.
  • a two-component PIV (d-PIV) is constructed (Fig. 2).
  • Substrate cells are transfected with two defective viral RNAs, one with a deletion in the C gene and another lacking the prM-E envelope protein genes.
  • the two defective genomes complement each other, resulting in accumulation of two types of PIVs in the cell culture medium (Shustov et al., J. Virol. 21:11737-11748, 2007; Suzuki et al., J. Virol. 82:6942-6951, 2008).
  • the two PIVs can be manufactured separately in appropriate helper cell lines and then mixed in a two-component formulation.
  • This type of PIV vaccine should be able to undergo a limited spread in vivo due to coinfection of some cells at the site of inoculation with both components.
  • the spread is expected to be self-limiting as there are more cells in tissues than viral particles produced by initially coinfected cells.
  • a relatively high MOI is necessary for efficient co-infection, and cells outside of the inoculation site are not expected to be efficiently coinfected (e.g., in draining lymph nodes).
  • Cells infected with the AC PIV alone produce the highly immunogenic VLPs.
  • Coinfected cells produce the two types of packaged defective viral particles, which also stimulate neutralizing antibodies.
  • viral sequences can be modified in both s-PIVs and d-PIVs using, e.g., synonymous codon replacements, to reduce nucleotide sequence homologies, and mutating the complementary cyclization 5' and 3' elements.
  • the invention provides replication-deficient or defective pseudoinfectious flaviviruses including a flavivirus genome that includes (i) one or more deletions or mutations in nucleotide sequences encoding one or more proteins selected from the group consisting of capsid (C), pre- membrane (prM), envelope (E), non-structural protein 1 (NSl), non-structural protein 3 (NS3), and non-structural protein 5 (NS5), and (ii) sequences encoding one or more heterologous pathogen, cancer, or allergy-related immunogens.
  • C capsid
  • prM pre- membrane
  • E envelope
  • NSl non-structural protein 1
  • NS3 non-structural protein 3
  • NS5 non-structural protein 5
  • the deletion/mutation can be within capsid (C) sequences; pre-membrane (prM) and/or envelope (E) sequences; capsid (C), pre-membrane (prM), and envelope (E) sequences; or non-structural protein 1 (NSl) sequences.
  • the heterologous immunogen can be, for example, from a pathogen selected from the group consisting of a rabies virus (e.g., a rabies virus G protein epitope), Borrelia burgdorferi (e.g., OspA immunogen or an immunogenic fragment thereof), a tick (e.g., a tick saliva protein selected from the group consisting of 64TRP, Isac, and Salp20, or an immunogenic fragment thereof), an influenza virus (e.g., an influenza virus M2, hemaglutinnin (HA), or neuraminidase (NA) epitope, or an immunogenic fragment thereof), a human immunodeficiency virus (e.g., a codon-optimized HIV gag, pol, tat/nef, pro, or variants of Env protein, such as gpl60, gpl45, gpl40, gpl20, gp41, etc., or immunogenic fragments thereof), a
  • the replication-deficient pseudoinfectious flaviviruses can include sequences encoding a pre-membrane (prM) and/or envelope (E) protein. Further, the replication-deficient
  • pseudoinfectious flavivirus genomes can be selected from those of yellow fever virus, West Nile virus, tick-borne encephalitis virus, Langat virus, Japanese encephalitis virus, dengue virus, and St. Louis encephalitis virus, attenuated strains thereof, and chimeras thereof (also see below).
  • the chimeras include pre-membrane (prM) and envelope (E) sequences of a first flavivirus (e.g., a tick-borne encephalitis virus or a Langat virus (e.g., Langat E5)), and capsid (C) and non-structural sequences of a second, different flavivirus (e.g., a yellow fever, a West Nile, or Langat (e.g., Langat E5) virus).
  • a first flavivirus e.g., a tick-borne encephalitis virus or a Langat virus (e.g., Langat E5)
  • C capsid
  • non-structural sequences of a second, different flavivirus e.g., a yellow fever, a West Nile, or Langat (e.g., Langat E5) virus.
  • a second flavivirus e.g., a yellow fever, a West Nile, or Langat (e.g., Langat E5) virus.
  • the replication-deficient pseudoinfectious flavivirus genomes can be packaged in particles including pre-membrane (prM) and envelope (E) sequences from a flavivirus that is the same or different from that of the genomes. Further, the sequences encoding the heterologous immunogens can be inserted in the place of, or in combination with, the deletion(s) or mutation(s) of the one or more proteins.
  • prM pre-membrane
  • E envelope sequences from a flavivirus that is the same or different from that of the genomes.
  • sequences encoding the heterologous immunogens can be inserted in the place of, or in combination with, the deletion(s) or mutation(s) of the one or more proteins.
  • sequences encoding the heterologous immunogens can be inserted in the flavivirus genomes within sequences encoding the envelope (E) protein, within sequences encoding the nonstructural 1 (NS1) protein, within sequences encoding the pre-membrane (prM) protein,
  • the replication-deficient pseudoinfectious flavivirus genomes include heterologous immunogen sequences from HIV, SIV, or influenza virus, such as any one or more of those described in Appendices 6-8.
  • the replication-deficient pseudoinfectious virus is selected from any one of the SIV constructs 1-11 of Sequence Appendix 6, a construct having at least 50% sequence identity (e.g., 50%, 60%, 70%, 85%, 90%, 95%, or 99% or more sequence identity) to the nucleic acid or amino acid sequences described therein, or a construct that includes homologs and/or other naturally occurring variants of the SIV protein(s).
  • the replication-deficient pseudoinfectious virus is selected from the HIV Gag construct (PIV-WN (AprME)-HIV Gag ) of Sequence Appendix 7, a construct having at least 50% sequence identity (e.g., 50%, 60%, 70%, 85%, 90%, 95%, or 99% or more sequence identity) to the nucleic acid or amino acid sequences described therein, or a construct that includes homologs and/or other naturally occurring variants of the HIV Gag protein.
  • the replication-deficient pseudoinfectious virus is selected from the HIV Env construct (PIV-WN (AprME)-HIV Env Gpl40) of Sequence Appendix 7, a construct having at least 50% sequence identity (e.g., 50%, 60%, 70%, 85%, 90%, 95%, or 99% or more sequence identity) to the nucleic acid or amino acid sequences described therein, or a construct that includes homologs and/or other naturally occurring variants of the HIV Env protein.
  • the replication- deficient pseudoinfectious virus is selected from construct 1 or 2 of Sequence Appendix 8, a construct having at least 50% sequence identity (e.g., 50%, 60%, 70%, 85%, 90%, 95%, or 99% or more sequence identity) to the nucleic acid or amino acid sequences described therein, or a construct that includes homologs and/or other naturally occurring variants of the HA protein.
  • compositions including a first replication-deficient
  • pseudoinfectious flavivirus as described above, and a second (or further), different replication- deficient pseudoinfectious flavivirus including a genome that includes one or more deletions or mutations in nucleotide sequences encoding one or more proteins selected from the group consisting of capsid (C), pre-membrane (prM), envelope (E), non-structural protein 1 (NS1), non-structural protein 3 (NS3), and non-structural protein 5 (NS5).
  • the one or more proteins encoded by the sequences in which the deletion(s) or mutation(s) occur in the second, different replication-deficient pseudoinfectious flavivirus are different from the one or more proteins encoded by the sequences in which the deletion(s) occur in the first replication-deficient pseudoinfectious flavivirus.
  • the invention further includes methods of inducing immune responses to an immunogen in a subject, which involves administering to the subject one or more replication-deficient
  • the replication-deficient pseudoinfectious flavivirus and/or composition includes any one or more of those described in Appendices 6-8, constructs having at least 50% sequence identity (e.g., 50%, 60%, 70%, 85%, 90%, 95%, or 99% or more sequence identity) to the nucleic acid or amino acid sequences described therein (or individual proteins or corresponding nucleic acid sequences therein), or constructs that include homologs and/or other naturally occurring variants of the immunogenic SIV, HIV, and or HA proteins.
  • sequence identity e.g., 50%, 60%, 70%, 85%, 90%, 95%, or 99% or more sequence identity
  • the subject is at risk of but does not have an infection by the pathogen or a disease or condition associated with the cancer or allergy-related immunogen.
  • the subject has an infection by the pathogen or a disease or condition associated with the cancer or allergy-related immunogen.
  • the invention thus includes prophylactic and therapeutic methods.
  • the immunogen can be from, for example, a pathogen selected from the group consisting of a rabies virus, Borrelia burgdorferi, a tick, an influenza virus, a human immunodeficiency virus, a simian immunodeficiency virus, a human papilloma virus, a respiratory syncytial virus, malaria parasite, and Mycobacterium tuberculosis (also see below).
  • the methods can be for inducing an immune response against a protein encoded by the flavivirus genome, in addition to the source of the immunogen.
  • the subject is at risk of but does not have an infection by the flavivirus corresponding to the genome of the pseudoinfectious flavivirus, which includes sequences encoding a flavivirus pre-membrane and/or envelope protein.
  • the subject has an infection by the flavivirus corresponding to the genome of the pseudoinfectious flavivirus, which includes sequences encoding a flavivirus pre-membrane and/or envelope protein.
  • the invention also includes live, attenuated chimeric flaviviruses including a yellow fever virus in which sequences encoding pre-membrane and envelope proteins are replaced with sequences encoding pre-membrane and envelope proteins of a tick-borne encephalitis virus or a Langat virus, and the signal sequence between the capsid and pre-membrane proteins of the chimeric flavivirus includes a hybrid of yellow fever virus and tick-borne encephalitis or Langat virus capsid/pre-membrane signal sequences, or a variant thereof.
  • the capsid/pre- membrane signal sequence of the chimeric flavivirus includes yellow fever virus sequences in the amino terminal region and tick-borne encephalitis or Langat virus sequences in the carboxy terminal region (see below).
  • the invention includes live, attenuated chimeric flaviviruses including a West Nile virus in which sequences encoding pre-membrane and envelope proteins are replaced with sequences encoding pre-membrane and envelope proteins of a tick-borne encephalitis or a Langat virus, and the signal sequence between the capsid and pre-membrane proteins of the chimeric flavivirus includes a tick-borne encephalitis or a Langat virus capsid/pre-membrane signal sequence, or a variant thereof.
  • the invention also includes pharmaceutical compositions including one or more
  • compositions can include an adjuvant.
  • replication-deficient pseudoinfectious flaviviruses including a flavivirus genome including one or more deletion(s) or mutation(s) in nucleotide sequences encoding non-structural protein 1 (NSl), non-structural protein 3 (NS3), or non-structural protein 5 (NS5).
  • the invention includes nucleic acid molecules corresponding to the genome of a pseudoinfectious flavivirus, or the genome of the live, attenuated flavivirus, as described herein, and complements thereof.
  • the invention also provides methods of making replication-deficient pseudoinfectious flaviviruses as described herein, involving introducing one or more nucleic acid molecules, as described above, into a cell that expresses the protein(s) corresponding to any sequences deleted from the flavivirus genome of the replication-deficient pseudoinfectious flaviviruses.
  • the protein can be expressed in the cell from the genome of a second (or further), different, replication-deficient pseudoinfectious flavivirus.
  • the protein is expressed from a replicon (e.g., an alphavirus replicon, such as a Venezuelan Equine Encephalitis virus replicon; see below).
  • the invention also includes compositions containing two or more replication-deficient pseudoinfectious flaviviruses, in which two of the replication-deficient pseudoinfectious flaviviruses are selected from the groups consisting of: (a) a replication-deficient pseudoinfectious flavivirus including a genome containing Japanese encephalitis virus sequences, and a replication-deficient pseudoinfectious flavivirus including a genome containing dengue virus sequences; (b) a replication- deficient pseudoinfectious flavivirus including a genome containing yellow fever virus sequences, and a replication-deficient pseudoinfectious flavivirus including a genome containing dengue virus sequences; and (c) a replication-deficient pseudoinfectious flavivirus including a genome containing tick-borne encephalitis or Langat virus sequences and an inserted sequence encoding a Borrelia burgdorferi immunogen, and a replication-deficient pseudoinfectious flavivirus including a genome containing tick-borne encephalitis or Langat virus sequences and
  • compositions including the live, attenuated chimeric flaviviruses described herein are also included in the invention. Further, the invention includes methods of inducing an immune response to tick-borne encephalitis virus or Langat virus in a subject, involving
  • the subject does not have but is at risk of developing infection by tick-borne encephalitis virus or Langat virus.
  • the subject is infected with tick-borne encephalitis virus or Langat virus.
  • the invention further includes replication-deficient pseudoinfectious flaviviruses including a flavivirus genome including one or more deletions or mutations in nucleotide sequences encoding one or more proteins selected from the group consisting of capsid (C), pre-membrane (prM), envelope (E), non-structural protein 1 (NS1), non-structural protein 3 (NS3), and non-structural protein 5 (NS5), wherein the flavivirus genome includes yellow fever virus sequences in which sequences encoding pre-membrane and envelope proteins are replaced with sequences encoding pre- membrane and envelope proteins of a tick-borne encephalitis virus or a Langat virus, and sequences encoding the signal sequence between the capsid and pre-membrane proteins of the flavivirus genome include a hybrid of sequences encoding yellow fever virus and tick-borne encephalitis or Langat virus capsid/pre-membrane
  • the invention includes replication-deficient pseudoinfectious flaviviruses including a flavivirus genome including one or more deletions or mutations in nucleotide sequences encoding one or more proteins selected from the group consisting of capsid (C), pre-membrane (prM), envelope (E), non-structural protein 1 (NS1), non-structural protein 3 (NS3), and non-structural protein 5 (NS5), wherein the flavivirus genome includes West Nile virus sequences in which sequences encoding pre-membrane and envelope proteins are replaced with sequences encoding pre- membrane and envelope proteins of a tick-borne encephalitis or a Langat virus, and the sequences encoding the signal sequence between the capsid and pre-membrane proteins of the flavivirus genome include sequences encoding a tick-borne encephalitis or a Langat virus capsid/pre- membrane signal sequence, or a variant thereof.
  • the invention includes replication-deficient pseudoinfectious flaviviruses including a flavivirus genome including one or more deletions or mutations in nucleotide sequences encoding one or more proteins selected from the group consisting of capsid (C), pre-membrane (prM), envelope (E), non-structural protein 1 (NS1), non-structural protein 3 (NS3), and nonstructural protein 5 (NS5), wherein any capsid (C) and non-structural (NS) proteins in the flavivirus genome are from Langat virus and any pre-membrane (prM) and envelope (E) proteins are from a tick-borne encephalitis virus.
  • the invention also includes use of the constructs, PIVs, LAVs, and combinations thereof for inducing immune responses, as described herein, or for preparation of medicaments as described herein.
  • replication-deficient pseudoinfectious flavivirus or “PIV” is meant a flavivirus that is replication-deficient due to a deletion or mutation in the flavivirus genome.
  • the deletion or mutation can be, for example, a deletion of a large sequence, such as most of the capsid protein, as described herein (with the cyclization sequence remaining; see below).
  • sequences encoding different proteins e.g., prM, E, NS1, NS3, and/or NS5; see below
  • combinations of proteins e.g., prM-E or C-prM-E
  • deletion may be advantageous if the PIV is to be used a vector to deliver a heterologous immunogen, as the deletion can permit insertion of sequences that may be, for example, at least up to the size of the deleted sequence.
  • the mutation can be, for example, a point mutation, provided that it results in replication deficiency, as discussed above. Because of the deletion or mutation, the genome does not encode all proteins necessary to produce a full flavivirus particle.
  • the missing sequences can be provided in trans by a complementing cell line that is engineered to express the missing sequence (e.g., by use of a replicon; s-PrV; see below), or by co-expression of two replication-deficient genomes in the same cell, where the two replication-deficient genomes, when considered together, encode all proteins necessary for production (d-PIV system; see below).
  • a complementing cell line that is engineered to express the missing sequence
  • s-PrV e.g., by use of a replicon; s-PrV; see below
  • VLPs including prM- E proteins are released from the cells. Because of the lack of capsid protein, the VLPs lack capsid and a nucleic acid genome. In the case of the d-PrV approach, production of further PIVs is possible in cells that are infected with two PIVs that complement each other with respect to the production of all required proteins (see below).
  • replication-defective pseudoinfectious flaviviruses including multiple heterologous immunogens from, e.g., a human immunodeficiency virus or a simian immunodeficiency virus.
  • the multiple immunogens can include heterologous transmembrane and/or signal sequences (from, e.g., a rabies virus G protein).
  • the PIV vectors and PIVs of the invention are highly attenuated and highly efficacious after one-to-two doses, providing durable immunity.
  • PIVs mimic whole virus infection, which can result in increased efficacy due to the induction of robust B- and T-cell responses, higher durability of immunity, and decreased dose requirements.
  • PIV vaccines target antigen-presenting cells, such as dendritic cells, stimulate toll-like receptors (TLRs), and induce balanced Thl/Tti2 immunity.
  • PrV constructs have also been shown to grow to high titers in substrate cells, with little or no CPE, allowing for high-yield manufacture, optionally employing multiple harvests and/or expansion of infected substrate cells. Further, the PIV vectors of the invention provide an option for developing vaccines against non-flavivirus pathogens for which no vaccines are currently available.
  • Figs. 1 A and IB are schematics illustrating single component PIV (s-PIV) technology.
  • the replication-deficient pseudoinfectious flavivirus genomes can be selected from, for example, those of yellow fever virus, West Nile virus, tick-borne encephalitis virus, Langat virus, Japanese encephalitis virus, dengue virus, and St. Louis encephalitis virus, attenuated strains thereof, and chimeras thereof.
  • Fig. 2 is a schematic illustration of two-component PIV (d-PIV) technology.
  • Figs. 3A and 3B are schematics illustrating general experimental designs
  • Fig. 4 is a graph comparing the humoral immune response induced by PIV-WN (RV-WN) with that of chimeric YF/WN LAV (CV-WN) in mice.
  • Fig. 5 is a series of graphs showing the results of challenging hamsters immunized with PIV-YF (RV-YF), YF17D, PIV-WN (RV-WN), and YF/WN LAV (CVWN) with hamster-adapted Asibi (PIV-YF and YF 17D vaccinees) and wild type WN-NY99 (PIV-WN and YF/WN LAV vaccinees).
  • Fig. 6 is a table showing YF/TBE and YF/LGT virus titers and plaque morphology obtained with the indicated chimeric flaviviruses.
  • Fig. 7A is a table showing WN/TBE PIV titers and examples of
  • FIG. 7B is a schematic showing differences in the PIV-WN/TBE constructs p39, p39I, p40, and p98.
  • Figs. 8C and 8D are graphs showing the effect that modification of the prM signal has on PIV-WN/TBE replication in vitro (Fig. 8C) and on immunogenicity (Fig. 8D) in mice.
  • Fig. 10 is a graph showing survival of mice inoculated TP with PIV-WN/TBE(Hypr) (RV- WN/Hypr), YF/TBE(Hypr) LAV (CV-Hypr), and YF/LGT LAV (CV-LGT) constructs and YF17D in a neuroinvasiveness test (3.5 week old ICR mice).
  • Fig. 11 is a series of graphs illustrating morbidity in mice measured by dynamics of body weight loss after TBE virus challenge, for groups immunized with s-PIV-TBE candidates (upper left panel), YF/TBE and YF/LGT chimeric viruses (upper right panel), and controls (YF 17D, human killed TBE vaccine, and mock; bottom panel).
  • Figs. 12A and 12B are schematic representations of ⁇ 7 constructs expressing rabies virus G protein.
  • Fig. 12A also shows an illustration of packaging of the constructs to make
  • Fig. 13 is a schematic representation of insertion designs resulting in viable/expressing constructs (exemplified by rabies G).
  • Fig. 14 is series of images showing immunofluorescence analysis and graphs showing growth curves of cells transfected with the indicated PIV-WN constructs (AC-Rabies G, APrM-E- Rabies G, and AC-PrM-E-Rabies G).
  • Fig. 15 is a series of images showing immunofluorescence analysis of RabG expressed on the plasma membranes of Vero cells transfected with the indicated PIV constructs (AC-Rabies G, APrM-E-Rabies G, and AC-PrM-E-Rabies G).
  • Fig. 16 is a schematic illustration of a PIV-WN-rabies G construct and a series of images showing that this construct spreads in helper cells, but not in naive cells.
  • Fig. 17 is a series of graphs showing stability of the rabies G protein gene in PIV-WN vectors.
  • Fig. 18 is a set of images showing a comparison of spread of single-component vs. two- component PIV-WN-rabies G variants in Vero cells.
  • Fig. 19 is a graph showing the results of rapid fluorescent focus inhibition test (RFFIT) using the indicated constructs.
  • RFFIT rapid fluorescent focus inhibition test
  • Fig. 20 is a set of immunofluorescence images showing expression of full-length RSV F protein (strain A2) by the AprM-E component of d-PIV- WN in helper cells after transfection.
  • Fig. 21 is a graph showing RSV-F neutralization titers.
  • Fig. 22 is a schematic representaiton of an artificial cassette containing SIV (GenBank accession number ADM52218.1) gpl20 (the native signal sequence in the gene was replaced with the tPA signal and gp41 was truncated to contain only the TM domain), Gag, and Pro (protease) genes.
  • Fig. 23 is a schematic representation of inserts of the first three constructs in Fig. 22 (the three top constructs shown in Fig. 23), starting with the Env glycoprotein that were designed similarly to the PrV WN-rabies G vectors described herein (see, e.g., Figures 12-14 and
  • gpl20 signal is fused with a portion of the signal sequence for prM (e.g., at the end of the C gene or downstream from AC deletion depending on vector).
  • a portion of the signal sequence for prM e.g., at the end of the C gene or downstream from AC deletion depending on vector.
  • schematic representations of alternate dC RV230 Env PIV constructs are shown (the three bottom constructs shown in Fig. 23).
  • Fig. 24 is a schematic representation of Gag and Gag-Pro PIV construct designs, in which Gag and Gag-Pro were cloned in place of the AprM-E or AC-prM-E deletions.
  • Fig. 25 is a photograph of a Western blot using anti-Gag antibodies, which shows correct processing of the polyprotein in recovered SIV Gag and SIV Gag/Pro PIVs grown in helper cells.
  • Figs. 26A-26C are photomicrographs showing that immunostaining of naive Vera cells infected with the Gag PIVs, showed individual stained cells as expected from sPIV.
  • Fig. 26A is a negative control
  • Fig. 26B shows immunostaining of nai ' ve Vero cells infected with RV230 9AA- FMD-Gag PIV
  • Fig. 26C shows immunostaining of nai ' ve Vero cells infected with RV230 FMD-Gag PIV.
  • the two constructs are illustrated schematically in Fig. 26D.
  • Figs. 27A-F are graphs showing growth curves of SIV Gag PIV variants after transfection of helper cells with in vitro synthesized PIV RNA (P0 passage) indicating efficient replication in vivo. Immunofluorescence images of Vero cells infected with the variants are shown inset.
  • Fig. 28 is a graph showing growth curves in nai ' ve Vero cells of SIV Gag PIV as a two- component formulation (d-PIV, sometimes also designated as tc-PIV) together with PIV-WN helper with AC deletion (RV909).
  • Fig. 29 is a graph showing high insert stability for one of the SIV Gag PIV variants (RV230- Gag variant, containing Gag gene in place of large AprM-E deletion, in helper BHK-CprME(WN) cells at MOI 0.1 FFU/cell ) when examined by ten serial passages.
  • Figs. 30A-G are immunofluorescence images showing efficient expression of SIV Env (gpl20) in Vero cells using PIV-(WN)-SIV Env variants. Efficient intracellular expression of the original gpl20 was observed in Vero cells infected with packaged dC230Env PIV variant as determined by immunostaining using anti-SIV Env antibody after methanol fixation (Fig. 30D), although transport of gpl20 to the surface of infected Vero cells was inefficient, as determined following formalin fixation (Fig. 30B). In contrast, the dC230Env/RabG anchor PIV construct (see Fig.
  • Fig. 31 is an immunofluorescence image showing expression of SIV Env on the surface of PIV-SrV Env/RabG TM infected Vero cells.
  • Fig. 32A is a table showing single dose ICLD 5 o and D34 plaque reduction neutralization assay 50 (PRNT 50 ) results using the indicated vectors in 2 day old suckling mice (or 8 day old sucklings for RV-TBE and YF17D).
  • PRNT 50 plaque reduction neutralization assay 50
  • Figs. 32B-32C are photographs showing brain histology in 2 day old suckling mice administered sPIV-WN (day 17 post 6 logio PFU IC; Fig. 32 A), tcPTV-RabG (day 11 post 6 log 10 PFU; Fig. 32B), and sPTV-RabG (day 11 post 6 log 10 PFU; Fig. 32C).
  • Fig. 33 is a graph showing individual sera endpoint titers following prime & boost with the indicated constructs. ALVAC is shown as a control.
  • Fig. 34 is a graph showing the results of IgG isotyping in mice treated with RV Env/RabG vector and with ALVAC EGP.
  • Figs. 35 is a series of three graphs showing that RV-Gag expressing constructs are capable of eliciting detectable T cell responses as measured by interferon gamma (IFNg) secretion upon peptide stimulation ex vivo.
  • IFNg interferon gamma
  • Fig. 36 is a schematic representation of PrV-flu HA construct designs, in which the full- length HA gene of Flu strain New Caledonia was cloned in place of AprM-E and AC-prM-E deletions of PIV-WN vectors in the same fashion as described for Rabies G, RSV F and SIV Env (as is described herein).
  • Figs. 37A-B are graphs showing growth curves in BHK 363 helper cells transfected at P14 with RNA from RV230 HA New Caledonia PIV clones 6 (Fig. 37 A) and 10 (Fig. 37B), as determined by immunostaining with anti-WN and anti-HA antibodies.
  • Figs. 38A-D are graphs showing growth curves in BHK 363 helper cells transfected at P14 with RNA from RV230 HA New Caledonia PIV clones 1 , 6, and 10 (Figs. 38A-C, respectively) and from dC RV230 HA New Caledonia PIV clone 6 (Fig. 38D), as determined by immunostaining with anti-WN and anti-HA antibodies.
  • Figs. 39A-F are immunofluorescence images showing surface expression (Figs. 39A-C) and intracellular expression (Figs. 39D-F) of HA in Vero cells infected with RV230 HA New Caledonia PIV clones 1, 6, and 10, respectively.
  • Figs. 40A-B are immunofluorescence images showing surface expression (Fig. 40A) and intracellular expression (Fig. 40B) of HA in Vero cells infected with dC RV230 HA New Caledonia PIV clone 6.
  • Fig. 41 shows immunofluorescence images confirming surface expression (Figs. 41 B and D) and intracellular expression (Figs. 41F and H) of HA in Vero cells infected with RV230 HA New Caledonia PIV.
  • Figs. 41 A, C, E, and G are negative controls showing the lack surface expression (Figs. 41 A and C) and intracellular expression (Figs. 41E and G) of HA in uninfected Vero cells.
  • the immunofluorescence images in Figs. 4 IB and F were produced using antibodies against the stem of HA, while the immunofluorescence images in Figs. 4 ID and H were produced using antibodies against the HA globular head.
  • Figs. 4 IB, D, F, and H confirm the correct, native protein confirmation of HA.
  • Figs. 42A-D are immunofluorescence images showing surface expression (Figs. 42A and B) and intracellular expression (Figs. 42C and D) of HA in Vero cells infected with RV230 HA New Caledonia PIV clones 6 and 10, respectively, 48 hours post infection. Staining was performed with a mix of HA stem and globular head antibodies.
  • Fig. 43 A is an immunofluorescence image showing staining of RV230-HA PIV infected Vero cells by HA stem-specific antibodies.
  • Fig. 43B is an immunofluorescence image showing staining of RV230-HA Pr infected Vero cells by HA globular head-specific antibodies.
  • Figs. 44A and 44B are schematics showing PIV constructs of the invention.
  • Fig. 44A is a replication defective (single-cycle) virus obtained by deletion of the capsid protein gene and incorporation of the prM-E from TBEV. The construct is produced in helper cells providing deleted gene(s) in trans.
  • Fig. 44B shows a chimera construct that is a recombinant virus between an attenuated backbone (CV-YFV 17D vaccine or PDK-53 DENV-2 viruses) and a target (TBEV) flavivirus obtained by replacing prM-E envelope protein genes.
  • CV-YFV 17D vaccine or PDK-53 DENV-2 viruses attenuated backbone
  • TBEV target flavivirus
  • Fig. 45 is a table showing that the PIV-TBE construct, which generates high yields on helper cells expressing WNV C protein in trans, is highly genetically stable after 10 passages at MOI 0.01 and is highly attenuated, showing no evidence of recombination in a single round of replication
  • Fig. 46 is a table showing neurovirulence and neuroinvasiveness of live chimeras (CV) and replication defective (PIV) viruses in 3.5 weeks old ICR mice.
  • TBE chimeric viruses- based on dengue and YFV 17D- are less attenuated than the replication defective PIV viruses.
  • PIV TBE (Hypr) is highly attenuated in adult and suckling mice. *Not determined in 3.5 week old mice, but shown to be >5 in 8-day old suckling mice. ⁇ 100% mortality at the lowest dose tested. * No mortality observed at the highest dose tested. ⁇ Partial mortality at the highest or lowest dose tested.
  • Figs. 47A-B are graphs showing the replication of PIV (Fig. 47 A) and chimera (Fig. 46B) constructs in vitro.
  • Fig. 46A shows growth curves of PIV variants in helper BHK or Vero cells supplying the indicated C protein in trans (MOI 0.1).
  • Fig. 47B shows growth curves of live chimeras in Vero cells (MOI 0.001).
  • Figs. 48A-B are graphs showing dose responses and durability of immunity of the indicated constructs in mice.
  • Fig. 48 A Mice were immunized IP with graded doses of PIV-WN/TBE or YF/TBE chimera (or 2 doses of dilutions of INV on days 0 and 14 (human dose is 2.4 mg); shown in insert), and challenged on day 21 with 500 LD 50 of TBE Hypr, to determine protective dose 50% values (PD 50 ).
  • Fig. 48B Mice were immunized with 10 PD 50 doses of PIV-WN/TBE, YF/TBE, or
  • Fig. 49 is a graph showing that pre-immunization with PIV-WN/TBE resulted in some reduction of TBE specific response compared to that in naive animals. Mice were preimmunized with YF 17D and then immunized 3 weeks or 6 months later with Chimera- JE or YF/TBE viruses.
  • mice were similarly preimmunized with PIV-WN and immunized with PIV-WN/TBE. All doses were 5 log 10 by IP. Vaccine-specific N Ab titers were measured 21 days after immunization.
  • Figs. 50A-B are graphs showing the immunogenicity and efficacy of PIV-WN/TBE compared to 3 doses of INV in non-human primate (NHP) study 2.
  • Fig. 50A Post-challenge LGT 1674 viremia (peak titers on day 2) determined by a sensitive RT-qPCR method.
  • Fig. 50B Post-challenge LGT 1674 viremia (peak titers on day 2) determined by a sensitive RT-qPCR method.
  • Fig. 50B :
  • Fig. 51 is a schematic and table showing a design for a NHP study using the indicated constucts.
  • Fig. 52 is a schematic showing a short term (70 days) segment of a NHP study using the indicated constructs. Day 51 after immunization, animals will be administered a heterologous challenge with LGV 1674.
  • Fig. 53 is a schematic showing a long term (6 months+) segment of a NHP study using the indicated constructs. Month 6 after immunization, animals will be administered a heterologous challenge with LGV 1674.
  • Figs. 54A-D Post-challenge viremia using LGT 1 74 virus determined using plaque assay.
  • Fig. 54A Establishing the model using 6 log 10 PFU dose. To achieve better resolution of viremia, the dose was reduced to 5 logi 0 PFU for challenge of animals.
  • Fig. 54B Challenge in NHP study 1 (see Figs. 51 and 52). Only Mock animals showed viremia, while immunized monkeys in all other groups showed no detectabale viremia.
  • Fig. 54C Postchallenge viremia in the short-term segment of Study #2 (see Figs. 51 and 52).
  • Fig. 54d Postchallenge viremia in the long-term segment of Study #2 (see Figs. 51 and 53).
  • the invention provides replication-defective or deficient pseudoinfectious virus (PIV) vectors including flavivirus sequences, which can be used in methods for inducing immunity against heterologous pathogen, cancer, and allergy-related immunogens inserted into the vectors as well as, optionally, the vectors themselves.
  • PSV pseudoinfectious virus
  • the invention also includes compositions including
  • the invention includes particular PIVs and live, attenuated chimeric flaviviruses including tick-borne encephalitis virus sequences, and related vectors, compositions, and methods of use.
  • the PIV vectors, PIVs, live attenuated chimeric flaviviruses, compositions, and methods of the invention are described further below.
  • the PIV vectors and PIVs of the invention can be based on the single- or two-component PIVs described above (also see WO 2007/098267 and WO 2008/137163).
  • the PIV vectors and PIVs can include a genome including a large deletion in capsid protein encoding sequences and be produced in a complementing cell line that produces capsid protein in trans (single component; Fig. 1 and Figs. 12A and 12B). According to this approach, most of the capsid-encoding region is deleted, which prevents the PIV genome from producing infectious progeny in normal cell lines (i.e., cell lines not expressing capsid sequences) and vaccinated subjects.
  • the capsid deletion typically does not disrupt RNA sequences required for genome cyclization (i.e., the sequence encoding amino acids in the region of positions 1-26), and/or the prM sequence required for maturation of prM to M.
  • the deleted sequences correspond to those encoding amino acids 26-100, 26-93, 31-100, or 31-93 of the C protein.
  • Single component PIV vectors and PIVs can be propagated in cell lines that express either C or a C-prM-E cassette, where they replicate to high levels.
  • Exemplary cell lines that can be used for expression of single component PIV vectors and PIVs include BHK-21 (e.g., ATCC CCL-10), Vero (e.g., ATCC CCL-81), C7/10, and other cells of vertebrate or mosquito origin.
  • the C or C-prM-E cassette can be expressed in such cells by use of a viral vector-derived replicon, such as an alphavirus replicon (e.g., a replicon based on Venezuelan Equine Encephalitis virus (VEEV), Sindbis virus, Semliki Forest virus (SFV), Eastern Equine Encephalitis virus (EEEV), Western Equine Encephalitis virus (WEEV), or Ross River virus).
  • VEEV Venezuelan Equine Encephalitis virus
  • Sindbis virus Sindbis virus
  • Semliki Forest virus SFV
  • EEEV Eastern Equine Encephalitis virus
  • WEEV Western Equine Encephalitis virus
  • Ross River virus a viral vector-derived replicon
  • sequences encoding a complementing C protein can include an unnatural cyclization sequence.
  • the mutations can result from codon optimization, which can provide an additional benefit with respect to PIV yield.
  • the PIV vectors and PIVs of the invention can also be based on the two-component genome technology described above.
  • This technology employs two partial genome constructs, each of which is deficient in expression of at least one protein required for productive replication (capsid or prM/E) but, when present in the same cell, result in the production of all components necessary to make a PIV.
  • the first component includes a large deletion of C, as described above in reference to single component PIVs
  • the second component includes a deletion of prM and E (Fig. 2 and Fig. 12A).
  • the first component includes a deletion of C, prM, and E
  • the second component includes a deletion of NS1 (Fig. 12 A).
  • Both components can include cis-acting promoter elements required for RNA replication and a complete set of non-structural proteins, which form the replicative enzyme complex.
  • both defective genomes can include a 5 '-untranslated region and at least about 60 nucleotides (Element 1) of the following, natural protein-coding sequence, which comprises an ammo-terminal fragment of the capsid protein.
  • This sequence can be followed by a protease cleavage sequence such as, for example, a ubiquitine or foot-and-mouth disease virus (FAMDV)- specific 2
  • a protease sequence which can be fused with either capsid or envelope (prM-E) coding sequences.
  • d-PIV approaches that can be used in the invention are based on use of complementing genomes including deletions in NS3 or NS5 sequences.
  • a deletion in, e.g., NS1, NS3, or NS5 proteins can be used as long as several hundred amino acids in the ORF, removing the entire chosen protein sequence, or as short as 1 amino acid inactivating protein enzymatic activity (e.g., NS5 RNA polymerase activity, NS3 helicase activity, etc.).
  • point amino acid changes (as few as 1 amino acid mutation, or optionally more mutations) can be introduced into any NS protein, inactivating enzymatic activity.
  • several ANS deletions can be combined in one helper molecule.
  • the same heterologous gene i.e., expressed by the first d-PIV component, can be expressed in place or in combination with the NS deletion(s) in the second component, increasing the amount of expressed immunogen.
  • the insertion capacity of the helper will increase proportionally to the size of NS deletion(s).
  • a different foreign immunogen(s) can be inserted in place of deletion(s) of the helper to produce multivalent vaccines.
  • the PIV vectors and Pr s of the invention can be comprised of sequences from a single flavivirus type (e.g., tick-borne encephalitis (TBE, e.g., strain Hypr), Langat (LGT), yellow fever (e.g., YF17D), West Nile, Japanese encephalitis, dengue (serotype 1-4), St.
  • TBE tick-borne encephalitis
  • LGT Langat
  • yellow fever e.g., YF17D
  • West Nile e.g., Japanese encephalitis, dengue (serotype 1-4), St.
  • the sequences can be those of a chimeric flavivirus, as described above (also see, e.g., U.S. Patent No. 6,962,708; U.S. Patent No. 6,696,281; and U.S. Patent No. 6,184,024).
  • the chimeras include pre-membrane and envelope sequences from one flavivirus (such as a flavivirus to which immunity may be desired), and capsid and non-structural sequences from a second, different flavivirus.
  • the second flavivirus is a yellow fever virus, such as the vaccine strain YF17D.
  • LGT/TBE chimeras examples include the YF/TBE, YF/LGT, WN/TBE, and WN/LGT chimeras described below.
  • Another example is an LGT/TBE chimera based on LGT virus backbone containing TBE virus prM-E proteins.
  • a PIV vaccine based on this genetic background would have an advantage, because LGT replicates very efficiently in vitro and is highly attenuated and immunogenic for humans.
  • a chimeric LGT/TBE PIV vaccine is expected to provide a robust specific immune response in humans against TBE, particularly due to inclusion of TBE prM-E genes.
  • Vectors of the invention can be based on PIV constructs or live, attenuated chimeric fiaviviruses as described herein (in particular, YF/TBE, YF LGT, WN/TBE, and WN LGT; see below).
  • Use of PIV constructs as vectors provides particular advantages in certain circumstances, because these constructs by necessity include large deletions, which render the constructs more amenable to accommodation of insertions that are at least up to the size of the deleted sequences, without there being a loss in replication efficiency.
  • PIV vectors in general can comprise very small insertions (e.g., in the range 6-10, 11-20, 21-100, 101-500, or more amino acid residues combined with the AC deletion or other deletions), as well as relatively large insertions or insertions of intermediate size (e.g., in the range 501-1000, 1001-1700, 1701-3000, or 3001-4000 or more residues).
  • non-flavivirus immunogens in PIVs and chimeric flaviviruses of the invention can result in dual vaccines that elicit protective immunity against both a flavivirus vector virus pathogen and a target heterologous immunogen (e.g., a pathogen (such as a bacterial, viral, parasite, or fungal pathogen), cancer, or allergy-related immunogen).
  • a target heterologous immunogen e.g., a pathogen (such as a bacterial, viral, parasite, or fungal pathogen), cancer, or allergy-related immunogen).
  • the PIV vectors and PIVs of the invention can comprise sequences of chimeric flaviviruses, for example, chimeric flaviviruses including pre-membrane and envelope sequences of a first flavivirus (e.g., a flavivirus to which immunity is sought), and capsid and nonstructural sequences of a second, different flavivirus, such as a yellow fever virus (e.g., YF17D; see above and also U.S. Patent No. 6,962,708; U.S. Patent No. 6,696,281; and U.S. Patent No.
  • a yellow fever virus e.g., YF17D
  • chimeric flaviviruses of the invention used as a source for constructing PIVs, or as vaccines/vaccine vectors per se, can optionally include one or more specific attenuating mutations (e.g., E protein mutations, prM protein mutations, deletions in the C protein, and/or deletions in the 3TJTR), such as any of those described in WO 2006/116182.
  • the C protein or 3'UTR deletions can be directly applied to YF/TBE or YF/LGT chimeras. Similar deletions can be designed and introduced in other chimeric LAV candidates such as based on LGT/TBE, WN/TBE, and WN/LGT genomes.
  • Attenuating mutations similar to those described for YF/WN chimera in WO 2006/116182 can be designed, e.g., based on the knowledge of crystal structure of the E protein (Rey et al., Nature 375(6529):291-298, 1995), and employed. Further, additional examples of attenuating E protein mutations described for TBE virus and other flaviviruses are provided in Table 10. These can be similarly introduced into chimeric vaccine candidates.
  • the invention also provides new, particular chimeric flaviviruses, which can be used as a basis for the design of PrV vectors and PIVs, as live attenuated chimeric flavivirus vectors, and as vaccines against the source(s) of the pre-membrane and envelope components of the chimeras.
  • These chimeras include tick-borne encephalitis (TBE) virus or related prM-E sequences.
  • TBE tick-borne encephalitis
  • the chimeras can include prM-E sequences from, for example, the Hypr strain of TBE or Langat (LGT) virus.
  • Capsid and non-structural proteins of the chimeras can include those from yellow fever virus (e.g., YF17D) or West Nile virus (e.g., NY99).
  • a central feature of these exemplary YF/TBE, YF/LGT, WN/TBE, and WN/LGT chimeras is the signal sequence between the capsid and prM proteins. As is shown in the Examples, below, we have found that, in the case of YF-based PIV chimeras, it is advantageous to use a signal sequence comprising yellow fever and TBE sequences (see below).
  • the signal sequence includes yellow fever sequences in the amino terminal region (e.g., SHDVLTVQFLIL; SEQ ID NO:l) and TBE sequences in the carboxy terminal region (e.g., GMLGMTIA; SEQ ID NO:2), resulting in the sequence SHDVLTVQFLILGMLGMTIA (SEQ ID NO:3).
  • the invention thus includes YF/TBE, YF/LGT, WN/TBE, and WN/LGT chimeras, both PIVs and LAVs, which include the above-noted signal sequences, or variants thereof having, e.g., 1-8, 2-7, 3-6, or 4-5 amino acid substitutions, deletions, or insertions, which do not substantially interfere with processing at the signal sequence.
  • the substitutions are "conservative substitutions," which are characterized by replacement of one amino acid residue with another, biologically similar residue.
  • Examples of conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine, or methionine for another, or the substitution of one polar residue for another, such as between arginine and lysine, between glutamic and aspartic acids, or between glutamine and asparagine and the like.
  • Examples of exemplary PIVs of the present invention include those described in Appendices 6-8, constructs having at least 50% sequence identity (e.g., 50%, 60%, 70%, 85%, 90%, 95%, or 99% or more sequence identity) to the nucleic acid or amino acid sequences described therein, or constructs that include homologs and or other naturally occurring variants of the SIV, HIV, and/or HA proteins. Additional information concerning these and other chimeras is provided below, in the Examples.
  • Sequences encoding immunogens can be inserted at one or more different sites within the vectors of the invention.
  • Relatively short peptides can be delivered on the surface of PIV or LAV glycoproteins (e.g., prM, E, and or NS 1 proteins) and/or in the context of other proteins (to induce predominantly B-cell and T-cell responses, respectively).
  • inserts including larger portions of foreign proteins, as well as complete proteins, can be expressed intergenically, at the N- and C- termini of the polyprotein, or bicistronically (e.g., within the ORF under an IRES or in the 3'UTR under an IRES; see, e.g., WO 02/102828, WO 2008/036146, WO 2008/094674, WO 2008/100464, WO 2008/115314, and below for further details).
  • PIV constructs there is an additional option of inserting a foreign amino acid sequence directly in place of introduced deletion(s). Insertions can be made in, for example, AC, AprM-E, AC-prM-E, ANSI, ANS3, and ANS5.
  • imrnunogen-encoding sequences can be inserted in place of deleted capsid sequences.
  • Imrnunogen-encoding sequences can also, optionally, be inserted in place of deleted prM-E sequences in the AprM-E component of d-PIVs.
  • the sequences are inserted in place of or combined with deleted sequences in AC-prM-E constructs. Examples of such insertions are provided in the Examples section, below.
  • the insertions can be made with a few (e.g., 1, 2, 3, 4, or 5) additional vector-specific residues at the N- and/or C-termini of the foreign immunogen, if the sequence is simply fused in-frame (e.g., ⁇ 20 first a.a. and a few last residues of the C protein if the sequence replaces the AC deletion), or without, if the foreign immunogen is flanked by appropriate elements well known in the field (e.g., viral protease cleavage sites; cellular protease cleavage sites, such as signalase, furin, etc.; autoprotease; termination codon; and/or IRES elements).
  • appropriate elements well known in the field e.g., viral protease cleavage sites; cellular protease cleavage sites, such as signalase, furin, etc.; autoprotease; termination codon; and/or IRES elements.
  • a protein is expressed outside of the continuous viral open reading frame (ORF), e.g., if vector and non-vector sequences are separated by an internal ribosome entry site (IRES), cytoplasmic expression of the product can be achieved or the product can be directed towards the secretory pathway by using appropriate signal/anchor segments, as desired.
  • ORF continuous viral open reading frame
  • IVS internal ribosome entry site
  • cytoplasmic expression of the product can be achieved or the product can be directed towards the secretory pathway by using appropriate signal/anchor segments, as desired.
  • important considerations include cleavage of the foreign protein from the nascent polyprotein sequence, and maintaining correct topology of the foreign protein and all viral proteins (to ensure vector viability) relative to the ER membrane, e.g., translocation of secreted proteins into the ER lumen, or keeping cytoplasmic proteins or membrane-associated proteins in the cytoplasm in association with the ER membrane.
  • the above-described approaches to making insertions can employ the use of, for instance, appropriate vector-derived, insert-derived, or unrelated signal and anchor sequences included at the N and C termini of glycoprotein inserts.
  • appropriate vector-derived, insert-derived, or unrelated signal and anchor sequences included at the N and C termini of glycoprotein inserts can be used in place of all or a portion of the signal and/or anchor sequences for glycoprotein inserts (e.g., one or more of the SIV, HIV, or influenza virus proteins described herein) to produce a heterologous polypeptide sequence.
  • Standard autoproteases such as, for example, FMDV 2A autoprotease (-20 amino acids) or ubiquitin (gene ⁇ 500 nt), or flanking viral NS2B/NS3 protease cleavage sites can be used to direct cleavage of an expressed product from a growing polypeptide chain, to release a foreign protein from a vector polyprotein, and to ensure viability of the construct.
  • growth of the polyprotein chain can be terminated by using a termination codon, e.g., following a foreign gene insert, and synthesis of the remaining proteins in the constructs can be re-initiated by incorporation of an IRES element, e.g., the encephalomyocarditis virus (EMCV) IRES commonly used in the field of RNA virus vectors.
  • IRES element e.g., the encephalomyocarditis virus (EMCV) IRES commonly used in the field of RNA virus vectors.
  • Viable recombinants can be recovered from helper cells (or regular cells for d-PIV versions).
  • backbone PIV sequences can be rearranged, e.g., if the latter results in more efficient expression of a foreign gene.
  • a gene rearrangement has been applied to TBE virus, in which the prM-E genes were moved to the 3' end of the genome under the control of an IRES (Orlinger et al., J. Virol. 80:12197-12208, 2006).
  • Translocation of prM-E or any other genes can be applied to PIV flavivirus vaccine candidates and expression vectors, according to the invention.
  • Peptide sequences can be inserted within the envelope protein, which is the principle target for neutralizing antibodies.
  • the sequences can be inserted into the envelope in, for example, positions corresponding to amino acid positions 59, 207, 231, 277, 287, 340, and/or 436 of the Japanese encephalitis virus envelope protein (see, e.g., WO 2008/115314 and WO 02/102828).
  • the flavivirus sequences are aligned with that of Japanese encephalitis virus.
  • the site of insertion may vary by, for example, 1, 2, 3, 4, or 5 amino acids, in either direction.
  • the identification of such sites as being permissive in JE they can also vary in JE by, for example, 1, 2, 3, 4, or 5 amino acids, in either direction. Additional permissive sites can be identified using methods such as transposon mutagenesis (see, e.g., WO 02/102828 and WO 2008/036146).
  • the insertions can be made at the indicated amino acids by insertion just C-terminal to the indicated amino acids (i.e., between amino acids 51-52, 207-208, 231-232, 277-278, 287-288, 340-341, and 436-437), or in place of short deletions (e.g., deletions of 1, 2, 3, 4, 5, 6, 7, or 8 amino acids) beginning at the indicated amino acids (or within 1-5 positions thereof, in either direction).
  • short deletions e.g., deletions of 1, 2, 3, 4, 5, 6, 7, or 8 amino acids
  • insertions can be made into other virus proteins including, for example, the membrane/pre-membrane protein and NSl (see, e.g., WO 2008/036146).
  • insertions can be made into a sequence preceding the capsid/pre-membrane cleavage site (at, e.g., -4, -2, or -1) or within the first 50 amino acids of the pre-membrane protein (e.g., at position 26), and/or between amino acids 236 and 237 of NS1 (or in regions surrounding the indicated sequences, as described above).
  • insertions can be made intergenically.
  • an insertion can be made between E and NS 1 proteins and/or between NS2B and NS3 proteins (see, e.g., WO 2008/100464).
  • the inserted sequence can be fused with the C-terminus of the E protein of the vector, after the C-terminal signal/anchor sequence of the E protein, and the insertion can include a C-terminal anchor/signal sequence, which is fused with vector NS 1 sequences.
  • flanking protease cleavage sites e.g., YF 17D cleavage sites
  • flanking protease cleavage sites e.g., YF 17D cleavage sites
  • a sequence can be inserted in the context of an internal ribosome entry site (IRES, e.g., an IRES derived from encephalomyocarditis virus; EMCY), as noted above, such as inserted in the 3 '-untranslated region (WO 2008/094674).
  • IRES internal ribosome entry site
  • EMCY encephalomyocarditis virus
  • an IRES-immunogen cassette can be inserted into a multiple cloning site engineered into the 3 '-untranslated region of the vector, e.g., in a deletion (e.g., a 136 nucleotide deletion in the case of a yellow fever virus-based example) after the polyprotein stop codon (WO 2008/094674).
  • Example 3 Details concerning the insertion of rabies virus G protein and full-length respiratory syncytial virus (RSV) F protein into s-PIV and d-PIV vectors of the invention are provided below in Example 3.
  • the information provided in Example 3 can be applied in the context of other vectors and immunogens described herein.
  • PIVs s-PrVs and d-PIVs
  • flavivirus sequences and live, attenuated chimeric flaviviruses e.g., YF/TBE, YF/LGT, WN/TBE, and WN/LGT
  • foreign pathogen e.g., viral, bacterial, fungal, and parasitic pathogens
  • cancer e.g., cancer, and allergy-related immunogens.
  • allergy-related immunogens e.g., in certain examples, it may be advantageous to target several pathogens occupying the same ecological niche, in a particular geographical region. Specific, non-limiting examples of such immunogens are provided as follows.
  • PIVs of the invention such as PIVs including TBE/LGT sequences, as well as chimeric flaviviruses including TBE sequences (e.g., YF/TBE, YF/LGT, WN/TBE, LGT/TBE, and WN/LGT; in all instances where "TBE” is indicated, this includes the option of using the Hypr strain), can be used as vectors to deliver protective immunogens of the causative agent of Lyme disease (tick-borne spirochete Borrelia burgdorferi). This combination, targeting both infectious agents (TBE and B.
  • TBE and Lyme disease are both tick- borne diseases.
  • the PIV approaches can be applied to chimeras (e.g., YF/TBE, YF/LGT, WN/TBE, or WN/LGT), according to the invention, as well as to non-chimeric TBE and LGT viruses.
  • An exemplary immunogen from B. burgdorferi that can be used in the invention is OspA (Gipson et al., Vaccine 21:3875-3884, 2003).
  • OspA can be mutated to reduce chances of autoimmune responses and/or to eliminate sites for unwanted post- translational modification in vertebrate animal cells, such as N-linked glycosylation, which may affect immunogenicity of the expression product.
  • Mutations that decrease autoimmunity can include, e.g., those described by Willett et al., Proc. Natl. Acad. Sci. U.S.A. 101 : 1303-1308, 2004.
  • FTK-OspA a putative cross-reactive T cell epitope, Bb OspA ⁇ s-m (YVLEGTLTA; SEQ ID NO: 5) is altered to resemble the corresponding peptide sequence of Borrelia afzelli
  • FTKVAN SEQ ID NO:6
  • FTLEGKLTA SEQ ID NO:7
  • OspA The sequence of OspA is as follows (SEQ ID NO: 8):
  • the full-length sequence and/or immunogenic fragments of the full-length sequence can be used in the present invention.
  • Exemplary fragments can include one or more of domains 1 (amino acids 34- 41), 2 (amino acids 65-75), 3 (amino acids 190-220), and 4 (amino acids 250-270) (Jiang et al., Clin. Diag. Lab. Immun. 1(4):406-412, 1994).
  • a peptide comprising any one (or more) of the following sequences (which include sequence variations that can be included in the sequence listed above, in any combination) can be delivered (SEQ ID NOs:9-12): LPGE/GM/IK/T/GVL; GTSDKN/S/DNGSGV/T;
  • tick saliva proteins such as 64TRP, Isac, and Salp20
  • tick saliva proteins can be expressed, e.g., to generate a vaccine candidate of trivalent-specificity (TBE+Lyme disease+ticks).
  • tick saliva proteins can be expressed instead of B. burgdorferi immunogens in TBE sequence-containing vectors.
  • tick saliva proteins there are many other candidate tick saliva proteins that can be used for tick vector vaccine development according to the invention (Francischetti et al., Insect Biochem. Mol. Biol. 35:1142-1161, 2005).
  • One or more of these immunogens can be expressed in s-PIV-TBE.
  • d-PIV-TBE may also be selected, because of its large insertion capacity.
  • other PIV vaccines can be used as vectors, e.g., to protect from Lyme disease and another flavivirus disease, such as West Nile virus.
  • Immunogens of other pathogens can be similarly expressed, in addition to Lyme disease and tick immunogens, with the purpose of making multivalent vaccine candidates.
  • Exemplary tick saliva immunogens that can be used in the invention include the following:
  • TBE-related PIVs and LAVs Additional details concerning the TBE-related PIVs and LAVs are provided in Example 2, below.
  • the invention further provides PIV and LAV-vectored vaccines against other non-flavivirus pathogens, including vaccines having dual action, eliciting protective immunity against both flavivirus (as specified by the vector envelope proteins) and non-flavivirus pathogens (as specified by expressed immunologic determinant(s)).
  • vaccines having dual action eliciting protective immunity against both flavivirus (as specified by the vector envelope proteins) and non-flavivirus pathogens (as specified by expressed immunologic determinant(s)).
  • dual-action vaccines can be developed against a broad range of pathogens by expression of immunogens from, for example, viral, bacterial, fungal, and parasitic pathogens, and immunogens associated with cancer and allergy.
  • Example 3 we describe herein the design and biological properties of PIV vectored-rabies and -respiratory syncytial virus (RSV) vaccine candidates constructed by expression of rabies virus G protein or full-length RSV F protein in place of or in combination with various deletions in one- and two-component PIV vectors (see Example 3, below). Also described in Example 4 are SrV/HIV-based PIV vectors. Example 5 provides influenza virus HA-based PIV vectors.
  • s-PIV constructs may be advantageously used to stably deliver relatively short foreign immunogens (similar to Lyme disease agent OspA protein and tick saliva proteins), because insertions are combined with a relatively short AC deletion.
  • Two- component PIV vectors may be advantageously used to stably express relatively large immunogens, such as rabies G protein and RSV F, as the insertions in such vectors are combined with, for example, large AprM-E, AC-prM-E, and/or ANS 1 deletions.
  • Some of the d-PIV components can be manufactured and used as vaccines individually, for instance, the PIV-RSV F construct described below containing a AC-prM-E deletion.
  • the vaccine induces an immune response (e.g., neutralizing antibodies) predominantly against the expressed protein, but not against the flavivirus vector virus pathogen.
  • an immune response e.g., neutralizing antibodies
  • dual immunity is obtained by having immunity induced both to vector and insert components.
  • PIV vectors offer the opportunity to target several non-flavivirus pathogens simultaneously, e.g., by expressing foreign immunogens from two different non-flavivirus pathogens in the two components of a
  • foreign immunogens can be expressed to target respective diseases including, for example, influenza virus type A and B immunogens.
  • a few short epitopes and/or whole genes of viral particle proteins can be used, such as the M2, HA, and NA genes of influenza A, and/or the NB or BM2 genes of influenza B (see, e.g., the PIV constructs of Example 5 below).
  • Shorter fragments of M2, NB, and BM2, corresponding for instance to M2e, the extracellular fragment of M2, can also be used.
  • fragments of the HA gene including epitopes identified as HA0 (23 amino acids in length, corresponding to the cleavage site in HA) can be used.
  • Specific examples of influenza- related sequences that can be used in the invention include PAKLLKERGFFGAIAGFLE (HAO; SEQ ID NO: 16), P AKLLKERGFFG AIAGFLEGS GC (HAO; SEQ ID NO: 17),
  • NNATFNYTNVNPISHIRGS (NBe; SEQ ID NO: 18), MSLLTEVETPIRNE WGCRCNDS SD (M2e; SEQ ID NO: 19), MSLLTEVETPTRNEWECRCSDSSD (M2e; SEQ ID NO:20),
  • MSLLTEVETLTRNGWGCRCSDSSD (M2e; SEQ ID NO:21), EVETPTRN (M2e; SEQ ID NO:21),
  • M2e SLLTEVETPIRNEWGCR
  • Additional M2e sequences that can be used in the invention include sequences from the extracellular domain of BM2 protein of influenza B (consensus MLEPFQ (SEQ ID NO:25), e.g., LEPFQILSISGC (SEQ ID NO:26)), and the M2e peptide from the H5N1 avian flu (MSLLTEVETLTRNGWGCRCSDSSD; SEQ ID NO:27).
  • sequences can be inserted in combination within a vector as described herein and be separated by, e.g., autoprotease sequences, as described herein.
  • the invention includes, for example, vectors including combinations of the HIV sequences noted above (gag (55 kDa), gpl20, gpl40, gpl45, gp41, gpl60, SIV mac239 pol/-rev/tat/nef/pro genes or analogs or homologs and/or other naturally occurring variants from SIV and/or HIV, and other SIV and/or ⁇ immunogens; e.g., gpl20, gag, and/or pro).
  • these constructs can optionally employ heterologous TM and/or signal sequences, and are, optionally, codon- optimized.
  • pathogen immunogens include immunogens from HPV viruses, such as HPV16, HPV18, etc., e.g., the capsid protein LI which self-assembles into HPV-like particles, the capsid protein L2 or its immunodominant portions (e.g., amino acids 1-200, 1-88, or 17-36), the E6 and E7 proteins which are involved in transforming and immortalizing mammalian cells fused together and appropriately mutated (fusion of the two genes creates a fusion protein, referred to as E6E7Rb " , that is about 10-fold less capable of transforming fibroblasts, and mutations of the E7 component at 2 residues renders the resulting fusion protein mutant incapable of inducing transformation (Boursnell et al., Vaccine 14:1485-1494, 1996).
  • HPV viruses such as HPV16, HPV18, etc.
  • the capsid protein LI which self-assembles into HPV-like particles
  • immunogens include protective immunogens from HCV, CMV, HS V2, viruses, malaria parasite, Mycobacterium tuberculosis causing tuberculosis, C. difficile, and other nosocomial infections, that are known in the art, as well as fungal pathogens, cancer immunogens, and proteins associated with allergy that can be used as vaccine targets.
  • Foreign immunogen inserts of the invention can be modified in various ways. For instance, codon optimization is used to increase the level of expression and eliminate long repeats in nucleotide sequences to increase insert stability in the RNA genome of PIV vectors.
  • Immunogenicity can be increased by chimerization of proteins with immunostimulatory moieties well known in the art, such as TLR agonists, stimulatory cytokines, components of complement, heat-shock proteins, etc. (e.g., reviewed in "Immunopotentiators in Modern Vaccines," Schijns and O'Hagan Eds., 2006, Elsevier Academic Press: Amsterdam, Boston).
  • immunostimulatory moieties well known in the art, such as TLR agonists, stimulatory cytokines, components of complement, heat-shock proteins, etc.
  • non-flavivirus non-rabies signals for secretion, intracellular transport determinants, inclusion of or fusion with immunostimulatory moieties such as cytokines, TLR agonists such as flagellin, multimerization components such as leucine zipper, and peptides that increase the period of protein circulation in the blood
  • immunostimulatory moieties such as cytokines, TLR agonists such as flagellin, multimerization components such as leucine zipper, and peptides that increase the period of protein circulation in the blood
  • cytokines cytokines
  • TLR agonists such as flagellin
  • multimerization components such as leucine zipper
  • peptides that increase the period of protein circulation in the blood can be used to facilitate antigen presentation and increase immunogenicity.
  • such designs can be applied to s-PIV and d-PIV vaccine candidates based on vector genomes of other flaviviruses, and expressing immunogens of other pathogens, e.g., including but not limited
  • PIV and LAV vectors of the invention including combination vaccines such as DEN+Chikungunya virus (CHIKV) and YF+CHIKV.
  • CHIKV an alphavirus
  • CHIKV an alphavirus
  • It causes serious disease primarily associated with severe pain (arthritis, other symptoms similar to DEN) and long-lasting sequelae in the majority of patients (Simon et al., Med. Clin. North Am. 92:1323-1343, 2008; Seneviratne et al., J. Travel Med. 14:320-325, 2007).
  • PIV and LAV vectors of the invention include YF+Ebola or DEN+Ebola, which co-circulate in Africa.
  • Immunogens for the above-noted non-flavivirus pathogens may include glycoprotein B or a pp65/IEl fusion protein of CMV (Reap et al., Vaccine 25(42):7441-7449, 2007; and references therein), several TB proteins (reviewed in Skeiky et al., Nat. Rev. Microbiol. 4(6):469-476, 2006), malaria parasite antigens such as RTS,S (a pre- erythrocytic circumsporozoite protein, CSP) and others (e.g., reviewed in Li et al., Vaccine
  • the vectors described herein may include one or more immunogen(s) derived from or that direct an immune response against one or more viruses (e.g., viral target antigen(s)) including, for example, a dsDNA virus (e.g., adenovirus, herpesvirus, epstein-barr virus, herpes simplex type 1, herpes simplex type 2, human herpes virus simplex type 8, human cytomegalovirus, varicella-zoster virus, poxvirus); ssDNA virus (e.g., parvovirus, papillomavirus (e.g., El, E2, E3, E4, E5, E6, E7, E8, BPV1, BPV2, BPV3, BPV4, BPV5, and BPV6 ⁇ In Papillomavirus and Human Cancer, edited by H.
  • viruses e.g., viral target antigen(s)
  • viruses e.g., viral target antigen(s)
  • viruses e.g.
  • dsRNA viruses e.g., reovirus
  • (+)ssRNA viruses e.g., picomavirus, coxsackie virus, hepatitis A virus, poliovirus, togavirus, rubella virus, flavivirus, hepatitis C virus, yellow fever virus, dengue virus, west Nile virus
  • (+)ssRNA viruses e.g., orthomyxovirus, influenza virus, rhabdovirus, paramyxovirus, measles virus, mumps virus, parainfluenza virus, rhabdovirus, rabies virus
  • ssRNA-RT viruses e.g., retrovirus, human immunodeficiency virus (HIV)
  • dsDNA-RT viruses e.
  • immunogens may be selected from any HIV isolate.
  • HIV isolates are now classified into discrete genetic subtypes.
  • HIV-1 is known to comprise at least ten subtypes (A, B, C, D, E, F, G, H, J, and K).
  • HIV-2 is known to include at least five subtypes (A, B, C, D, and E).
  • Subtype B has been associated with the HIV epidemic in homosexual men and intravenous drug users worldwide.
  • Most HIV-1 immunogens, laboratory adapted isolates, reagents and mapped epitopes belong to subtype B.
  • HIV-1 subtype B In sub-Saharan Africa, India, and China, areas where the incidence of new HIV infections is high, HIV-1 subtype B accounts for only a small minority of infections, and subtype HIV-1 C appears to be the most common infecting subtype. Thus, in certain embodiments, it may be desirable to select immunogens from HIV-1 subtypes B and/or C. It may be desirable to include immunogens from multiple HIV subtypes (e.g., HIV-1 subtypes B and C, HIV-2 subtypes A and B, or a combination of HIV-1 and HIV-2 subtypes) in a single immunological composition. Suitable HIV immunogens include E V, GAG, PRO, POL, NEF, as well as variants, derivatives, and fusion proteins thereof, for example.
  • the invention includes constructs including multiple different proteins in a single precursor, wherein the open reading frames may be, optionally, separated by protease cleavage sites, such as FMDV 2A cleavage sites, as described herein.
  • a cassette may include gpl20 (e.g., modified as described in Example 4), gag, and pro genes from SIV or HIV.
  • the invention includes the hybrid sequences including, e.g., heterologous transmembrane and.or signal sequences, as described in detail in Example 4.
  • the invention includes the use of rabies virus G protien- specific signale and/or anchor sequences in the contect of gpl20-containing PIV constructs, as described herein.
  • Immunogens may also be derived from or direct an immune response against one or more bacterial species (spp.) (e.g., bacterial target antigen(s)) including, for example, Bacillus spp. (e.g., Bacillus anthracis), Bordetella spp. (e.g., Bordetella pertussis), Borrelia spp. (e.g., Borrelia burgdorferi), Brucella spp. (e.g., Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis), Campylobacter spp. (e.g., Campylobacter jejuni), Chlamydia spp.
  • Bacillus spp. e.g., Bacillus anthracis
  • Bordetella spp. e.g., Bordetella pertussis
  • Borrelia spp. e.g., Borrelia burgdorf
  • Clostridium spp. e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani
  • Corynebacterium spp. e.g., Corynebacterium diptheriae
  • Enterococcus spp. e.g., Enterococcus faecalis, enterococcus faecum
  • Escherichia spp. e.g., Escherichia coli
  • Haemophilus spp. e.g., Haemophilus influenza
  • Helicobacter spp. e.g., Helicobacter pylori
  • Legionella spp. e.g., Legionella pneumophila
  • Leptospira spp. e.g., Leptospira interrogans
  • Listeria spp. e.g., Listeria monocytogenes
  • Mycobacterium spp. e.g., Mycobacterium leprae, Mycobacterium tuberculosis
  • Mycoplasma spp. e.g., Mycoplasma pneumoniae
  • Neisseria gonorrhea Neisseria meningitidis
  • Pseudomonas spp. e.g., Pseudomonas aeruginosa
  • Rickettsia spp. e.g., Rickettsia rickettsii
  • Salmonella spp. e.g., Salmonella typhi, Salmonella typhinurium
  • Shigella spp. e.g., Shigella sonnei
  • Staphylococcus spp. e.g., Staphylococcus aureus,
  • Staphylococcus epidermidis Staphylococcus saprophyticus, coagulase negative staphylococcus (e.g., U.S. Patent No. 7,473,762)
  • Streptococcus spp. e.g., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyrogenes
  • Treponema spp. e.g., Treponema pallidum
  • Vibrio spp. e.g., Vibrio cholerae
  • Yersinia spp. Yersinia pestis
  • Immunogens may also be derived from or direct the immune response against other bacterial species not listed above but available to those of skill in the art.
  • Immunogens may also be derived from or direct an immune response against one or more parasitic organisms (spp.) (e.g., parasite target antigen(s)) including, for example, Ancylostoma spp. (e.g., A. duodenale), Anisakis spp., Ascaris lumbricoides, Balantidium coli, Cestoda spp., Cimicidae spp., Clonorchis sinensis, Dicrocoelium dendriticum, Dicrocoelium hospes, Diphyllobothrium latum, Dracunculus spp., Echinococcus spp. (e.g., E. granulosus, E. multilocularis), Entamoeba histolytica, Enterobius vermicularis, Fasciola spp. (e.g., F. hepatica, F. magna, F. gigantica, F.
  • Plasmodium spp. e.g., P. falciparum
  • Protofasciola robusta Parafasciolopsis fasciomorphae
  • Paragonimus westermani Schistosoma spp.
  • S. mansoni S. japonicum
  • S. mekongi S. haematobium
  • Spirometra erinaceieuropaei Strongyloides stercoralis
  • Taenia spp. e.g., T. saginata, T. solium
  • Toxocara spp. e.g., T. canis, T.
  • Immunogens may also be derived from or direct the immune response against other parasitic organisms not listed above but available to those of skill in the art.
  • Immunogens may be derived from or direct the immune response against tumor target antigens (e.g., tumor target antigens).
  • tumor target antigen may include both tumor- associated antigens (TAAs) and tumor-specific antigens (TSAs), where a cancerous cell is the source of the antigen.
  • TSA tumor-specific antigens
  • a TA may be an antigen that is expressed on the surface of a tumor cell in higher amounts than is observed on normal cells or an antigen that is expressed on normal cells during fetal development.
  • a TSA is typically an antigen that is unique to tumor cells and is not expressed on normal cells.
  • TAs are typically classified into five categories according to their expression pattern, function, or genetic origin: cancer-testis (CT) antigens (i.e., MAGE, NY-ESO-1); melanocyte differentiation antigens (e.g., Melan A/MART-1, tyrosinase, gplOO); mutational antigens (e.g., MUM-1, p53, CD -4); overexpressed 'self antigens (e.g., HER-2/neu, p53); and viral antigens (e.g., HPV, EBV).
  • CT cancer-testis
  • MAGE MAGE
  • NY-ESO-1 melanocyte differentiation antigens
  • mutational antigens e.g., MUM-1, p53, CD -4
  • overexpressed 'self antigens e.g., HER-2/neu, p53
  • viral antigens e.g., HPV, EBV
  • Suitable TAs include, for example, gplOO (Cox et al., Science 264:716-719, 1994), MART-l/Melan A (Kawakami et al., J. Exp. Med., 180:347-352, 1994), gp75 (TRP-1) (Wang et al., J. Exp. Med., 186:1131-1140, 1996), tyrosinase (Wolfel et al., Eur. J. Immunol., 24:759-764, 1994), NY-ESO-1 (WO 98/14464; WO 99/18206), melanoma proteoglycan (Hellstrom et al., J.
  • MAGE family antigens e.gl, MAGE-1, 2, 3, 4, 6, and 12; Van der Bruggen et al., Science 254: 1643-1647, 1991 ; U.S. Patent No. 6,235,525)
  • BAGE family antigens Boel et al., Immunity 2: 167-175, 1995
  • GAGE family antigens e.g., GAGE-1,2; Van den Eynde et al, J. Exp. Med. 182:689-698, 1995; U.S. Patent No.
  • RAGE family antigens e.g., RAGE-1; Gaugler et al., Immunogenetics 44:323-330, 1996; U.S. Patent No. 5,939,526), N- acetylglucosaminyltransferase-V (Guilloux et al., J. Exp. Med. 183:1173-1183, 1996), pl5 (Robbins et al., J. Immunol. 154:5944-5950, 1995), ⁇ -catenin (Robbins et al., J. Exp. Med., 183: 1185-1192, 1996), MUM-1 (Coulie et al., Proc. Natl.
  • carcinoma-associated mutated mucins e.g., MUC-1 gene products; Jerome et al., J. Immunol., 151:1654-1662, 1993
  • EBNA gene products of EBV e.g., EBNA-1 ; Rickinson et al, Cancer Surveys 13:53-80, 1992
  • E7 E6 proteins of human papillomavirus (Ressing et al., J. Immunol.
  • PSA prostate specific antigen
  • PSMA prostate specific membrane antigen
  • idiotypic epitopes or antigens for example, immunoglobulin idiotypes or T cell receptor idiotypes (Chen et al., J. Immunol. 153:4775-4787, 1994); SA (U.S. Patent No. 5,348,887), kinesin 2 (Dietz, et al., Biochem. Biophys. Res. Commun. 275(3):731-738,
  • Immunogens may also be derived from or direct the immune response against include TAs not listed above but available to one of skill in the art.
  • the invention also includes the use of analogs of the sequences.
  • analogs include sequences that are, for example, at least 80%, 90%, 95%, or 99% identical to the reference sequences, or fragments thereof.
  • the analogs also include fragments of the reference sequences that include, for example, one or more immunogenic epitopes of the sequences.
  • the analogs include truncations or expansions of the sequences (e.g., insertion of additional/repeat immunodominant/helper epitopes) by, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-20, etc., amino acids on either or both ends.
  • Truncation may remove immunologically unimportant or interfering sequences, e.g., within known structural/immunologic domains, or between domains; or whole undesired domains can be deleted; such modifications can be in the ranges 21-30, 31-50, 51-100, 101-400, etc. amino acids.
  • the ranges also include, e.g., 20-400, 30- 100, and 50-100 amino acids.
  • the invention also includes compositions including mixtures of two or more PIVs and/or PrV vectors, as described herein.
  • use of such mixtures or cocktails may be particularly advantageous when induction of immunity to more than one immunogen and/or pathogen is desired. This may be useful, for example, in vaccination against different flaviviruses that may be endemic to the region in which the vaccine recipient resides. This may also be useful in the context of administration of multiple immunogens against the same target.
  • Non-limiting examples of Pr cocktails included in the invention are those including PIV-JE
  • the PIVs for either or both components can be single or dual component PIVs, as described above.
  • the PIV-DEN can include sequences of just one dengue serotype selected from the group consisting of dengue serotypes 1-4, or the cocktail can include PIVs expressing sequences from two, three, or all four of the serotypes.
  • the ⁇ /Borrelia burgdorferi/tick saliva protein (e.g., 64TRP, Isac, Salp20) vaccines described herein can be based on including the different immunogens within a single PIV or live attenuated flavivirus, or can be based on mixtures of PIVs (or LAVs), which each include one or more of the immunogens.
  • the cocktails of the invention can be formulated as such or can be mixed just prior to administration. Use, Formulation, and Administration
  • the invention includes the PIV vectors, PIVs, LAV vectors, and LAVs, as well as corresponding nucleic acid molecules, pharmaceutical or vaccine compositions, and methods of their use and preparation.
  • the PIV vectors, PIVs, LAV vectors, and LAVs of the invention can be used, for example, in vaccination methods to induce an immune response to TBE or other flavivirus, and/or another expressed immunogen, as described herein. These methods can be prophylactic, in which case they are carried out on subjects (e.g., human subjects or other mammalian subjects) not having, but at risk of developing infection or disease caused by TBE or another flavivirus and/or a pathogen from which the other expressed immunogen is derived.
  • the methods can also be therapeutic, in which they are carried out on subjects already having an infection by one or more of the relevant pathogens.
  • the viruses and vectors can be used individually or in combination with one another or other vaccines.
  • the subjects treated according to the methods of the invention include humans, as well as non-human mammals (e.g., livestock, such as, cattle, pigs, horses, sheep, and goats, and domestic animals, including dogs and cats).
  • Formulation of the PIV vectors, PIVs, LAV vectors, and LAVs of the invention can be carried out using methods that are standard in the art. Numerous pharmaceutically acceptable solutions for use in vaccine preparation are well known and can readily be adapted for use in the present invention by those of skill in this art (see, e.g., Remington 's Pharmaceutical Sciences (18 th edition), ed. A. Gennaro, 1990, Mack Publishing Co., Easton, PA). In two specific examples, the PIV vectors, PIVs, LAV vectors, and LAVs are formulated in Minimum Essential Medium Earle's Salt (MEME) containing 7.5% lactose and 2.5% human serum albumin or MEME containing 10% sorbitol. However, the PIV vectors, PIVs, LAV vectors, and LAVs can simply be diluted in a physiologically acceptable solution, such as sterile saline or sterile buffered saline.
  • MEME Minimum Essential Medium Earle's Salt
  • the PIV vectors, PIVs, LAV vectors, and LAVs of the invention can be administered using methods that are well known in the art, and appropriate amounts of the viruses and vectors to be administered can readily be determined by those of skill in the art. What is determined to be an appropriate amount of virus to administer can be determined by consideration of factors such as, e.g., the size and general health of the subject to whom the virus is to be administered.
  • the viruses can be formulated as sterile aqueous solutions containing between 10 2 and 10 8 , e.g., 10 3 to 10 7 , infectious units (e.g., plaque- forming units or tissue culture infectious doses) in a dose volume of 0.1 to 1.0 ml.
  • infectious units e.g., plaque- forming units or tissue culture infectious doses
  • PIVs can be administered at similar doses and in similar volumes; PIV titers however are usually measured in, e.g., focus-forming units determined by immunostaining of foci, as these defective constructs tend not to form virus-like plaques.
  • Doses can range between 10 2 and 10 8 FFU and administered in volumes of 0.1 to 1.0 ml.
  • All viruses and vectors of the invention can be administered by, for example, intradermal, subcutaneous, intramuscular, intraperitoneal, or oral routes.
  • dendritic cells are targeted by intradermal or transcutaneous administration, by use of, for example, microneedles or microabrasion devices.
  • the vaccines of the invention can be administered in a single dose or, optionally, administration can involve the use of a priming dose followed by a booster dose that is administered, e.g., 2-6 months later, as determined to be appropriate by those of skill in the art.
  • PIV vaccines can be administered via DNA or RNA immunization using methods known to those skilled in the art (Chang et al., Nat. Biotechnol. 26:571-577, 2008; Kofler et al., Proc. Natl. Acad. Sci. U.S.A. 101:1951-1956, 2004).
  • adjuvants that are known to those skilled in the art can be used in the
  • Adjuvants that can be used to enhance the immunogenicity of the viruses include, for example, liposomal formulations, synthetic adjuvants, such as (e.g., QS21), muramyl dipeptide, monophosphoryl lipid A, polyphosphazine, CpG oligonucleotides, or other molecules that appear to work by activating Toll-like Receptor (TLR) molecules on the surface of cells or on nuclear membranes within cells.
  • TLR Toll-like Receptor
  • these adjuvants are typically used to enhance immune responses to inactivated vaccines, they can also be used with live or replication-defective vaccines. Both agonists of TLRs or antagonists may be useful in the case of live or replication-defective vaccines.
  • the vaccine candidates can be designed to express TLR agonists.
  • mucosal adjuvants such as the heat-labile toxin of E. coli (LT) or mutant derivations of LT can be used as adjuvants.
  • genes encoding cytokines that have adjuvant activities can be inserted into the vaccine candidates.
  • genes encoding desired cytokines such as GM-CSF, IL- 2, IL-12, IL-13, IL-5, etc.
  • desired cytokines such as GM-CSF, IL- 2, IL-12, IL-13, IL-5, etc.
  • foreign immunogen genes can be inserted together with foreign immunogen genes to produce a vaccine that results in enhanced immune responses, or to modulate immunity directed more specifically towards cellular, humoral, or mucosal responses (e.g., reviewed in "Immunopotentiators in Modern Vaccines", Schijns and O'Hagan Eds., 2006, Elsevier Academic Press: Amsterdam,
  • a patch containing a layer of an appropriate toxin-derived adjuvant can be applied over the injection site.
  • Toxin promotes local inflammation attracting lymphocytes, which leads to a more robust immune response.
  • the C/prM junction is an important location in the flavivirus polyprotein orchestrating the formation of viral envelope and synthesis of viral proteins (Yamshchikov and Compans, Virology 192:38-51, 1993; Amberg and Rice, J. Virol. 73:8083-8094, 1999; Stocks and Lobigs, J. Virol. 72:2141-2149, 1998).
  • cleavage efficiency can be achieved by analysis of specific amino acid substitutions near the cleavage site with SignalP 3.0 (e.g., as described in application WO 2008/ 100464), followed by incorporation of chosen mutation(s) into PIV genomes, recovery of PIV progeny and measuring VLP secretion.
  • Non-fiavivirus signals are inserted by methods standard in the art.
  • Uncoupling between the viral protease and signalase cleavages can be achieved by ablating the viral cleavage site by any non-conservative mutation (e.g., RRS in YF17D C to RRA or GRS or RSS, etc.), or deletion of the entire site or some of its 3 residues.
  • formation of free N-terminus of the signal of foreign protein can be achieved by using such elements as autoprotease, or termination codon followed by an IRES.
  • the native AUG initiation codon of C can be ablated (in constructs where C protein sequence is unnecessary, e.g., AC PIV) and AUG placed in front of foreign gene.
  • Optimization of vector signal can be performed by random mutagenesis, e.g., by insertion of synthetic randomized sequence followed by identification of viable PIV variants with increased VLP secretion.
  • PIV constructs were substantially more immunogenic in hamsters when administered by the IP route, as compared to the subcutaneous route. We concluded that this was most likely due to better targeting of antigen presenting cells in lymphoid tissues, which are abundant in the abdomen, but not abundant in tissues underlying the skin. Based on these observations, we concluded that efficient targeting of PIVs to dendritic cells, abundant in the skin, can be achieved by cutaneous inoculation, e.g., via skin microabrasion or intradermal injection using microneedles (Dean et al., Hum Vaccin. 1: 106-111, 2005).
  • cocktail PIV vaccines are feasible.
  • Such formulations may be of particular significance in geographical regions where different flaviviruses co-circulate. This could be also used to simultaneously administer several PIV-based vaccines against non-flavivirus pathogens.
  • s-PIV-WN based on wt WN virus strain NY99 sequences
  • s-PIV-JE based on wt WN virus backbone and prM-E genes from wt JE virus Nakayama strain
  • s-PIV-YF/WN YF 17D backbone and prM-E genes from WN virus
  • s-PIV-YF based on YF 17D sequences
  • Additional materials include d-PIV-YF (YF d-PIV, grown in regular BHK cells (Shustov et al., J. Virol. 21:11737-11748, 2007), and two-component d-PIV- WN (grown in regular Vero cells; Suzuki et al., J. Virol. 82:6942-6951, 2008).
  • Attenuation of these PIV prototypes was compared to LAVs YF 17D, a chimeric YF/JE virus, and a chimeric YF/WN virus in suckling mouse NV test (IC inoculation) using highly susceptible 5 -day old ICR mice (the chimeric viruses include yellow fever capsid and non-structural sequences, and JE or WN prM-E sequences). None of the animals that received PIV constructs showed clinical signs or died, while mortality was observed in animals inoculated with LAVs (Table 2).
  • the YF 17D virus is neurovirulent for mice of all ages, while the chimeric vaccines are not neurovirulent for adult mice, but can cause dose-dependent mortality in more sensitive suckling mice (Guirakhoo et al., Virology 257:363-372, 1999; Arroyo et al., J. Virol. 78: 12497-12507, 2004). Accordingly, 90%- 100% of suckling mice that received doses as low as 1 PFU of YF 17D died. YF/JE and YF WN LAVs caused partial mortality at much higher doses (> 2 log 10 PFU and 3 log 10 PFU, respectively), with longer average survival time (AST) of animals that died, as expected.
  • AST average survival time
  • PIV constructs are completely avirulent in this sensitive model (at least 20,000 - 200,000 times less neurovirulent than the licensed YF 17D vaccine).
  • the YF d-PIV and WN d-PIV caused no mortality or clinical signs.
  • the two- component PIV variants that theoretically could spread within brain tissue from cells co-infected by both of their components did not cause disease.
  • we tried to detect the d-PIVs in the brains of additional animals in this experiment sacrificed on day 6 post-inoculation by titration, and detected none (brain tissues from 10 and 11 mice that received 4 log 10 FFU of YF d-PIV and WN d- PIV, respectively, were homogenized and used for titration).
  • the d-PFVs did not cause spreading infection characteristic of whole virus.
  • YF/JE LAV has been shown to replicate in the brain of adult ICR mice inoculated by the IC route with a peak titer of ⁇ 6 log 10 PFU/g on day 6, albeit without clinical signs (Guirakhoo et al., Virology 257:363-372, 1999).
  • Co-infection of cells with components of a d-PIV is clearly a less efficient process than infection with whole virus.
  • the data show that d-PIV replication in vivo is quickly brought under control by innate immune responses (and adaptive responses in older animals).
  • Neutralizing antibody responses were determined in animal sera by standard PRNT 50 against YF/WN or /JE LAVs, or YF 17D viruses.
  • PIV-WN induced very high WN-specific neutralizing antibody responses in all groups, with or without boost, as evidenced by PRNT 50 titers determined in pools of sera from immunized animals on days 20 and 34, which was comparable to that in the YF/WN LAV control group. Accordingly, animals immunized with both PIV-WN and YF/WN LAV were protected from lethal challenge on day 35 with wt WN virus (IP, 270 LD 50 ), but not mock-immunized animals (Table 3).
  • PIV-JE was also highly immunogenic (black mice), while immunogenicity of PIV-YF was significantly lower compared to the YF 17D control (ICR mice). Yet, dose-dependent protection of PIV-YF immunized animals (but not mock-immunized animals) was observed following a severe lethal IC challenge with wt YF strain Asibi virus (500 LD 50 ) (Table 3), which is in agreement with the knowledge that neutralizing antibody titers as low 1 : 10 are protective against flavivirus infections.
  • the YF 17D control virus was highly immunogenic (e.g., PRNT 50 titer 1:1,280 on day 34), and thus it is able to infect cells and replicate efficiently in vivo, and its envelope is a strong immunogen. Therefore, it is unlikely that low immunogenicity of PIV-YF was due to its inability to infect cells or replicate efficiently in infected cells in vivo.
  • the low immunogenicity of PIV-YF e.g., compared to PIV-W
  • immunogenicity of PIV-YF can be significantly increased, e.g., by appropriate modifications at the C/prM junction, e.g., by uncoupling the two protease cleavages that occur at this junction (viral protease and signalase cleavages), and/or by using a strong heterologous signal [e.g., rabies virus G protein signal, or eukaryotic tissue plasminogen activator (tPA) signal (Malin et al., Microbes and Infection, 2:1677-1685, 2000), etc.] in place of the YF signal for prM.
  • a strong heterologous signal e.g., rabies virus G protein signal, or eukaryotic tissue plasminogen activator (tPA) signal (Malin et al., Microbes and Infection, 2:1677-1685, 2000, etc.
  • PIV-JE and -YF induced detectable specific neutralizing antibody responses, albeit with lower titers compared to YF/JE LAV and YF 17D controls.
  • All animals immunized with PIV-WN and YF/WN were solidly protected from lethal challenge with wt WN virus as evidenced by the absence of mortality and morbidity (e.g., loss of body weight after challenge), as well as absence or a significant reduction of postchallenge WN virus viremia. Mock-immunized animals were not protected (Table 4).
  • PIV-JE and -WN protected animals from respective challenge in dose-dependent fashion. Protective efficacy in this experiment is additionally illustrated in Fig. 5.
  • viremia was observed in mock immunized animals, peaking on day 3 at a titer of> 8 logio PFU/ml (upper left panel); all of the animals lost weight, and 1 out of 4 died (upper right panel).
  • viremia was significantly reduced or absent in hamsters immunized with PIV- YF (two doses; despite relatively low neutralizing titers) or YF 17D; none of these animals lost weight.
  • mice were immunized with PIV constructs by the IP route, with two doses.
  • Table 5 compares neutralizing immune responses (specific for each vaccine) determined in pooled sera of hamsters in the above-described experiment (SC inoculation) to those after IP immunization, for PIV-WN, - YF/WN, -WN/JE, and-YF after the first dose (days 20-21) and second dose (days 34-38).
  • a clear effect of the immunization route was observed both after the 1 st and 2 nd doses.
  • PIV vaccines can be efficiently administered as cocktails, inducing immunity against two or more flavivirus pathogens.
  • various cocktails can be made between non-flavivirus PIV vaccines, or between any of flavivirus and non-flavivirus PIV vaccines.
  • PIVs or in regular Vero cells, for d-PFVs.
  • Samples harvested after each passage were titrated in Vero cells by immunostaining. Constructs grew to high titers, and no recombination restoring whole virus was observed.
  • PIV-WN consistently grew to titers 7-8 log ]0 FFU/ml in BHK- CprME(WN) helper cells (containing a VEE replicon expressing the WN virus C-prM-E proteins), and WN d-PrV grew to titers exceeding 8 log 10 FFU/ml in Vero cells, without recombination.
  • PIV-TBE vaccine candidates can be assembled based entirely on sequences from wt TBE virus or the closely serologically related Langat (LGT) virus (naturally attenuated virus, e.g., wt strain TP-21 or its empirically attenuated variant, strain E5), or based on chimeric sequences containing the backbone (capsid and non-structural sequences) from YF 17D or other flaviviruses, such as WN virus, and the prM-E envelope protein genes from TBE, LGT, or other serologically related flaviviruses from the TBE serocomplex.
  • YF/TBE LAV candidates are constructed based on the backbone from YF 17D and the prM-E genes from TBE or related viruses (e.g., the E5 strain of LGT), similar to other chimeric LAV vaccines.
  • Plasmids for PIV-WN (Mason et al., Virology 351 :432-443, 2006; Suzuki et al., J. Virol. 82:6942-6951, 2008), or plasmids for chimeric LAVs (e.g., pBSA-ARl, a single-plasmid version of infectious clone of YF/JE LAV; WO 2008/036146), respectively, using standard methods in the art of reverse genetics.
  • the prM-E sequences of TBE virus strain Hypr (GenBank accession number U39292) and LGT strain E5 (GenBank accession number AF253420) were first computer codon-optimized to conform to the preferential codon usage in the human genome, and to eliminate nucleotide sequence repeats longer than 8 nt to ensure high genetic stability of inserts (if determined to be necessary, further shortening of nt sequence repeats can be performed).
  • the genes were chemically synthesized and cloned into plasmids for PIV-WN and YF/JE LAV, in place of corresponding prM-E genes. Resulting plasmids were in vitro transcribed and appropriate cells (V ero for chimeric viruses, and helper BH cells for PIV) were transfected with RNA transcripts to generate virus/PIV samples.
  • E5 (plasmid P43) prM-E genes two different types of the C/prM junction were first examined (see in Fig. 6; C/prM junctions only are shown in Sequence Appendix 1, and complete 5'-terminal sequences covering the 5'UTR-C-prM-E-beginning of NSl region are shown in Sequence Appendix 2).
  • the p42 -derived YF17D/Hypr chimera contained a hybrid YF17D/Hypr signal peptide for the prM protein, while the p45-derived YF17D/Hypr chimera contained a hybrid YF17D/WN signal peptide for prM (Sequence Appendix 1).
  • the former chimeric virus produced very high titers at both P0 (immediately after transfection) and PI (the next passage in Vero cells), up to 7.9 log 10 PFU/ml, which were 0.5 log 10 times higher, compared to the latter virus; in addition it formed significantly larger plaques in Vero cells (Fig. 6).
  • TBE-specific residues in the signal peptide for prM conferred a significant growth advantage over the signal containing WN-specific residues.
  • the p43-derived YF17D/LGT chimera had the same prM signal as the p42 -derived virus; it also produced very high titers at P0 and PI passages (up to 8.1 log 10 PFU/ml) and formed large plaques.
  • a derivative of the p42-derived virus was also produced from plasmid p59, which contained a strong attenuating mutation characterized previously in the context of a YF/WN LAV vaccine virus, specifically, a 3 -a. a. deletion in the YF17D-specific C protein (PSR, residues 40-42 in the beginning of a-Helix I; WO 2006/116182).
  • the p59 virus grew to lower titers (5.6 and 6.5 logio PFU/ml at P0 and PI, respectively), and formed small plaques (determined in a separate titration experiment and thus not shown in Fig. 6), compared to the parent p42-derived chimera.
  • PIV-WN/TBE variants were constructed, and packaged PIV samples were derived from plasmids p39 and p40 (Figs. 7A-B; Sequence Appendix 1 for C/prM junction sequences, and Sequence Appendix 3 for complete 5'UTR-AC-prM-E-beginning of NS1 sequences). These contained complete Hypr or WN prM signals, respectively. Both PIVs were successfully recovered and propagated in BHK-CprME(W ) or BHK-C(WN) helper cells (Mason et al., Virology 351:432- 443, 2006; Widman et al., Vaccine 26:2762-2771, 2008).
  • the P0 and PI sample titers of the p39 variant were 0.2 - 1.0 log 10 times, higher than p40 variant.
  • Vero cells infected with p39 variant were stained brighter in immunofluorescence assay using a polyclonal TBE-specific antibody, compared to p40, indicative of more efficient replication (Fig. 7A).
  • the higher rate of replication of the p39 candidate than p40 candidate was confirmed in a growth curve experiment (Fig. 8).
  • the invention also includes the use of other flavivirus signals, including with appropriate mutations, the uncoupling the viral protease and signalase cleavages at the C/prM junction, e.g., by mutating or deleting the viral protease cleavage site at the C-terminus of C preceding the prM signal, the use of strong non-flavivirus signals (e.g., tPA signal, etc.) in place of prM signal, as well as optimization of sequences downstream from the signalase cleavage site.
  • flavivirus signals including with appropriate mutations, the uncoupling the viral protease and signalase cleavages at the C/prM junction, e.g., by mutating or deleting the viral protease cleavage site at the C-terminus of C preceding the prM signal, the use of strong non-flavivirus signals (e.g., tPA signal, etc.) in place of prM signal, as well as
  • PIV-TBE variants based entirely on wt TBE (Hypr strain) and LGT virus (TP21 wild type strain or attenuated E5 strain), and chimeric YF 17D backbone/prM-E (TBE or LGT) sequences are also included in the invention.
  • Helper cells providing appropriate C, C-prM-E, etc., proteins (e.g., TBE-specific) for trans-complementation can be constructed by means of stable DNA transfection or through the use of an appropriate vector, e.g., an alphavirus replicon, such as based on VEE strain TC-83, with antibiotic selection of replicon-containing cells.
  • Vero and BHK21 cells can be used in practice of the invention.
  • the former are an approved substrate for human vaccine manufacture; any other cell line acceptable for human and/or veterinary vaccine manufacturing can be also used.
  • d-PIV constructs can also be assembled. To additionally ascertain safety for vaccinees and the environment, appropriate modifications can be employed, including the use of degenerate codons and complementary mutations in the 5' and 3' CS elements, to minimize chances of recombination that theoretically could result in viable virus.
  • mice inoculated IC with YF 17D control (1 - 3 log 10 PFU) showed dose-dependent mortality, while all animals inoculated TP (5 log 10 PFU) survived, in accord with the knowledge that YF 17D virus is not neuroinvasive. All animals that received graded IC doses (2 - 4 logio PFU) of YF/TBE LAV prototypes p42, p45, p43, and p59 died (moribund animals were humanely euthanized). These variants appear to be less attenuated than YF 17D, e.g., as evidenced by complete mortality and shorter AST at the 2 log 10 PFU dose, the lowest dose tested for YF/TBE LAV candidates.
  • Fig. 9 The non- neuro virulent phenotype of PIV-TBE, virulent phenotype of YF/TBE LAV and intermediate- virulence phenotype of YF 17D are also illustrated in Fig. 9, showing survival curves of mice after IC inoculation. It should be noted that the p43 (LGT prM-E genes) and p59 (the dC2 deletion variant of YF/Hypr LAV) were less neurovirulent than p42 and p45 YF Hypr LAV constructs as evidenced by larger AST values for corresponding doses (Table 7; Fig. 45).
  • TBE-specific neutralizing antibody responses in mice immunized IP with one or two doses of the PIV-TBE or YF/TBE LAV variants described above, or a human formalin-inactivated TBE vaccine control (1 :30 of human dose) are being measured.
  • Animals have been challenged with a high IP dose (500 PFU) of wt Hypr TBE virus; morbidity (e.g., weight loss), and mortality after challenge are monitored.
  • Titers in individual test samples as well as GMTs for each group are provided in Tables 8 and 9. Titers in test samples were similar within each group, e.g., in groups immunized with PIVs, indicating high uniformity of immune response in animals. As expected, no TBE-specific neutralizing antibodies were detected in negative control groups (YF 17D and Mock; GMTs ⁇ 1:10); accordingly, animals in these groups were not protected from challenge on day 21 post-immunization with a high ⁇ dose (500 PFU) of wt Hypr TBE virus. Mortalities from partial observation (on day 9 post-challenge; observation being continued) are provided in Tables 8 and 9, and dynamics of average post-challenge body weights indicative of morbidity are shown in Fig. 11.
  • Neutralizing antibodies were detected in killed vaccine controls, which were particularly high after two doses (GMT 1 : 1 ,496); animals in the 2-dose group were completely protected in that there was no mortality or body weight loss (but not animals in the 1- dose group). Animals that received both one and two doses of PIV-Hypr p39 had very high antibody titers (GMTs 1 :665 and 1 : 10,584) and were solidly protected, demonstrating that robust protective immunity can be induced by s-PIV-TBE defective vaccine. The two animals that survived immunization with YF/Hypr p42 chimera (see in Table 7; see also Fig.
  • genes of interest were codon optimized (e.g., for efficient expression in a target vaccination host) and to eliminate long nt sequence repeats to increase insert stability (> 8 nt long; additional shortening of repeats can be performed if necessary), and then chemically synthesized.
  • the genes were cloned into PIV-WN vector plasmids using standard methods of molecular biology well known in the art, and packaged PIVs were recovered following in vitro transcription and transfection of appropriate helper (for s- PIVs) or regular (for d-PIVs) cells.
  • Rabies virus Rhabdoviridae family
  • Rabies virus is a significant human and veterinary pathogen.
  • Rabies virus glycoprotein G mediates entry of the virus into cells and is the main immunogen. It has been expressed in other vectors with the purpose of developing veterinary vaccines (e.g., Pastoret and Brochier, Epidemio. Infect. 1 16:235-240, 1996; Li et al., Virology 356: 147-154, 2006).
  • Full length rabies virus G protein (original Pasteur virus isolate, GenBank accession number NC 001542) was codon-optimized, chemically synthesized, and inserted adjacent to the AC, AprM-E and AC-prM-E deletions in PIV-WN vectors (Figs. 12A and 12B).
  • the sequences of constructs are provided in Sequence Appendix 4. General designs of the constructs are illustrated in Figs. 12 and 13.
  • the entire G protein containing its own signal peptide was inserted in- frame downstream from the WN C protein either with the AC deletion (AC and AC-prM-E constricts) or without (AprM-E) and a few residues from the prM signal.
  • FMDV 2 A autoprotease was placed downstream from the transmembrane C-terminal anchor of G to provide cleavage of C-terminus of G from the viral polyprotein during translation.
  • the FMDV 2A element is followed by WN-specific signal for prM and prM-E-NS 1-5 genes in the AC construct, or signal for NS1 and NS1-5 genes in AprM-E and AC-prM-E constructs.
  • WN(AC)-rabiesG, WN(AprME)-rabiesG, and WN(ACprME)-rabiesG PIVs were produced by transfection of helper BHK cells complementing the PIV vector deletion [containing a Venezuelan equine encephalitis virus (strain TC-83) replicon expressing WN virus structural proteins for trans-complementation].
  • rabies G protein Efficient replication and expression of rabies G protein was demonstrated for the three constructs by transfection/infection of BHK-C(WN) and/or BHK-C-prM-E(WN) helper cells, as well as regular BHK cells, by immunostaining and immunofluorescence assay (IF A) using anti- Rabies G monoclonal antibody (RabG-Mab) (Fig. 14). Titers were determined in Vero cells by immunostaining with the Mab or an anti-WN virus polyclonal antibody. Growth curves of the constructs in BHK-CprME(WN) cells after transfection with in vitro RNA transcripts are shown in Fig. 14, bottom panels.
  • VSV Vesicular stomatitis virus
  • SFV Semliki Forest virus
  • the stability of the rabies G insert in the three PIVs was demonstrated by serial passages in helper BHK-CprME(WN) cells at high or low MOI (0.1 or 0.001 FFU/cell). At each passage, cell supernatants were harvested and titrated in regular cells (e.g., Vero cells) using immunostaining with an anti-WN polyclonal antibody to determine total PIV titer, or anti-rabies G monoclonal antibody to determine titer of particles containing the G gene (illustrated for MOI 0.1 in Fig. 17; similar results were obtained at MOI 0.001).
  • regular cells e.g., Vero cells
  • the WN(AC)-rabiesG Pr was stable for 5 passages, while the titer of insert-containing PIV started declining at passage 6, indicative of insert instability. This could be expected, because in this construct, large G gene insert ( ⁇ 1500 nt) is combined with a small AC deletion ( ⁇ 200 nt), significantly increasing the overall size of the recombinant RNA genome. In contrast, in WN(AprME)-rabiesG, and
  • the WN(AC)-rabiesG s-PIV is expected to induce strong neutralizing antibody immune responses against both rabies and WN viruses, as well as T-cell responses.
  • the WN(AprME)-rabiesG and WN(ACprME)-rabiesG PIVs will induce humoral immune response only against rabies because they do not encode the WN prM-E genes.
  • WN(AC)-rabiesG s-PrV construct can be also co-inoculated with WN(AprME)-rabiesG construct in a d-PIV formulation (see in Figs. 12A and 12B), increasing the dose of expressed G protein, and with enhanced immunity against both pathogens due to limited spread.
  • titration results in Vero cells of a s-PIV sample, WN(AprME)-rabiesG, and a d-PIV sample, WN(AprME)-rabiesG + WN(AC) PIV are shown in Fig. 18.
  • Infection of naive Vero cells with s-PIV gave only individual cells stainable with RabG-Mab (or small clusters formed due to division of cells).
  • large foci were observed following infection with the d-PIV sample (Fig. 18, right panel) that were products of coinfection with the two PIV types.
  • the WN(ACprME)-rabiesG construct can be also used in a d-PIV formulation, if it is co- inoculated with a helper genome providing C-prM-E in trans (see in Figs. 12A and 12B).
  • a helper genome providing C-prM-E in trans see in Figs. 12A and 12B.
  • it can be a WN virus genome containing a deletion of one of the NS proteins, e.g., NS1, NS3, or NS5, which are known to be trans-complementable (Khromykh et al., J. Virol. 73:10272-10280, 1999;
  • rabies G protein can be also inserted and expressed in helper genome, e.g., WN-ANS1 genome, to increase the amount of expressed rabies G protein resulting in an increased anti-rabies immune response.
  • one immunogen can be from one pathogen (e.g., rabies G) and the other from a second pathogen, resulting in three antigenic specificities of vaccine.
  • pathogen e.g., rabies G
  • ANS 1 deletions can be replaced with or used in combination with ANS3 and/or ANS5 deletions/mutations, in other examples.
  • Respiratory syncytial virus member of Paramyxoviridae family, is the leading cause of severe respiratory tract disease in young children worldwide (Collins and Crowe, Respiratory Syncytial Virus and Metapneumovirus, In: Knipe et al. Eds., Fields Virology, 5 th ed., Philadelphia: Wolters Kluwer/Lippincott Williams and Wilkins, 2007: 1601-1646). Fusion protein F of the virus is a lead viral antigen for developing a safe and effective vaccine.
  • a balanced Thl/Th2 response to F is required which can be achieved by better TLR stimulation, a prerequisite for induction of high-affinity antibodies (Delgado et al., Nat. Med. 15:34-41, 2009), which should be achievable through delivering F in a robust virus-based vector.
  • both yellow fever virus-based chimeric LAVs and PIV vectors are used for delivering RSV F to induce optimal immune response profile.
  • Other LAVs and PIV vectors described herein can also be used for this purpose.
  • RSV F protein Efficient replication and expression of RSV F protein was first demonstrated by immunostainmg of transfected cells with an anti-RSV F Mab, as illustrated for the WN(AprME)- RSV F construct in Figs. 19 and 20.
  • the presence of packaged PIVs in the superaatants from transfected cells was determined by titration in Vero cells with immunostainmg (Fig. 21).
  • similar constructs can be used that contain a modified full length F protein gene. Specifically, the N-terminal native signal peptide of F is replaced in modified F protein with the one from rabies virus G protein. The modification is intended to elucidate whether the use of a heterologous signal can increase the rate of F protein synthesis and/or replication of PfVs.
  • mice that died; na, not applicable.
  • Numbers in parenthesis correspond to number of mice in each pooled serum sample tested.
  • RV-TBE/TBE 5 2464 3237 ⁇ 749 0/8 (0%)
  • TBE specific N Ab titers were determined in individual sera or pools from two animals on day 20 (*) or 30, followed by challenge the next day with 500 LD 50 of wt TBE Hypr.
  • ⁇ INV was given at 1/20 of a human dose; in the 2-dose group, the second dose was on day 14.
  • V385R III conserved, -2 position to deleted RGD in D2 Hiramatsu et al, 1996,
  • SIV GenBank accession number ADM52218.1
  • g l20 a modified gene where the native signal sequence was replaced with the tPA signal and gp41 was truncated to contain only the TM domain
  • Gag, and Pro protease genes
  • the cassette was designed in a way that would allow its expression in the recombinant PIV ORF as a single precursor (different from SIV or ⁇ gene organization).
  • the genes are separated by FMDV 2A autoprotease sequences (see above).
  • nucleotide sequence of the entire cassette was optimized by silent nucleotide changes to eliminate direct sequence repeats (e.g., all repeats longer than 8 nt were eliminated) to increase insert stability (using optimization algorithms at DNA 2.0) and by incorporating monkey codon preference to enable efficient protein translation in primate cells.
  • the codon-optimized cassette was chemically synthesized, followed by in-frame insertion of the genes, alone or in different combinations, in PIV-WN vectors in place of the AC (RV909 vector), AprM-E (RV230 vector) or AC-prM-E (dC RV230 vector) deletions.
  • PIV-WN vectors in place of the AC (RV909 vector), AprM-E (RV230 vector) or AC-prM-E (dC RV230 vector) deletions.
  • sequences of the constructs are provided in Sequence Appendix 6. Inserts of the first three constructs in Fig. 22, starting with the Env glycoprotein, were designed similarly to the PIV WV-rabies G described hereinabove (gpl20 signal fused with a portion of the signal sequence for prM at the end of the C gene or downstream from AC deletion depending on vector), as is also additionally illustrated for individual Env constructs in Fig. 23.
  • dC RV230 Env constructs were generated, in which the tPA signal and/or the S IV Env TM region of the gpl20 gene were replaced with rabies virus G protein-specific signal and/or anchor sequences (three bottom constructs in Fig. 23), to determine whether these heterologous rabies G-derived sequences will have a beneficial effect on gpl20 presentation or recombinant PIV replication.
  • Gag and Gag-Pro insertions were designed to start with and end with FMDV 2 A autoprotease sequences, to free the island C-termini of the cytoplasmically synthesized Gag protein. They were cloned in place of the AprM-E or AC-prM-E deletions (Figs. 22 and 24).
  • the N-terminal FMDV 2A was positioned either downstream from the viral cleavage site in C, or downstream from additional 9 or 18 amino acids following the cleavage site (from the prM signal) in the RV230 and dC RV230 vectors (Fig. 24) in order to determine which fusion type is preferable for efficient cleavage of FMDV 2A preceding
  • Gag which theoretically can be important in terms of both transgene expression and PIV replication.
  • High insert stability is illustrated for one of the SIV Gag PIV variants in Fig. 29.
  • the stability of Gag was examined by ten serial passages of a RV230-Gag variant, containing Gag gene in place of large AprM-E deletion, in helper BHK-CprME(WN) cells at MOI 0.1 FFU/cell. At each passage, cell supernatants were harvested and titrated in regular Vero cells using immunostaining with an anti-WN antibody to determine total PIV titer, or an anti-SIV Gag antibody to determine titer of particles containing the Gag gene.
  • Viable PIV-(WN)-SIV Env variants were also recovered in helper BHK cells transfected with in vitro RNA transcripts and efficient expression of gpl20 was demonstrated by immunofluorescence (Figs. 30A-F and Fig. 31).
  • efficient intracellular expression of the original gpl20 was observed in Vero cells infected with packaged dC230Env variant as determined by immunostaining using anti-SIV Env antibody after methanol fixation (Fig. 30D), but little gpl20 was detected on the surface of the infected cells fixed by formalin (Fig.
  • SIV/HIV VLPs e.g., Gag with or without Env
  • HIV Env immunogen in addition to g l20, other variants of the HIV Env immunogen, such as the full- length gpl60, gpl40, gpl45, gp41, etc., with or without desired mutations, truncations, deletions, or insertions (e.g., of dominant CD4 T cell epitopes, etc., including of non-HIV origin) in expressed molecules increasing immunogenicity and/or breadth of immune response against the variable HIV genotypes/strains, can be expressed without changing the meaning of this invention. Examples of possible modifications of Env are discussed below.
  • the Envelope (Env) protein is one of the primary targets of the humoral immune response upon infection with HIV.
  • the Env protein has a number of defenses which prevent an effective antibody response from being mounted. These defenses include high degree of sequence variability, protection of functionally important domains through the use of variable loops and quaternary interactions, and high levels of glycosylation to shield the underlying protein backbone.
  • these modifications include high degree of sequence variability, protection of functionally important domains through the use of variable loops and quaternary interactions, and high levels of glycosylation to shield the underlying protein backbone.
  • In order to overcome this researchers have attempted a number of methods to increase the potency and breadth of antibody responses to Eriv. These modifications begin with an alteration of the underlying protein backbone itself.
  • Example 5 Immunogenicity of SIV Gag and Env proteins (HIV prototypes) in WN s-PIV and d-PIV.
  • RV-Gag expressing constructs are capable of eliciting detectable T cell responses as measured by interferon gamma (IFNg) secretion upon peptide stimulation ex vivo (Fig. 35).
  • IFNg interferon gamma
  • Splenocytes harvested 7 days after the second immunizing dose were stimulated with a known CD8 gag specific epitope and assayed for IFNg secretion by ELISPOT.
  • IFNg secreting cells in the RV 9AA Anchor Gag construct were detected and proved to be statistically greater than that of the ALVAC-Gag expressing control virus against the same peptide.
  • Example 6 Prime-boost vaccination regimens with PIV- WN/TBE constructs.
  • RV-WN/TBE and INV vaccines could be interchangeable in prime-boost vaccination regimens.
  • the highest PRNT 50 titers were observed in INV prime - RV-WN/TBE boost, RV-WN/TBE prime - RV- WN/TBE boost, and RV-WN/TBE prime - INV boost groups (GMTs 3,287, 6,291 and 14,205, respectively). Animals in all groups primed or boosted with sPIV- WN/TBE were protected from challenge (Table 1 1).
  • RV-WN/TBE was administered at 5 log 10 FFU/dose, and INV at 1/20 of a human dose.
  • Challenge was on day 43 with 500 LD 50 of TBE Hypr.
  • Example 7 A novel safe single-dose vaccine against tick-borne encephalitis.
  • Tick-borne encephalitis is an acute viral infection of the central nervous system and the most important disease of humans transmitted by ticks. It is endemic in eastern, central and northern European countries and Russia. TBE virus is also present in parts of several Asian countries (northern China and Mongolia, Japan). The virus is an emerging pathogen spreading rapidly in Europe. The disease is causing more than 10,000 hospitalizations annually, with case- fatality rates of 1-2% in Europe and up to 40% in Siberia and the Far East of Russia. The public awareness of TBE is high due to the severity of the disease and long-lasting neuropsychiatric sequelae occurring in up to ⁇ 40% of patients (World Health Organization. 2011. Wkly Epidemiol Rec. 86:241 -256.).
  • TBE virus belongs to the Flavivirus genus of small enveloped plus-strand RNA viruses, which also includes such major mosquito-transmitted pathogens as yellow fever (YF), Japanese encephalitis (JE), West Nile (WN) and dengue types 1-4 (DEN 1-4) viruses (Burke and Monath. 2001. Flaviviruses, p. 1043-1 126. In Knipe et al. (ed.), Fields Virology, 4th ed. Lippincott Williams and Wilkins , Philadelphia, PA.).
  • YF yellow fever
  • JE Japanese encephalitis
  • WN West Nile
  • DEN 1-4 dengue types 1-4
  • the flavivirus genomic RNA of ⁇ 11,000 nucleotides contains a single open reading frame which encodes the three structural proteins of the virion, capsid C, premembrane prM and envelope E, followed by seven nonstructural (NS) proteins involved in virus replication (Lindenbach et al., In: Knipe et al., editors. Fields Virology, 5th ed. Philadelphia:
  • the E protein is the main immunogen, eliciting neutralizing antibodies which are considered to be the main correlate of protection from flavivirus infection.
  • Available INV and live attenuated (LAV) flavivirus vaccines have been reviewed, including the single-dose YF 17D LAV that has been used in >500 million persons worldwide, and the recently developed ChimeriVax vaccines against JE, dengue and WN obtained by chimerization with YF 17D virus (Pugachev et al., In: Levine et al., editors. New generation vaccines, 4 th ed. New York: Informa Healthcare USA, 2010, p. 557-69; Guy et al., Vaccine 2010; 28:632-49.).
  • VLP virus like particles
  • PIV-TBE variants containing the prM-E genes from TBE Hypr virus were constructed based on the WN, TBE Hypr, LGT E5 and YF 17D backbones (RV-WN/TBE, RV-TBE/TBE, RV-YF/TBE and RV-LGT/TBE).
  • Live chimeras based on the backbones of attenuated YF 17D, DEN2 PD -53 and LGT E5 viruses genes, and one additional virus, YF/LGT, contained the prM-E genes from LGT E5 (Fig. 44).
  • the PIV-WN/TBE variant (based on the WN backbone, with the TBE specific prM signal) grew very efficiently in both BH and Vero (WN C) helper cells (Fig. 47 A), with peak titers as high as 8 log 10 FFU/ml in some experiments. High titers, also up to 8 log 10 FFU/ml, were observed in a broad range of MOIs (0.001, 0.01 and 0.1 FFU/cell) (Fig. XXX). This variant was also used to examine whether modifications in the prM signal could increase the secretion of TBE VLPs (to increase immunogenicity). Three amino acid changes shown previously to increase VLP secretion in a AC RNA vaccine candidate against TBE ( ofler et al., Proc Natl Acad Sci U S A. 2004 Feb
  • PIV-WN/TBE was serially passaged eleven times in two independent passage series in BHK WN C helper cells at an MOI of 0.01 FFU/cell, and the full genomes of the PI 1 samples were sequenced by consensus sequencing. Only a few mutations were detected, E122Q in the E protein in the first passage series, and K3M in the prM signal and R122L in NS2A in the second passage series. The mutants replicated efficiently in helper cells. Importantly, no recombination (replication competent virus) was detected in these samples. The absence of recombination was also confirmed by titration of numerous other PIV-TBE samples, as well as titration of mouse brain tissue samples harvested after IC inoculation.
  • BH WN C helper cells were found to provide equally high yields of PIV- WN/TBE and the PIV-WN prototype at cell passages > P20 (including after 5 passages in puromycin-free medium), and early passages (Fig. XXX).
  • mice Analysis of neurovirulence and neuroinvasiveness of PIV-TBE variants and live chimeras in mice. 3.5 week-old ICR mice were inoculated with graded doses of PIV-TBE constructs and chimeric viruses by the IC route to measure neurovirulence or IP route to measure neuroinvasiveness (Fig. 45). All animals that received doses up to 5 log 10 FFU of RV-WN/TBE by both routes survived without any signs of sickness.
  • live chimeras are less attenuated.
  • chimerization resulted in some attenuation, particularly for neuroinvasiveness, compared to TBE Hypr virus (IP LD 50 1 PFU), with most chimeras exhibiting neurovirulence/neuroinvasiveness not higher than that of the naturally attenuated LGT TP21 virus (Fig. 45).
  • IP LD 50 1 PFU TBE Hypr virus
  • mice Immunogenicity and efficacy of candidates in mice, Th type of response, effect of anti- vector preimmunity, and prime-boost regimens with INV. All immunizations of mice (3.5 week old ICR) were by the IP route. TBE-specific PRNT 50 titers were measured in sera collected on days 20 or 30. Challenge was done the following day after bleeding. The PIV-TBE candidates RV- (reciprocal GMTs 1,778 and 3,237, respectively), which were comparable to neutralizing titers in sera of mice that survived after receiving 5 log 10 PFU of YF/TBE chimera (GMT 6,615).
  • mice in the negative control groups were solidly protected from a severe IP challenge with TBE Hypr virus (500 LD50), while mice in the negative control groups (mock, YF 17D, DEN2 PDK-53) died (Table 9).
  • RV- YF/TBE was poorly immunogenic and only 50% efficacious. Lower immunogenicity and efficacy were observed for DEN2/TBE and particularly YF/LGT chimeras.
  • a single dose of the human INV control resulted in low neutralizing antibody titers (GMT 13; with 50% protection), which significantly increased, as expected, after the second dose (GMT 1,826; 100% protection).
  • RV-WN/TBE which is the preferred PIV-TBE variant because of its efficient replication in vitro, is essentially as immunogenic and efficacious in mice as the underattenuated YF/TBE virus after one dose.
  • concentrations of IgG isotypes were determined in sera from mice immunized with RV-WN/TBE, YF/TBE and INV by IgG type specific ELBA.
  • the IgG2a/IgGl antibody ratios were found to be 4: 1 and 16:1 for RV-WN/TBE and YF/TBE, respectively, indicative of a Thl biased immune response.
  • the ratio was 1 :4, indicative of a Th2 bias.
  • Anti-vector (YF) preimmunity is not a concern for ChimeriVax vaccines as was previously shown in humans (Monath et al, Vaccine. 2002 Jan 15;20(7-8): 1004-18; Guirakhoo et al., Hum Vaccin. 2006 Mar-Apr;2(2):60-7), although this aspect has not been examined in mice.
  • mice were preimmunized with YF 17D and then immunized 3 weeks (short interval) or 6 months (long interval) later with YF/JE and YF/TBE.
  • mice were similarly preimmunized with RV-WN to induce by YF and WN PRNT 50 .
  • JE or TBE neutralizing antibody titers in these groups were determined 21 days after vaccinations and compared to parallel groups of naive animals vaccinated with YF/JE, YF/TBE or RV-WN/TBE.
  • pre-immunization resulted in some reduction of TBE specific response compared to that in naive animals.
  • it was less pronounced compared to the effect of YF 17D pre-immunization on immunogenicity of YF/JE and YF/TBE viruses (Fig. 49) suggesting that anti- vector immunity should not be a concern for PIV-TBE in humans.
  • LGT TP21 caused no detectable viremia
  • YF/TBE induced a low-level viremia in some animals, ⁇ 1.6 log 10 PFU/ml, on days 5 - 6.
  • a reduction of inoculation dose delays the onset of viremia in Rhesus monkeys without affecting the peak titer (Monath et al, J Virol. 2000 Feb;74(4): 1742-51).
  • LGT T1674 virus was given at a lower SC dose of 5 log 10 PFU, which indeed allowed for a better resolution of viremia (in naive animals), peaking on day 2 (Fig. 54B-D). This is a stringent virus.
  • a single IM 7-log 10 dose of RV-WN/TBE was also highly effective (GMTs 955 and 707 on days 29 and 50).
  • a single SC 7-log 10 dose of RV- WN/TBE induced a lower antibody response (e.g., GMT 232 on day 50) which was significantly boosted by a second SC dose (GMT 3,357 on day 50 in the SC two-dose group).
  • a single ID inoculation of RV-WN TBE given at 6 and 5 log 10 FFU doses induced appreciable neutralizing antibody responses which however were significantly lower compared to a 7-logio ID dose. This may indicate that the immunizing dose should be above 6 log 10 FFU in humans.
  • WN preimmunity reduced the TBE specific neutralizing titers by approximately 5 fold compared to the 7-logio ID group of immunized WN-nai've animals [GMT 306 on day 50 in all four monkeys (two monkeys naturally WN virus immune, and two monkeys preimmunized with RV-WN)].
  • the effect of WN preimmunity was less pronounced compared to the effect of YF preimmunity on
  • CONCLUSION TBE virus is the most important human pathogen in Eurasia transmitted by ticks. Inactivated vaccines (INV) are available but require multiple doses and frequent boosters to induce and maintain immunity. Thus far the goal of developing a safe, live attenuated vaccine (LAV) effective after a single dose has remained elusive.
  • LAV live attenuated vaccine
  • PIV-TBE constructs discussed above to generate a safe, single-dose TBE vaccine relying on immunologic mechanisms similar to LAVs.
  • PIV-TBE candidates attenuated by a deletion in the capsid gene were constructed based on different flaviviras backbones containing the envelope genes of TBE virus.
  • PIV-TBE constructed using a West Nile virus backbone PIV-WN/TBE
  • PIV-WN/TBE West Nile virus backbone
  • YF 17D backbones a West Nile virus backbone
  • Live chimeric YF 17D/TBE, Dengue 2/TBE and Langat E5/TBE candidates were also constructed but found to be less attenuated than PIV-WN/TBE. Similar to YF 17D/TBE virus, PIV-WN/TBE elicited a Thl biased response.
  • PIV-WN/TBE was demonstrated to be highly immunogenic in Rhesus macaques after a single dose, inducing a significantly more durable response compared to 3 doses of a human INV. Immunogenicity was not significantly affected by pre-existing immunity against WN. Immunized monkeys were protected from a stringent surrogate challenge. These results indicate that we have developed a novel TBE vaccine with a superior product profile to existing inactivated vaccines, which could lead to improved vaccine coverage and control of TBE disease.
  • MATERIALS AND METHODS obtained by transfection with a Venezuelan equine encephalitis virus replicon (rVEE, based TC-83 vaccine strain) expressing the WN virus C protein and a puromycin N-acetyl-transferase selective marker (Mason et al., Virology 2006; 351:432-43; and Widman et al., Adv Virus Res 2008; 72:77-126), or an additionally constructed rVEE helper expressing the C protein of TBE Hypr. The cells were maintained in puromycin-containing selective media.
  • rVEE Venezuelan equine encephalitis virus replicon
  • PIV-TBE titers were determined by immuno-focus assay in Vero cells (Rumyantsev et al, 2011, supra) using anti-TBE mouse hyperimmune ascitic fluid (ATCC) or polyclonal rabbit antibodies raised against an inactivated human vaccine against TBE (FSME, Baxter) as primary antibodies.
  • the FSME vaccine was used as INV control in animal studies. Live viruses and chimeras were propagated in regular Vero cells in media without puromycin.
  • YF 17D virus (YF-VAX, Sanofi Pasteur) used in animal experiments was prepared by amplification in Vero cells.
  • YF/JE and YF/WN chimeras were described previously (Guirakhoo et al., 1999; and Monath et al., 2006).
  • LGT virus strains TP21 isolated in Malaysia from Ixodes granulatus ticks in 1956
  • Tl 674-73 isolated in Thailand from Haemaphysalis papuana ticks in 1973
  • wild type TBE strain Hypr were from R. Tesh (World Reference Center for Emerging Viruses and Arboviruses, University of Texas, Galveston, TX). Infectious titers/doses of PIV constructs and live viruses are expressed in FFU (focus forming units) and PFU (plaque forming units), respectively.
  • RV-WN/TBE was constructed by replacing the WN-specific prM-E genes in the PIV-WN prototype (Mason et al, 2006) with those from TBE virus strain Hypr (GenBank accession number U39292; the nt sequence was optimized to have preferential human-genome codon usage and to eliminate repeats > 8 nt).
  • the TBE genes were synthesized by DNA2.0, Inc. (Menlo Park, CA).
  • a variant with the TBE-specific signal for prM was used in all experiments. (Another variant, with WN-specific prM signal, was also constructed but not used because it replicated to several fold lower titers in helper cells).
  • RV-YF/TBE was constructed by replacing the YF 17D prM-E genes in PIV-YF prototype (Mason et al., 2006) with the TBE prM-E genes.
  • RV-LGT/TBE was constructed using a synthetic infectious clone of Langat E5 virus (LGT E5, an attenuated strain of LGT TP21) assembled from three DNA fragments synthesized based on published LGT E5 sequence (GenBank accession number ).
  • RV-TBE/TBE based entirely on the TBE Hypr sequence was assembled from three DNA fragments synthesized based on published TBE Hypr sequence (GenBank accession number U39292).
  • LGT/TBE chimera was made by inserting the complete C protein gene of LGT E5 into the RV-LGT/TBE construct.
  • Vero for chimeric viruses were transfected with RNA transcripts to generate infectious PIV and chimeric viruses.
  • RNA transcripts For DEN2/TBE chimera (the genome split in two plasmids), the full length cDNA template was prepared by two-fragment in vitro ligation followed by transcription and transfection of Vero cells.
  • mice were from Taconic. Inoculation routes/doses, and bleeding/challenge days were as described in Results. Challenge was done by the IP route with 500 LD 50 (500 PFU) of TBE Hypr. In neurovirulence/neuroinvasiveness tests and after challenge, mice were monitored for 21 days for survival. Doses causing 50% mortality (LD50) were calculated using the Reed and
  • Neutralizing antibody (PRNT 50 ) titers were determined in heat-inactivated serum samples collected by mandibular bleeding against wt Hypr or YF/TBE viruses. Titers of IgG
  • isotypes were determined by isotype-specific ELISA as described (Rumyantsev, 2011 , supra) using YF/TBE virus as a coating agent.
  • the second NHP study consisted of two parts, the short-term (Fig. 52) and long-term (Fig. 53) parts.
  • Rhesus monkeys prescreened to be seronegative for JE, WN and YF were used, except for two animals found to be seropositive for WN which were assigned to a group addressing the effect preexisting anti-vector immunity.
  • the schedule of steps in the first part was similar to the first NHP study.
  • Two groups received 6 and 5 log 10 FFU/dose of RV-WN/TBE by the ID route to evaluate dose- responses.
  • animals received 7 log 10 FFU of RV-WN/TBE by the ID route on day 0 (the two naturally WN-immune animals) or day 30 (two additional animals that first received 7 log 10 FFU/dose of PIV-WN on day 0).
  • the dynamics of TBE neutralizing antibody responses were monitored for 6 months following
  • Example 7 Delivery of HA protein of influenza H1N1 virus (strain New Caledonia) in WN s- PIV (which optionally can be used in d-PIV).
  • flu antigens can be similarly delivered, such as NA, M2 (e.g., M2e), etc., or fragments thereof.
  • NA e.g., NA2
  • M2e e.g., M2e
  • fragments thereof e.g., N-(2-aminoe)
  • HA e.g., N-(2-aminoe)
  • modifications can be introduced, and modified antigens then expressed in PIV vaccine vectors, without changing the meaning of this invention.
  • PIV-SIV and PIV-Flu vaccine candidates described in Examples 4 and 5 can be tested for immunogenicity and efficacy in animal models.
  • Earlier in vivo data have demonstrated that PIV vaccines expressing transgenes are highly immunogenic in animals, as has been shown for PIV-RSV F (see, e.g., WO 2010/107847, incorporated herein by reference), and more recent experiments for PIV-Rabies G.
  • CV-TBEV Hypr or CV-LGT E5 with YFV/TBEV chimeric signal (p42, p59, and p43 constructs; SEQ ID NOs:28-30)
  • CV-TBEV Hypr with YFV/WNV chimeric signal (p45; SEQ ID NOs:31-33)
  • RV-WNV/TBEV Hypr with TBEV signal (p39; SEQ ID NOs:34-36)
  • RV-WNVTBEV Hypr with WNV signal (p40; SEQ ID NOs:37-39)
  • PIV-WNTBEV Hypr with TBEV signal (p39; SEQ ID NOs:52-54)
  • TAACTGCCAA TAGCCGCTCG TGCGTACCCT AAAACCAAGG CGTCCCCCTA AGGACAGAAG ATAACCATTC CGTGACGTAT GGCACGACCC CCCGCGTAAG

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Abstract

This invention provides replication-defective flavivirus vaccines and vectors, and corresponding compositions and methods.

Description

REPLICATION-DEFECTIVE FLAVr TRUS VACCINES
AND VACCINE VECTORS
Cross-Reference to Related Applications This application is a continuation of U.S. Serial No. 13/633,436, filed October 2, 2012, which is a continuation of U.S. Serial No. 13/364,187, filed February 1, 2012. This application also claims benefit of U.S. Serial No. 61/674,768, filed July 23, 2012. The prior applications are incorporated herein by reference. Field of the Invention
This invention relates to replication-defective fiavivirus vaccines and vaccine vectors, and corresponding compositions and methods.
Background of the Invention
Flaviviruses are distributed worldwide and represent a global public health problem.
Flaviviruses also have a significant impact as veterinary pathogens. Fiavivirus pathogens include yellow fever (YF), dengue types 1-4 (DEN1-4), Japanese encephalitis (JE), West Nile (WN), tick- borne encephalitis (TBE), and other viruses from the TBE serocomplex, such as Kyasanur Forest disease (KFD) and Omsk hemorrhagic fever (OHF) viruses. Vaccines against YF [live attenuated vaccine (LAV) strain 17D], JE [inactivated vaccines (INV) and LAV], and TBE (INV) are available. No licensed human vaccines are currently available against DEN and WN. Veterinary vaccines have been in use including, for example, vaccines against WN in horses (INV, recombinant and live chimeric vaccines), JE (INV and LAV) to prevent encephalitis in horses and stillbirth in pigs in Asia, louping ill fiavivirus (INV) to prevent neurologic disease in sheep in the UK, and TBE (INV) used in farm animals in Czech Republic (INV) (Monath and Heinz, Flaviviruses, in Fields et al. Eds., Fields Virology, 3rd Edition, Philadelphia, New York, Lippincott-Raven Publishers, 1996, pp. 961-1034).
Tick-borne encephalitis (TBE) is the most important tick-borne viral disease of humans. It is endemic in parts of Europe and Northern Asia, causing more than 10,000 hospitalizations annually, with a case-fatality rate 0.5-1.5% in Europe and 6-40% in Siberia and the Far East. A significant proportion of patients suffer from long-lasting neuropsychiatric sequelae. Inactivated vaccines produced in chick embryo cell cultures have proven effective in preventing the disease. For example, an 86% vaccination coverage of the Austrian population (the highest among European countries) has resulted in an approximately 90% reduction of hospitalized cases (Heinz and Kunz, Arch. Virol. Suppl. 18:201-205, 2004). The inactivated vaccines are expensive and require three inoculations for primary immunization. Periodic boosters (every 2-5 years) are required to maintain immunity. Therefore, a less costly TBE vaccine, which is effective after one-two doses and provides durable, such as life-long immunity (similar to that achieved by YF 17D immunization) is needed, and indeed has been identified by the WHO as a major priority. Development of TBE LAV candidates in the past several decades by means of empirical or rational attenuation of TBE virus parent per se or chimerization of TBE or Langat (LGT, a naturally attenuated flavi virus that is closely related (serologically) to TBE) viruses with dengue 4 virus has faced difficulties due to problems with residual virulence of candidates and/or low immunogenicity/overattenuation (Wright et al., Vaccine 26:882-890, 2008; Maximova et al, J. Virol. 82:5255-5268, 2008; Rumyantsev et al., Vaccine 24:133-143, 2006; Kofler et al., Arch. Virol. Suppl. 18:191-200, 2004; and references therein).
Flaviviruses are small, enveloped, plus-strand RNA viruses transmitted primarily by arthropod vectors (mosquitoes or ticks) to natural hosts, which are primarily vertebrate animals, such as various mammals, including humans, and birds. The flavivirus genomic RNA molecule is about 11,000 nucleotides (nt) in length and encompasses a long open reading frame (ORF) flanked by 5' and 3' untranslated terminal regions (UTRs) of about 120 and 500 nucleotides in length,
respectively. The ORF encodes a polyprotein precursor that is cleaved co- and post-translationally to generate individual viral proteins. The proteins are encoded in the order: C-prM/M-E-NSl- NS2A/2B-NS3-NS4A/4B-NS5, where C (core/capsid), prM/M (pre-membrane/membrane), and E (envelope) are the structural proteins, i.e., the components of viral particles, and the NS proteins are non-structural proteins, which are involved in intracellular virus replication. Flavivirus replication occurs in the cytoplasm. Upon infection of cells and translation of genomic RNA, processing of the polyprotein starts with translocation of the prM portion of the polyprotein into the lumen of endoplasmic reticulum (ER) of infected cells, followed by translocation of E and NS1 portions, as directed by the hydrophobic signals for the prM, E, and NS 1 proteins. Amino-termini of prM, E, and NS1 proteins are generated by cleavage with cellular signalase, which is located on the luminal side of the ER membrane, and the resulting individual proteins remain carboxy-terminally anchored in the membrane. Most of the remaining cleavages, in the nonstructural region, are carried out by the viral NS2B/NS3 serine protease. The viral protease is also responsible for generating the C- terminus of the mature C protein found in progeny virions. Newly synthesized genomic RNA molecules and the C protein form a dense spherical nucleocapsid, which becomes surrounded by cellular membrane in which the E and prM proteins are embedded. The mature M protein is produced by cleavage of prM shortly prior to virus release by cellular furin or a similar protease. E, the major protein of the envelope, is the principal target for neutralizing antibodies, the main correlate of immunity against flavivirus infection. Virus-specific cytotoxic T-lymphocyte (CTL) response is the other key attribute of immunity. Multiple CD8+ and CD4+ CTL epitopes have been characterized in various flavivirus structural and non-structural proteins. In addition, innate immune responses contribute to both virus clearance and regulating the development of adaptive immune responses and immunologic memory.
In addition to the inactivated (INV) and live-attenuated (LAV) vaccines against flaviviruses discussed above, other vaccine platforms have been developed. One example is based on chimeric flaviviruses that include yellow fever virus capsid and non-structural sequences and prM-E proteins from other flaviviruses, to which immunity is sought. This technology has been used to develop vaccine candidates against dengue (DEN), Japanese encephalitis (JE), West Nile (WN), and St. Louis encephalitis (SLE) viruses (see, e.g., U.S. Patent Nos. 6,962,708 and 6,696,281). Yellow fever virus-based chimeric flaviviruses have yielded highly promising results in clinical trials.
Another flavivirus vaccine platform is based on the use of pseudoinfectious virus (PIV) technology (Mason et al., Virology 351:432-443, 2006; Shustov et al., J. Virol. 21: 11737-11748, 2007; Widman et al., Adv. Virus. Res. 72:77-126, 2008; Suzuki et al., J. Virol. 82:6942-6951, 2008; Suzuki et al., J. Virol. 83: 1870-1880, 2009; Ishikawa et al., Vaccine 26:2772-2781, 2008; Widman et al., Vaccine 26:2762-2771, 2008). PIVs are replication-defective viruses attenuated by a deletion(s). Unlike live flavivirus vaccines, they undergo a single round replication in vivo (or optionally limited rounds, for two-component constructs; see below), which may provide benefits with respect to safety. PIVs also do not induce viremia and systemic infection. Further, unlike inactivated vaccines, PIVs mimic whole virus infection, which can result in increased efficacy due to the induction of robust B- and T-cell responses, higher durability of immunity, and decreased dose requirements. Similar to whole viruses, PIV vaccines target antigen-presenting cells, such as dendritic cells, stimulate toll-like receptors (TLRs), and induce balanced Thl/Th2 immunity. In addition, PIV constructs have been shown to grow to high titers in substrate cells, with little or no cytopathic effect (CPE), allowing for high-yield manufacture, optionally employing multiple harvests and/or expansion of infected substrate cells. The principles of the PIV technology are illustrated in Figs. 1 and 2. There are two variations of the technology. In the first variation, a single-component pseudoinfectious virus (s- PIV) is constructed with a large deletion in the capsid protein (C), rendering mutant virus unable to form infectious viral particles in normal cells (Fig. 1). The deletion does not remove the first -20 codons of the C protein, which contain an RNA cyclization sequence, and a similar number of codons at the end of C, which encode a viral protease cleavage site and the signal peptide for prM. The s-PrV can be propagated, e.g., during manufacture, in substrate (helper) cell cultures in which the C protein is supplied in trans, e.g., in stably transfected cells producing the C protein (or a larger helper cassette including C protein), or in cells containing an alphavirus replicon [e.g., a Venezuelan equine encephalitis virus (VEE) replicon] expressing the C protein or another intracellular expression vector expressing the C protein. Following inoculation in vivo, e.g., after immunization, the PIV undergoes a single round of replication in infected cells in the absence of trans- complementation of the deletion, without spread to surrounding cells. The infected cells produce empty virus-like particles (VLPs), which are the product of the prM-E genes in the Pr , resulting in the induction of neutralizing antibody response. A T-cell response should also be induced via MHCI presentation of viral epitopes. This approach has been applied to YF 17D virus and WN viruses and WN/JE and WN/DEN2 chimeric viruses (Mason et al., Virology 351 :432-443, 2006; Suzuki et al., J. Virol. 83:1870-1880, 2009; Ishikawa et al, Vaccine 26:2772-2781, 2008; Widman et al, Vaccine 26:2762-2771, 2008; WO 2007/098267; WO 2008/137163).
In the second variation, a two-component PIV (d-PIV) is constructed (Fig. 2). Substrate cells are transfected with two defective viral RNAs, one with a deletion in the C gene and another lacking the prM-E envelope protein genes. The two defective genomes complement each other, resulting in accumulation of two types of PIVs in the cell culture medium (Shustov et al., J. Virol. 21:11737-11748, 2007; Suzuki et al., J. Virol. 82:6942-6951, 2008). Optionally, the two PIVs can be manufactured separately in appropriate helper cell lines and then mixed in a two-component formulation. The latter may offer an advantage of adjusting relative concentrations of the two components, increasing immunogenicity and efficacy. This type of PIV vaccine should be able to undergo a limited spread in vivo due to coinfection of some cells at the site of inoculation with both components. The spread is expected to be self-limiting as there are more cells in tissues than viral particles produced by initially coinfected cells. In addition, a relatively high MOI is necessary for efficient co-infection, and cells outside of the inoculation site are not expected to be efficiently coinfected (e.g., in draining lymph nodes). Cells infected with the AC PIV alone produce the highly immunogenic VLPs. Coinfected cells produce the two types of packaged defective viral particles, which also stimulate neutralizing antibodies. The limited infection is expected to result in a stronger neutralizing antibody response and T-cell response compared to s-PIVs. To decrease chances of recombination during manufacture or in vivo, including with circulating flaviviruses, viral sequences can be modified in both s-PIVs and d-PIVs using, e.g., synonymous codon replacements, to reduce nucleotide sequence homologies, and mutating the complementary cyclization 5' and 3' elements.
Summary of the Invention
The invention provides replication-deficient or defective pseudoinfectious flaviviruses including a flavivirus genome that includes (i) one or more deletions or mutations in nucleotide sequences encoding one or more proteins selected from the group consisting of capsid (C), pre- membrane (prM), envelope (E), non-structural protein 1 (NSl), non-structural protein 3 (NS3), and non-structural protein 5 (NS5), and (ii) sequences encoding one or more heterologous pathogen, cancer, or allergy-related immunogens. For example, the deletion/mutation can be within capsid (C) sequences; pre-membrane (prM) and/or envelope (E) sequences; capsid (C), pre-membrane (prM), and envelope (E) sequences; or non-structural protein 1 (NSl) sequences.
The heterologous immunogen can be, for example, from a pathogen selected from the group consisting of a rabies virus (e.g., a rabies virus G protein epitope), Borrelia burgdorferi (e.g., OspA immunogen or an immunogenic fragment thereof), a tick (e.g., a tick saliva protein selected from the group consisting of 64TRP, Isac, and Salp20, or an immunogenic fragment thereof), an influenza virus (e.g., an influenza virus M2, hemaglutinnin (HA), or neuraminidase (NA) epitope, or an immunogenic fragment thereof), a human immunodeficiency virus (e.g., a codon-optimized HIV gag, pol, tat/nef, pro, or variants of Env protein, such as gpl60, gpl45, gpl40, gpl20, gp41, etc., or immunogenic fragments thereof), a simian immunodeficiency virus (e.g., a codon-optimized SIV gag, pol, tat/nef, pro, or variants of Env, or immunogenic fragments or combinations of two or more (e.g., 3, 4, or 5) thereof), a human papilloma virus (e.g., an HPV16 or HPV18 capsid protein LI or L2, or an immunogenic fragment thereof), a respiratory syncytial virus (e.g., a respiratory syncytial virus F or G glycoprotein), malaria parasite, and Mycobacterium tuberculosis (also see below).
The replication-deficient pseudoinfectious flaviviruses can include sequences encoding a pre-membrane (prM) and/or envelope (E) protein. Further, the replication-deficient
pseudoinfectious flavivirus genomes can be selected from those of yellow fever virus, West Nile virus, tick-borne encephalitis virus, Langat virus, Japanese encephalitis virus, dengue virus, and St. Louis encephalitis virus, attenuated strains thereof, and chimeras thereof (also see below). In various examples, the chimeras include pre-membrane (prM) and envelope (E) sequences of a first flavivirus (e.g., a tick-borne encephalitis virus or a Langat virus (e.g., Langat E5)), and capsid (C) and non-structural sequences of a second, different flavivirus (e.g., a yellow fever, a West Nile, or Langat (e.g., Langat E5) virus). One specific example includes a West Nile virus backbone with a C protein deletion and TBE Hypr prM-E sequences inserted in place of corresponding WN sequences (and TBE-specific prM signal).
The replication-deficient pseudoinfectious flavivirus genomes can be packaged in particles including pre-membrane (prM) and envelope (E) sequences from a flavivirus that is the same or different from that of the genomes. Further, the sequences encoding the heterologous immunogens can be inserted in the place of, or in combination with, the deletion(s) or mutation(s) of the one or more proteins.
The sequences encoding the heterologous immunogens can be inserted in the flavivirus genomes within sequences encoding the envelope (E) protein, within sequences encoding the nonstructural 1 (NS1) protein, within sequences encoding the pre-membrane (prM) protein,
intergenically between sequences encoding the envelope (E) protein and non-structural protein 1 (NS1), intergenically between non- structural protein 2B (NS2B) and non-structural protein 3 (NS3), and/or as a bicistronic insertion in the 3' untranslated region of the flavivirus genome.
In several embodiments, the replication-deficient pseudoinfectious flavivirus genomes include heterologous immunogen sequences from HIV, SIV, or influenza virus, such as any one or more of those described in Appendices 6-8. In particular embodiments, the replication-deficient pseudoinfectious virus is selected from any one of the SIV constructs 1-11 of Sequence Appendix 6, a construct having at least 50% sequence identity (e.g., 50%, 60%, 70%, 85%, 90%, 95%, or 99% or more sequence identity) to the nucleic acid or amino acid sequences described therein, or a construct that includes homologs and/or other naturally occurring variants of the SIV protein(s). In other embodiments, the replication-deficient pseudoinfectious virus is selected from the HIV Gag construct (PIV-WN (AprME)-HIV Gag ) of Sequence Appendix 7, a construct having at least 50% sequence identity (e.g., 50%, 60%, 70%, 85%, 90%, 95%, or 99% or more sequence identity) to the nucleic acid or amino acid sequences described therein, or a construct that includes homologs and/or other naturally occurring variants of the HIV Gag protein. In still other embodiments, the replication-deficient pseudoinfectious virus is selected from the HIV Env construct (PIV-WN (AprME)-HIV Env Gpl40) of Sequence Appendix 7, a construct having at least 50% sequence identity (e.g., 50%, 60%, 70%, 85%, 90%, 95%, or 99% or more sequence identity) to the nucleic acid or amino acid sequences described therein, or a construct that includes homologs and/or other naturally occurring variants of the HIV Env protein. In yet other embodiments, the replication- deficient pseudoinfectious virus is selected from construct 1 or 2 of Sequence Appendix 8, a construct having at least 50% sequence identity (e.g., 50%, 60%, 70%, 85%, 90%, 95%, or 99% or more sequence identity) to the nucleic acid or amino acid sequences described therein, or a construct that includes homologs and/or other naturally occurring variants of the HA protein.
The invention also includes compositions including a first replication-deficient
pseudoinfectious flavivirus, as described above, and a second (or further), different replication- deficient pseudoinfectious flavivirus including a genome that includes one or more deletions or mutations in nucleotide sequences encoding one or more proteins selected from the group consisting of capsid (C), pre-membrane (prM), envelope (E), non-structural protein 1 (NS1), non-structural protein 3 (NS3), and non-structural protein 5 (NS5). In these compositions, the one or more proteins encoded by the sequences in which the deletion(s) or mutation(s) occur in the second, different replication-deficient pseudoinfectious flavivirus are different from the one or more proteins encoded by the sequences in which the deletion(s) occur in the first replication-deficient pseudoinfectious flavivirus.
The invention further includes methods of inducing immune responses to an immunogen in a subject, which involves administering to the subject one or more replication-deficient
pseudoinfectious flavivirus and/or composition as described herein to the subject. In particular embodiments, the replication-deficient pseudoinfectious flavivirus and/or composition includes any one or more of those described in Appendices 6-8, constructs having at least 50% sequence identity (e.g., 50%, 60%, 70%, 85%, 90%, 95%, or 99% or more sequence identity) to the nucleic acid or amino acid sequences described therein (or individual proteins or corresponding nucleic acid sequences therein), or constructs that include homologs and/or other naturally occurring variants of the immunogenic SIV, HIV, and or HA proteins.
In various examples, the subject is at risk of but does not have an infection by the pathogen or a disease or condition associated with the cancer or allergy-related immunogen. In other examples, the subject has an infection by the pathogen or a disease or condition associated with the cancer or allergy-related immunogen. The invention thus includes prophylactic and therapeutic methods. In these methods, the immunogen can be from, for example, a pathogen selected from the group consisting of a rabies virus, Borrelia burgdorferi, a tick, an influenza virus, a human immunodeficiency virus, a simian immunodeficiency virus, a human papilloma virus, a respiratory syncytial virus, malaria parasite, and Mycobacterium tuberculosis (also see below). Further, the methods can be for inducing an immune response against a protein encoded by the flavivirus genome, in addition to the source of the immunogen. In various examples, the subject is at risk of but does not have an infection by the flavivirus corresponding to the genome of the pseudoinfectious flavivirus, which includes sequences encoding a flavivirus pre-membrane and/or envelope protein. In other examples, the subject has an infection by the flavivirus corresponding to the genome of the pseudoinfectious flavivirus, which includes sequences encoding a flavivirus pre-membrane and/or envelope protein.
The invention also includes live, attenuated chimeric flaviviruses including a yellow fever virus in which sequences encoding pre-membrane and envelope proteins are replaced with sequences encoding pre-membrane and envelope proteins of a tick-borne encephalitis virus or a Langat virus, and the signal sequence between the capsid and pre-membrane proteins of the chimeric flavivirus includes a hybrid of yellow fever virus and tick-borne encephalitis or Langat virus capsid/pre-membrane signal sequences, or a variant thereof. In various examples, the capsid/pre- membrane signal sequence of the chimeric flavivirus includes yellow fever virus sequences in the amino terminal region and tick-borne encephalitis or Langat virus sequences in the carboxy terminal region (see below).
Further, the invention includes live, attenuated chimeric flaviviruses including a West Nile virus in which sequences encoding pre-membrane and envelope proteins are replaced with sequences encoding pre-membrane and envelope proteins of a tick-borne encephalitis or a Langat virus, and the signal sequence between the capsid and pre-membrane proteins of the chimeric flavivirus includes a tick-borne encephalitis or a Langat virus capsid/pre-membrane signal sequence, or a variant thereof.
The invention also includes pharmaceutical compositions including one or more
pseudoinfectious flavivirus, composition, or live, attenuated flavivirus as described herein, and a pharmaceutically acceptable carrier or diluent. Further, the compositions can include an adjuvant.
Also included in the invention are replication-deficient pseudoinfectious flaviviruses including a flavivirus genome including one or more deletion(s) or mutation(s) in nucleotide sequences encoding non-structural protein 1 (NSl), non-structural protein 3 (NS3), or non-structural protein 5 (NS5). Further, the invention includes nucleic acid molecules corresponding to the genome of a pseudoinfectious flavivirus, or the genome of the live, attenuated flavivirus, as described herein, and complements thereof.
The invention also provides methods of making replication-deficient pseudoinfectious flaviviruses as described herein, involving introducing one or more nucleic acid molecules, as described above, into a cell that expresses the protein(s) corresponding to any sequences deleted from the flavivirus genome of the replication-deficient pseudoinfectious flaviviruses. In these methods, the protein can be expressed in the cell from the genome of a second (or further), different, replication-deficient pseudoinfectious flavivirus. In other examples, the protein is expressed from a replicon (e.g., an alphavirus replicon, such as a Venezuelan Equine Encephalitis virus replicon; see below).
The invention also includes compositions containing two or more replication-deficient pseudoinfectious flaviviruses, in which two of the replication-deficient pseudoinfectious flaviviruses are selected from the groups consisting of: (a) a replication-deficient pseudoinfectious flavivirus including a genome containing Japanese encephalitis virus sequences, and a replication-deficient pseudoinfectious flavivirus including a genome containing dengue virus sequences; (b) a replication- deficient pseudoinfectious flavivirus including a genome containing yellow fever virus sequences, and a replication-deficient pseudoinfectious flavivirus including a genome containing dengue virus sequences; and (c) a replication-deficient pseudoinfectious flavivirus including a genome containing tick-borne encephalitis or Langat virus sequences and an inserted sequence encoding a Borrelia burgdorferi immunogen, and a replication-deficient pseudoinfectious flavivirus including a genome containing tick-borne encephalitis or Langat virus sequences and an inserted sequence encoding a tick saliva protein immunogen, or a replication-deficient pseudoinfectious flavivirus including a genome containing
tick-borne encephalitis or Langat virus sequences and inserted sequences encoding a Borrelia burgdorferi immunogen and a tick saliva protein immunogen.
Pharmaceutical compositions including the live, attenuated chimeric flaviviruses described herein are also included in the invention. Further, the invention includes methods of inducing an immune response to tick-borne encephalitis virus or Langat virus in a subject, involving
administering to the subject such a pharmaceutical composition. In various examples, the subject does not have but is at risk of developing infection by tick-borne encephalitis virus or Langat virus.
In other examples, the subject is infected with tick-borne encephalitis virus or Langat virus. The invention further includes replication-deficient pseudoinfectious flaviviruses including a flavivirus genome including one or more deletions or mutations in nucleotide sequences encoding one or more proteins selected from the group consisting of capsid (C), pre-membrane (prM), envelope (E), non-structural protein 1 (NS1), non-structural protein 3 (NS3), and non-structural protein 5 (NS5), wherein the flavivirus genome includes yellow fever virus sequences in which sequences encoding pre-membrane and envelope proteins are replaced with sequences encoding pre- membrane and envelope proteins of a tick-borne encephalitis virus or a Langat virus, and sequences encoding the signal sequence between the capsid and pre-membrane proteins of the flavivirus genome include a hybrid of sequences encoding yellow fever virus and tick-borne encephalitis or Langat virus capsid/pre-membrane signal sequences, or a variant thereof. In various examples, the sequences encoding the capsid/pre-membrane signal sequence of the flavivirus genome include yellow fever virus sequences in the 5' region and tick-borne encephalitis or Langat virus sequences in the 3' region.
Further, the invention includes replication-deficient pseudoinfectious flaviviruses including a flavivirus genome including one or more deletions or mutations in nucleotide sequences encoding one or more proteins selected from the group consisting of capsid (C), pre-membrane (prM), envelope (E), non-structural protein 1 (NS1), non-structural protein 3 (NS3), and non-structural protein 5 (NS5), wherein the flavivirus genome includes West Nile virus sequences in which sequences encoding pre-membrane and envelope proteins are replaced with sequences encoding pre- membrane and envelope proteins of a tick-borne encephalitis or a Langat virus, and the sequences encoding the signal sequence between the capsid and pre-membrane proteins of the flavivirus genome include sequences encoding a tick-borne encephalitis or a Langat virus capsid/pre- membrane signal sequence, or a variant thereof.
In addition, the invention includes replication-deficient pseudoinfectious flaviviruses including a flavivirus genome including one or more deletions or mutations in nucleotide sequences encoding one or more proteins selected from the group consisting of capsid (C), pre-membrane (prM), envelope (E), non-structural protein 1 (NS1), non-structural protein 3 (NS3), and nonstructural protein 5 (NS5), wherein any capsid (C) and non-structural (NS) proteins in the flavivirus genome are from Langat virus and any pre-membrane (prM) and envelope (E) proteins are from a tick-borne encephalitis virus.
The invention also includes use of the constructs, PIVs, LAVs, and combinations thereof for inducing immune responses, as described herein, or for preparation of medicaments as described herein.
By "replication-deficient pseudoinfectious flavivirus" or "PIV" is meant a flavivirus that is replication-deficient due to a deletion or mutation in the flavivirus genome. The deletion or mutation can be, for example, a deletion of a large sequence, such as most of the capsid protein, as described herein (with the cyclization sequence remaining; see below). In other examples, sequences encoding different proteins (e.g., prM, E, NS1, NS3, and/or NS5; see below) or combinations of proteins (e.g., prM-E or C-prM-E) are deleted. This type of deletion may be advantageous if the PIV is to be used a vector to deliver a heterologous immunogen, as the deletion can permit insertion of sequences that may be, for example, at least up to the size of the deleted sequence. In other examples, the mutation can be, for example, a point mutation, provided that it results in replication deficiency, as discussed above. Because of the deletion or mutation, the genome does not encode all proteins necessary to produce a full flavivirus particle. The missing sequences can be provided in trans by a complementing cell line that is engineered to express the missing sequence (e.g., by use of a replicon; s-PrV; see below), or by co-expression of two replication-deficient genomes in the same cell, where the two replication-deficient genomes, when considered together, encode all proteins necessary for production (d-PIV system; see below).
Upon introduction into cells that do not express complementing proteins, the genomes replicate and, in some instances, generate "virus-like particles," which are released from the cells and are able to leave the cells and be immunogenic, but cannot infect other cells and lead to the generation of further particles. For example, in the case of a PIV including a deletion in capsid protein encoding sequences, after infection of cells that do not express capsid, VLPs including prM- E proteins are released from the cells. Because of the lack of capsid protein, the VLPs lack capsid and a nucleic acid genome. In the case of the d-PrV approach, production of further PIVs is possible in cells that are infected with two PIVs that complement each other with respect to the production of all required proteins (see below).
Also included in the invention are replication-defective pseudoinfectious flaviviruses including multiple heterologous immunogens from, e.g., a human immunodeficiency virus or a simian immunodeficiency virus. In various examples, the multiple immunogens can include heterologous transmembrane and/or signal sequences (from, e.g., a rabies virus G protein).
The invention provides several advantages. For example, the PIV vectors and PIVs of the invention are highly attenuated and highly efficacious after one-to-two doses, providing durable immunity. Further, unlike inactivated vaccines, PIVs mimic whole virus infection, which can result in increased efficacy due to the induction of robust B- and T-cell responses, higher durability of immunity, and decreased dose requirements. In addition, similar to whole viruses, PIV vaccines target antigen-presenting cells, such as dendritic cells, stimulate toll-like receptors (TLRs), and induce balanced Thl/Tti2 immunity. PrV constructs have also been shown to grow to high titers in substrate cells, with little or no CPE, allowing for high-yield manufacture, optionally employing multiple harvests and/or expansion of infected substrate cells. Further, the PIV vectors of the invention provide an option for developing vaccines against non-flavivirus pathogens for which no vaccines are currently available.
Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims.
Brief Description of the Drawings
Figs. 1 A and IB are schematics illustrating single component PIV (s-PIV) technology. The replication-deficient pseudoinfectious flavivirus genomes can be selected from, for example, those of yellow fever virus, West Nile virus, tick-borne encephalitis virus, Langat virus, Japanese encephalitis virus, dengue virus, and St. Louis encephalitis virus, attenuated strains thereof, and chimeras thereof.
Fig. 2 is a schematic illustration of two-component PIV (d-PIV) technology.
Figs. 3A and 3B are schematics illustrating general experimental designs and
immunization schedules for testing immunogenicity and efficacy of PIVs in mice.
Fig. 4 is a graph comparing the humoral immune response induced by PIV-WN (RV-WN) with that of chimeric YF/WN LAV (CV-WN) in mice.
Fig. 5 is a series of graphs showing the results of challenging hamsters immunized with PIV-YF (RV-YF), YF17D, PIV-WN (RV-WN), and YF/WN LAV (CVWN) with hamster-adapted Asibi (PIV-YF and YF 17D vaccinees) and wild type WN-NY99 (PIV-WN and YF/WN LAV vaccinees).
Fig. 6 is a table showing YF/TBE and YF/LGT virus titers and plaque morphology obtained with the indicated chimeric flaviviruses.
Figs. 7A-B. Fig. 7A is a table showing WN/TBE PIV titers and examples of
immunofluorescence of cells containing the indicated PIVs. Fig. 7B is a schematic showing differences in the PIV-WN/TBE constructs p39, p39I, p40, and p98. Figs. 8A-D. Figs. 8A-B are graphs showing the replication kinetics of YF/TBE LAV (Fig. 8A) and PIV-WN/TBE (Fig. 8B) in Vera and BHK cell lines, respectively (CV-Hypr = YF/Hypr LAV; CV-LGT = YF/LGT LAV; RV-WN/TBEV = PIV-WN/TBEV). Figs. 8C and 8D are graphs showing the effect that modification of the prM signal has on PIV-WN/TBE replication in vitro (Fig. 8C) and on immunogenicity (Fig. 8D) in mice.
Fig. 9 is a series of graphs showing survival of mice inoculated IC with PIV-TBE and YF/TBE LAV constructs in a neurovirulence test (3.5 week old ICR mice; RV- WN/Hypr = PIV- WN/TBE(Hypr); CV-Hypr = YF/TBE(Hypr) LAV; CV-LGT = YF/LGT LAV).
Fig. 10 is a graph showing survival of mice inoculated TP with PIV-WN/TBE(Hypr) (RV- WN/Hypr), YF/TBE(Hypr) LAV (CV-Hypr), and YF/LGT LAV (CV-LGT) constructs and YF17D in a neuroinvasiveness test (3.5 week old ICR mice).
Fig. 11 is a series of graphs illustrating morbidity in mice measured by dynamics of body weight loss after TBE virus challenge, for groups immunized with s-PIV-TBE candidates (upper left panel), YF/TBE and YF/LGT chimeric viruses (upper right panel), and controls (YF 17D, human killed TBE vaccine, and mock; bottom panel).
Figs. 12A and 12B are schematic representations of ΡΓ7 constructs expressing rabies virus G protein. Fig. 12A also shows an illustration of packaging of the constructs to make
pseudoinfectious virus and immunization.
Fig. 13 is a schematic representation of insertion designs resulting in viable/expressing constructs (exemplified by rabies G).
Fig. 14 is series of images showing immunofluorescence analysis and graphs showing growth curves of cells transfected with the indicated PIV-WN constructs (AC-Rabies G, APrM-E- Rabies G, and AC-PrM-E-Rabies G).
Fig. 15 is a series of images showing immunofluorescence analysis of RabG expressed on the plasma membranes of Vero cells transfected with the indicated PIV constructs (AC-Rabies G, APrM-E-Rabies G, and AC-PrM-E-Rabies G).
Fig. 16 is a schematic illustration of a PIV-WN-rabies G construct and a series of images showing that this construct spreads in helper cells, but not in naive cells.
Fig. 17 is a series of graphs showing stability of the rabies G protein gene in PIV-WN vectors.
Fig. 18 is a set of images showing a comparison of spread of single-component vs. two- component PIV-WN-rabies G variants in Vero cells. Fig. 19 is a graph showing the results of rapid fluorescent focus inhibition test (RFFIT) using the indicated constructs.
Fig. 20 is a set of immunofluorescence images showing expression of full-length RSV F protein (strain A2) by the AprM-E component of d-PIV- WN in helper cells after transfection.
Fig. 21 is a graph showing RSV-F neutralization titers.
Fig. 22 is a schematic representaiton of an artificial cassette containing SIV (GenBank accession number ADM52218.1) gpl20 (the native signal sequence in the gene was replaced with the tPA signal and gp41 was truncated to contain only the TM domain), Gag, and Pro (protease) genes.
Fig. 23 is a schematic representation of inserts of the first three constructs in Fig. 22 (the three top constructs shown in Fig. 23), starting with the Env glycoprotein that were designed similarly to the PrV WN-rabies G vectors described herein (see, e.g., Figures 12-14 and
hereinbelow), in which the gpl20 signal is fused with a portion of the signal sequence for prM (e.g., at the end of the C gene or downstream from AC deletion depending on vector). In addition, schematic representations of alternate dC RV230 Env PIV constructs are shown (the three bottom constructs shown in Fig. 23).
Fig. 24 is a schematic representation of Gag and Gag-Pro PIV construct designs, in which Gag and Gag-Pro were cloned in place of the AprM-E or AC-prM-E deletions.
Fig. 25 is a photograph of a Western blot using anti-Gag antibodies, which shows correct processing of the polyprotein in recovered SIV Gag and SIV Gag/Pro PIVs grown in helper cells.
Figs. 26A-26C are photomicrographs showing that immunostaining of naive Vera cells infected with the Gag PIVs, showed individual stained cells as expected from sPIV. Fig. 26A is a negative control, Fig. 26B shows immunostaining of nai've Vero cells infected with RV230 9AA- FMD-Gag PIV, and Fig. 26C shows immunostaining of nai've Vero cells infected with RV230 FMD-Gag PIV. The two constructs are illustrated schematically in Fig. 26D.
Figs. 27A-F are graphs showing growth curves of SIV Gag PIV variants after transfection of helper cells with in vitro synthesized PIV RNA (P0 passage) indicating efficient replication in vivo. Immunofluorescence images of Vero cells infected with the variants are shown inset.
Fig. 28 is a graph showing growth curves in nai've Vero cells of SIV Gag PIV as a two- component formulation (d-PIV, sometimes also designated as tc-PIV) together with PIV-WN helper with AC deletion (RV909). Fig. 29 is a graph showing high insert stability for one of the SIV Gag PIV variants (RV230- Gag variant, containing Gag gene in place of large AprM-E deletion, in helper BHK-CprME(WN) cells at MOI 0.1 FFU/cell ) when examined by ten serial passages.
Figs. 30A-G are immunofluorescence images showing efficient expression of SIV Env (gpl20) in Vero cells using PIV-(WN)-SIV Env variants. Efficient intracellular expression of the original gpl20 was observed in Vero cells infected with packaged dC230Env PIV variant as determined by immunostaining using anti-SIV Env antibody after methanol fixation (Fig. 30D), although transport of gpl20 to the surface of infected Vero cells was inefficient, as determined following formalin fixation (Fig. 30B). In contrast, the dC230Env/RabG anchor PIV construct (see Fig. 23), in which the SIV Env TM domain was replaced with the TM anchor sequence from rabies virus G protein, showed efficient intracellular (Figs. 30C and 30F) and extracellular expression (Figs. 30A and E). Efficient intracellular expression of dC230Gag PIV variant was also observed (Fig. 30G). Improved cell surface staining was observed with RV Env/RabG relative to RV-Env.
Fig. 31 is an immunofluorescence image showing expression of SIV Env on the surface of PIV-SrV Env/RabG TM infected Vero cells.
Fig. 32A is a table showing single dose ICLD5o and D34 plaque reduction neutralization assay 50 (PRNT50) results using the indicated vectors in 2 day old suckling mice (or 8 day old sucklings for RV-TBE and YF17D).
Figs. 32B-32C are photographs showing brain histology in 2 day old suckling mice administered sPIV-WN (day 17 post 6 logio PFU IC; Fig. 32 A), tcPTV-RabG (day 11 post 6 log10 PFU; Fig. 32B), and sPTV-RabG (day 11 post 6 log10 PFU; Fig. 32C).
Fig. 33 is a graph showing individual sera endpoint titers following prime & boost with the indicated constructs. ALVAC is shown as a control.
Fig. 34 is a graph showing the results of IgG isotyping in mice treated with RV Env/RabG vector and with ALVAC EGP.
Figs. 35 is a series of three graphs showing that RV-Gag expressing constructs are capable of eliciting detectable T cell responses as measured by interferon gamma (IFNg) secretion upon peptide stimulation ex vivo.
Fig. 36 is a schematic representation of PrV-flu HA construct designs, in which the full- length HA gene of Flu strain New Caledonia was cloned in place of AprM-E and AC-prM-E deletions of PIV-WN vectors in the same fashion as described for Rabies G, RSV F and SIV Env (as is described herein). Figs. 37A-B are graphs showing growth curves in BHK 363 helper cells transfected at P14 with RNA from RV230 HA New Caledonia PIV clones 6 (Fig. 37 A) and 10 (Fig. 37B), as determined by immunostaining with anti-WN and anti-HA antibodies.
Figs. 38A-D are graphs showing growth curves in BHK 363 helper cells transfected at P14 with RNA from RV230 HA New Caledonia PIV clones 1 , 6, and 10 (Figs. 38A-C, respectively) and from dC RV230 HA New Caledonia PIV clone 6 (Fig. 38D), as determined by immunostaining with anti-WN and anti-HA antibodies.
Figs. 39A-F are immunofluorescence images showing surface expression (Figs. 39A-C) and intracellular expression (Figs. 39D-F) of HA in Vero cells infected with RV230 HA New Caledonia PIV clones 1, 6, and 10, respectively.
Figs. 40A-B are immunofluorescence images showing surface expression (Fig. 40A) and intracellular expression (Fig. 40B) of HA in Vero cells infected with dC RV230 HA New Caledonia PIV clone 6.
Fig. 41 shows immunofluorescence images confirming surface expression (Figs. 41 B and D) and intracellular expression (Figs. 41F and H) of HA in Vero cells infected with RV230 HA New Caledonia PIV. Figs. 41 A, C, E, and G are negative controls showing the lack surface expression (Figs. 41 A and C) and intracellular expression (Figs. 41E and G) of HA in uninfected Vero cells. The immunofluorescence images in Figs. 4 IB and F were produced using antibodies against the stem of HA, while the immunofluorescence images in Figs. 4 ID and H were produced using antibodies against the HA globular head. Figs. 4 IB, D, F, and H confirm the correct, native protein confirmation of HA.
Figs. 42A-D are immunofluorescence images showing surface expression (Figs. 42A and B) and intracellular expression (Figs. 42C and D) of HA in Vero cells infected with RV230 HA New Caledonia PIV clones 6 and 10, respectively, 48 hours post infection. Staining was performed with a mix of HA stem and globular head antibodies.
Fig. 43 A is an immunofluorescence image showing staining of RV230-HA PIV infected Vero cells by HA stem-specific antibodies. Fig. 43B is an immunofluorescence image showing staining of RV230-HA Pr infected Vero cells by HA globular head-specific antibodies.
Figs. 44A and 44B are schematics showing PIV constructs of the invention. Fig. 44A is a replication defective (single-cycle) virus obtained by deletion of the capsid protein gene and incorporation of the prM-E from TBEV. The construct is produced in helper cells providing deleted gene(s) in trans. Fig. 44B shows a chimera construct that is a recombinant virus between an attenuated backbone (CV-YFV 17D vaccine or PDK-53 DENV-2 viruses) and a target (TBEV) flavivirus obtained by replacing prM-E envelope protein genes.
Fig. 45 is a table showing that the PIV-TBE construct, which generates high yields on helper cells expressing WNV C protein in trans, is highly genetically stable after 10 passages at MOI 0.01 and is highly attenuated, showing no evidence of recombination in a single round of replication
(tested in vitro and in vivo).
Fig. 46 is a table showing neurovirulence and neuroinvasiveness of live chimeras (CV) and replication defective (PIV) viruses in 3.5 weeks old ICR mice. TBE chimeric viruses- based on dengue and YFV 17D- are less attenuated than the replication defective PIV viruses. PIV TBE (Hypr) is highly attenuated in adult and suckling mice. *Not determined in 3.5 week old mice, but shown to be >5 in 8-day old suckling mice.† 100% mortality at the lowest dose tested. * No mortality observed at the highest dose tested. § Partial mortality at the highest or lowest dose tested.
^ Could not be calculated (partial mortality in all tested doses).
Figs. 47A-B are graphs showing the replication of PIV (Fig. 47 A) and chimera (Fig. 46B) constructs in vitro. Fig. 46A shows growth curves of PIV variants in helper BHK or Vero cells supplying the indicated C protein in trans (MOI 0.1). Fig. 47B shows growth curves of live chimeras in Vero cells (MOI 0.001).
Figs. 48A-B are graphs showing dose responses and durability of immunity of the indicated constructs in mice. Fig. 48 A: Mice were immunized IP with graded doses of PIV-WN/TBE or YF/TBE chimera (or 2 doses of dilutions of INV on days 0 and 14 (human dose is 2.4 mg); shown in insert), and challenged on day 21 with 500 LD50 of TBE Hypr, to determine protective dose 50% values (PD50). Fig. 48B: Mice were immunized with 10 PD50 doses of PIV-WN/TBE, YF/TBE, or
2x INV and N Ab titers in sera were monitored for up to ~ 5 months. % survivals after challenge with 500 LD5o of TBE Hypr are indicated.
Fig. 49 is a graph showing that pre-immunization with PIV-WN/TBE resulted in some reduction of TBE specific response compared to that in naive animals. Mice were preimmunized with YF 17D and then immunized 3 weeks or 6 months later with Chimera- JE or YF/TBE viruses.
Mice were similarly preimmunized with PIV-WN and immunized with PIV-WN/TBE. All doses were 5 log10 by IP. Vaccine-specific N Ab titers were measured 21 days after immunization.
Figs. 50A-B are graphs showing the immunogenicity and efficacy of PIV-WN/TBE compared to 3 doses of INV in non-human primate (NHP) study 2. Fig. 50A: Post-challenge LGT 1674 viremia (peak titers on day 2) determined by a sensitive RT-qPCR method. Fig. 50B:
Durability of N Ab responses.
Fig. 51 is a schematic and table showing a design for a NHP study using the indicated constucts.
Fig. 52 is a schematic showing a short term (70 days) segment of a NHP study using the indicated constructs. Day 51 after immunization, animals will be administered a heterologous challenge with LGV 1674.
Fig. 53 is a schematic showing a long term (6 months+) segment of a NHP study using the indicated constructs. Month 6 after immunization, animals will be administered a heterologous challenge with LGV 1674.
Figs. 54A-D. Post-challenge viremia using LGT 1 74 virus determined using plaque assay. Fig. 54A: Establishing the model using 6 log10 PFU dose. To achieve better resolution of viremia, the dose was reduced to 5 logi0 PFU for challenge of animals. Fig. 54B: Challenge in NHP study 1 (see Figs. 51 and 52). Only Mock animals showed viremia, while immunized monkeys in all other groups showed no detectabale viremia. Fig. 54C: Postchallenge viremia in the short-term segment of Study #2 (see Figs. 51 and 52). Fig. 54d: Postchallenge viremia in the long-term segment of Study #2 (see Figs. 51 and 53).
Detailed Description of the Invention
The invention provides replication-defective or deficient pseudoinfectious virus (PIV) vectors including flavivirus sequences, which can be used in methods for inducing immunity against heterologous pathogen, cancer, and allergy-related immunogens inserted into the vectors as well as, optionally, the vectors themselves. The invention also includes compositions including
combinations of PIVs and/or PIV vectors, as described herein, and methods of using such compositions to induce immune responses against inserted immunogen sequences and/or sequences of the PIVs themselves. Further, the invention includes particular PIVs and live, attenuated chimeric flaviviruses including tick-borne encephalitis virus sequences, and related vectors, compositions, and methods of use. The PIV vectors, PIVs, live attenuated chimeric flaviviruses, compositions, and methods of the invention are described further below. PIV Vectors and PIVs
The PIV vectors and PIVs of the invention can be based on the single- or two-component PIVs described above (also see WO 2007/098267 and WO 2008/137163). Thus, for example, in the case of single component PIVs, the PIV vectors and PIVs can include a genome including a large deletion in capsid protein encoding sequences and be produced in a complementing cell line that produces capsid protein in trans (single component; Fig. 1 and Figs. 12A and 12B). According to this approach, most of the capsid-encoding region is deleted, which prevents the PIV genome from producing infectious progeny in normal cell lines (i.e., cell lines not expressing capsid sequences) and vaccinated subjects. The capsid deletion typically does not disrupt RNA sequences required for genome cyclization (i.e., the sequence encoding amino acids in the region of positions 1-26), and/or the prM sequence required for maturation of prM to M. In specific examples, the deleted sequences correspond to those encoding amino acids 26-100, 26-93, 31-100, or 31-93 of the C protein.
Single component PIV vectors and PIVs can be propagated in cell lines that express either C or a C-prM-E cassette, where they replicate to high levels. Exemplary cell lines that can be used for expression of single component PIV vectors and PIVs include BHK-21 (e.g., ATCC CCL-10), Vero (e.g., ATCC CCL-81), C7/10, and other cells of vertebrate or mosquito origin. The C or C-prM-E cassette can be expressed in such cells by use of a viral vector-derived replicon, such as an alphavirus replicon (e.g., a replicon based on Venezuelan Equine Encephalitis virus (VEEV), Sindbis virus, Semliki Forest virus (SFV), Eastern Equine Encephalitis virus (EEEV), Western Equine Encephalitis virus (WEEV), or Ross River virus). To decrease the possibility of productive recombination between the PIV vectors/PIVs and complementing sequences, the sequences in the replicons (encoding C, prM, and/or E) can include nucleotide mutations. For example, sequences encoding a complementing C protein can include an unnatural cyclization sequence. The mutations can result from codon optimization, which can provide an additional benefit with respect to PIV yield. Further, in the case of complementing cells expressing C protein sequences (and not a C- prM-E cassette), it may be beneficial to include an anchoring sequence at the carboxy terminus of the C protein including, for example, about 20 amino acids of prM (see, e.g., WO 2007/098267).
The PIV vectors and PIVs of the invention can also be based on the two-component genome technology described above. This technology employs two partial genome constructs, each of which is deficient in expression of at least one protein required for productive replication (capsid or prM/E) but, when present in the same cell, result in the production of all components necessary to make a PIV. Thus, in one example of the two-component genome technology, the first component includes a large deletion of C, as described above in reference to single component PIVs, and the second component includes a deletion of prM and E (Fig. 2 and Fig. 12A). In another example, the first component includes a deletion of C, prM, and E, and the second component includes a deletion of NS1 (Fig. 12 A). Both components can include cis-acting promoter elements required for RNA replication and a complete set of non-structural proteins, which form the replicative enzyme complex. Thus, both defective genomes can include a 5 '-untranslated region and at least about 60 nucleotides (Element 1) of the following, natural protein-coding sequence, which comprises an ammo-terminal fragment of the capsid protein. This sequence can be followed by a protease cleavage sequence such as, for example, a ubiquitine or foot-and-mouth disease virus (FAMDV)- specific 2 A protease sequence, which can be fused with either capsid or envelope (prM-E) coding sequences. Further, artificial, codon optimized sequences can be used to exclude the possibility of recombination between the two defective viral genomes, which could lead to formation of replication-competent viruses (see, e.g., WO 2008/137163). Use of the two-component genome approach does not require the development of cell lines expressing complementing genomes, such as the cells transformed with replicons, as discussed above in reference to the single component PIV approach. Exemplary cell lines that can be used in the two-component genome approach include Vera (e.g., ATCC CCL-81), BHK-21 (e.g., ATCC CCL-10), C7/10, and other cells of vertebrate or mosquito origin.
Additional examples of d-PIV approaches that can be used in the invention are based on use of complementing genomes including deletions in NS3 or NS5 sequences. A deletion in, e.g., NS1, NS3, or NS5 proteins can be used as long as several hundred amino acids in the ORF, removing the entire chosen protein sequence, or as short as 1 amino acid inactivating protein enzymatic activity (e.g., NS5 RNA polymerase activity, NS3 helicase activity, etc.). Alternatively, point amino acid changes (as few as 1 amino acid mutation, or optionally more mutations) can be introduced into any NS protein, inactivating enzymatic activity. In addition, several ANS deletions can be combined in one helper molecule. The same heterologous gene, i.e., expressed by the first d-PIV component, can be expressed in place or in combination with the NS deletion(s) in the second component, increasing the amount of expressed immunogen. Notably, the insertion capacity of the helper will increase proportionally to the size of NS deletion(s). Alternatively, a different foreign immunogen(s) can be inserted in place of deletion(s) of the helper to produce multivalent vaccines. Further, additional approaches that can be used in making PIV vectors and PIVs for use in the present invention are described, for example, in WO 99/28487, WO 03/046189, WO
2004/108936, US 2004/0265338, US 2007/0249032, and U.S. Patent No. 7,332,322.
The PIV vectors and Pr s of the invention can be comprised of sequences from a single flavivirus type (e.g., tick-borne encephalitis (TBE, e.g., strain Hypr), Langat (LGT), yellow fever (e.g., YF17D), West Nile, Japanese encephalitis, dengue (serotype 1-4), St. Louis encephalitis, Kunjin, Rocio encephalitis, Ilheus, Central European encephalitis, Siberian encephalitis, Russian Spring-Summer encephalitis, Kyasanur Forest Disease, Omsk Hemorrhagic fever, Louping ill, Powassan, Negishi, Absettarov, Hansalova, and Apoi viruses), or can comprise sequences from two or more different fiaviviruses. Sequences of some strains of these viruses are readily available from generally accessible sequence databases; sequences of other strains can be easily determined by methods well known in the art. In the case of PIV vectors and PIVs including sequences of more than one flavivirus, the sequences can be those of a chimeric flavivirus, as described above (also see, e.g., U.S. Patent No. 6,962,708; U.S. Patent No. 6,696,281; and U.S. Patent No. 6,184,024). In certain examples, the chimeras include pre-membrane and envelope sequences from one flavivirus (such as a flavivirus to which immunity may be desired), and capsid and non-structural sequences from a second, different flavivirus. In one specific example, the second flavivirus is a yellow fever virus, such as the vaccine strain YF17D. Other examples include the YF/TBE, YF/LGT, WN/TBE, and WN/LGT chimeras described below. Another example is an LGT/TBE chimera based on LGT virus backbone containing TBE virus prM-E proteins. A PIV vaccine based on this genetic background would have an advantage, because LGT replicates very efficiently in vitro and is highly attenuated and immunogenic for humans. Thus, a chimeric LGT/TBE PIV vaccine is expected to provide a robust specific immune response in humans against TBE, particularly due to inclusion of TBE prM-E genes.
Vectors of the invention can be based on PIV constructs or live, attenuated chimeric fiaviviruses as described herein (in particular, YF/TBE, YF LGT, WN/TBE, and WN LGT; see below). Use of PIV constructs as vectors provides particular advantages in certain circumstances, because these constructs by necessity include large deletions, which render the constructs more amenable to accommodation of insertions that are at least up to the size of the deleted sequences, without there being a loss in replication efficiency. Thus, PIV vectors in general can comprise very small insertions (e.g., in the range 6-10, 11-20, 21-100, 101-500, or more amino acid residues combined with the AC deletion or other deletions), as well as relatively large insertions or insertions of intermediate size (e.g., in the range 501-1000, 1001-1700, 1701-3000, or 3001-4000 or more residues). In contrast, in certain examples, it may be advantageous to express relatively short sequences in live attenuated viruses, particularly if the insertions are made in the absence of a corresponding deletion. Additional information concerning insertion sites that can be used in the invention is provided below. In addition, as discussed further below, expression of non-flavivirus immunogens in PIVs and chimeric flaviviruses of the invention can result in dual vaccines that elicit protective immunity against both a flavivirus vector virus pathogen and a target heterologous immunogen (e.g., a pathogen (such as a bacterial, viral, parasite, or fungal pathogen), cancer, or allergy-related immunogen).
As discussed above, the PIV vectors and PIVs of the invention can comprise sequences of chimeric flaviviruses, for example, chimeric flaviviruses including pre-membrane and envelope sequences of a first flavivirus (e.g., a flavivirus to which immunity is sought), and capsid and nonstructural sequences of a second, different flavivirus, such as a yellow fever virus (e.g., YF17D; see above and also U.S. Patent No. 6,962,708; U.S. Patent No. 6,696,281; and U.S. Patent No.
6,184,024). Further, chimeric flaviviruses of the invention, used as a source for constructing PIVs, or as vaccines/vaccine vectors per se, can optionally include one or more specific attenuating mutations (e.g., E protein mutations, prM protein mutations, deletions in the C protein, and/or deletions in the 3TJTR), such as any of those described in WO 2006/116182. For example, the C protein or 3'UTR deletions can be directly applied to YF/TBE or YF/LGT chimeras. Similar deletions can be designed and introduced in other chimeric LAV candidates such as based on LGT/TBE, WN/TBE, and WN/LGT genomes. With respect to E protein mutations, attenuating mutations similar to those described for YF/WN chimera in WO 2006/116182 can be designed, e.g., based on the knowledge of crystal structure of the E protein (Rey et al., Nature 375(6529):291-298, 1995), and employed. Further, additional examples of attenuating E protein mutations described for TBE virus and other flaviviruses are provided in Table 10. These can be similarly introduced into chimeric vaccine candidates.
The invention also provides new, particular chimeric flaviviruses, which can be used as a basis for the design of PrV vectors and PIVs, as live attenuated chimeric flavivirus vectors, and as vaccines against the source(s) of the pre-membrane and envelope components of the chimeras. These chimeras include tick-borne encephalitis (TBE) virus or related prM-E sequences. Thus, the chimeras can include prM-E sequences from, for example, the Hypr strain of TBE or Langat (LGT) virus. Capsid and non-structural proteins of the chimeras can include those from yellow fever virus (e.g., YF17D) or West Nile virus (e.g., NY99).
A central feature of these exemplary YF/TBE, YF/LGT, WN/TBE, and WN/LGT chimeras is the signal sequence between the capsid and prM proteins. As is shown in the Examples, below, we have found that, in the case of YF-based PIV chimeras, it is advantageous to use a signal sequence comprising yellow fever and TBE sequences (see below). In one example, the signal sequence includes yellow fever sequences in the amino terminal region (e.g., SHDVLTVQFLIL; SEQ ID NO:l) and TBE sequences in the carboxy terminal region (e.g., GMLGMTIA; SEQ ID NO:2), resulting in the sequence SHDVLTVQFLILGMLGMTIA (SEQ ID NO:3). We have also found that, in the case of WN-based PIV chimeras, it is advantageous to use a signal sequence comprising TBE sequences (e.g., GGTDWMSWLLVIGMLGMTIA; SEQ ID NO:4). The invention thus includes YF/TBE, YF/LGT, WN/TBE, and WN/LGT chimeras, both PIVs and LAVs, which include the above-noted signal sequences, or variants thereof having, e.g., 1-8, 2-7, 3-6, or 4-5 amino acid substitutions, deletions, or insertions, which do not substantially interfere with processing at the signal sequence. In various examples, the substitutions are "conservative substitutions," which are characterized by replacement of one amino acid residue with another, biologically similar residue. Examples of conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine, or methionine for another, or the substitution of one polar residue for another, such as between arginine and lysine, between glutamic and aspartic acids, or between glutamine and asparagine and the like. Examples of exemplary PIVs of the present invention include those described in Appendices 6-8, constructs having at least 50% sequence identity (e.g., 50%, 60%, 70%, 85%, 90%, 95%, or 99% or more sequence identity) to the nucleic acid or amino acid sequences described therein, or constructs that include homologs and or other naturally occurring variants of the SIV, HIV, and/or HA proteins. Additional information concerning these and other chimeras is provided below, in the Examples.
Insertion Sites
Sequences encoding immunogens can be inserted at one or more different sites within the vectors of the invention. Relatively short peptides can be delivered on the surface of PIV or LAV glycoproteins (e.g., prM, E, and or NS 1 proteins) and/or in the context of other proteins (to induce predominantly B-cell and T-cell responses, respectively). Other inserts, including larger portions of foreign proteins, as well as complete proteins, can be expressed intergenically, at the N- and C- termini of the polyprotein, or bicistronically (e.g., within the ORF under an IRES or in the 3'UTR under an IRES; see, e.g., WO 02/102828, WO 2008/036146, WO 2008/094674, WO 2008/100464, WO 2008/115314, and below for further details). In PIV constructs, there is an additional option of inserting a foreign amino acid sequence directly in place of introduced deletion(s). Insertions can be made in, for example, AC, AprM-E, AC-prM-E, ANSI, ANS3, and ANS5. Thus, in one example, in the case of s-PIVs and the AC component of d-PIVs, imrnunogen-encoding sequences can be inserted in place of deleted capsid sequences. Imrnunogen-encoding sequences can also, optionally, be inserted in place of deleted prM-E sequences in the AprM-E component of d-PIVs. In another example, the sequences are inserted in place of or combined with deleted sequences in AC-prM-E constructs. Examples of such insertions are provided in the Examples section, below.
In the case of making insertions into PIV deletions, the insertions can be made with a few (e.g., 1, 2, 3, 4, or 5) additional vector-specific residues at the N- and/or C-termini of the foreign immunogen, if the sequence is simply fused in-frame (e.g., ~ 20 first a.a. and a few last residues of the C protein if the sequence replaces the AC deletion), or without, if the foreign immunogen is flanked by appropriate elements well known in the field (e.g., viral protease cleavage sites; cellular protease cleavage sites, such as signalase, furin, etc.; autoprotease; termination codon; and/or IRES elements).
If a protein is expressed outside of the continuous viral open reading frame (ORF), e.g., if vector and non-vector sequences are separated by an internal ribosome entry site (IRES), cytoplasmic expression of the product can be achieved or the product can be directed towards the secretory pathway by using appropriate signal/anchor segments, as desired. If the protein is expressed within the vector ORF, important considerations include cleavage of the foreign protein from the nascent polyprotein sequence, and maintaining correct topology of the foreign protein and all viral proteins (to ensure vector viability) relative to the ER membrane, e.g., translocation of secreted proteins into the ER lumen, or keeping cytoplasmic proteins or membrane-associated proteins in the cytoplasm in association with the ER membrane.
In more detail, the above-described approaches to making insertions can employ the use of, for instance, appropriate vector-derived, insert-derived, or unrelated signal and anchor sequences included at the N and C termini of glycoprotein inserts. For example, all or a portion of the rabies G-derived signal and/or anchor sequences can be used in place of all or a portion of the signal and/or anchor sequences for glycoprotein inserts (e.g., one or more of the SIV, HIV, or influenza virus proteins described herein) to produce a heterologous polypeptide sequence. Standard autoproteases, such as, for example, FMDV 2A autoprotease (-20 amino acids) or ubiquitin (gene ~ 500 nt), or flanking viral NS2B/NS3 protease cleavage sites can be used to direct cleavage of an expressed product from a growing polypeptide chain, to release a foreign protein from a vector polyprotein, and to ensure viability of the construct. Optionally, growth of the polyprotein chain can be terminated by using a termination codon, e.g., following a foreign gene insert, and synthesis of the remaining proteins in the constructs can be re-initiated by incorporation of an IRES element, e.g., the encephalomyocarditis virus (EMCV) IRES commonly used in the field of RNA virus vectors. Viable recombinants can be recovered from helper cells (or regular cells for d-PIV versions).
Optionally, backbone PIV sequences can be rearranged, e.g., if the latter results in more efficient expression of a foreign gene. For example, a gene rearrangement has been applied to TBE virus, in which the prM-E genes were moved to the 3' end of the genome under the control of an IRES (Orlinger et al., J. Virol. 80:12197-12208, 2006). Translocation of prM-E or any other genes can be applied to PIV flavivirus vaccine candidates and expression vectors, according to the invention.
Additional details concerning different insertion sites that can be used in the invention are as follows (also see WO 02/102828, WO 2008/036146, WO 2008/094674, WO 2008/100464, WO 2008/115314, as noted above). Peptide sequences can be inserted within the envelope protein, which is the principle target for neutralizing antibodies. The sequences can be inserted into the envelope in, for example, positions corresponding to amino acid positions 59, 207, 231, 277, 287, 340, and/or 436 of the Japanese encephalitis virus envelope protein (see, e.g., WO 2008/115314 and WO 02/102828). To identify the corresponding loci in different flaviviruses, the flavivirus sequences are aligned with that of Japanese encephalitis virus. As there may not be an exact match, it should be understood that, in non-JE viruses, the site of insertion may vary by, for example, 1, 2, 3, 4, or 5 amino acids, in either direction. Further, given the identification of such sites as being permissive in JE, they can also vary in JE by, for example, 1, 2, 3, 4, or 5 amino acids, in either direction. Additional permissive sites can be identified using methods such as transposon mutagenesis (see, e.g., WO 02/102828 and WO 2008/036146). The insertions can be made at the indicated amino acids by insertion just C-terminal to the indicated amino acids (i.e., between amino acids 51-52, 207-208, 231-232, 277-278, 287-288, 340-341, and 436-437), or in place of short deletions (e.g., deletions of 1, 2, 3, 4, 5, 6, 7, or 8 amino acids) beginning at the indicated amino acids (or within 1-5 positions thereof, in either direction).
Pn addition to the envelope protein, insertions can be made into other virus proteins including, for example, the membrane/pre-membrane protein and NSl (see, e.g., WO 2008/036146). For example, insertions can be made into a sequence preceding the capsid/pre-membrane cleavage site (at, e.g., -4, -2, or -1) or within the first 50 amino acids of the pre-membrane protein (e.g., at position 26), and/or between amino acids 236 and 237 of NS1 (or in regions surrounding the indicated sequences, as described above). In other examples, insertions can be made intergenically. For example, an insertion can be made between E and NS 1 proteins and/or between NS2B and NS3 proteins (see, e.g., WO 2008/100464). In one example of an intergenic insertion, the inserted sequence can be fused with the C-terminus of the E protein of the vector, after the C-terminal signal/anchor sequence of the E protein, and the insertion can include a C-terminal anchor/signal sequence, which is fused with vector NS 1 sequences. In another example of an intergenic insertion, the inserted sequences, with flanking protease cleavage sites (e.g., YF 17D cleavage sites), can be inserted into a unique restriction site introduced at the NS2B NS3 junction (WO 2008/100464).
In other examples, a sequence can be inserted in the context of an internal ribosome entry site (IRES, e.g., an IRES derived from encephalomyocarditis virus; EMCY), as noted above, such as inserted in the 3 '-untranslated region (WO 2008/094674). In one example of such a vector, employing, for example, yellow fever virus sequences, an IRES-immunogen cassette can be inserted into a multiple cloning site engineered into the 3 '-untranslated region of the vector, e.g., in a deletion (e.g., a 136 nucleotide deletion in the case of a yellow fever virus-based example) after the polyprotein stop codon (WO 2008/094674).
Details concerning the insertion of rabies virus G protein and full-length respiratory syncytial virus (RSV) F protein into s-PIV and d-PIV vectors of the invention are provided below in Example 3. The information provided in Example 3 can be applied in the context of other vectors and immunogens described herein.
Immunogens
PIVs (s-PrVs and d-PIVs) based on flavivirus sequences and live, attenuated chimeric flaviviruses (e.g., YF/TBE, YF/LGT, WN/TBE, and WN/LGT), as described above, can be used in the invention to deliver foreign (e.g., non-flavivirus) pathogen (e.g., viral, bacterial, fungal, and parasitic pathogens), cancer, and allergy-related immunogens. As discussed further below, in certain examples, it may be advantageous to target several pathogens occupying the same ecological niche, in a particular geographical region. Specific, non-limiting examples of such immunogens are provided as follows. In addition to TBE virus, ticks are known to transmit another major disease, Lyme disease. Thus, in a first example, PIVs of the invention, such as PIVs including TBE/LGT sequences, as well as chimeric flaviviruses including TBE sequences (e.g., YF/TBE, YF/LGT, WN/TBE, LGT/TBE, and WN/LGT; in all instances where "TBE" is indicated, this includes the option of using the Hypr strain), can be used as vectors to deliver protective immunogens of the causative agent of Lyme disease (tick-borne spirochete Borrelia burgdorferi). This combination, targeting both infectious agents (TBE and B. burgdorferi) is advantageous, because TBE and Lyme disease are both tick- borne diseases. The PIV approaches can be applied to chimeras (e.g., YF/TBE, YF/LGT, WN/TBE, or WN/LGT), according to the invention, as well as to non-chimeric TBE and LGT viruses. An exemplary immunogen from B. burgdorferi that can be used in the invention is OspA (Gipson et al., Vaccine 21:3875-3884, 2003). Optionally, to increase safety and/or immunogenicity, OspA can be mutated to reduce chances of autoimmune responses and/or to eliminate sites for unwanted post- translational modification in vertebrate animal cells, such as N-linked glycosylation, which may affect immunogenicity of the expression product. Mutations that decrease autoimmunity can include, e.g., those described by Willett et al., Proc. Natl. Acad. Sci. U.S.A. 101 : 1303-1308, 2004. In one example, FTK-OspA, a putative cross-reactive T cell epitope, Bb OspA^s-m (YVLEGTLTA; SEQ ID NO: 5) is altered to resemble the corresponding peptide sequence of Borrelia afzelli
(FTLEGKVAN; SEQ ID NO:6). In FTK-OspA, the corresponding sequence is FTLEGKLTA (SEQ ID NO:7).
The sequence of OspA is as follows (SEQ ID NO: 8):
1 mkkyllgigl ilaliackqn vssldeknsv svdlpgemkv lvskeknkdg kydliatvdk 61 lelkgtsdkn ngsgvlegvk adkskvklti sddlgqttle vfkedgktlv skkvtskdks 121 steekfnekg evsekiitra dgtrleytgi ksdgsgkake vlkgyvlegt Itaekttlvv 181 kegtvtlskn isksgevsve lndtdssaat kktaawnsgt stltitvnsk ktkdlvftke 241 ntitvqqyds ngtklegsav eitkldeikn alk
The full-length sequence and/or immunogenic fragments of the full-length sequence can be used in the present invention. Exemplary fragments can include one or more of domains 1 (amino acids 34- 41), 2 (amino acids 65-75), 3 (amino acids 190-220), and 4 (amino acids 250-270) (Jiang et al., Clin. Diag. Lab. Immun. 1(4):406-412, 1994).
Thus, for example, a peptide comprising any one (or more) of the following sequences (which include sequence variations that can be included in the sequence listed above, in any combination) can be delivered (SEQ ID NOs:9-12): LPGE/GM/IK/T/GVL; GTSDKN/S/DNGSGV/T;
N/H/EIS/P/L/A/SK/NSGEV/IS/TV/AE/ALN/DDT/SD/NS/
TS/TA/Q/RATKKTA/GA/K TWN/DS/AG/N/KT; SN/AGTK/NLEGS/N/K TAVEIT/ K/TLD/KEI/LKN.
In addition to B. burgdorferi immunogens, tick saliva proteins, such as 64TRP, Isac, and Salp20, can be expressed, e.g., to generate a vaccine candidate of trivalent-specificity (TBE+Lyme disease+ticks). Alternatively, tick saliva proteins can be expressed instead of B. burgdorferi immunogens in TBE sequence-containing vectors. In addition, there are many other candidate tick saliva proteins that can be used for tick vector vaccine development according to the invention (Francischetti et al., Insect Biochem. Mol. Biol. 35:1142-1161, 2005). One or more of these immunogens can be expressed in s-PIV-TBE. However, d-PIV-TBE may also be selected, because of its large insertion capacity. In addition to PIV-TBE, other PIV vaccines can be used as vectors, e.g., to protect from Lyme disease and another flavivirus disease, such as West Nile virus.
Expression of these immunogens can be evaluated in cell culture, and immunogenicity/protection examined in available animal models (e.g., as described in Gipson et al., Vaccine 21:3875-3884, 2003; Labuda et al., Pathog. 2(e27):0251-0259, 2006). Immunogens of other pathogens can be similarly expressed, in addition to Lyme disease and tick immunogens, with the purpose of making multivalent vaccine candidates. Exemplary tick saliva immunogens that can be used in the invention include the following:
64TRP (AF469170) (SEP ID NO: 13)
MKAFFVLSLL STAALTNAAR AGRLGSDLDT FGRVHGNLYA GIERAGPRGY PGLTASIGGE VGARLGGRAG VGVSSYGYGY PSWGYPYGGY GGYGGYGGYG GYDQGFGSAY GGYPGYYGYY YPSGYGGGYG GSYGGSYGGS YTYPNVRASA GAAA
Isac (AF270496) (SEP ID NO: 14)
RTAFTCALL AISFLGSPCS SSEDGLEQDT IVETTTQNLY ERHYRNHSGL CGAQYRNSSH AEAVYNCTLN HLPPWNATW EGIRHRINKT IPQFVKLICN FTVAMPQEFY LVYMGSDGNS DFEEDKESTG TDEDSNTGSS AAAKVTEALI IEAEENCTAH ITGWTTETPT TLEPTTESQF EAIP
Salp20 (EU008559) (SEP ID NO: 15)
MRTALTCALL AISFLGSPCS SSEGGLEKDS RVETTTQNLY ERYYRKHPGL CGAQYRNSSH AEAVYNCTLS LLPLSVNTTW EGIRHRINKT IPEFVNLICN FTVAMPDQFY LVYMGSNGNS YSEEDEDGKT GSSAAVQVTE QLIIQAEENC TAHITGWTTE APTTLEPTTE TQFEAIS
Additional details concerning the TBE-related PIVs and LAVs are provided in Example 2, below.
The invention further provides PIV and LAV-vectored vaccines against other non-flavivirus pathogens, including vaccines having dual action, eliciting protective immunity against both flavivirus (as specified by the vector envelope proteins) and non-flavivirus pathogens (as specified by expressed immunologic determinant(s)). These are similar to the example of PIV-TBE-Lyme disease-tick vector vaccines described above. As mentioned above, such dual-action vaccines can be developed against a broad range of pathogens by expression of immunogens from, for example, viral, bacterial, fungal, and parasitic pathogens, and immunogens associated with cancer and allergy. As specific non-limiting examples, we describe herein the design and biological properties of PIV vectored-rabies and -respiratory syncytial virus (RSV) vaccine candidates constructed by expression of rabies virus G protein or full-length RSV F protein in place of or in combination with various deletions in one- and two-component PIV vectors (see Example 3, below). Also described in Example 4 are SrV/HIV-based PIV vectors. Example 5 provides influenza virus HA-based PIV vectors.
As is demonstrated in the Examples, below, s-PIV constructs may be advantageously used to stably deliver relatively short foreign immunogens (similar to Lyme disease agent OspA protein and tick saliva proteins), because insertions are combined with a relatively short AC deletion. Two- component PIV vectors may be advantageously used to stably express relatively large immunogens, such as rabies G protein and RSV F, as the insertions in such vectors are combined with, for example, large AprM-E, AC-prM-E, and/or ANS 1 deletions. Some of the d-PIV components can be manufactured and used as vaccines individually, for instance, the PIV-RSV F construct described below containing a AC-prM-E deletion. In this case, the vaccine induces an immune response (e.g., neutralizing antibodies) predominantly against the expressed protein, but not against the flavivirus vector virus pathogen. In other examples of the invention, dual immunity is obtained by having immunity induced both to vector and insert components. Additionally, because of the large insertion capacity of PIV vectors, and the option of using two-component genomes, PIV vectors offer the opportunity to target several non-flavivirus pathogens simultaneously, e.g., by expressing foreign immunogens from two different non-flavivirus pathogens in the two components of a
d-PIV.
In addition to the RSV F protein, rabies G protein, Lyme disease protective immunogens, and tick saliva proteins, as examples of foreign immunogens described above, other foreign immunogens can be expressed to target respective diseases including, for example, influenza virus type A and B immunogens. In these examples, a few short epitopes and/or whole genes of viral particle proteins can be used, such as the M2, HA, and NA genes of influenza A, and/or the NB or BM2 genes of influenza B (see, e.g., the PIV constructs of Example 5 below). Shorter fragments of M2, NB, and BM2, corresponding for instance to M2e, the extracellular fragment of M2, can also be used. In addition, fragments of the HA gene, including epitopes identified as HA0 (23 amino acids in length, corresponding to the cleavage site in HA) can be used. Specific examples of influenza- related sequences that can be used in the invention include PAKLLKERGFFGAIAGFLE (HAO; SEQ ID NO: 16), P AKLLKERGFFG AIAGFLEGS GC (HAO; SEQ ID NO: 17),
NNATFNYTNVNPISHIRGS (NBe; SEQ ID NO: 18), MSLLTEVETPIRNE WGCRCNDS SD (M2e; SEQ ID NO: 19), MSLLTEVETPTRNEWECRCSDSSD (M2e; SEQ ID NO:20),
MSLLTEVETLTRNGWGCRCSDSSD (M2e; SEQ ID NO:21), EVETPTRN (M2e; SEQ ID
NO:22), SLLTEVETPIRNEWGCRCNDSSD (M2e; SEQ ID NO:23), and
SLLTEVETPIRNEWGCR (M2e; SEQ ID NO:24). Additional M2e sequences that can be used in the invention include sequences from the extracellular domain of BM2 protein of influenza B (consensus MLEPFQ (SEQ ID NO:25), e.g., LEPFQILSISGC (SEQ ID NO:26)), and the M2e peptide from the H5N1 avian flu (MSLLTEVETLTRNGWGCRCSDSSD; SEQ ID NO:27).
Other examples of pathogen immunogens that can be delivered in the vectors of the invention include optionally codon optimized SIV or HIV gag (55 kDa), g l20, gpl40, gpl45, gp41, gpl60, SIV mac239 pol/-rev/tat/nef/pro genes or analogs or homologs and/or other naturally occurring variants from SIV and/or HIV, and other SIV and/or HIV immunogens (see, e.g., the PIV vectors described in Example 4 below). Sequences of these antigens can be inserted into PIV vectors, as described herein, in place of C, prM-E, and/or C-prM-E sequences. Further, these sequences (and other sequences described herein) can be inserted in combination within a vector as described herein and be separated by, e.g., autoprotease sequences, as described herein. Thus, the invention includes, for example, vectors including combinations of the HIV sequences noted above (gag (55 kDa), gpl20, gpl40, gpl45, gp41, gpl60, SIV mac239 pol/-rev/tat/nef/pro genes or analogs or homologs and/or other naturally occurring variants from SIV and/or HIV, and other SIV and/or ΗΓν immunogens; e.g., gpl20, gag, and/or pro). As described elsewhere herein, these constructs can optionally employ heterologous TM and/or signal sequences, and are, optionally, codon- optimized.
Additional examples of pathogen immunogens include immunogens from HPV viruses, such as HPV16, HPV18, etc., e.g., the capsid protein LI which self-assembles into HPV-like particles, the capsid protein L2 or its immunodominant portions (e.g., amino acids 1-200, 1-88, or 17-36), the E6 and E7 proteins which are involved in transforming and immortalizing mammalian cells fused together and appropriately mutated (fusion of the two genes creates a fusion protein, referred to as E6E7Rb", that is about 10-fold less capable of transforming fibroblasts, and mutations of the E7 component at 2 residues renders the resulting fusion protein mutant incapable of inducing transformation (Boursnell et al., Vaccine 14:1485-1494, 1996). Other immunogens include protective immunogens from HCV, CMV, HS V2, viruses, malaria parasite, Mycobacterium tuberculosis causing tuberculosis, C. difficile, and other nosocomial infections, that are known in the art, as well as fungal pathogens, cancer immunogens, and proteins associated with allergy that can be used as vaccine targets.
Foreign immunogen inserts of the invention can be modified in various ways. For instance, codon optimization is used to increase the level of expression and eliminate long repeats in nucleotide sequences to increase insert stability in the RNA genome of PIV vectors.
Immunogenicity can be increased by chimerization of proteins with immunostimulatory moieties well known in the art, such as TLR agonists, stimulatory cytokines, components of complement, heat-shock proteins, etc. (e.g., reviewed in "Immunopotentiators in Modern Vaccines," Schijns and O'Hagan Eds., 2006, Elsevier Academic Press: Amsterdam, Boston).
With respect to construction of dual vaccines against rabies and other flavivirus diseases, other combinations, such as TBE + rabies, YF + rabies, etc., can be of interest both for human and veterinary use in corresponding geographical regions, and thus can be similarly generated. Possible designs of expression constructs are not limited to those described herein. For example deletions and insertions can be modified, genetic elements can be rearranged, or other genetic elements (e.g. non-flavivirus, non-rabies signals for secretion, intracellular transport determinants, inclusion of or fusion with immunostimulatory moieties such as cytokines, TLR agonists such as flagellin, multimerization components such as leucine zipper, and peptides that increase the period of protein circulation in the blood) can be used to facilitate antigen presentation and increase immunogenicity. Further, such designs can be applied to s-PIV and d-PIV vaccine candidates based on vector genomes of other flaviviruses, and expressing immunogens of other pathogens, e.g., including but not limited to pathogens described in elsewhere herein.
Other examples of PIV and LAV vectors of the invention including combination vaccines such as DEN+Chikungunya virus (CHIKV) and YF+CHIKV. CHIKV, an alphavirus, is endemic in Africa, South East Asia, Indian subcontinent and the Islands, and the Pacific Islands and shares ecological/geographical niches with YF and DEN 1-4. It causes serious disease primarily associated with severe pain (arthritis, other symptoms similar to DEN) and long-lasting sequelae in the majority of patients (Simon et al., Med. Clin. North Am. 92:1323-1343, 2008; Seneviratne et al., J. Travel Med. 14:320-325, 2007). Other examples of PIV and LAV vectors of the invention include YF+Ebola or DEN+Ebola, which co-circulate in Africa. Immunogens for the above-noted non-flavivirus pathogens, sequences of which are well known in the art, may include glycoprotein B or a pp65/IEl fusion protein of CMV (Reap et al., Vaccine 25(42):7441-7449, 2007; and references therein), several TB proteins (reviewed in Skeiky et al., Nat. Rev. Microbiol. 4(6):469-476, 2006), malaria parasite antigens such as RTS,S (a pre- erythrocytic circumsporozoite protein, CSP) and others (e.g., reviewed in Li et al., Vaccine
25(14):2567-2574, 2007), CHIKV envelope proteins El and E2 (or the C-E2-E1, E2-E1 cassettes), HCV structural proteins C-E1-E2 forming VLPs (Ezelle et al., J. Virol. 76(23):12325-12334, 2002) or other proteins to induce T-cell responses, Ebola virus glycoprotein GP (Yang et al., Virology 377(2):255-264, 2008).
In addition to the immunogens described above, the vectors described herein may include one or more immunogen(s) derived from or that direct an immune response against one or more viruses (e.g., viral target antigen(s)) including, for example, a dsDNA virus (e.g., adenovirus, herpesvirus, epstein-barr virus, herpes simplex type 1, herpes simplex type 2, human herpes virus simplex type 8, human cytomegalovirus, varicella-zoster virus, poxvirus); ssDNA virus (e.g., parvovirus, papillomavirus (e.g., El, E2, E3, E4, E5, E6, E7, E8, BPV1, BPV2, BPV3, BPV4, BPV5, and BPV6 {In Papillomavirus and Human Cancer, edited by H. Pfister (CRC Press, Inc. 1990)); Lancaster et al., Cancer Metast. Rev. pp. 6653-6664, 1987; Pfister et al., Adv. Cancer Res. 48: 113-147, 1987)); dsRNA viruses (e.g., reovirus); (+)ssRNA viruses (e.g., picomavirus, coxsackie virus, hepatitis A virus, poliovirus, togavirus, rubella virus, flavivirus, hepatitis C virus, yellow fever virus, dengue virus, west Nile virus); (-)ssRNA viruses (e.g., orthomyxovirus, influenza virus, rhabdovirus, paramyxovirus, measles virus, mumps virus, parainfluenza virus, rhabdovirus, rabies virus); ssRNA-RT viruses (e.g., retrovirus, human immunodeficiency virus (HIV)); and dsDNA-RT viruses (e.g. hepadnavirus, hepatitis B). Immunogens may also be derived from other viruses not listed above but available to those of skill in the art.
With respect to HIV, immunogens may be selected from any HIV isolate. As is well-known in the art, HIV isolates are now classified into discrete genetic subtypes. HIV-1 is known to comprise at least ten subtypes (A, B, C, D, E, F, G, H, J, and K). HIV-2 is known to include at least five subtypes (A, B, C, D, and E). Subtype B has been associated with the HIV epidemic in homosexual men and intravenous drug users worldwide. Most HIV-1 immunogens, laboratory adapted isolates, reagents and mapped epitopes belong to subtype B. In sub-Saharan Africa, India, and China, areas where the incidence of new HIV infections is high, HIV-1 subtype B accounts for only a small minority of infections, and subtype HIV-1 C appears to be the most common infecting subtype. Thus, in certain embodiments, it may be desirable to select immunogens from HIV-1 subtypes B and/or C. It may be desirable to include immunogens from multiple HIV subtypes (e.g., HIV-1 subtypes B and C, HIV-2 subtypes A and B, or a combination of HIV-1 and HIV-2 subtypes) in a single immunological composition. Suitable HIV immunogens include E V, GAG, PRO, POL, NEF, as well as variants, derivatives, and fusion proteins thereof, for example.
Further, as described in Example 4 in reference to particular constructs, the invention includes constructs including multiple different proteins in a single precursor, wherein the open reading frames may be, optionally, separated by protease cleavage sites, such as FMDV 2A cleavage sites, as described herein. Thus, in one example, a cassette may include gpl20 (e.g., modified as described in Example 4), gag, and pro genes from SIV or HIV. Further, the invention includes the hybrid sequences including, e.g., heterologous transmembrane and.or signal sequences, as described in detail in Example 4. Thus, for example, the invention includes the use of rabies virus G protien- specific signale and/or anchor sequences in the contect of gpl20-containing PIV constructs, as described herein.
Immunogens may also be derived from or direct an immune response against one or more bacterial species (spp.) (e.g., bacterial target antigen(s)) including, for example, Bacillus spp. (e.g., Bacillus anthracis), Bordetella spp. (e.g., Bordetella pertussis), Borrelia spp. (e.g., Borrelia burgdorferi), Brucella spp. (e.g., Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis), Campylobacter spp. (e.g., Campylobacter jejuni), Chlamydia spp. (e.g., Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis), Clostridium spp. (e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani), Corynebacterium spp. (e.g., Corynebacterium diptheriae), Enterococcus spp. (e.g., Enterococcus faecalis, enterococcus faecum), Escherichia spp. (e.g., Escherichia coli), Francisella spp. (e.g., Francisella tularensis), Haemophilus spp. (e.g., Haemophilus influenza), Helicobacter spp. (e.g., Helicobacter pylori), Legionella spp. (e.g., Legionella pneumophila), Leptospira spp. (e.g., Leptospira interrogans), Listeria spp. (e.g., Listeria monocytogenes), Mycobacterium spp. (e.g., Mycobacterium leprae, Mycobacterium tuberculosis), Mycoplasma spp. (e.g., Mycoplasma pneumoniae), Neisseria spp. (e.g., Neisseria gonorrhea, Neisseria meningitidis), Pseudomonas spp. (e.g., Pseudomonas aeruginosa), Rickettsia spp. (e.g., Rickettsia rickettsii), Salmonella spp. (e.g., Salmonella typhi, Salmonella typhinurium), Shigella spp. (e.g., Shigella sonnei), Staphylococcus spp. (e.g., Staphylococcus aureus,
Staphylococcus epidermidis, Staphylococcus saprophyticus, coagulase negative staphylococcus (e.g., U.S. Patent No. 7,473,762)), Streptococcus spp. (e.g., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyrogenes), Treponema spp. (e.g., Treponema pallidum), Vibrio spp. (e.g., Vibrio cholerae), and Yersinia spp. (Yersinia pestis). Immunogens may also be derived from or direct the immune response against other bacterial species not listed above but available to those of skill in the art.
Immunogens may also be derived from or direct an immune response against one or more parasitic organisms (spp.) (e.g., parasite target antigen(s)) including, for example, Ancylostoma spp. (e.g., A. duodenale), Anisakis spp., Ascaris lumbricoides, Balantidium coli, Cestoda spp., Cimicidae spp., Clonorchis sinensis, Dicrocoelium dendriticum, Dicrocoelium hospes, Diphyllobothrium latum, Dracunculus spp., Echinococcus spp. (e.g., E. granulosus, E. multilocularis), Entamoeba histolytica, Enterobius vermicularis, Fasciola spp. (e.g., F. hepatica, F. magna, F. gigantica, F.
jacksoni), Fasciolopsis buski, Giardia spp. (Giardia lamblia), Gnathostoma spp., Hymenolepis spp. (e.g., H. nana, H. diminuta), Leishmania spp., Loa loa, Metorchis spp. (M. conjunctus, M. albidus), Necator americanus, Oestroidea spp. (e.g., botfly), Onchocercidae spp., Opisthorchis spp. (e.g., O. viverrini, O. felineus, O. guayaquilensis, and O. noverca), Plasmodium spp. (e.g., P. falciparum), Protofasciola robusta, Parafasciolopsis fasciomorphae, Paragonimus westermani, Schistosoma spp. (e.g., S. mansoni, S. japonicum, S. mekongi, S. haematobium), Spirometra erinaceieuropaei, Strongyloides stercoralis, Taenia spp. (e.g., T. saginata, T. solium), Toxocara spp. (e.g., T. canis, T. cati), Toxoplasma spp. (e.g., T. gondii), Trichobilharzia regenti, Trichinella spiralis, Trichuris trichiura, Trombiculidae spp., Trypanosoma spp., Tunga penetrans, and/or Wuchereria bancrofti. Immunogens may also be derived from or direct the immune response against other parasitic organisms not listed above but available to those of skill in the art.
Immunogens may be derived from or direct the immune response against tumor target antigens (e.g., tumor target antigens). The term tumor target antigen (TA) may include both tumor- associated antigens (TAAs) and tumor-specific antigens (TSAs), where a cancerous cell is the source of the antigen. A TA may be an antigen that is expressed on the surface of a tumor cell in higher amounts than is observed on normal cells or an antigen that is expressed on normal cells during fetal development. A TSA is typically an antigen that is unique to tumor cells and is not expressed on normal cells. TAs are typically classified into five categories according to their expression pattern, function, or genetic origin: cancer-testis (CT) antigens (i.e., MAGE, NY-ESO-1); melanocyte differentiation antigens (e.g., Melan A/MART-1, tyrosinase, gplOO); mutational antigens (e.g., MUM-1, p53, CD -4); overexpressed 'self antigens (e.g., HER-2/neu, p53); and viral antigens (e.g., HPV, EBV). Suitable TAs include, for example, gplOO (Cox et al., Science 264:716-719, 1994), MART-l/Melan A (Kawakami et al., J. Exp. Med., 180:347-352, 1994), gp75 (TRP-1) (Wang et al., J. Exp. Med., 186:1131-1140, 1996), tyrosinase (Wolfel et al., Eur. J. Immunol., 24:759-764, 1994), NY-ESO-1 (WO 98/14464; WO 99/18206), melanoma proteoglycan (Hellstrom et al., J. Immunol., 130: 1467-1472, 1983), MAGE family antigens (e.gl, MAGE-1, 2, 3, 4, 6, and 12; Van der Bruggen et al., Science 254: 1643-1647, 1991 ; U.S. Patent No. 6,235,525), BAGE family antigens (Boel et al., Immunity 2: 167-175, 1995), GAGE family antigens (e.g., GAGE-1,2; Van den Eynde et al, J. Exp. Med. 182:689-698, 1995; U.S. Patent No. 6,013,765), RAGE family antigens (e.g., RAGE-1; Gaugler et al., Immunogenetics 44:323-330, 1996; U.S. Patent No. 5,939,526), N- acetylglucosaminyltransferase-V (Guilloux et al., J. Exp. Med. 183:1173-1183, 1996), pl5 (Robbins et al., J. Immunol. 154:5944-5950, 1995), β-catenin (Robbins et al., J. Exp. Med., 183: 1185-1192, 1996), MUM-1 (Coulie et al., Proc. Natl. Acad. Sci. U.S.A. 92:7976-7980, 1995), cyclin dependent kinase-4 (CDK4) (Wolfel et al., Science 269:1281-1284, 1995), p21-ras (Fossum et al., Int. J.
Cancer 56:40-45, 1994), BCR-abl (Bocchia et al., Blood 85:2680-2684, 1995), p53 (Theobald et al., Proc. Natl. Acad. Sci. U.S.A. 92: 11993-11997, 1995), pl85 HER2/neu (erb-Bl; Fisk et al., J. Exp. Med., 181:2109-2117, 1995), epidermal growth factor receptor (EGFR) (Harris et al., Breast Cancer Res. Treat, 29:1-2, 1994), carcinoembryonic antigens (CEA) (Kwong et al., J. Natl. Cancer Inst., 85:982-990, 1995) U.S. Patent Nos. 5,756,103; 5,274,087; 5,571,710; 6,071,716; 5,698,530;
6,045,802; EP 263933; EP 346710; and EP 784483; carcinoma-associated mutated mucins (e.g., MUC-1 gene products; Jerome et al., J. Immunol., 151:1654-1662, 1993); EBNA gene products of EBV (e.g., EBNA-1 ; Rickinson et al, Cancer Surveys 13:53-80, 1992); E7, E6 proteins of human papillomavirus (Ressing et al., J. Immunol. 154:5934-5943, 1995); prostate specific antigen (PSA; Xue et al., The Prostate 30:73-78, 1997); prostate specific membrane antigen (PSMA; Israeli et al., Cancer Res. 54: 1807-1811, 1994); idiotypic epitopes or antigens, for example, immunoglobulin idiotypes or T cell receptor idiotypes (Chen et al., J. Immunol. 153:4775-4787, 1994); SA (U.S. Patent No. 5,348,887), kinesin 2 (Dietz, et al., Biochem. Biophys. Res. Commun. 275(3):731-738,
2000) , HIP-55, TGF -l anti-apoptotic factor (Toomey et al., Br. J. Biomed. Sci. 58(3): 177-183,
2001) , tumor protein D52 (Bryne et al., Genomics 35:523-532, 1996), HI FT, NY-BR-1 (WO 01/47959), NY-BR-62, NY-BR-75, NY-BR-85, NY-BR-87, and NY-BR-96 (Scanlan, M. Serologic and Bioinforaiatic Approaches to the Identification of Human Tumor Antigens, in Cancer Vaccines 2000, Cancer Research Institute, New York, NY), and/or pancreatic cancer antigens (e.g., SEQ ID NOs: 1-288 of U.S. Patent No. 7,473,531). Immunogens may also be derived from or direct the immune response against include TAs not listed above but available to one of skill in the art. In addition to the specific immunogen sequences listed above, the invention also includes the use of analogs of the sequences. Such analogs include sequences that are, for example, at least 80%, 90%, 95%, or 99% identical to the reference sequences, or fragments thereof. The analogs also include fragments of the reference sequences that include, for example, one or more immunogenic epitopes of the sequences. Further, the analogs include truncations or expansions of the sequences (e.g., insertion of additional/repeat immunodominant/helper epitopes) by, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-20, etc., amino acids on either or both ends. Truncation may remove immunologically unimportant or interfering sequences, e.g., within known structural/immunologic domains, or between domains; or whole undesired domains can be deleted; such modifications can be in the ranges 21-30, 31-50, 51-100, 101-400, etc. amino acids. The ranges also include, e.g., 20-400, 30- 100, and 50-100 amino acids.
Cocktails
The invention also includes compositions including mixtures of two or more PIVs and/or PrV vectors, as described herein. As discussed above, use of such mixtures or cocktails may be particularly advantageous when induction of immunity to more than one immunogen and/or pathogen is desired. This may be useful, for example, in vaccination against different flaviviruses that may be endemic to the region in which the vaccine recipient resides. This may also be useful in the context of administration of multiple immunogens against the same target.
Non-limiting examples of Pr cocktails included in the invention are those including PIV-JE
+ PIV-DEN, and PIV-YF + PIV-DEN. In both of these examples, the PIVs for either or both components can be single or dual component PIVs, as described above. In addition, in the case of the PIV-DEN, the PIV can include sequences of just one dengue serotype selected from the group consisting of dengue serotypes 1-4, or the cocktail can include PIVs expressing sequences from two, three, or all four of the serotypes. Further, the ΊΒΕ/Borrelia burgdorferi/tick saliva protein (e.g., 64TRP, Isac, Salp20) vaccines described herein can be based on including the different immunogens within a single PIV or live attenuated flavivirus, or can be based on mixtures of PIVs (or LAVs), which each include one or more of the immunogens. The cocktails of the invention can be formulated as such or can be mixed just prior to administration. Use, Formulation, and Administration
The invention includes the PIV vectors, PIVs, LAV vectors, and LAVs, as well as corresponding nucleic acid molecules, pharmaceutical or vaccine compositions, and methods of their use and preparation. The PIV vectors, PIVs, LAV vectors, and LAVs of the invention can be used, for example, in vaccination methods to induce an immune response to TBE or other flavivirus, and/or another expressed immunogen, as described herein. These methods can be prophylactic, in which case they are carried out on subjects (e.g., human subjects or other mammalian subjects) not having, but at risk of developing infection or disease caused by TBE or another flavivirus and/or a pathogen from which the other expressed immunogen is derived. The methods can also be therapeutic, in which they are carried out on subjects already having an infection by one or more of the relevant pathogens. Further, the viruses and vectors can be used individually or in combination with one another or other vaccines. The subjects treated according to the methods of the invention include humans, as well as non-human mammals (e.g., livestock, such as, cattle, pigs, horses, sheep, and goats, and domestic animals, including dogs and cats).
Formulation of the PIV vectors, PIVs, LAV vectors, and LAVs of the invention can be carried out using methods that are standard in the art. Numerous pharmaceutically acceptable solutions for use in vaccine preparation are well known and can readily be adapted for use in the present invention by those of skill in this art (see, e.g., Remington 's Pharmaceutical Sciences (18th edition), ed. A. Gennaro, 1990, Mack Publishing Co., Easton, PA). In two specific examples, the PIV vectors, PIVs, LAV vectors, and LAVs are formulated in Minimum Essential Medium Earle's Salt (MEME) containing 7.5% lactose and 2.5% human serum albumin or MEME containing 10% sorbitol. However, the PIV vectors, PIVs, LAV vectors, and LAVs can simply be diluted in a physiologically acceptable solution, such as sterile saline or sterile buffered saline.
The PIV vectors, PIVs, LAV vectors, and LAVs of the invention can be administered using methods that are well known in the art, and appropriate amounts of the viruses and vectors to be administered can readily be determined by those of skill in the art. What is determined to be an appropriate amount of virus to administer can be determined by consideration of factors such as, e.g., the size and general health of the subject to whom the virus is to be administered. For example, in the case of live, attenuated viruses of the invention, the viruses can be formulated as sterile aqueous solutions containing between 102 and 108, e.g., 103 to 107, infectious units (e.g., plaque- forming units or tissue culture infectious doses) in a dose volume of 0.1 to 1.0 ml. PIVs can be administered at similar doses and in similar volumes; PIV titers however are usually measured in, e.g., focus-forming units determined by immunostaining of foci, as these defective constructs tend not to form virus-like plaques. Doses can range between 102 and 108 FFU and administered in volumes of 0.1 to 1.0 ml.
All viruses and vectors of the invention can be administered by, for example, intradermal, subcutaneous, intramuscular, intraperitoneal, or oral routes. In specific examples, dendritic cells are targeted by intradermal or transcutaneous administration, by use of, for example, microneedles or microabrasion devices. Further, the vaccines of the invention can be administered in a single dose or, optionally, administration can involve the use of a priming dose followed by a booster dose that is administered, e.g., 2-6 months later, as determined to be appropriate by those of skill in the art. Optionally, PIV vaccines can be administered via DNA or RNA immunization using methods known to those skilled in the art (Chang et al., Nat. Biotechnol. 26:571-577, 2008; Kofler et al., Proc. Natl. Acad. Sci. U.S.A. 101:1951-1956, 2004).
Optionally, adjuvants that are known to those skilled in the art can be used in the
administration of the viruses and vectors of the invention. Adjuvants that can be used to enhance the immunogenicity of the viruses include, for example, liposomal formulations, synthetic adjuvants, such as (e.g., QS21), muramyl dipeptide, monophosphoryl lipid A, polyphosphazine, CpG oligonucleotides, or other molecules that appear to work by activating Toll-like Receptor (TLR) molecules on the surface of cells or on nuclear membranes within cells. Although these adjuvants are typically used to enhance immune responses to inactivated vaccines, they can also be used with live or replication-defective vaccines. Both agonists of TLRs or antagonists may be useful in the case of live or replication-defective vaccines. The vaccine candidates can be designed to express TLR agonists. In the case of a virus delivered via a mucosal route, for example, orally, mucosal adjuvants such as the heat-labile toxin of E. coli (LT) or mutant derivations of LT can be used as adjuvants. In addition, genes encoding cytokines that have adjuvant activities can be inserted into the vaccine candidates. Thus, genes encoding desired cytokines, such as GM-CSF, IL- 2, IL-12, IL-13, IL-5, etc., can be inserted together with foreign immunogen genes to produce a vaccine that results in enhanced immune responses, or to modulate immunity directed more specifically towards cellular, humoral, or mucosal responses (e.g., reviewed in "Immunopotentiators in Modern Vaccines", Schijns and O'Hagan Eds., 2006, Elsevier Academic Press: Amsterdam,
Boston, etc.). Optionally, a patch containing a layer of an appropriate toxin-derived adjuvant, can be applied over the injection site. Toxin promotes local inflammation attracting lymphocytes, which leads to a more robust immune response.
Examples
Additional details concerning the invention are provided in the Examples, below. In the
Examples, experiments are described in which PIVs based on WN, JE, and YF viruses (see, e.g., WO 2007/098267 and WO 2008/137163) were tested. Firstly, we demonstrated that the constructs are significantly more attenuated in a sensitive suckling mouse neurovirulence model (zero mortality at all tested doses) as compared to available LAV controls (YF 17D, YF/JE LAV, and YF/WN LAV). We demonstrated for the first time that d-PIV constructs were avirulent in this model and thus that two-component PIVs do not undergo uncontrolled (unlimited) spread in vivo and cannot cause clinical signs. Secondly, we performed comparisons of the immunogenicity and efficacy of the PIVs and the LAVs, and demonstrated that PIV vaccines can induce immune response comparable to LAVs and be equally efficacious (e.g., as observed for PIV-WN and YF/WN LAV pair of vaccines). In one pair examined, YF 17D LAV was significantly more immunogenic than PrV-YF. Thus, production of VLPs can vary between different, similarly designed PIV constructs. Specifically, we propose that PFV-YF does not generate a large amount of YF VLPs compared to PIV-WN (WN VLPs), and that increased production of VLPs can be achieved by genetic modifications at the C/prM junction in suboptimal PIV constructs. Specifically, the C/prM junction is an important location in the flavivirus polyprotein orchestrating the formation of viral envelope and synthesis of viral proteins (Yamshchikov and Compans, Virology 192:38-51, 1993; Amberg and Rice, J. Virol. 73:8083-8094, 1999; Stocks and Lobigs, J. Virol. 72:2141-2149, 1998). We propose that secretion of VLPs in PIV infected cells (in contrast to production of viral particles in whole viruses) can be increased by uncoupling of the viral protease and signalase cleavages at the junction, or use of a strong heterologous signal peptide (tPA, etc.) in place of the signal for prM, or by mutagenesis of the signal for prM. The efficiency of signalase cleavage at the C/prM junction of flaviviruses is low (Stocks and Lobigs, J. Virol. 72:2141-2149, 1998), e.g., as predicted by SignalP 3.0 on-line program. It is expected that more efficient cleavage efficiency can be achieved by analysis of specific amino acid substitutions near the cleavage site with SignalP 3.0 (e.g., as described in application WO 2008/ 100464), followed by incorporation of chosen mutation(s) into PIV genomes, recovery of PIV progeny and measuring VLP secretion. Non-fiavivirus signals are inserted by methods standard in the art. Uncoupling between the viral protease and signalase cleavages can be achieved by ablating the viral cleavage site by any non-conservative mutation (e.g., RRS in YF17D C to RRA or GRS or RSS, etc.), or deletion of the entire site or some of its 3 residues. If necessary, formation of free N-terminus of the signal of foreign protein can be achieved by using such elements as autoprotease, or termination codon followed by an IRES. Alternatively, the native AUG initiation codon of C can be ablated (in constructs where C protein sequence is unnecessary, e.g., AC PIV) and AUG placed in front of foreign gene. Optimization of vector signal can be performed by random mutagenesis, e.g., by insertion of synthetic randomized sequence followed by identification of viable PIV variants with increased VLP secretion.
We also discovered that PIV constructs were substantially more immunogenic in hamsters when administered by the IP route, as compared to the subcutaneous route. We concluded that this was most likely due to better targeting of antigen presenting cells in lymphoid tissues, which are abundant in the abdomen, but not abundant in tissues underlying the skin. Based on these observations, we concluded that efficient targeting of PIVs to dendritic cells, abundant in the skin, can be achieved by cutaneous inoculation, e.g., via skin microabrasion or intradermal injection using microneedles (Dean et al., Hum Vaccin. 1: 106-111, 2005).
Further, we have carried out experiments to show the feasibility of administering mixtures, or cocktails, of different PIVs, such as those described herein (e.g., JE+DEN and YF+DEN). In order to administer cocktails, it is important to verify that there is no interference between co- administered components, and that a balanced immune response is induced. Several PIV mixtures were used to immunize rodents and immune responses were compared to PIV constructs
administered individually. No interference was observed in mixtures, and thus cocktail PIV vaccines are feasible. Such formulations may be of particular significance in geographical regions where different flaviviruses co-circulate. This could be also used to simultaneously administer several PIV-based vaccines against non-flavivirus pathogens.
Further, we have demonstrated that no neutralizing antibody response is induced against packaging envelope after at least two doses of PIV (and thus antibodies are elicited against VLPs secreted from infected cells). This was demonstrated using the helper (AprM-E) component of a d- PIV (see in Fig. 2) packaged individually, or by measuring neutralizing antibodies to heterologous packaging envelopes (e.g., to the WN envelope used to package PIV-JE in helper cells providing WN-specific C-prM-E proteins in trans). The latter observations support sequential use of different PIV vaccines manufactured in a universal helper packaging cells line, and sequential use of different recombinant PlV-vectored vaccines in the same individual, as discussed above. In addition, we confirmed previous observations that PIV constructs can be stably propagated to high yields in vitro, and that no recombination restoring whole virus occurs after prolonged passaging in substrate cells (Mason et al, Virology 351 :432-443, 2006; Shustov et al., J. Virol. 21:11737-11748, 2007).
These and other aspects of the invention are further described in the Examples, below.
Example 1. Pseudoinfectious virus platform development studies
Attenuation in suckling mouse neurovirulence (NV) model
Materials used in the studies described below are described in Table 1 and the references cited therein. These include s-PIV-WN (based on wt WN virus strain NY99 sequences), s-PIV-JE, s-PIV-WN/JE (based on wt WN virus backbone and prM-E genes from wt JE virus Nakayama strain), s-PIV-YF/WN (YF 17D backbone and prM-E genes from WN virus), and s-PIV-YF (based on YF 17D sequences). Additional materials include d-PIV-YF (YF d-PIV, grown in regular BHK cells (Shustov et al., J. Virol. 21:11737-11748, 2007), and two-component d-PIV- WN (grown in regular Vero cells; Suzuki et al., J. Virol. 82:6942-6951, 2008).
Attenuation of these PIV prototypes was compared to LAVs YF 17D, a chimeric YF/JE virus, and a chimeric YF/WN virus in suckling mouse NV test (IC inoculation) using highly susceptible 5 -day old ICR mice (the chimeric viruses include yellow fever capsid and non-structural sequences, and JE or WN prM-E sequences). None of the animals that received PIV constructs showed clinical signs or died, while mortality was observed in animals inoculated with LAVs (Table 2). The YF 17D virus is neurovirulent for mice of all ages, while the chimeric vaccines are not neurovirulent for adult mice, but can cause dose-dependent mortality in more sensitive suckling mice (Guirakhoo et al., Virology 257:363-372, 1999; Arroyo et al., J. Virol. 78: 12497-12507, 2004). Accordingly, 90%- 100% of suckling mice that received doses as low as 1 PFU of YF 17D died. YF/JE and YF WN LAVs caused partial mortality at much higher doses (> 2 log10 PFU and 3 log10 PFU, respectively), with longer average survival time (AST) of animals that died, as expected.
Thus, PIV constructs are completely avirulent in this sensitive model (at least 20,000 - 200,000 times less neurovirulent than the licensed YF 17D vaccine).
The YF d-PIV and WN d-PIV caused no mortality or clinical signs. Thus, the two- component PIV variants that theoretically could spread within brain tissue from cells co-infected by both of their components did not cause disease. Moreover, we tried to detect the d-PIVs in the brains of additional animals in this experiment, sacrificed on day 6 post-inoculation by titration, and detected none (brain tissues from 10 and 11 mice that received 4 log10 FFU of YF d-PIV and WN d- PIV, respectively, were homogenized and used for titration). Thus, the d-PFVs did not cause spreading infection characteristic of whole virus. YF/JE LAV has been shown to replicate in the brain of adult ICR mice inoculated by the IC route with a peak titer of ~ 6 log10 PFU/g on day 6, albeit without clinical signs (Guirakhoo et al., Virology 257:363-372, 1999). Co-infection of cells with components of a d-PIV is clearly a less efficient process than infection with whole virus. The data show that d-PIV replication in vivo is quickly brought under control by innate immune responses (and adaptive responses in older animals).
Immunogenicity /efficacy in mice and hamsters
Immunogenicity/efficacy of the PIV prototypes described above was compared to that of chimeric LAV counterparts and YF 17D in mice and Syrian hamsters. The general experiment design is illustrated in Fig. 3 (mice, IP immunization). Experiments in hamsters were performed similarly (plus-minus a few days, SC or IP inoculation with doses indicated below). 3.5-week old ICR mice (for s-PIV-WN and -YF, YF/WN LAV, and YF 17D groups) or C57/BL6 mice (for s-PIV- JE and YF/JE LAV groups) were immunized IP with graded doses of PIV constructs (4-6 log10 FFU/dose) or chimeric LAV and YF 17D LAV controls (4 log10 PFU). Select PIV-WN, -JE and - YF groups were boosted on day 21 with 5 log10 FFU of corresponding constructs (Table 3).
Neutralizing antibody responses were determined in animal sera by standard PRNT50 against YF/WN or /JE LAVs, or YF 17D viruses. PIV-WN induced very high WN-specific neutralizing antibody responses in all groups, with or without boost, as evidenced by PRNT50 titers determined in pools of sera from immunized animals on days 20 and 34, which was comparable to that in the YF/WN LAV control group. Accordingly, animals immunized with both PIV-WN and YF/WN LAV were protected from lethal challenge on day 35 with wt WN virus (IP, 270 LD50), but not mock-immunized animals (Table 3). When WN neutralizing antibodies were measured in sera from individual mice, high uniformity of immune responses was observed (Fig. 4). Thus, single-round PIV vaccines can be as immunogenic and efficacious as corresponding LAVs. PIV-JE was also highly immunogenic (black mice), while immunogenicity of PIV-YF was significantly lower compared to the YF 17D control (ICR mice). Yet, dose-dependent protection of PIV-YF immunized animals (but not mock-immunized animals) was observed following a severe lethal IC challenge with wt YF strain Asibi virus (500 LD50) (Table 3), which is in agreement with the knowledge that neutralizing antibody titers as low 1 : 10 are protective against flavivirus infections.
The YF 17D control virus was highly immunogenic (e.g., PRNT50 titer 1:1,280 on day 34), and thus it is able to infect cells and replicate efficiently in vivo, and its envelope is a strong immunogen. Therefore, it is unlikely that low immunogenicity of PIV-YF was due to its inability to infect cells or replicate efficiently in infected cells in vivo. We believe that the low immunogenicity of PIV-YF (e.g., compared to PIV-W ) was most likely due to a low-level production of YF- specific VLPs in PIV-YF infected cells (while VLP secretion is high in PIV-WN infected cells). As discussed above, we propose that immunogenicity of PIV-YF can be significantly increased, e.g., by appropriate modifications at the C/prM junction, e.g., by uncoupling the two protease cleavages that occur at this junction (viral protease and signalase cleavages), and/or by using a strong heterologous signal [e.g., rabies virus G protein signal, or eukaryotic tissue plasminogen activator (tPA) signal (Malin et al., Microbes and Infection, 2:1677-1685, 2000), etc.] in place of the YF signal for prM.
A similar experiment was performed in ~ 4.5-week old Syrian hamsters, to compare immunogenicity of PIV constructs to LAV controls in this model. Animals were immunized SC with graded doses of the test articles (Table 4). PIV-WN was highly immunogenic, e.g., WN- specific PRNT50 titers on day 38 (pre-challenge) were 1:320, 1 :640, and 1 : 1280 in groups that received 5, 6, and 6 (prime)+5 (boost) logio FFU doses, respectively. This was somewhat lower compared to YF/WN LAV 4 log10 PFU control (> 1 :2560). PIV-JE and -YF induced detectable specific neutralizing antibody responses, albeit with lower titers compared to YF/JE LAV and YF 17D controls. All animals immunized with PIV-WN and YF/WN were solidly protected from lethal challenge with wt WN virus as evidenced by the absence of mortality and morbidity (e.g., loss of body weight after challenge), as well as absence or a significant reduction of postchallenge WN virus viremia. Mock-immunized animals were not protected (Table 4). PIV-JE and -WN protected animals from respective challenge in dose-dependent fashion. Protective efficacy in this experiment is additionally illustrated in Fig. 5. For example, high post-challenge YF virus (hamster adapted Asibi strain) viremia was observed in mock immunized animals, peaking on day 3 at a titer of> 8 logio PFU/ml (upper left panel); all of the animals lost weight, and 1 out of 4 died (upper right panel). In contrast, viremia was significantly reduced or absent in hamsters immunized with PIV- YF (two doses; despite relatively low neutralizing titers) or YF 17D; none of these animals lost weight. Similarly, animals immunized with PIV-WN or YF/WN LAV were significantly or completely protected in terms of post-challenge WN virus viremia and body weigh loss/mortality, in contrast to mock controls (compare in bottom panels). Thus, high immunogenicity/efficacy of PIV was demonstrated in a second animal model.
In another hamster experiment, animals were immunized with PIV constructs by the IP route, with two doses. Table 5 compares neutralizing immune responses (specific for each vaccine) determined in pooled sera of hamsters in the above-described experiment (SC inoculation) to those after IP immunization, for PIV-WN, - YF/WN, -WN/JE, and-YF after the first dose (days 20-21) and second dose (days 34-38). A clear effect of the immunization route was observed both after the 1st and 2nd doses. For instance, for PIV-WN after 1st dose, SC immunization resulted in WN- specific PRNT50 titer of 1 :40, while IP inoculation resulted in much higher titer 1 :320 (and after the 2nd dose, titers were similar). A more pronounced effect was observed for other constructs after both the 1st and 2nd doses. Interestingly, PIV- YF/WN was very highly immunogenic by IP route (titer 1 :320 after 1st IP dose vs. 1 :20 by SC, and 1 : 1 ,280 after 2nd dose vs. 1 : 160 by SC). Similarly, immunogenicity of PIV-JE was significantly increased (e.g., JE-specific titer of 1:640 after two IP poses). Thus, better targeting of lymphoid cells, specifically antigen-presenting cells (which are more abundant in the abdomen as opposed to tissues under the skin), is an important consideration for use of PIV vaccines. In humans, efficient targeting of dendritic cells of the skin, increasing the magnitude of immune response, can be achieved by intradermal delivery, which we thus propose for a route for PIV immunization of humans.
In the above-described experiments, we also determined whether a neutralizing antibody response was induced against packaging envelopes (as opposed to response to VLPs encoded by PIV constructs and secreted by infected cells). No WN-specific neutralizing antibodies were detected by PRNT50 in animals immunized with 5 logio FFU of the second component of WN d-PIV, containing the AC-prM-E deletion and thus not encoding VLPs, but packaged into the WN envelope in BHK- CprME(WN) helper cells, and no YF-specific neutralizing activity was found in sera from animals immunized with 4 log10 FFU of the second component of YF d-PIV packaged in YF envelope. No YF-specific neutralizing response was induced by two doses of PIV- YF/WN packaged into YF envelope, and similarly, no WN-specific response was induced by two doses of PFV-JE packaged into WN envelope. The absence of neutralizing response against packaging envelopes permits manufacturing different ΡΓ vaccines in one (universal) manufacturing helper cell line, or immunization of one individual with different recombinant vaccines based on the same vector, according to the present invention. PIV cocktails
Because PIVs undergo a single (optionally several, but limited) round(s) of replication in vivo, we considered that mixtures of different PIV vaccines can be administered without interference between individual constructs in the mixture (cocktail). To elucidate whether PIV vaccines can be used in cocktail formulations, immune responses in mice and hamsters to several PIV constructs given as mixtures were compared to the same constructs given individually. Similar results were obtained in both animal models. Results of mouse experiments are shown in Table 6. Similar anti-JE neutralizing antibody titers were observed in pools of sera from animals that were given one or two doses of either PIV-JE + PIV-WN mixture or PIV-JE alone (1:20 vs. 1:80 and 1 :640 vs. 1: 160, for one and two doses, respectively). Similarly, WN-specific titers against PIV-JE + PIV-WN mixture and PIV-WN alone were similar (1:320 vs. 1:640 and 1:5,120 vs. 1:5,120 for one and 2 doses, respectively). No or little cross-specific response was induced by either PIV-JE or -WN. The result was also confirmed by measuring PRNT5o titers in sera from individual animals. Thus, it is clear that PIV vaccines can be efficiently administered as cocktails, inducing immunity against two or more flavivirus pathogens. In addition, as discussed above, various cocktails can be made between non-flavivirus PIV vaccines, or between any of flavivirus and non-flavivirus PIV vaccines.
In vitro studies
Different PIV prototypes were serially passaged up to 10 times in helper BHK cells, for s-
PIVs, or in regular Vero cells, for d-PFVs. Samples harvested after each passage were titrated in Vero cells by immunostaining. Constructs grew to high titers, and no recombination restoring whole virus was observed. For instance, PIV-WN consistently grew to titers 7-8 log]0 FFU/ml in BHK- CprME(WN) helper cells (containing a VEE replicon expressing the WN virus C-prM-E proteins), and WN d-PrV grew to titers exceeding 8 log10 FFU/ml in Vero cells, without recombination.
Example 2. PIV-TBE
PIV-TBE vaccine candidates can be assembled based entirely on sequences from wt TBE virus or the closely serologically related Langat (LGT) virus (naturally attenuated virus, e.g., wt strain TP-21 or its empirically attenuated variant, strain E5), or based on chimeric sequences containing the backbone (capsid and non-structural sequences) from YF 17D or other flaviviruses, such as WN virus, and the prM-E envelope protein genes from TBE, LGT, or other serologically related flaviviruses from the TBE serocomplex. YF/TBE LAV candidates are constructed based on the backbone from YF 17D and the prM-E genes from TBE or related viruses (e.g., the E5 strain of LGT), similar to other chimeric LAV vaccines.
Construction of PIV-TBE and YF/TBE LAV vaccine prototypes was performed by cloning of appropriate genetic elements into plasmids for PIV-WN (Mason et al., Virology 351 :432-443, 2006; Suzuki et al., J. Virol. 82:6942-6951, 2008), or plasmids for chimeric LAVs (e.g., pBSA-ARl, a single-plasmid version of infectious clone of YF/JE LAV; WO 2008/036146), respectively, using standard methods in the art of reverse genetics. The prM-E sequences of TBE virus strain Hypr (GenBank accession number U39292) and LGT strain E5 (GenBank accession number AF253420) were first computer codon-optimized to conform to the preferential codon usage in the human genome, and to eliminate nucleotide sequence repeats longer than 8 nt to ensure high genetic stability of inserts (if determined to be necessary, further shortening of nt sequence repeats can be performed). The genes were chemically synthesized and cloned into plasmids for PIV-WN and YF/JE LAV, in place of corresponding prM-E genes. Resulting plasmids were in vitro transcribed and appropriate cells (V ero for chimeric viruses, and helper BH cells for PIV) were transfected with RNA transcripts to generate virus/PIV samples.
YF/TBE LA V constructs
In YF/TBE constructs containing either the TBE Hypr (plasmids p42, p45, and p59) or LGT
E5 (plasmid P43) prM-E genes, two different types of the C/prM junction were first examined (see in Fig. 6; C/prM junctions only are shown in Sequence Appendix 1, and complete 5'-terminal sequences covering the 5'UTR-C-prM-E-beginning of NSl region are shown in Sequence Appendix 2). The p42 -derived YF17D/Hypr chimera contained a hybrid YF17D/Hypr signal peptide for the prM protein, while the p45-derived YF17D/Hypr chimera contained a hybrid YF17D/WN signal peptide for prM (Sequence Appendix 1). The former chimeric virus produced very high titers at both P0 (immediately after transfection) and PI (the next passage in Vero cells), up to 7.9 log10 PFU/ml, which were 0.5 log10 times higher, compared to the latter virus; in addition it formed significantly larger plaques in Vero cells (Fig. 6). Thus, use of TBE-specific residues in the signal peptide for prM conferred a significant growth advantage over the signal containing WN-specific residues. The p43-derived YF17D/LGT chimera had the same prM signal as the p42 -derived virus; it also produced very high titers at P0 and PI passages (up to 8.1 log10 PFU/ml) and formed large plaques. A derivative of the p42-derived virus was also produced from plasmid p59, which contained a strong attenuating mutation characterized previously in the context of a YF/WN LAV vaccine virus, specifically, a 3 -a. a. deletion in the YF17D-specific C protein (PSR, residues 40-42 in the beginning of a-Helix I; WO 2006/116182). As expected, the p59 virus grew to lower titers (5.6 and 6.5 logio PFU/ml at P0 and PI, respectively), and formed small plaques (determined in a separate titration experiment and thus not shown in Fig. 6), compared to the parent p42-derived chimera. These initial observations of growth properties of YF/TBE LAV prototypes, and correlation of replication in vitro with plaque morphologies, have been confirmed in growth curve experiments (Fig. 8).
PIV-TBE constructs
PIV-WN/TBE variants were constructed, and packaged PIV samples were derived from plasmids p39 and p40 (Figs. 7A-B; Sequence Appendix 1 for C/prM junction sequences, and Sequence Appendix 3 for complete 5'UTR-AC-prM-E-beginning of NS1 sequences). These contained complete Hypr or WN prM signals, respectively. Both PIVs were successfully recovered and propagated in BHK-CprME(W ) or BHK-C(WN) helper cells (Mason et al., Virology 351:432- 443, 2006; Widman et al., Vaccine 26:2762-2771, 2008). The P0 and PI sample titers of the p39 variant were 0.2 - 1.0 log10 times, higher than p40 variant. In addition, Vero cells infected with p39 variant were stained brighter in immunofluorescence assay using a polyclonal TBE-specific antibody, compared to p40, indicative of more efficient replication (Fig. 7A). The higher rate of replication of the p39 candidate than p40 candidate was confirmed in a growth curve experiment (Fig. 8). In the latter experiment, both candidates appeared to grow better in the BHK-C(WN) helper cells compared to BHK-CprME(WN), with the p39 variant reaching titer of ~ 7 logio PFU/ml on day 5 (note that peak titers have not been reached). The discovery of the effect of prM signal on replication rates of both PIV and chimeric LAV vaccine candidates, and head-to-head comparison of different signals to generate the most efficiently replicating and immunogenic (see above) construct, are a distinguishing feature of our approach. As discussed above, the invention also includes the use of other flavivirus signals, including with appropriate mutations, the uncoupling the viral protease and signalase cleavages at the C/prM junction, e.g., by mutating or deleting the viral protease cleavage site at the C-terminus of C preceding the prM signal, the use of strong non-flavivirus signals (e.g., tPA signal, etc.) in place of prM signal, as well as optimization of sequences downstream from the signalase cleavage site. Other PIV-TBE variants based entirely on wt TBE (Hypr strain) and LGT virus (TP21 wild type strain or attenuated E5 strain), and chimeric YF 17D backbone/prM-E (TBE or LGT) sequences are also included in the invention. Helper cells providing appropriate C, C-prM-E, etc., proteins (e.g., TBE-specific) for trans-complementation can be constructed by means of stable DNA transfection or through the use of an appropriate vector, e.g., an alphavirus replicon, such as based on VEE strain TC-83, with antibiotic selection of replicon-containing cells. Vero and BHK21 cells can be used in practice of the invention. The former are an approved substrate for human vaccine manufacture; any other cell line acceptable for human and/or veterinary vaccine manufacturing can be also used. In addition to s-PIV constructs, d-PIV constructs can also be assembled. To additionally ascertain safety for vaccinees and the environment, appropriate modifications can be employed, including the use of degenerate codons and complementary mutations in the 5' and 3' CS elements, to minimize chances of recombination that theoretically could result in viable virus.
Following construction, all vaccine candidates can be evaluated in vitro for
manufacturability/stability, and in vivo for attenuation and immunogenicity/efficacy, in available pre-clinical animal models, such as those used in development and quality control of TBE and YF vaccines.
Neurovirulence and neuroinvasiveness in mice of PIV-TBE and YF /TBE LAV constructs
Young adult ICR mice (~ 3.5 week-old), were inoculated with graded doses of PIV-TBE and YF/TBE LAV candidates by the IC route to measure neurovirulence, or IP route to measure neuroinvasiveness (and later immunogenicity/efficacy). Animals that received 5 log10 FFU of ΡΓ7- Hypr (p39 and p40) variants by both routes survived and showed no signs of sickness, similar to mock-inoculated animals (Table 7; Fig. 45), and thus PIV-TBE vaccines are completely avirulent. Mice inoculated IC with YF 17D control (1 - 3 log10 PFU) showed dose-dependent mortality, while all animals inoculated TP (5 log10 PFU) survived, in accord with the knowledge that YF 17D virus is not neuroinvasive. All animals that received graded IC doses (2 - 4 logio PFU) of YF/TBE LAV prototypes p42, p45, p43, and p59 died (moribund animals were humanely euthanized). These variants appear to be less attenuated than YF 17D, e.g., as evidenced by complete mortality and shorter AST at the 2 log10 PFU dose, the lowest dose tested for YF/TBE LAV candidates. The non- neuro virulent phenotype of PIV-TBE, virulent phenotype of YF/TBE LAV and intermediate- virulence phenotype of YF 17D are also illustrated in Fig. 9, showing survival curves of mice after IC inoculation. It should be noted that the p43 (LGT prM-E genes) and p59 (the dC2 deletion variant of YF/Hypr LAV) were less neurovirulent than p42 and p45 YF Hypr LAV constructs as evidenced by larger AST values for corresponding doses (Table 7; Fig. 45). In addition, p43 and p59 candidates were non-neuroinvasive, while p42 and p45 caused partial mortality after IP inoculation (5 logi0 PFU/dose) (Table 7; Fig. 10 and Fig. 45). It should be noted however that all the YF/TBE LAV constructs were significantly attenuated as compared to wt TBE viruses, e.g., compared to wt TBE Hypr virus, which is uniformly highly virulent for mice, both at very low IC (LD50 ~ 0.1 PFU) and IP (LD50 < 10 PFU) doses (Wallner et al., J. Gen. Virol. 77:1035-1042, 1996; Mandl et al., J. Virol. 72:2132-2140, 1998; Mandl et al., J. Gen. Virol. 78:1049-1057, 1997 Immunogenicity/efficacy of PIV-TBE and YF/TBE LA V constructs in mice
TBE-specific neutralizing antibody responses in mice immunized IP with one or two doses of the PIV-TBE or YF/TBE LAV variants described above, or a human formalin-inactivated TBE vaccine control (1 :30 of human dose) are being measured. Animals have been challenged with a high IP dose (500 PFU) of wt Hypr TBE virus; morbidity (e.g., weight loss), and mortality after challenge are monitored.
Immunogenicity/efficacy of PIV-TBE and YF/TBE LAV constructs in mice
TBE-specific neutralizing antibody responses in mice immunized IP with one or two doses of the PIV-TBE or YF/TBE LAV variants described above (from experiment in Table 7; see also Fig. 45), or a human formalin-inactivated TBE vaccine control (1 :20 of human dose; one or two doses), or YF 17D and mock controls, were measured on day 20 by PRNT50 against wt TBE Hypr virus (Tables 8 and 9; second dose of indicated test articles was given on day 14). [Titers were determined in individual sera, or pooled sera from two animals in most cases, or pooled sera from 4 animals for the YF17D and Mock negative controls]. Titers in individual test samples as well as GMTs for each group are provided in Tables 8 and 9. Titers in test samples were similar within each group, e.g., in groups immunized with PIVs, indicating high uniformity of immune response in animals. As expected, no TBE-specific neutralizing antibodies were detected in negative control groups (YF 17D and Mock; GMTs < 1:10); accordingly, animals in these groups were not protected from challenge on day 21 post-immunization with a high ΓΡ dose (500 PFU) of wt Hypr TBE virus. Mortalities from partial observation (on day 9 post-challenge; observation being continued) are provided in Tables 8 and 9, and dynamics of average post-challenge body weights indicative of morbidity are shown in Fig. 11. Neutralizing antibodies were detected in killed vaccine controls, which were particularly high after two doses (GMT 1 : 1 ,496); animals in the 2-dose group were completely protected in that there was no mortality or body weight loss (but not animals in the 1- dose group). Animals that received both one and two doses of PIV-Hypr p39 had very high antibody titers (GMTs 1 :665 and 1 : 10,584) and were solidly protected, demonstrating that robust protective immunity can be induced by s-PIV-TBE defective vaccine. The two animals that survived immunization with YF/Hypr p42 chimera (see in Table 7; see also Fig. 45) also had high antibody titers (GMT 1 :6,085) and were protected (Tables 8 and 9; Fig. 11). Interestingly, PIV-Hypr p40 and YF/Hypr p45 were poorly immunogenic (GMTs 1 :15 and 1 : 153 for one and two doses, and 1 :68, respectively). As discussed above, these contained WN-specific sequences in the signal for prM, while the highly immunogenic PIV-Hypr p39 and YF/Hypr p42 constructs contained TBE-specific signal sequences. In agreement with discussion above, this result demonstrates the importance of choosing the right prM signal, e.g., the TBE-specific signal, to achieve high-level replication/VLP secretion, which in this experiment in vivo resulted in drastically different immune responses.
Immunogenicity of YF/LGT p43 and YF/Hypr dC2 p59 chimeras was relatively low which could be expected, because of the use of a heterologous envelope (LGT, different from challenge TBE virus) and high attenuating effect of the dC2 deletion, respectively.
Example 3. Foreign gene expression
In the examples of recombinant PIV constructs described below, genes of interest were codon optimized (e.g., for efficient expression in a target vaccination host) and to eliminate long nt sequence repeats to increase insert stability (> 8 nt long; additional shortening of repeats can be performed if necessary), and then chemically synthesized. The genes were cloned into PIV-WN vector plasmids using standard methods of molecular biology well known in the art, and packaged PIVs were recovered following in vitro transcription and transfection of appropriate helper (for s- PIVs) or regular (for d-PIVs) cells.
Expression of rabies virus G protein in WN s-PIV and d-PJV
Rabies virus, Rhabdoviridae family, is a significant human and veterinary pathogen.
Despite the availability of several (killed) vaccines, improved vaccines are still needed for both veterinary and human use (e.g. as an inexpensive pre-exposure prophylactic vaccines). Rabies virus glycoprotein G mediates entry of the virus into cells and is the main immunogen. It has been expressed in other vectors with the purpose of developing veterinary vaccines (e.g., Pastoret and Brochier, Epidemio. Infect. 1 16:235-240, 1996; Li et al., Virology 356: 147-154, 2006).
Full length rabies virus G protein (original Pasteur virus isolate, GenBank accession number NC 001542) was codon-optimized, chemically synthesized, and inserted adjacent to the AC, AprM-E and AC-prM-E deletions in PIV-WN vectors (Figs. 12A and 12B). The sequences of constructs are provided in Sequence Appendix 4. General designs of the constructs are illustrated in Figs. 12 and 13. The entire G protein containing its own signal peptide was inserted in- frame downstream from the WN C protein either with the AC deletion (AC and AC-prM-E constricts) or without (AprM-E) and a few residues from the prM signal. Foot and mouth disease virus (FMDV) 2 A autoprotease was placed downstream from the transmembrane C-terminal anchor of G to provide cleavage of C-terminus of G from the viral polyprotein during translation. The FMDV 2A element is followed by WN-specific signal for prM and prM-E-NS 1-5 genes in the AC construct, or signal for NS1 and NS1-5 genes in AprM-E and AC-prM-E constructs.
Packaged WN(AC)-rabiesG, WN(AprME)-rabiesG, and WN(ACprME)-rabiesG PIVs were produced by transfection of helper BHK cells complementing the PIV vector deletion [containing a Venezuelan equine encephalitis virus (strain TC-83) replicon expressing WN virus structural proteins for trans-complementation]. Efficient replication and expression of rabies G protein was demonstrated for the three constructs by transfection/infection of BHK-C(WN) and/or BHK-C-prM-E(WN) helper cells, as well as regular BHK cells, by immunostaining and immunofluorescence assay (IF A) using anti- Rabies G monoclonal antibody (RabG-Mab) (Fig. 14). Titers were determined in Vero cells by immunostaining with the Mab or an anti-WN virus polyclonal antibody. Growth curves of the constructs in BHK-CprME(WN) cells after transfection with in vitro RNA transcripts are shown in Fig. 14, bottom panels. The PIVs grew efficiently to titers ~ 6 to >7 logio FFU/ml. Importantly, nearly identical titers were detected by both RabG-Mab and WN-antibody staining, which was the first evidence of genetic stability of the insert. In PIV-infected Vero cells, which were fixed but not permeabilized, strong membrane staining was observed by RabG-Mab staining, demonstrating that the product was efficiently delivered to the cell surface (Fig. 15). The latter is known to be the main prerequisite for high immunogenicity of expressed G. Individual packaged PIVs can spread following infection of helper BHK cells, but cannot spread in regular cells as illustrated for WN(AC)-rabiesG PIV in Fig. 16. The fact that there is no spread in naive BHK cells demonstrates that the recombinant RNA genomes cannot be non-specifically packaged into membrane vesicles containing the G protein, if produced by PIV infected cells. An identical result was obtained with the G protein of another rhabdo virus, Vesicular stomatitis virus (VSV), contrary to previous observations of non-specific packaging of Semliki Forest virus (SFV) replicon expressing VSV G protein (Rolls et al, Cell 79:497- 506, 1994). The latter is a desired safety feature. [Alternatively, some non-specific packaging could result in a limited spread of PIV in vivo, potentially enhancing anti-rabies immune response. The latter could be also a beneficial feature, given that such PIV is demonstrated to be safe]. The stability of the rabies G insert in the three PIVs was demonstrated by serial passages in helper BHK-CprME(WN) cells at high or low MOI (0.1 or 0.001 FFU/cell). At each passage, cell supernatants were harvested and titrated in regular cells (e.g., Vero cells) using immunostaining with an anti-WN polyclonal antibody to determine total PIV titer, or anti-rabies G monoclonal antibody to determine titer of particles containing the G gene (illustrated for MOI 0.1 in Fig. 17; similar results were obtained at MOI 0.001). The WN(AC)-rabiesG Pr was stable for 5 passages, while the titer of insert-containing PIV started declining at passage 6, indicative of insert instability. This could be expected, because in this construct, large G gene insert (~ 1500 nt) is combined with a small AC deletion (~ 200 nt), significantly increasing the overall size of the recombinant RNA genome. In contrast, in WN(AprME)-rabiesG, and
W (ACprME)-rabiesG PIVs, the insert is combined with a much larger deletion (~ 2000 nt). Therefore, these constructs stably maintained the insert for all 10 passages examined (Fig. 17). Further, it can be seen in Fig. 17 that at some passages, titers as high as 8 log10 FFU/ml, or higher, were attained for all three PIVs, additionally demonstrating that PIVs can be easily propagated to high yields.
Following inoculation in vivo individually, the WN(AC)-rabiesG s-PIV is expected to induce strong neutralizing antibody immune responses against both rabies and WN viruses, as well as T-cell responses. The WN(AprME)-rabiesG and WN(ACprME)-rabiesG PIVs will induce humoral immune response only against rabies because they do not encode the WN prM-E genes. WN(AC)-rabiesG s-PrV construct can be also co-inoculated with WN(AprME)-rabiesG construct in a d-PIV formulation (see in Figs. 12A and 12B), increasing the dose of expressed G protein, and with enhanced immunity against both pathogens due to limited spread. As an example of spread, titration results in Vero cells of a s-PIV sample, WN(AprME)-rabiesG, and a d-PIV sample, WN(AprME)-rabiesG + WN(AC) PIV (the latter did not encode rabies G protein), are shown in Fig. 18. Infection of naive Vero cells with s-PIV gave only individual cells stainable with RabG-Mab (or small clusters formed due to division of cells). In contrast, large foci were observed following infection with the d-PIV sample (Fig. 18, right panel) that were products of coinfection with the two PIV types.
The WN(ACprME)-rabiesG construct can be also used in a d-PIV formulation, if it is co- inoculated with a helper genome providing C-prM-E in trans (see in Figs. 12A and 12B). For example it can be a WN virus genome containing a deletion of one of the NS proteins, e.g., NS1, NS3, or NS5, which are known to be trans-complementable (Khromykh et al., J. Virol. 73:10272-10280, 1999;
Khromykh et al., J. Virol. 74:3253-3263, 2000). We have constructed a WN-ANS1 genome (sequence provided in Sequence Appendix 4) and obtained evidence of co-infection with WN(AprME)-rabiesG or WN(ACprME)-rabiesG constructs, and spread in vitro, by immunostaining. In the case of such d-PIVs, rabies G protein can be also inserted and expressed in helper genome, e.g., WN-ANS1 genome, to increase the amount of expressed rabies G protein resulting in an increased anti-rabies immune response. As with any d-PIV versions, one immunogen can be from one pathogen (e.g., rabies G) and the other from a second pathogen, resulting in three antigenic specificities of vaccine. As discussed above, ANS 1 deletions can be replaced with or used in combination with ANS3 and/or ANS5 deletions/mutations, in other examples.
Expression of RSV F protein in WN s-PIV and d-PIV
Respiratory syncytial virus (RSV), member of Paramyxoviridae family, is the leading cause of severe respiratory tract disease in young children worldwide (Collins and Crowe, Respiratory Syncytial Virus and Metapneumovirus, In: Knipe et al. Eds., Fields Virology, 5th ed., Philadelphia: Wolters Kluwer/Lippincott Williams and Wilkins, 2007: 1601-1646). Fusion protein F of the virus is a lead viral antigen for developing a safe and effective vaccine. To avoid post- vaccination exacerbation of RSV infection observed previously with a formalin-inactivated vaccine candidate, a balanced Thl/Th2 response to F is required which can be achieved by better TLR stimulation, a prerequisite for induction of high-affinity antibodies (Delgado et al., Nat. Med. 15:34-41, 2009), which should be achievable through delivering F in a robust virus-based vector. We have previously demonstrated the capacity of yellow fever virus-based chimeric LAV vectors to induce a strong, balanced Thl/Th2 response in vivo against an influenza antigen (WO 2008/036146). In the present invention, both yellow fever virus-based chimeric LAVs and PIV vectors are used for delivering RSV F to induce optimal immune response profile. Other LAVs and PIV vectors described herein can also be used for this purpose.
Full-length RSV F protein of A2 strain of the virus (GenBank accession number P03420) was codon optimized as described above, synthesized, and cloned into plasmids for PIV-WN s-PIV and d- PIV, using the insertion schemes shown in Figs. 12A 12B and 13 for rabies G protein, by applying standard methods of molecular biology. Exact sequences of the insertions and surrounding genetic elements are provided in Sequence Appendix 5. In vitro RNA transcripts of resulting WN(AC)-RSV F, WN(AprME)- RSV F, and WN(ACprME)- RSV F PIV constructs were used to transfect helper BHK- CprME(W ) cells. Efficient replication and expression of RSV F protein was first demonstrated by immunostainmg of transfected cells with an anti-RSV F Mab, as illustrated for the WN(AprME)- RSV F construct in Figs. 19 and 20. The presence of packaged PIVs in the superaatants from transfected cells (titer as high as 7 log 10 FFU/ml) was determined by titration in Vero cells with immunostainmg (Fig. 21). Additionally, similar constructs can be used that contain a modified full length F protein gene. Specifically, the N-terminal native signal peptide of F is replaced in modified F protein with the one from rabies virus G protein. The modification is intended to elucidate whether the use of a heterologous signal can increase the rate of F protein synthesis and/or replication of PfVs.
Table 1. PIV prototype constructs used in platform development studies
Tabl e 2.
Safe ty:
Sue klin g mou se neur
Figure imgf000056_0001
ovir ulence
Figure imgf000056_0002
Single dose, IC inoculation, ICR 5-day old mice, graded log doses administered.
'AST for mice that died; na, not applicable.
Table 3. PIV highly immunogenic and efficacious in mice1
Group Dose PRNT PRNT Post-challenge
Day 20 Day 34 mortality (%)
PIV-WN 10s 640 1280 0/8 (0%)
106 1280 2560 1/8 (12.5%)
106 +105 2560 2560 0/6 (0% i
Figure imgf000057_0001
1IP immunization (dO prime, and d21 boost in select groups); challenge on d35: wt WN
LD50; wt YF Asibi, 3 log10 PFU IC, 500 LD50: N/D, not determined.
5
Figure imgf000057_0002
JE Nakayama 5.8 log]0 PFU IC, or hamster-adapted YF Asibi 7 logio PFU IP (McArthur et al., J. Virol. 77: 1462-1468, 2003; McArthur et al., Virus Res. 110:65-71, 2005).
Table 5. Immunization of hamsters with PIV: comparison of SC and routes
Figure imgf000058_0001
Table 6. Immune res onses to PIV cocktails mice 1
Figure imgf000058_0002
C57/BL6 mice, IP inoculations on days 0 and 21; pooled serum PRNT titers.
Table 7. Neuro virulence (IC inoculation) and neuroinvasiveness (IP inoculation) of PIV-TBE YF/TBE vaccine constructs in adult ICR mice
Figure imgf000058_0003
AST for mice that died. Table 8. Neutralizing antibody titers (PRNT50) in mice immunized IP (determined against wt TBE virus Hypr), and protection from challenge (postchallenge observation, day 9)
Immunogen Dose(s), PR Tjo titer, PR Tjo GMT Postchallenge
logio individ. samples1 mortality (%)
on day 92
PIV-Hypr p39, 1 dose 5 1746 (2) 665 0/8 (0%)
1187 (2)
164 (2)
574 (2)
PIV-Hypr p39, 2 doses 5+5 16229 (2) 10,584 0/8 (0%)
12928 (2)
12927 (2)
4627 (2)
PIV-Hypr p40, 1 dose 5 <10 (2) 15 6/8 (75%)
<10 (2)
18 (2)
33 (2)
PIV-Hypr p40, 2 doses 5+5 169 (2) 153 1/8 (12.5%)
638 (2)
26 (2)
192 (2)
YF/Hypr p42 5 9210 (1) 6,085 0/2 (0%)
4020 (1)
YF/LGT p43 5 123 (2) 64 1/8 (12.5%)
32 (2)
96 (2)
45 (2)
YF/Hypr p45 5 292 (2) 68 0/3 (0%)
16 (1)
YF/Hypr dC2 p59 5 194 (2) 68 0/8 (0%)
93 (2)
45 (2)
26 (2)
Killed human TBE vaccine, 1 1/20 19 (2) 12 1/8 (12.5%) dose (at 1/20 of human dose) <10 (2)
13 (2)
<10 (2)
Killed human TBE vaccine, 2 1/20+1/20 3435 (2) 1,496 0/6 (0%) doses (each at 1/20 of human 1267 (2)
dose) 770 (2)
YF 17D control 5 <10 (4) <10 5/8 (62.5%)
11 (4)
Mock none <10 (4) <10 4/8 (50%)
<10 (4)
Numbers in parenthesis correspond to number of mice in each pooled serum sample tested.
2Mortalities on day 9 are shown. Table 9
Immunogenicity and efficacy of PIV variants and live chimeras in mice.
Dose log FFU PRNT PRNT50 Post-challenge
Immunogen 50
GMT GMT ± SE mortality
RV-WN/TBE 5 1368 1778 ± 469 0/8 (0%)
RV-YF/TBE 5 17 126 ± 101 4/8 (50%)
RV-TBE/TBE 5 2464 3237 ± 749 0/8 (0%)
YF/TBE 5 6,085 6615 ± 2595* 0/2 (0%)
YF/LGT 5 64 74 ± 21 * 3/8 (37.5%)
DEN2/TBE 5 337 885 ± 370 1/7 (14.3%)
INV, 1 dose† - 12 13 ± 2* 4/8 (50%)
INV, 2 doses† - 1,496 1824 ± 812* 0/6 (0%)
YF 17D 5 <10* 8/8 (100%)
DEN2 PDK-53 6 <10 8/8 (100%)
LGT TP21 5 1177 ± 412* 0/3 (100%)
Mock - <10 8/8 (100%)
All immunizations were by the IP route. TBE specific N Ab titers were determined in individual sera or pools from two animals on day 20 (*) or 30, followed by challenge the next day with 500 LD50 of wt TBE Hypr.
† INV was given at 1/20 of a human dose; in the 2-dose group, the second dose was on day 14.
Table 10. Examples of published attenuating E protein mutations that can be used for attenuation of chimeric TBE LAV candidates
Figure imgf000060_0001
S310K III putative cell attachment, change from E to JE Jiang et al, 1993, Gao et al, 1994,
G in JE reduced virulence Wu et al, 1997
K311E III highly conserved, putative cell attachment TBE, YF Rey et al, 995, Jennings, 1994
T333L III putative cell attachment YF, LGT Raynman et al, 1998
G334 III putative cell attachment YF Chambers and Nickells, 2001
S335K III putative cell attachment JE Wu et al, 1997
K336D III putative cell attachment JE Cecilia and Gould, 1991
P337D III putative cell attachment JE Cecilia and Gould, 1991
G368R III putative cell attachment TBE, JE Holzman et al 1997,
Hasegawa et al 1992
Y384H III change to H attenuated TBE, putative cell TBE Holzmann et al, 1990
attachment, -3 position to deleted RGD in
TBE
V385R III conserved, -2 position to deleted RGD in D2 Hiramatsu et al, 1996,
TBE, putative cell attachment Lobigs, 1990
G386R III highly conserved, -1 position to deleted D2, VE Hiramatsu, 1996, Lobigs et al,
RGD in TBE, putative cell attachment 1990
E387R III conserved, +2 position to deleted RGD in D2, MVE Hiramatsu, 1996, Lobigs et al,
TBE, putative cell attachment 1990
F403K none highly conserved, C-terminal region not D-2, D-4 Kawano et al, 1993, Bray et al, included in crystal structure sE 1998
H438Y None highly conserved, C-terminal region not LGT Campbell and Pletnev 2000
included in crystal structure sE
H496R none highly conserved, C-terminal region not TBE Gritsun et al, 2001
included in crystal structure sE
References: Hasegawa et al., Virology 191(1): 158-165; Schlesinger et al., J. Gen. Virol. 1996, 77 (
Pt 6):1277-1285, 1996; Labuda et al., Virus Res. 31(3):305-315, 1994; Wu et al., Virus Res.
51(2):173-181, 1997; Holzmann et al., J. Gen. Virol. 78 (Pt l):31-37, 1997; Bray et al., J. Virol. 72(2): 1647-1651, 1998; Guirakhoo et al., Virology 194(1):219-223, 1993; Pletnev et al., J. Virol.
67(8):4956-4963, 1993; Kawano et al., J. Virol. 67(11):6567-6575, 1993; Jennings et al., J. Infect.
Dis. 169(3):512-518, 1994; Mandl et al., J. Virol. 63(2):564-571, 1989; Chambers et al., J. Virol.
75(22): 10912-10922, 2001; Cecilia et al., Virology 181(l):70-77, 1991; Jiang et al., J. Gen. Virol.
74 (Pt 5):931-935, 1993; Gao et al., J. Gen. Virol. 75 (Pt 3):609-614, 1994; Holzmann et al., J. Virol. 64(10):5156-5159, 1990; Hiramatsu et al., Virology 224(2):437-445, 1996; Lobigs et al.,
Virology 176(2):587-595, 1990; Campbell et al., Virology 269(l):225-237, 2000; Gritsun et al., J.
Gen. Virol. 82(Pt 7): 1667-1675, 2001. Example 4. Delivery of SIV Gag and Env proteins (HIV prototypes) in WN s-PIV and d-PIV.
An artificial cassette containing SIV (GenBank accession number ADM52218.1) g l20 (a modified gene where the native signal sequence was replaced with the tPA signal and gp41 was truncated to contain only the TM domain), Gag, and Pro (protease) genes is shown in Fig. 22. The cassette was designed in a way that would allow its expression in the recombinant PIV ORF as a single precursor (different from SIV or ΗΓν gene organization). To allow for cleavage into individual SIV proteins, the genes are separated by FMDV 2A autoprotease sequences (see above). The nucleotide sequence of the entire cassette (~ 4 kb in length) was optimized by silent nucleotide changes to eliminate direct sequence repeats (e.g., all repeats longer than 8 nt were eliminated) to increase insert stability (using optimization algorithms at DNA 2.0) and by incorporating monkey codon preference to enable efficient protein translation in primate cells.
The codon-optimized cassette was chemically synthesized, followed by in-frame insertion of the genes, alone or in different combinations, in PIV-WN vectors in place of the AC (RV909 vector), AprM-E (RV230 vector) or AC-prM-E (dC RV230 vector) deletions. Examples of sequences of the constructs are provided in Sequence Appendix 6. Inserts of the first three constructs in Fig. 22, starting with the Env glycoprotein, were designed similarly to the PIV WV-rabies G described hereinabove (gpl20 signal fused with a portion of the signal sequence for prM at the end of the C gene or downstream from AC deletion depending on vector), as is also additionally illustrated for individual Env constructs in Fig. 23. In addition, alternate dC RV230 Env constructs were generated, in which the tPA signal and/or the S IV Env TM region of the gpl20 gene were replaced with rabies virus G protein-specific signal and/or anchor sequences (three bottom constructs in Fig. 23), to determine whether these heterologous rabies G-derived sequences will have a beneficial effect on gpl20 presentation or recombinant PIV replication. Gag and Gag-Pro insertions were designed to start with and end with FMDV 2 A autoprotease sequences, to free the island C-termini of the cytoplasmically synthesized Gag protein. They were cloned in place of the AprM-E or AC-prM-E deletions (Figs. 22 and 24). The N-terminal FMDV 2A was positioned either downstream from the viral cleavage site in C, or downstream from additional 9 or 18 amino acids following the cleavage site (from the prM signal) in the RV230 and dC RV230 vectors (Fig. 24) in order to determine which fusion type is preferable for efficient cleavage of FMDV 2A preceding
Gag, which theoretically can be important in terms of both transgene expression and PIV replication.
Correct processing of the polyprotein in recovered SIV Gag and Sr Gag/Pro PIVs grown in helper cells was confirmed by Western blot using anti-Gag antibodies (Fig. 25). Constructs expressing Gag alone showed the correct individual p58 Gag band of ~58 kDa, and constructs that also included Pro also showed an additional band of p28 which is a product of Gag cleavage by Pro. Immunostaining of naive Vero cells infected with the Gag PIVs (constructs shown in Fig. 26D), showed individual stained cells as expected from sPIV (Figs. 26A-C).
Efficient replication in vivo is illustrated by growth curves of SIV Gag PIV variants after transfection of helper cells with in vitro synthesized PIV RNA (P0 passage) (Figs. 27A-F). Some of the PIV variants grew efficiently to titers in excess of 7 log10 FFU/ml, and nearly identical titers were detected by both anti-Gag and anti-WN antibody staining, which was the first evidence of genetic stability of the Gag insert. When SIV Gag PIV was propagated in naive Vero cells as a two- component formulation (d-PIV, sometimes also designated as tc-PIV), together with PIV-WN helper with AC deletion (RV909), titers in the excess of 8 log10 FFU/ml were observed (Fig. 28). These results confirm that this formulation does not require helper cells for production (the principle of dPrV is described above).
High insert stability is illustrated for one of the SIV Gag PIV variants in Fig. 29. The stability of Gag was examined by ten serial passages of a RV230-Gag variant, containing Gag gene in place of large AprM-E deletion, in helper BHK-CprME(WN) cells at MOI 0.1 FFU/cell. At each passage, cell supernatants were harvested and titrated in regular Vero cells using immunostaining with an anti-WN antibody to determine total PIV titer, or an anti-SIV Gag antibody to determine titer of particles containing the Gag gene. Similar WN and Gag titers were observed after all 10 passages and no significant progressive decline in Gag positive titers was observed, e.g., as compared to the WN(AC)-rabies G PIV expressing the G insert in place of the very short AC deletion (see above).
Viable PIV-(WN)-SIV Env variants (Figs. 22 and 23) were also recovered in helper BHK cells transfected with in vitro RNA transcripts and efficient expression of gpl20 was demonstrated by immunofluorescence (Figs. 30A-F and Fig. 31). Interestingly, efficient intracellular expression of the original gpl20 was observed in Vero cells infected with packaged dC230Env variant as determined by immunostaining using anti-SIV Env antibody after methanol fixation (Fig. 30D), but little gpl20 was detected on the surface of the infected cells fixed by formalin (Fig. 30B), indicating inefficient transport of the translation product through the secretory pathway or cleavage of the TM domain away from the gpl20 molecule. The dC230Env/RabG anchor construct (Fig. 23), in which the SIV Env TM domain was replaced with the TM anchor sequence from rabies virus G protein, not only provided efficient intracellular expression of gpl20 (Fig. 28C), but also enabled its efficient cell surface delivery (Figs. 30A/30E and Fig. 31). Better surface expression/secretion of Env variants should result in higher immunogenicity of vaccine candidates. Therefore, the results presented with these constructs confirm the beneficial effect of using heterologous TM and/or signal sequences to increase immunogenicity of HIV Env glycoproteins.
Examples of sequences of similar PIV-HIV vaccine designs, using HIV-1 Clade C gene sequences, are provided in Sequence Appendix 7.
These examples demonstrate the feasibility of robust delivery of SIV (HIV) glycoproteins
(e.g., variants of Env) as well as cytoplasmic antigens (Gag, Pol, Nef and any other desired intracellular antigens), some of which can be secreted as SIV/HIV VLPs (e.g., Gag with or without Env), by PIV vaccine vectors.
In addition to g l20, other variants of the HIV Env immunogen, such as the full- length gpl60, gpl40, gpl45, gp41, etc., with or without desired mutations, truncations, deletions, or insertions (e.g., of dominant CD4 T cell epitopes, etc., including of non-HIV origin) in expressed molecules increasing immunogenicity and/or breadth of immune response against the variable HIV genotypes/strains, can be expressed without changing the meaning of this invention. Examples of possible modifications of Env are discussed below.
The Envelope (Env) protein is one of the primary targets of the humoral immune response upon infection with HIV. However, the Env protein has a number of defenses which prevent an effective antibody response from being mounted. These defenses include high degree of sequence variability, protection of functionally important domains through the use of variable loops and quaternary interactions, and high levels of glycosylation to shield the underlying protein backbone. In order to overcome this researchers have attempted a number of methods to increase the potency and breadth of antibody responses to Eriv. These modifications begin with an alteration of the underlying protein backbone itself. Attempts to minimize the genetic distance between immunizing isolates and those seen in the wild have led to the use of centralized sequences (consensus and ancestral) as immunogens (Kothe et al., Virology 2007, 360:218-234; Liao et al., Virology 2006, 353: 268-282), Modifying specific glycosylations has also been attempted. In some instances, hyperglycosylation of Env to mask unwanted epitopes in order to focus the humoral response on neutralizing domains has been utilized (Selvarajah et al., J. Virol. 2005, 79-12148-12163). Others have attempted to eliminate specific glycans to increase the availability of critical domains and hence increase Env immunogenicity (Li et al., J. Virol. 2008, 82:638-651). Altering the total glycosylation of the Env protein with expression in different systems has also been investigated (Kong et al., J. Mol. Biol. 2010, 403: 131-147). Outside of post translational modifications other groups have focused on manipulating Env variable loops as a means to increase immunogenicity. These modifications include shortening or deletion of variable loops (Ching and Stamatatos, J. Virol. 2010, 84:9932-9946; Yang et al., J. Virol. 2004, 78:4029-4036) as means to expose underlying domains. On the surface of virions, functional Env spikes exist as non-covalently linked trimers. However, these trimers are highly unstable making them difficult to use as immunogens. To overcome this hurdle attempts have been made to stabilize these trimers through mutagenesis (Beddows et al., J. Virol. 2005, 79:8812-8827) and introduction of heterologous trimerization domains (Yang et al., J. Virol. 2002, 76:4634-4642). Attempts have also been made to graft known epitopes recognized by mAbs to heterologous scaffolds (Phogat et al., Virology 2008, 373: 72-84; Zolla-Pazner et al., J. Virol. 2011, 85:9887-9898). Others have attempted to overcome the low immunogenicity of HIV Env by combining Env with immunostimulatory molecules in an effort to nonspecifically raise the immunogenicity of immunization (Melchers et al., J. Virol. 2011 , published ahead of print, doi:101128/JVI.06259-11).
If necessary, these and/or any other modifications of Env or other expressed HIV
immunogens leading to increased immunogenicity and/or breadth of humoral or cellular responses can be incorporated in the HIV antigenic moieties of PrV-HIV without changing the meaning of this invention.
Example 5. Immunogenicity of SIV Gag and Env proteins (HIV prototypes) in WN s-PIV and d-PIV.
Animal studies in Balb/c mice were performed to evaluate the immunogenicity of PIV-SIV Envelope (Env) and Gag expressing constructs. Dosing, route, and immunizing schedule of various experimental PIV (RV) and control ALVAC constructs are shown in Fig. 3B. Analysis of Env specific antibody titers (Fig. 33) revealed several interesting trends. Antibody responses to the RV construct expressing the SIV Env fused to the Rabies G transmembrane (TM) and cytoplasmic domain (CD) tended to elicit higher Env specific antibody titers at a 106 FFU/mouse dose compared to the ALVAC virus expressing the SIV Env administered at a 107 FFU/mouse dose. This phenomenon is seen more clearly when both the RV-Env and the ALVAC Env expressing construct are given at the same 106 FFU/mouse dose. In this comparison the RV-Env/RabG construct elicited a geometric mean titer greater than 100 times higher than the ALVAC Env expressing positive control virus. This phenomenon may be due to a combination of increased immunogenicity provided by the PIV platform and the enhanced surface expression of the SIV Env when the RabG TM & CD are present, as demonstrated in vitro (see Figs. 30A-G). We also demonstrated that RV-Gag expressing constructs are capable of eliciting detectable T cell responses as measured by interferon gamma (IFNg) secretion upon peptide stimulation ex vivo (Fig. 35). Splenocytes harvested 7 days after the second immunizing dose were stimulated with a known CD8 gag specific epitope and assayed for IFNg secretion by ELISPOT. IFNg secreting cells in the RV 9AA Anchor Gag construct were detected and proved to be statistically greater than that of the ALVAC-Gag expressing control virus against the same peptide. Example 6. Prime-boost vaccination regimens with PIV- WN/TBE constructs.
We also investigated in mice whether RV-WN/TBE and INV vaccines could be interchangeable in prime-boost vaccination regimens. A single dose of INV induced low TBE specific neutralizing antibody titers and provided only 60% protection from challenge. A second dose of INV significantly increased the titers (PRNT50 GMT 1,019 on day 42) which resulted in 100% protection. RV-WN/TBE was efficient in prime-boost vaccination regimens. The highest PRNT50 titers were observed in INV prime - RV-WN/TBE boost, RV-WN/TBE prime - RV- WN/TBE boost, and RV-WN/TBE prime - INV boost groups (GMTs 3,287, 6,291 and 14,205, respectively). Animals in all groups primed or boosted with sPIV- WN/TBE were protected from challenge (Table 1 1).
Table 11. Evaluation of prime-boost vaccination regimens with RV-WN/TBE and INV in mice.
Prime Day 21 Boost Day 42 Post- Day 0 PRNTso Day 22 PRNTso challenge
GMT
GMT mortality
INV 48 INV 1,019 0%
INV 57 Diluent 115 40%
INV 59 RV-WN/TBE 3,287 0%
RV-WN/TBE 341 RV-WN/TBE 6,291 0%
RV-WN/TBE 558 INV 14,205 0%
RV-WN/TBE 565 Diluent 2,535 0%
Diluent <20 Diluent <10 100%
All immunizations were by the IP route. RV-WN/TBE was administered at 5 log10 FFU/dose, and INV at 1/20 of a human dose. Challenge was on day 43 with 500 LD50 of TBE Hypr.
Example 7: A novel safe single-dose vaccine against tick-borne encephalitis.
Tick-borne encephalitis (TBE) is an acute viral infection of the central nervous system and the most important disease of humans transmitted by ticks. It is endemic in eastern, central and northern European countries and Russia. TBE virus is also present in parts of several Asian countries (northern China and Mongolia, Japan). The virus is an emerging pathogen spreading rapidly in Europe. The disease is causing more than 10,000 hospitalizations annually, with case- fatality rates of 1-2% in Europe and up to 40% in Siberia and the Far East of Russia. The public awareness of TBE is high due to the severity of the disease and long-lasting neuropsychiatric sequelae occurring in up to ~ 40% of patients (World Health Organization. 2011. Wkly Epidemiol Rec. 86:241 -256.). Highly effective inactivated vaccines (INV) have been available for several 856.). Yet vaccination rates remain low in most endemic countries (6-22%), with a few exceptions (Austria 88%, Latvia 38%). Among people considered vaccinated, more than a half have not received all three doses required for primary vaccination or do not adhere to the recommended booster schedules (every 3-5 years) and thus may not be adequately protected. Among unvaccinated, many people resort to protecting themselves from TBE infection by taking precautions to avoid tick bites or refraining from certain outdoor activities altogether rather than vaccination (Kunze, Wien Med Wochenschr. 2011 Jul;161(13-14):361-4.), with the need for undergoing multiple
immunizations being an obvious confounding factor.
TBE virus belongs to the Flavivirus genus of small enveloped plus-strand RNA viruses, which also includes such major mosquito-transmitted pathogens as yellow fever (YF), Japanese encephalitis (JE), West Nile (WN) and dengue types 1-4 (DEN 1-4) viruses (Burke and Monath. 2001. Flaviviruses, p. 1043-1 126. In Knipe et al. (ed.), Fields Virology, 4th ed. Lippincott Williams and Wilkins , Philadelphia, PA.). The flavivirus genomic RNA of ~ 11,000 nucleotides (nt) contains a single open reading frame which encodes the three structural proteins of the virion, capsid C, premembrane prM and envelope E, followed by seven nonstructural (NS) proteins involved in virus replication (Lindenbach et al., In: Knipe et al., editors. Fields Virology, 5th ed. Philadelphia:
Wolters Kluwer, Lippincott Williams and Wilkins, 2007. p. 1101-52.). The E protein is the main immunogen, eliciting neutralizing antibodies which are considered to be the main correlate of protection from flavivirus infection. Available INV and live attenuated (LAV) flavivirus vaccines have been reviewed, including the single-dose YF 17D LAV that has been used in >500 million persons worldwide, and the recently developed ChimeriVax vaccines against JE, dengue and WN obtained by chimerization with YF 17D virus (Pugachev et al., In: Levine et al., editors. New generation vaccines, 4th ed. New York: Informa Healthcare USA, 2010, p. 557-69; Guy et al., Vaccine 2010; 28:632-49.).
A safe and effective LAV against TBE capable of eliciting durable immunity after a single dose has been highly desirable since the discovery of the virus in 1949, but all attempts thus far to develop one using empirical and rational attenuation approaches have not been successful. In the early 1970s, a naturally attenuated Langat (LGT) virus serologically related to TBE was used to vaccinate more than 600,000 people in the former Soviet Union. This experimental vaccine was abandoned because of cases of vaccine-induced encephalitis observed at a rate of 1/18,570. It is important however that a long term follow-up of the vaccinees confirmed a durable protective Prophylaxis" (AA Smorodincev, Ed.), pp. 190-211. Meditsina, Leningrad.). More recent attempts to develop a TBE LAV by means of molecular modifications of the TBE virus genome or
chimerization of TBE or LGT viruses with DEN4 virus also resulted in either insufficient attenuation or low immunogenicity (Mandl et al., 1998, J. Virol. 72:2132-2140; Kofler et al. 2004. Arch Virol Suppl. 18:191-200; Rumyantsev et al. 2006. Vaccine 24:133-143; Maximova et al. 2008. J Virol. 82:5255-5268; Wright et al., Vaccine. 2008 Feb 13;26(7):882-90.).
Here, we used a novel approach to flavivirus vaccines, PIV constructs, to develop a safe, single-dose TBE vaccine (Mason et al, Virology 2006; 351 :432-43; Ishikawa et al., Vaccine 2008; 26:2772-81 ; Suzuki et al, J Virol 2008; 82:6942-51; and Widman et al., Adv Virus Res 2008;
72:77-126). PIV constructs are engineered to have a deletion removing most of the capsid protein C gene. Therefore they are propagated in vitro in complementing cells supplying the C protein in trans. In normal cells, both in vitro and in vivo, they undergo a single round of replication, without spread to surrounding cells. Infected cells secrete empty virus like particles (VLP, the product of the prM-E genes) devoid of the nucleocapsid. VLPs are smaller than viral virions but have the same
architecture of the envelope (Ferlenghi et al., Mol Cell. 2001 Mar;7(3):593-602) and therefore are highly immunogenic. Recently, we performed direct head-to-head comparison of several PIV prototypes (PIV-WN, -JE and -YF) to available LAV and INV vaccines for safety, immunogenicity and efficacy in mice and hamsters and demonstrated that they are extremely attenuated (due to the single-cycle nature) but at the same time can be as immunogenic and efficacious as LAVs after a single dose (Rumyantsev et al., Vaccine. 2011 Jul 18;29(32):5184-94.). We report the construction and characterization in vitro and in vivo, including in nonhuman primates (NHP), of PIV-TBE and live chimeric vaccine candidates. The promising data for the selected PIV-TBE candidate indicate that a single-dose TBE vaccine suitable for human use and superior to INVs is finally in hand.
RESULTS
Replication in vitro of PIV-TBE variants and live chimeras. Several PIV -TBE variants containing the prM-E genes from TBE Hypr virus were constructed based on the WN, TBE Hypr, LGT E5 and YF 17D backbones (RV-WN/TBE, RV-TBE/TBE, RV-YF/TBE and RV-LGT/TBE). Live chimeras based on the backbones of attenuated YF 17D, DEN2 PD -53 and LGT E5 viruses genes, and one additional virus, YF/LGT, contained the prM-E genes from LGT E5 (Fig. 44).
The PIV-WN/TBE variant (based on the WN backbone, with the TBE specific prM signal) grew very efficiently in both BH and Vero (WN C) helper cells (Fig. 47 A), with peak titers as high as 8 log10 FFU/ml in some experiments. High titers, also up to 8 log10 FFU/ml, were observed in a broad range of MOIs (0.001, 0.01 and 0.1 FFU/cell) (Fig. XXX). This variant was also used to examine whether modifications in the prM signal could increase the secretion of TBE VLPs (to increase immunogenicity). Three amino acid changes shown previously to increase VLP secretion in a AC RNA vaccine candidate against TBE ( ofler et al., Proc Natl Acad Sci U S A. 2004 Feb
17; 101 (7) : 1951 -6) were introduced, or the entire signal was replaced with an idealized signal WWRLWW(L)8WPMVWA (Barash et al, Biochem Biophys Res Commun. 2002 Jun
21;294(4):835-42). These mutations did not increase VLP secretion in the context of PIV-WN/TBE, however it is interesting that both mutants were viable and replicated efficiently in helper cells (Figs. 7B, 8C and 8D). In the latter mutant, not only the signal was replaced with a non-flavivirus sequence, but the modification also abolished the viral protease cleavage site at the C/prM junction. Obviously, the AC deletion in PIV constructs uncouples the coordinated viral protease and signalase cleavages at the C/prM junction known to be critical for flavivirus replication (Yamshchikov et al., Virology 1993, 192:38-51; Amberg et al, J Virol 1999, 73:8083-8094).
Other PIV constructs, RV-TBE/TBE, RV-YF/TBE and RV-LGT/TBE, replicated less efficiently in helper cells than PIV-WN/TBE, with peak titers not higher 6 log10 FFU/ml (Fig. 47 A). Interestingly, PIV-YF/TBE variant grew better in WN C helper cells than in YF C helper cells. PIV- LGT/TBE replicated particularly poorly. Additional attempts to increase its replication by reducing the size of the AC deletion or using the TBE specific prM signal instead of LGT signal did not increase the yields.
To demonstrate genetic stability, PIV-WN/TBE was serially passaged eleven times in two independent passage series in BHK WN C helper cells at an MOI of 0.01 FFU/cell, and the full genomes of the PI 1 samples were sequenced by consensus sequencing. Only a few mutations were detected, E122Q in the E protein in the first passage series, and K3M in the prM signal and R122L in NS2A in the second passage series. The mutants replicated efficiently in helper cells. Importantly, no recombination (replication competent virus) was detected in these samples. The absence of recombination was also confirmed by titration of numerous other PIV-TBE samples, as well as titration of mouse brain tissue samples harvested after IC inoculation. tor ΐν manufacturing. BH WN C helper cells were found to provide equally high yields of PIV- WN/TBE and the PIV-WN prototype at cell passages > P20 (including after 5 passages in puromycin-free medium), and early passages (Fig. XXX).
Live chimeras, YF/TBE, YF/LGT, LGT/TBE and DEN2/TBE, were also constructed and found to replicate efficiently in Vero cells with peak titers of ~ 7 - 8 log10 PFU/ml (Fig. 47B).
Analysis of neurovirulence and neuroinvasiveness of PIV-TBE variants and live chimeras in mice. 3.5 week-old ICR mice were inoculated with graded doses of PIV-TBE constructs and chimeric viruses by the IC route to measure neurovirulence or IP route to measure neuroinvasiveness (Fig. 45). All animals that received doses up to 5 log10 FFU of RV-WN/TBE by both routes survived without any signs of sickness. Neurovirulence of all PIV-TBE variants (RV- WN/TBE, RV-TBE/TBE, RV- YF/TBE and RV-LGT/TBE) was also exmined in more sensitive 8 day-old ICR suckling mice, and 100% survival without sickness was observed at doses up to 5 logio FFU (in control groups, IC LD50 values of YF 17D and YF/JE LAVs were 0.5 and 316 PFU, respectively). Thus, PIV-TBE constructs are extremely highly attenuated in mice, as expected. In contrast, all live chimeras (YF/TBE, YF/LGT, LGT/TBE and DEN2/TBE) were found to be highly neurovirulent for 3.5 week-old ICR mice, with IC LD50 values below 2 logio PFU. YF/TBE, LGT/TBE and DEN2/TBE chimeras, but not YF/LGT, exhibited at least some degree of neuroinvasivennes. YF/TBE had an IP LD50 of 3.5 log10 PFU. A partial mortality was observed for LGT/TBE across all tested doses (1 - 6 log10 PFU), and DEN2/TBE caused a partial mortality at the highest tested dose of 6 log10 PFU. Thus, live chimeras, particularly with the TBE envelope, are less attenuated. It should be noted however that chimerization resulted in some attenuation, particularly for neuroinvasiveness, compared to TBE Hypr virus (IP LD50 1 PFU), with most chimeras exhibiting neurovirulence/neuroinvasiveness not higher than that of the naturally attenuated LGT TP21 virus (Fig. 45). We also tried to further attenuate the YF/TBE virus by introducing a small (3- amino acid) nonlethal deletion into its C protein, or various combinations of E protein mutations shown previously to attenuate LGT E5 virus (Rumyantsev et al., J Virol. 2006 Feb;80(3): 1427-39), with little success.
Immunogenicity and efficacy of candidates in mice, Th type of response, effect of anti- vector preimmunity, and prime-boost regimens with INV. All immunizations of mice (3.5 week old ICR) were by the IP route. TBE-specific PRNT50 titers were measured in sera collected on days 20 or 30. Challenge was done the following day after bleeding. The PIV-TBE candidates RV- (reciprocal GMTs 1,778 and 3,237, respectively), which were comparable to neutralizing titers in sera of mice that survived after receiving 5 log10 PFU of YF/TBE chimera (GMT 6,615). The animals were solidly protected from a severe IP challenge with TBE Hypr virus (500 LD50), while mice in the negative control groups (mock, YF 17D, DEN2 PDK-53) died (Table 9). RV- YF/TBE was poorly immunogenic and only 50% efficacious. Lower immunogenicity and efficacy were observed for DEN2/TBE and particularly YF/LGT chimeras. A single dose of the human INV control resulted in low neutralizing antibody titers (GMT 13; with 50% protection), which significantly increased, as expected, after the second dose (GMT 1,826; 100% protection).
Immunization of groups of mice with graded doses of RV-WN/TBE and YF/TBE followed by challenge on day 21 revealed that both candidates had the same 50%-protective dose (PD50) values of ~ 3 log10 FFU/PFU (Fig. 48 A). Durability of immunity was examined by inoculating mice with doses of RV-WN/TBE, YF/TBE and INV equivalent to 10 PD50 (two doses for INV) followed by monitoring neutralizing antibody titers for 4-5 months, and challenge. High level neutralizing titers were induced and maintained throughout the duration of the study in the three groups, and most animals were protected from challenge (100% protection for RV-WN/TBE and 2 x INV) (Fig. 48B). It should be noted that in contrast to primates, mice vaccinated with flavivirus INVs maintain high-level immunity throughout their short life span, as described previously (Rumyantsev, 2011, supra).
Thus, RV-WN/TBE which is the preferred PIV-TBE variant because of its efficient replication in vitro, is essentially as immunogenic and efficacious in mice as the underattenuated YF/TBE virus after one dose. To obtain evidence that a PIV-TBE and a TBE LAV share the same mode of action in vivo, concentrations of IgG isotypes were determined in sera from mice immunized with RV-WN/TBE, YF/TBE and INV by IgG type specific ELBA. The IgG2a/IgGl antibody ratios were found to be 4: 1 and 16:1 for RV-WN/TBE and YF/TBE, respectively, indicative of a Thl biased immune response. For the INV, the ratio was 1 :4, indicative of a Th2 bias.
Anti-vector (YF) preimmunity is not a concern for ChimeriVax vaccines as was previously shown in humans (Monath et al, Vaccine. 2002 Jan 15;20(7-8): 1004-18; Guirakhoo et al., Hum Vaccin. 2006 Mar-Apr;2(2):60-7), although this aspect has not been examined in mice. To compare the effect of anti-vector preimmunity on immunogenicity of chimeric viruses and PIV, mice were preimmunized with YF 17D and then immunized 3 weeks (short interval) or 6 months (long interval) later with YF/JE and YF/TBE. Mice were similarly preimmunized with RV-WN to induce by YF and WN PRNT50. JE or TBE neutralizing antibody titers in these groups were determined 21 days after vaccinations and compared to parallel groups of naive animals vaccinated with YF/JE, YF/TBE or RV-WN/TBE. For RV-WN/TBE, pre-immunization resulted in some reduction of TBE specific response compared to that in naive animals. However, it was less pronounced compared to the effect of YF 17D pre-immunization on immunogenicity of YF/JE and YF/TBE viruses (Fig. 49), suggesting that anti- vector immunity should not be a concern for PIV-TBE in humans.
We also investigated in mice whether RV-WN/TBE and INV vaccines could be
interchangeable in prime-boost vaccination regimens. A single dose of INV induced low TBE specific neutralizing antibody titers and provided only 60% protection from challenge. A second dose of INV significantly increased the titers (PRNT50 GMT 1,019 on day 42) which resulted in 100% protection. RV-WN/TBE was efficient in prime-boost vaccination regimens. The highest PRNTso titers were observed in INV prime - RV-WN/TBE boost, RV-WN/TBE prime - RV- WN/TBE boost, and RV-WN/TBE prime - INV boost groups (GMTs 3,287, 6,291 and 14,205, respectively). Animals in all groups primed or boosted with sPIV-WN/TBE were protected from challenge (Table 11).
Immunogenicity and efficacy of candidates in NHP, effect of vaccination routes, dose- responses, effect of anti-vector preimmunity, and durability of responses. Two studies in Rhesus monkeys were performed (Figs. 51-53). Because no lethal TBE virus challenge model exists (monkeys are mostly not susceptible to peripheral TBE virus infection), a surrogate challenge model was established. Animals were inoculated by the SC route with 6 log10 PFU of a plaque purified LGT T1674 virus (large plaque of LGT T1674-73), LGT TP21, or YF/TBE (N = 3, 3, and 4, respectively) and viremia were determined daily on days 1 - 8. Only animals, inoculated with the LGT T1674 virus developed readily detectable, uniform viremia with GMTs of 3.2 and 2.1 log 10 PFU/ml on days 1 and 2, respectively (Fig. 54A), and therefore this virus was selected for challenge in these experiments. LGT TP21 caused no detectable viremia, and YF/TBE induced a low-level viremia in some animals, <1.6 log10 PFU/ml, on days 5 - 6. We have observed previously for chimera- JE that a reduction of inoculation dose delays the onset of viremia in Rhesus monkeys without affecting the peak titer (Monath et al, J Virol. 2000 Feb;74(4): 1742-51). Therefore during challenge, LGT T1674 virus was given at a lower SC dose of 5 log10 PFU, which indeed allowed for a better resolution of viremia (in naive animals), peaking on day 2 (Fig. 54B-D). This is a stringent virus.
Monkeys (N=4 per group) immunized with RV-WN/TBE at a 7 log10 FFU dose by the ID route developed high TBE-specific neutralizing antibody titers measured on days 29 and 50 post- immunization (GMTs 1,954 and 1,677, respectively), which were comparable to 5 and 6 log10 PFU doses of YF/TBE chimera administered SC (the route used for the chimera vaccines), and 2 and 3 complete IM (the route used in humans) doses of the INV control (GMT 3,314 on day 29 for two doses and 4,111 on day 50 for three doses) (Table 12. A single IM 7-log10 dose of RV-WN/TBE was also highly effective (GMTs 955 and 707 on days 29 and 50). A single SC 7-log10 dose of RV- WN/TBE induced a lower antibody response (e.g., GMT 232 on day 50) which was significantly boosted by a second SC dose (GMT 3,357 on day 50 in the SC two-dose group). A single ID inoculation of RV-WN TBE given at 6 and 5 log10 FFU doses induced appreciable neutralizing antibody responses which however were significantly lower compared to a 7-logio ID dose. This may indicate that the immunizing dose should be above 6 log10 FFU in humans. Anti-vector (WN) preimmunity reduced the TBE specific neutralizing titers by approximately 5 fold compared to the 7-logio ID group of immunized WN-nai've animals [GMT 306 on day 50 in all four monkeys (two monkeys naturally WN virus immune, and two monkeys preimmunized with RV-WN)]. The effect of WN preimmunity was less pronounced compared to the effect of YF preimmunity on
ChimeriVax-DEN2 in NHP (Guirakhoo et al, J Virol. 2000 Jun;74(12):5477-85), and thus antivector immunity should not be a concern for use of PIV-TBE in humans. No adverse reactions were observed in the 1st study, including in the 6-log10 SC YF/TBE group. In the 2nd study, two of the 4 monkeys inoculated by a lower, 5 logio PFU SC dose of YF/TBE virus developed neurological signs (miscoordination, imbalance) and one animal had to be euthanized, while animals in other groups remained healthy. Induction of pathology at a lower virus dose was likely due to the
"prozone" effect. Thus the chimera is also less attenuated in NHP.
Figure imgf000074_0001
accuracy.
* Challenge was SC with 5 log10 PFU of LGT Tl 674 virus on day 58 (the 1st study) or day 51 (the short-term part of the 2nd study); animals were considered protected if no viremia was detected by plaque assay during 8 days post-challenge. † Second SC dose was given on day 30
* Two WN-positive monkey were given RV-WN/TBE on day 0 (day 29 PRNT50 is shown for these animals only), additional two monkeys were preimmunized with RV-WN on day 0 and given RV-WN/TBE on day 30 (day 50 PRNT50 is for all four animals).
§ The three complete human INV doses were given on days 0, 14 and 30.
Efficacy of vaccination was demonstrated by challenge with LGT T1674 virus on day 58 of the 1st study or day 51 in the short-term part of the 2nd study. All immunized animals had no detectable viremia on days 1-8 after challenge, as determined by plaque assay, with the exception of one animal in each of the 5- and 6-log10 ID RV-WN/TBE groups in agreement with pre-challenge PRNT50 titers (Table 12; Figs. 54B-D). Mock immunized animals were not protected. When examined by a more sensitive RT-qPCR method (sensitivity = 0.14 PFU/ml), the WN-preimmune group that received 7-log10 PFU of RV-WN/TBE by the ID route was found to be completely protected, while three out of 4 monkeys in the 5- and 6-log10 ID RV-WN/TBE groups were viremic (Fig. 50A).
Most importantly, in the long term part of the 2nd study, high-level neutralizing antibody titers were maintained in the 7-log10 ID and IM RV-WN/TBE groups during the 6 months of progressively declined (Fig. 50B). The difference in titers between the RV-WN/TBE groups and the 3 x INV group became statistically significant at 5 and 6 months (p < 0.03). The RV-WN/TBE vaccinated animals were protected from challenge done at 6 months, while a detectable post- challenge viremia was found in all monkeys in the 3 x INV group (Fig. 50A). Thus a single 7-log10 dose of RV-WN/TBE is superior to 3 doses of a human INV in terms of inducing a robust, long- lasting immunity in NHP.
CONCLUSION TBE virus is the most important human pathogen in Eurasia transmitted by ticks. Inactivated vaccines (INV) are available but require multiple doses and frequent boosters to induce and maintain immunity. Thus far the goal of developing a safe, live attenuated vaccine (LAV) effective after a single dose has remained elusive. Here we used a novel approach to replication-defective (single- cycle) flaviviras vaccines, the PIV-TBE constructs discussed above, to generate a safe, single-dose TBE vaccine relying on immunologic mechanisms similar to LAVs. Several PIV-TBE candidates attenuated by a deletion in the capsid gene were constructed based on different flaviviras backbones containing the envelope genes of TBE virus. PIV-TBE constructed using a West Nile virus backbone (PIV-WN/TBE) grew more efficiently in helper cells than candidates based on Langat E5, TBE, and yellow fever (YF) 17D backbones, and was found to be highly immunogenic and efficacious in mice. Live chimeric YF 17D/TBE, Dengue 2/TBE and Langat E5/TBE candidates were also constructed but found to be less attenuated than PIV-WN/TBE. Similar to YF 17D/TBE virus, PIV-WN/TBE elicited a Thl biased response. PIV-WN/TBE was demonstrated to be highly immunogenic in Rhesus macaques after a single dose, inducing a significantly more durable response compared to 3 doses of a human INV. Immunogenicity was not significantly affected by pre-existing immunity against WN. Immunized monkeys were protected from a stringent surrogate challenge. These results indicate that we have developed a novel TBE vaccine with a superior product profile to existing inactivated vaccines, which could lead to improved vaccine coverage and control of TBE disease.
MATERIALS AND METHODS obtained by transfection with a Venezuelan equine encephalitis virus replicon (rVEE, based TC-83 vaccine strain) expressing the WN virus C protein and a puromycin N-acetyl-transferase selective marker (Mason et al., Virology 2006; 351:432-43; and Widman et al., Adv Virus Res 2008; 72:77-126), or an additionally constructed rVEE helper expressing the C protein of TBE Hypr. The cells were maintained in puromycin-containing selective media. PIV-TBE titers were determined by immuno-focus assay in Vero cells (Rumyantsev et al, 2011, supra) using anti-TBE mouse hyperimmune ascitic fluid (ATCC) or polyclonal rabbit antibodies raised against an inactivated human vaccine against TBE (FSME, Baxter) as primary antibodies. The FSME vaccine was used as INV control in animal studies. Live viruses and chimeras were propagated in regular Vero cells in media without puromycin. YF 17D virus (YF-VAX, Sanofi Pasteur) used in animal experiments was prepared by amplification in Vero cells. YF/JE and YF/WN chimeras (ChimeriVax) were described previously (Guirakhoo et al., 1999; and Monath et al., 2006). LGT virus strains TP21 (isolated in Malaysia from Ixodes granulatus ticks in 1956) and Tl 674-73 (isolated in Thailand from Haemaphysalis papuana ticks in 1973), and wild type TBE strain Hypr were from R. Tesh (World Reference Center for Emerging Viruses and Arboviruses, University of Texas, Galveston, TX). Infectious titers/doses of PIV constructs and live viruses are expressed in FFU (focus forming units) and PFU (plaque forming units), respectively.
Construction of PIV-TBE variants and live chimeras. RV-WN/TBE was constructed by replacing the WN-specific prM-E genes in the PIV-WN prototype (Mason et al, 2006) with those from TBE virus strain Hypr (GenBank accession number U39292; the nt sequence was optimized to have preferential human-genome codon usage and to eliminate repeats > 8 nt). The TBE genes were synthesized by DNA2.0, Inc. (Menlo Park, CA). A variant with the TBE-specific signal for prM was used in all experiments. (Another variant, with WN-specific prM signal, was also constructed but not used because it replicated to several fold lower titers in helper cells). RV-YF/TBE was constructed by replacing the YF 17D prM-E genes in PIV-YF prototype (Mason et al., 2006) with the TBE prM-E genes. RV-LGT/TBE was constructed using a synthetic infectious clone of Langat E5 virus (LGT E5, an attenuated strain of LGT TP21) assembled from three DNA fragments synthesized based on published LGT E5 sequence (GenBank accession number ). RV-TBE/TBE based entirely on the TBE Hypr sequence was assembled from three DNA fragments synthesized based on published TBE Hypr sequence (GenBank accession number U39292).
YF/TBE and YF/LGT chimeras containing the TBE Hypr and LGT E5 prM-E genes,
respectively, in the YF 17D backbone were made by replacing the JE specific prM-E genes with the a synthetic infectious clone of DEN2 virus strain PDK-53 assembled from two DNA fragments synthesized based on published sequence (GenBank accession number ). LGT/TBE chimera was made by inserting the complete C protein gene of LGT E5 into the RV-LGT/TBE construct.
Resulting plasmids were transcribed in vitro and appropriate cells (helper BH cells for PIV and
Vero for chimeric viruses) were transfected with RNA transcripts to generate infectious PIV and chimeric viruses. For DEN2/TBE chimera (the genome split in two plasmids), the full length cDNA template was prepared by two-fragment in vitro ligation followed by transcription and transfection of Vero cells.
Mouse Studies. All procedures were performed under approved IACUC protocols in accordance with the National Institutes of Health (NIH) requirements for humane treatment of laboratory animals. ICR mice were from Taconic. Inoculation routes/doses, and bleeding/challenge days were as described in Results. Challenge was done by the IP route with 500 LD50 (500 PFU) of TBE Hypr. In neurovirulence/neuroinvasiveness tests and after challenge, mice were monitored for 21 days for survival. Doses causing 50% mortality (LD50) were calculated using the Reed and
Muench method. Neutralizing antibody (PRNT50) titers were determined in heat-inactivated serum samples collected by mandibular bleeding against wt Hypr or YF/TBE viruses. Titers of IgG
isotypes were determined by isotype-specific ELISA as described (Rumyantsev, 2011 , supra) using YF/TBE virus as a coating agent.
NHP studies. The in-life parts of the two described NHP studies were performed at Bioqual,
Inc. (Rockville, MD) in accordance with Animal Welfare Regulations (USD A), PHS Policy on
Humane Care and Use of Laboratory Animals, and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD).The first NHP study (design shown in Fig. 51) was done in 34 experimentally naive Rhesus monkeys of Chinese origin pre-screened to be
seronegative for JE, WN and YF by PRNT50. Three groups (3 - 4 animals/group) were inoculated by the SC route with 6 logio PFU/dose of two strains of LGT virus (TP21 and a plaque purified variant of T1674-73) and YF/TBE chimera to establish a surrogate challenge model based on viremia during days 1 - 8 after inoculation. Four groups (4 animals/group) were inoculated by the ID, IM and SC routes (1 or 2 doses for SC; in the two-dose group, inoculations were on days 0 and 30) with 7 log10 FFU/dose of RV-WN/TBE to evaluate immunogenicity (PRNT50 on days 29 and 50) and efficacy compared to YF/TBE virus, three human doses of the FSME INV administered IM on days 0, 14 and 30, and Mock. ID inoculations were performed using a Becton Dickinson ID inoculation devise with a 1-mm needle. Tl 674-73 challenge virus. Viremia was determined by plaque assay with immunostaining.
The second NHP study consisted of two parts, the short-term (Fig. 52) and long-term (Fig. 53) parts. Rhesus monkeys prescreened to be seronegative for JE, WN and YF were used, except for two animals found to be seropositive for WN which were assigned to a group addressing the effect preexisting anti-vector immunity. The schedule of steps in the first part was similar to the first NHP study. Two groups received 6 and 5 log10 FFU/dose of RV-WN/TBE by the ID route to evaluate dose- responses. In the group addressing the effect of anti-vector preimmunity, animals received 7 log10 FFU of RV-WN/TBE by the ID route on day 0 (the two naturally WN-immune animals) or day 30 (two additional animals that first received 7 log10 FFU/dose of PIV-WN on day 0). In the long-term part, the dynamics of TBE neutralizing antibody responses were monitored for 6 months following
immunization with 7 log10 FFU/dose of RV-WN/TBE by the ID and IM routes compared to YF/TBE (5 log10 PFU by SC) and three doses of FSME. PRNTso titers in sera were measured on days 29 and 50 and then at 3, 4, 5 and 6 months. The immunized animals and a control (unimmunized) group were challenged with LGT T 1674-73 following the final bleeding. In addition to plaque assay, post- challenge viremia was measured by a highly sensitive RT-qPCR method performed on RNA isolated from monkey sera.
Statistical analyses. Calculations of end point titers and analyses of statistical significance were performed using GraphPad Prism 5 (GraphPad Prism Software, Inc., San Diego, CA).
Example 7. Delivery of HA protein of influenza H1N1 virus (strain New Caledonia) in WN s- PIV (which optionally can be used in d-PIV).
The full-length HA gene of Flu strain New Caledonia was cloned in place of AprM-E and AC-prM-E deletions of PIV-WN vectors in the same fashion as described for Rabies G, RSV F, and SIV Env (as described above; Fig. 36). Examples of sequences are provided in Sequence Appendix 8. The variants were viable, and grew to high titers immediately after RNA transfections of helper cells (Figs. 37A-B and Figs. 38A-D). Identical titers in the growth curves using immunostaining with anti-WN and anti-HA antibodies provided evidence of insert stability.
All variants efficiently expressed the HA protein both intracellularly (methanol fixation) and on the cell surface (formalin fixation) of infected Vero cells as shown by immuno-fluorescence (Figs. 39A-F, 40A-B, 41A-H, 42A-D, and 43A-B). The latter is a known prerequisite for high HA immunogenicity. Importantly, the expressed HA was efficiently recognized by both antibodies 43A-B).
Other flu antigens can be similarly delivered, such as NA, M2 (e.g., M2e), etc., or fragments thereof. With respect to HA, various modifications can be introduced, and modified antigens then expressed in PIV vaccine vectors, without changing the meaning of this invention.
The PIV-SIV and PIV-Flu vaccine candidates described in Examples 4 and 5 can be tested for immunogenicity and efficacy in animal models. Earlier in vivo data have demonstrated that PIV vaccines expressing transgenes are highly immunogenic in animals, as has been shown for PIV-RSV F (see, e.g., WO 2010/107847, incorporated herein by reference), and more recent experiments for PIV-Rabies G.
Sequence Appendix 1
CV-TBEV Hypr or CV-LGT E5 with YFV/TBEV chimeric signal (p42, p59, and p43 constructs; SEQ ID NOs:28-30)
YF17D partial signal
TBEV partial signal
C protein YF17D Hypr or LGT E5 prM protein
R K R R S H D V L T V Q F L I L G M L G M T I A A T V R
401 A GGAAACGCCG TTCCCATGAT GTTCTGACTG TGCAATTCCT AATTTTGGGC ATGCTGGGCA TGACAATCGC AGCTACGGTT CGC
T CCTTTGCGGC AAGGGTACTA CAAGACTGAC ACGTTAAGGA TTAAAACCCG TACGACCCGT ACTGTTAGCG TCGATGCCAA GCG
CV-TBEV Hypr with YFV/WNV chimeric signal (p45; SEQ ID NOs:31-33)
C protein YF17D NV partial signal
YF 17D partial signal Hypr prM protein
R K R R S H D V L T V Q F L I L G M L A C V G A A T V R
401 A GGAAACGCCG TTCCCATGAT GTTCTGACTG TGCAATTCCT AATTTTGGGC ATGCTGGCTT GTGTCGGAGC AGCTACCGTG CGA
T CCTTTGCGGC AAGGGTACTA CAAGACTGAC ACGTTAAGGA TTAAAACCCG TACGACCGAA CACAGCCTCG TCGATGGCAC GCT
RV-WNV/TBEV Hypr with TBEV signal (p39; SEQ ID NOs:34-36)
TBEV signal
WNV C protein Hypr prM protein
Q K R G G T D W M S W L L V I G M L G M T I A A T V R
201 CAAAAGAAA CGGGGGGGAA CAGACTGGAT GAGCTGGCTG CTCGTAATCG GCATGCTGGG CATGACAATC GCAGCTACGG TTCGC
GTTTTCTTT GCCCCCCCTT GTCTGACCTA CTCGACCGAC GAGCATTAGC CGTACGACCC GTACTGTTAG CGTCGATGCC AAGCG
RV-WNVTBEV Hypr with WNV signal (p40; SEQ ID NOs:37-39)
WNV signal
WNV C protein Hypr prM protein
Q K K R G G K T G I A V M I G M L A C V G A A T V R
201 CAAAAGAAA CGCGGGGGAA AGACAGGCAT AGCTGTGATG ATAGGCATGC TGGCTTGTGT CGGAGCAGCT ACCGTGCGA
GTTTTCTTT GCGCCCCCTT TCTGTCCGTA TCGACACTAC TATCCGTACG ACCGAACACA GCCTCGTCGA TGGCACGCT
Sequence Appendix 2
CV-TBEV Hypr with YFV/TBEV chimeric signal (p42; SEQ ID NOs:40-43)
5' UTR
1 AGTAAATCCT GTGTGCTAAT TGAGGTGCAT TGGTCTGCAA ATCGAGTTGC TAGGCAATAA ACACATTTGG ATTAATTTTA ATCGTTCGTT GAGCGATTAG TCATTTAGGA CACACGATTA ACTCCACGTA ACCAGACGTT TAGCTCAACG ATCCGTTATT TGTGTAAACC TAATTAAAAT TAGCAAGCAA CTCGCTAATC
5' OTR
C protein
M S G R K A Q G K T L G V N M V R R G V R S L S N K I K
101 CAGAGAACTG ACCAGAACAT GTCTGGTCGT AAAGCTCAGG GAAAAACCCT GGGCGTCAAT ATGGTACGAC GAGGAGTTCG CTCCTTGTCA AACAAAATAA GTCTCTTGAC TGGTCTTGTA CAGACCAGCA TTTCGAGTCC CTTTTTGGGA CCCGCAGTTA TACCATGCTG CTCCTCAAGC GAGGAACAGT TTGTTTTATT
C protein
■ Q K T K Q I G N R P G P S R G V Q G F I F F F L F N I L T G K K I 201 AACAAAAAAC AAAACAAATT GGAAACAGAC CTGGACCTTC AAGAGGTGTT CAAGGATTTA TCTTTTTCTT TTTGTTCAAC ATTTTGACTG GAAAAAAGAT TTGTTTTTTG TTTTGTTTAA CCTTTGTCTG GACCTGGAAG TTCTCCACAA GTTCCTAAAT AGAAAAAGAA AAACAAGTTG TAAAACTGAC CTTTTTTCTA
C protein
• T A H L K R L W K M L D P R Q G L A V L R K V K R V V A S L M R G 301 CACAGCCCAC CTAAAGAGGT TGTGGAAAAT GCTGGACCCA AGACAAGGCT TGGCTGTTCT AAGGAAAGTC AAGAGAGTGG TGGCCAGTTT GATGAGAGGA GTGTCGGGTG GATTTCTCCA ACACCTTTTA CGACCTGGGT TCTGTTCCGA ACCGACAAGA TTCCTTTCAG TTCTCTCACC ACCGGTCAAA CTACTCTCCT
YF17D partial signal
TBEV partial signal
C protein prM protein
L S S R K R R S H D V L T V Q F L I L G M L G T I A A T V R K E R
401 TTGTCCTCAA GGAAACGCCG TTCCCATGAT GTTCTGACTG TGCAATTCCT AATTTTGGGC ATGCTGGGCA TGACAATCGC AGCTACGGTT CGCAAGGAAA
AACAGGAGTT CCTTTGCGGC AAGGGTACTA CAAGACTGAC ACGTTAAGGA TTAAAACCCG TACGACCCGT ACTGTTAGCG TCGATGCCAA GCGTTCCTTT
prM protein
■ D G S T V I R A E G D A A T Q V R V E N G T C V I L A T D G S · 501 GAGACGGCAG TACGGTCATA CGCGCGGAAG GTAAGGATGC CGCTACCCAA GTGAGAGTGG AAAATGGTAC CTGCGTCATT CTGGCCACCG ACATGGGCTC CTCTGCCGTC ATGCCAGTAT GCGCGCCTTC CATTCCTACG GCGATGGGTT CACTCTCACC TTTTACCATG GACGCAGTAA GACCGGTGGC TGTACCCGAG
prM protein
■ W C D D S L S Y E C V T I D Q G E E P V D V D C F C R N V D G V Y 601 TTGGTGTGAT GATAGCCTTT CTTATGAGTG CGTAACCA GATCAAGGTG AGGAACCTGT TGACGTTGAT TGCTTCTGCC GAAACGTGGA TGGGGTGTAT AACCACACTA CTATCGGAAA GAATACTCAC GCATTGGTAT CTAGTTCCAC TCCTTGGACA ACTGCAACTA ACGAAGACGG CTTTGCACCT ACCCCACATA
prM protein
L E Y G R C G K Q E G S R T R R S V L I P S H A Q G E L T G R G H K
701 CTCGAATATG GACGGTGTGG TAAACAAGAA GGAAGCAGAA CCAGACGCTC AGTGCTTATA CCCTCCCACG CTCAAGGAGA GCTGACCGGA CGGGGACATA GAGCTTATAC CTGCCACACC ATTTGTTCTT CCTTCGTCTT GGTCTGCGAG TCACGAATAT GGGAGGGTGC GAGTTCCTCT CGACTGGCCT GCCCCTGTAT
prM protein
• W L E G D S L R T H L T R V E G W V K K R L L A L A M V T V V 801 AATGGTTGGA GGGCGACTCA CTCCGAACAC ATTTGACCCG CGTCGAGGGC TGGGTCTGGA AAAATCGGCT GTTGGCCCTC GCTATGGTGA CAGTCGTTTG TTACCAACCT CCCGCTGAGT GAGGCTTGTG TAAACTGGGC GCAGCTCCCG ACCCAGACCT TTTTAGCCGA CAACCGGGAG CGATACCACT GTCAGCAAAC
Hypr E protein prM protein
• L T L E S V V T R V A V L V V L L C L A P V Y A S R C T H L E N R 901 GCTCACGCTG GAGTCTGTGG TTACTCGCGT GGCAGTGCTG GTGGTGCTCC TCTGTCTTGC CCCTGTCTAC GCGTCCAGGT GTACTCATTT GGAAAACAGA CGAGTGCGAC CTCAGACACC AATGAGCGCA CCGTCACGAC CACCACGAGG AGACAGAACG GGGACAGATG CGCAGGTCCA CATGAGTAAA CCTTTTGTCT
Hypr E protein
D F V T G T Q G T T R V T L V L E L G G C V T I T A E G K P S M D V
1001 GATTTTGTCA CCGGCACCCA GGGGACGACT CGGGTAACCC TGGTGCTTGA ACTGGGTGGT TGCGTTACTA TTACCGCTGA GGGCAAACCC TCTATGGATG CTAAAACAGT GGCCGTGGGT CCCCTGCTGA GCCCATTGGG ACCACGAACT TGACCCACCA ACGCAATGAT AATGGCGACT CCCGTTTGGG AGATACCTAC
Hypr E protein
• W L D A I Y Q E N P A Q T R E Y C L H A K L S D T K V A A R C P T 1101 TGTGGCTGGA TGCAATCTAT CAGGAGAATC CCGCACAAAC CAGGGAATAT TGCCTTCACG CAAAGCTGTC CGATACAAAG GTCGCGGCTA GGTGCCCAAC ACACCGACCT ACGTTAGATA GTCCTCTTAG GGCGTGTTTG GTCCCTTATA ACGGAAGTGC GTTTCGACAG GCTATGTTTC CAGCGCCGAT CCACGGGTTG
Hypr E protein
• M G P A T L A E E H Q G G T V C K R D Q S D R G W G N H C G L F G 1201 AATGGGACCG GCCACCCTGG CGGAGGAACA TCAGGGAGGT ACAGTGTGCA AACGGGACCA GAGTGATAGA GGCTGGGGTA ATCACTGCGG CCTGTTCGGC TTACCCTGGC CGGTGGGACC GCCTCCTTGT AGTCCCTCCA TGTCACACGT TTGCCCTGGT CTCACTATCT CCGACCCCAT TAGTGACGCC GGACAAGCCG
Hypr E protein
K G S I V A C V K A A C E A K K K A T G H V Y D A N K I V Y T V K V
1301 AAAGGAAGTA TTGTCGCTTG CGTCAAGGCA GCCTGTGAGG CCAAAAAGAA GGCTACTGGG CACGTCTATG ACGCCAACAA GATCGTTTAT ACAGTGAAAG TTTCCTTCAT AACAGCGAAC GCAGTTCCGT CGGACACTCC GGTTTTTCTT CCGATGACCC GTGCAGATAC TGCGGTTGTT CTAGCAAATA TGTCACTTTC
Hypr E protein
• E P H T G D Y V A A N E T H S G R K T A S F T V S S E K T I L T M 1401 TGGAACCACA CACAGGGGAT TACGTGGCGG CCAACGAGAC TCATTCCGGT CGCAAAACGG CCAGCTTCAC CGTGTCATCC GAAAAGACCA TCCTCACTAT ACCTTGGTGT GTGTCCCCTA ATGCACCGCC GGTTGCTCTG AGTAAGGCCA GCGTTTTGCC GGTCGAAGTG GCACAGTAGG CTTTTCTGGT AGGAGTGATA
Hypr E protein
• G E Y G D V S L L C R V A S G D L A Q T V I L E L D K T V E H L 1501 GGGGGAGTAT GGCGACGTTT CTCTGCTCTG CCGGGTGGCT AGCGGAGTCG ACCTGGCCCA GACAGTCATC CTGGAACTGG ATAAAACAGT TGAGCATCTG CCCCCTCATA CCGCTGCAAA GAGACGAGAC GGCCCACCGA TCGCCTCAGC TGGACCGGGT CTGTCAGTAG GACCTTGACC TATTTTGTCA ACTCGTAGAC
Hypr E protein
P T A W Q V H R D W F N D L A L P W K H E G A R N W N N A E R L V E
1601 CCTACCGCTT GGCAGGTGCA CAGGGATTGG TTTAACGACC TTGCCCTGCC ATGGAAACAT GAAGGAGCGA GAAACTGGAA TAATGCAGAG CGACTCGTAG GGATGGCGAA CCGTCCACGT GTCCCTAACC AAATTGCTGG AACGGGACGG TACCTTTGTA CTTCCTCGCT CTTTGACCTT ATTACGTCTC GCTGAGCATC
Hypr E protein
• F G A P H A V K M D V Y N L G D Q T G V L L K A L A G V P V A H I 1701 AATTCGGTGC CCCTCATGCC GTGAAGATGG ACGTCTACAA TCTGGGTGAT CAGACCGGCG TTCTCCTTAA AGCTCTCGCT GGCGTACCAG TTGCCCACAT TTAAGCCACG GGGAGTACGG CACTTCTACC TGCAGATGTT AGACCCACTA GTCTGGCCGC AAGAGGAATT TCGAGAGCGA CCGCATGGTC AACGGGTGTA
Hypr E protein
• E G T K Y H L S G H V T C E V G L E K L K M K G L T Y T M C D K 1801 CGAAGGAACG AAGTACCACC TGAAGTCAGG CCATGTAACT TGCGAGGTGG GCCTGGAGAA GTTGAAAATG AAAGGTCTTA CGTACACAAT GTGTGACAAG GCTTCCTTGC TTCATGGTGG ACTTCAGTCC GGTACATTGA ACGCTCCACC CGGACCTCTT CAACTTTTAC TTTCCAGAAT GCATGTGTTA CACACTGTTC
Hypr E protein
T K F T W K R A P T D S G H D T V V M E V T F S G T K P C R I P V R
1901 ACCAAGTTCA CATGGAAGAG GGCCCCCACA GATAGCGGCC ACGATACTGT GGTGATGGAG GTGACCTTTT CTGGAACAAA ACCCTGCAGA ATACCCGTGC TGGTTCAAGT GTACCTTCTC CCGGGGGTGT CTATCGCCGG TGCTATGACA CCACTACCTC CACTGGAAAA GACCTTGTTT TGGGACGTCT TATGGGCACG
Hypr E protein
• A V A H G S P D V N V A M L I T P N P T I E N N G G G F I E M Q L 2001 GGGCTGTAGC TCACGGATCT CCCGATGTCA ATGTTGCTAT GCTGATTACA CCTAACCCTA CCATCGAGAA TAACGGTGGT GGTTTTATTG AGATGCAGCT CCCGACATCG AGTGCCTAGA GGGCTACAGT TACAACGATA CGACTAATGT GGATTGGGAT GGTAGCTCTT ATTGCCACCA CCAAAATAAC TCTACGTCGA
Hypr E protein
• P P G D N I I Y V G E L S Y Q W F Q K G S S I G R V F Q K T K K G 2101 TCCGCCAGGC GATAACATCA TCTACGTGGG CGAACTCTCT TACCAGTGGT TTCAGAAAGG GAGTTCAATT GGGCGGGTCT TCCAAAAAAC GAAGAAGGGA AGGCGGTCCG CTATTGTAGT AGATGCACCC GCTTGAGAGA ATGGTCACCA AAGTCTTTCC CTCAAGTTAA CCCGCCCAGA AGGTTTTTTG CTTCTTCCCT
Hypr E protein
I E R L T V I G E H A W D F G S A G G F L S S I G K A L H T V L G G
2201 ATCGAACGAT TGACGGTTAT CGGCGAGCAC GCATGGGATT TTGGTTCCGC AGGGGGATTC CTGTCTTCTA TTGGTAAGGC ACTGCATACC GTGCTGGGGG TAGCTTGCTA ACTGCCAATA GCCGCTCGTG CGTACCCTAA AACCAAGGCG TCCCCCTAAG GACAGAAGAT AACCATTCCG TGACGTATGG CACGACCCCC
Hypr E protein
• A F N S I F G G V G F L P K L L L G V A L A W L G L N M R N P T M 2301 GCGCATTCAA TTCTATTTTC GGGGGCGTGG GGTTCCTGCC TAAACTCCTG CTGGGAGTAG CCCTGGCCTG GTTGGGACTG AATATGCGGA ATCCGACGAT CGCGTAAGTT AAGATAAAAG CCCCCGCACC CCAAGGACGG ATTTGAGGAC GACCCTCATC GGGACCGGAC CAACCCTGAC TTATACGCCT TAGGCTGCTA
Hypr E protein
NS1 gene of YF17D
• S M S F L L A G V L V L A M T L G V G A D Q G C A I N F G K R E L 2401 GTCCATGTCA TTCCTCTTGG CCGGCGTGCT TGTACTGGCC ATGACACTGG GCGTTGGCGC CGATCAAGGA TGCGCCATCA ACTTTGGCAA GAGAGAGCTC CAGGTACAGT AAGGAGAACC GGCCGCACGA ACATGACCGG TACTGTGACC CGCAACCGCG GCTAGTTCCT ACGCGGTAGT TGAAACCGTT CTCTCTCGAG
CV-TBEV Hypr with YFWWNV chimeric signal (p45; SEQ ID NOs:43-45)
5' OTR
1 AGTAAATCCT GTGTGCTAAT TGAGGTGCAT TGGTCTGCAA ATCGAGTTGC TAGGCAATAA ACACATTTGG ATTAATTTTA ATCGTTCGTT GAGCGATTAG TCATTTAGGA CACACGATTA ACTCCACGTA ACCAGACGTT TAGCTCAACG ATCCGTTATT TGTGTAAACC TAATTAAAAT TAGCAAGCAA CTCGCTAATC
5' UTR
C protein YF17D
M S G R K A Q G K T L G V N M V R R G V R S L S N K I K
101 CAGAGAACTG ACCAGAACAT GTCTGGTCGT AAAGCTCAGG GAAAAACCCT GGGCGTCAAT ATGGTACGAC GAGGAGTTCG CTCCTTGTCA AACAAAATAA GTCTCTTGAC TGGTCTTGTA CAGACCAGCA TTTCGAGTCC CTTTTTGGGA CCCGCAGTTA TACCATGCTG CTCCTCAAGC GAGGAACAGT TTGTTTTATT
C protein YF17D
Q K T K Q I G N R P G P S R G V Q G F I F F F L F N I L T G K K I
201 AACAAAAAAC AAAACAAATT GGAAACAGAC CTGGACCTTC AAGAGGTGTT CAAGGATTTA TCTTTTTCTT TTTGTTCAAC ATTTTGACTG GAAAAAAGAT TTGTTTTTTG TTTTGTTTAA CCTTTGTCTG GACCTGGAAG TTCTCCACAA GTTCCTAAAT AGAAAAAGAA AAACAAGTTG TAAAACTGAC CTTTTTTCTA
C protein YF17D
• T A H L R L M L D P R Q G L A V L R K V K R V V A S L M R G 301 CACAGCCCAC CTAAAGAGGT TGTGGAAAAT GCTGGACCCA AGACAAGGCT TGGCTGTTCT AAGGAAAGTC AAGAGAGTGG TGGCCAGTTT GATGAGAGGA GTGTCGGGTG GATTTCTCCA ACACCTTTTA CGACCTGGGT TCTGTTCCGA ACCGACAAGA TTCCTTTCAG TTCTCTCACC ACCGGTCAAA CTACTCTCCT
C protein YF17D WNV partial signal
YF 17D partial signal Hypr pr protein
L S S R K R R S H D V L T V Q F L I L G L A C V G A A T V R K E R
401 TTGTCCTCAA GGAAACGCCG TTCCCATGAT GTTCTGACTG TGCAATTCCT AATTTTGGGC ATGCTGGCTT GTGTCGGAGC AGCTACCGTG CGAAAAGAAC AACAGGAGTT CCTTTGCGGC AAGGGTACTA CAAGACTGAC ACGTTAAGGA TTAAAACCCG TACGACCGAA CACAGCCTCG TCGATGGCAC GCTTTTCTTG
Hypr prM protein
• D G S T V I R A E G K D A A T Q V R V E N G T C V I L A T D G S
501 GCGACGGAAG CACCGTGATA AGGGCTGAGG GTAAGGATGC GGCTACGCAG GTGAGAGTAG AGAATGGCAC TTGCGTAATA CTCGCGACTG ATATGGGATC
CGCTGCCTTC GTGGCACTAT TCCCGACTCC CATTCCTACG CCGATGCGTC CACTCTCATC TCTTACCGTG AACGCATTAT GAGCGCTGAC TATACCCTAG
Hypr prM protein
■ C D D S L S Y E C V T I D Q G E E P V D V D C F C R N V D G V Y 601 CTGGTGTGAC GATAGCCTCA GTTATGAATG CGTAACAATA GACCAGGGCG AAGAACCTGT GGACGTTGAC TGTTTCTGTA GAAATGTGGA TGGCGTTTAT GACCACACTG CTATCGGAGT CAATACTTAC GCATTGTTAT CTGGTCCCGC TTCTTGGACA CCTGCAACTG ACAAAGACAT CTTTACACCT ACCGCAAATA
Hypr prM protein
L E Y G R C G K Q E G S R T R R S V L I P S H A Q G E L T G R G H
701 CTGGAGTACG GCCGCTGTGG AAAACAGGAG GGCTCACGAA CTCGAAGATC TGTGCTGATT CCAAGTCACG CGCAAGGAGA GTTGACCGGT AGAGGCCACA GACCTCATGC CGGCGACACC TTTTGTCCTC CCGAGTGCTT GAGCTTCTAG ACACGACTAA GGTTCAGTGC GCGTTCCTCT CAACTGGCCA TCTCCGGTGT
Hypr prM protein
• W L E G D S L R T H L T R V E G V W K N R L L A L A M V T V V W 801 AGTGGCTTGA AGGGGACTCA TTGAGGACCC ACCTGACTAG GGTGGAGGGT TGGGTTTGGA AGAATCGGTT GCTCGCGCTC GCTATGGTCA CCGTCGTGTG TCACCGAACT TCCCCTGAGT AACTCCTGGG TGGACTGATC CCACCTCCCA ACCCAAACCT TCTTAGCCAA CGAGCGCGAG CGATACCAGT GGCAGCACAC
Hypr prM protein
Hypr E protein
• L T L E S V V T R V A V L V V L L C L A P V Y A S R C T H L E N R 901 GCTGACACTG GAGAGTGTCG TGACTCGGGT TGCTGTGTTG GTTGTCCTCC TCTGTTTGGC CCCAGTGTAC GCGTCCAGGT GTACTCATTT GGAAAACAGA CGACTGTGAC CTCTCACAGC ACTGAGCCCA ACGACACAAC CAACAGGAGG AGACAAACCG GGGTCACATG CGCAGGTCCA CATGAGTAAA CCTTTTGTCT
Hypr E protein
D F V T G T Q G T T R V T L V L E L G G C V T I T A E G K P S M D V
1001 GATTTTGTCA CCGGCACCCA GGGGACGACT CGGGTAACCC TGGTGCTTGA ACTGGGTGGT TGCGTTACTA TTACCGCTGA GGGCAAACCC TCTATGGATG CTAAAACAGT GGCCGTGGGT CCCCTGCTGA GCCCATTGGG ACCACGAACT TGACCCACCA ACGCAATGAT AATGGCGACT CCCGTTTGGG AGATACCTAC
Hypr E protein
- W L D A I Y Q E N P A Q T R E Y C L H A K L S D T K V A A R C P T 1101 TGTGGCTGGA TGCAATCTAT CAGGAGAATC CCGCACAAAC CAGGGAATAT TGCCTTCACG CAAAGCTGTC CGATACAAAG GTCGCGGCTA GGTGCCCAAC ACACCGACCT ACGTTAGATA GTCCTCTTAG GGCGTGTTTG GTCCCTTATA ACGGAAGTGC GTTTCGACAG GCTATGTTTC CAGCGCCGAT CCACGGGTTG
Hypr E protein
• M G P A T L A E E H Q G G T V C K R D Q S D R G W G N H C G L F G
1201 AATGGGACCG GCCACCCTGG CGGAGGAACA TCAGGGAGGT ACAGTGTGCA AACGGGACCA GAGTGATAGA GGCTGGGGTA ATCACTGCGG CCTGTTCGGC TTACCCTGGC CGGTGGGACC GCCTCCTTGT AGTCCCTCCA TGTCACACGT TTGCCCTGGT CTCACTATCT CCGACCCCAT TAGTGACGCC GGACAAGCCG
Hypr E protein
K G S I V A C V K A A C E A K K K A T G H V Y D A N K I V Y T V K V
1301 AAAGGAAGTA TTGTCGCTTG CGTCAAGGCA GCCTGTGAGG CCAAAAAGAA GGCTACTGGG CACGTCTATG ACGCCAACAA GATCGTTTAT ACAGTGAAAG TTTCCTTCAT AACAGCGAAC GCAGTTCCGT CGGACACTCC GGTTTTTCTT CCGATGACCC GTGCAGATAC TGCGGTTGTT CTAGCAAATA TGTCACTTTC
Hypr E protein
■ E P H T G D Y V A A N E T H S G R K T A S F T V S S E K T I L T M 1401 TGGAACCACA CACAGGGGAT TACGTGGCGG CCAACGAGAC TCATTCCGGT CGCAAAACGG CCAGCTTCAC CGTGTCATCC GAAAAGACCA TCCTCACTAT ACCTTGGTGT GTGTCCCCTA ATGCACCGCC GGTTGCTCTG AGTAAGGCCA GCGTTTTGCC GGTCGAAGTG GCACAGTAGG CTTTTCTGGT AGGAGTGATA
Hypr E protein
• G E Y G D V S L L C R V A S G V D L A Q T V I L E L D K T V E H L 1501 GGGGGAGTAT GGCGACGTTT CTCTGCTCTG CCGGGTGGCT AGCGGAGTCG ACCTGGCCCA GACAGTCATC CTGGAACTGG ATAAAACAGT TGAGCATCTG CCCCCTCATA CCGCTGCAAA GAGACGAGAC GGCCCACCGA TCGCCTCAGC TGGACCGGGT CTGTCAGTAG GACCTTGACC TATTTTGTCA ACTCGTAGAC
Hypr E protein
P T A W Q V H R D W F H D L A L P W K H E G A R N W N N A E R L V E
1601 CCTACCGCTT GGCAGGTGCA CAGGGATTGG TTTAACGACC TTGCCCTGCC ATGGAAACAT GAAGGAGCGA GAAACTGGAA TAATGCAGAG CGACTCGTAG GGATGGCGAA CCGTCCACGT GTCCCTAACC AAATTGCTGG AACGGGACGG TACCTTTGTA CTTCCTCGCT CTTTGACCTT ATTACGTCTC GCTGAGCATC
Hypr E protein
• F G A P H A V K M D V Y N L G D Q T G V L L K A L A G V P V A H I 1701 AATTCGGTGC CCCTCATGCC GTGAAGATGG ACGTCTACAA TCTGGGTGAT CAGACCGGCG TTCTCCTTAA AGCTCTCGCT GGCGTACCAG TTGCCCACAT TTAAGCCACG GGGAGTACGG CACTTCTACC TGCAGATGTT AGACCCACTA GTCTGGCCGC AAGAGGAATT TCGAGAGCGA CCGCATGGTC AACGGGTGTA
Hypr E protein
• E G T K Y H L S G H V T C E V G L E K L K M K G L T Y T M C D K 1801 CGAAGGAACG AAGTACCACC TGAAGTCAGG CCATGTAACT TGCGAGGTGG GCCTGGAGAA GTTGAAAATG AAAGGTCTTA CGTACACAAT GTGTGACAAG GCTTCCTTGC TTCATGGTGG ACTTCAGTCC GGTACATTGA ACGCTCCACC CGGACCTCTT CAACTTTTAC TTTCCAGAAT GCATGTGTTA CACACTGTTC
Hypr E protein
T K F T W K R A P T D S G H D T V V M E V T F S G T K P C R I P V R
1901 ACCAAGTTCA CATGGAAGAG GGCCCCCACA GATAGCGGCC ACGATACTGT GGTGATGGAG GTGACCTTTT CTGGAACAAA ACCCTGCAGA ATACCCGTGC TGGTTCAAGT GTACCTTCTC CCGGGGGTGT CTATCGCCGG TGCTATGACA CCACTACCTC CACTGGAAAA GACCTTGTTT TGGGACGTCT TATGGGCACG
Hypr E protein
• A V A H G S P D V K V A L I T P N P T I E N N G G G F I E M Q L 2001 GGGCTGTAGC TCACGGATCT CCCGATGTCA ATGTTGCTAT GCTGATTACA CCTAACCCTA CCATCGAGAA TAACGGTGGT GGTTTTATTG AGATGCAGCT CCCGACATCG AGTGCCTAGA GGGCTACAGT TACAACGATA CGACTAATGT GGATTGGGAT GGTAGCTCTT ATTGCCACCA CCAAAATAAC TCTACGTCGA
Hypr E protein
• P P G D N I I Y V G E L S Y Q W F Q K G S S I G R V F Q K T K K G 2101 TCCGCCAGGC GATAACATCA TCTACGTGGG CGAACTCTCT TACCAGTGGT TTCAGAAAGG GAGTTCAATT GGGCGGGTCT TCCAAAAAAC GAAGAAGGGA AGGCGGTCCG CTATTGTAGT AGATGCACCC GCTTGAGAGA ATGGTCACCA AAGTCTTTCC CTCAAGTTAA CCCGCCCAGA AGGTTTTTTG CTTCTTCCCT
Hypr E protein
I E R L T V I G E H A W D F G S A G G F L S S I G K A L H T V L G G
2201 ATCGAACGAT TGACGGTTAT CGGCGAGCAC GCATGGGATT TTGGTTCCGC AGGGGGATTC CTGTCTTCTA TTGGTAAGGC ACTGCATACC GTGCTGGGGG TAGCTTGCTA ACTGCCAATA GCCGCTCGTG CGTACCCTAA AACCAAGGCG TCCCCCTAAG GACAGAAGAT AACCATTCCG TGACGTATGG CACGACCCCC
Hypr E protein
• A F N S I F G G V G F L P K L L L G V A L A W L G L N M R N P T M 2301 GCGCATTCAA TTCTATTTTC GGGGGCGTGG GGTTCCTGCC TAAACTCCTG CTGGGAGTAG CCCTGGCCTG GTTGGGACTG AATATGCGGA ATCCGACGAT CGCGTAAGTT AAGATAAAAG CCCCCGCACC CCAAGGACGG ATTTGAGGAC GACCCTCATC GGGACCGGAC CAACCCTGAC TTATACGCCT TAGGCTGCTA
Hypr E protein
NS1 gene of YF17D
• S M S F L L A G V L V L A M T L G V G A D Q G C A I N F G K R E L 2401 GTCCATGTCA TTCCTCTTGG CCGGCGTGCT TGTACTGGCC ATGACACTGG GCGTTGGCGC CGATCAAGGA TGCGCCATCA ACTTTGGCAA GAGAGAGCTC CAGGTACAGT AAGGAGAACC GGCCGCACGA ACATGACCGG TACTGTGACC CGCAACCGCG GCTAGTTCCT ACGCGGTAGT TGAAACCGTT CTCTCTCGAG
CV-LGTV E5 with YFV/TBEV chimeric signal (p43; SEQ ID NOs:46-48)
5' OTR
1 AGTAAATCCT GTGTGCTAAT TGAGGTGCAT TGGTCTGCAA ATCGAGTTGC TAGGCAATAA ACACATTTGG ATTAATTTTA ATCGTTCGTT GAGCGATTAG TCATTTAGGA CACACGATTA ACTCCACGTA ACCAGACGTT TAGCTCAACG ATCCGTTATT TGTGTAAACC TAATTAAAAT TAGCAAGCAA CTCGCTAATC
5' UTR
C protein YF17D
M S G R K A Q G K T L G V K M V R R G V R S L S N I K
101 CAGAGAACTG ACCAGAACAT GTCTGGTCGT AAAGCTCAGG GAAAAACCCT GGGCGTCAAT ATGGTACGAC GAGGAGTTCG CTCCTTGTCA AACAAAATAA GTCTCTTGAC TGGTCTTGTA CAGACCAGCA TTTCGAGTCC CTTTTTGGGA CCCGCAGTTA TACCATGCTG CTCCTCAAGC GAGGAACAGT TTGTTTTATT
C protein YF17D
■ Q K T K Q I G N R P G P S R G V Q G F I F F F L F K I L T G K K I AACAAAAAAC AAAACAAATT GGAAACAGAC CTGGACCTTC AAGAGGTGTT CAAGGATTTA TCTTTTTCTT TTTGTTCAAC ATTTTGACTG GAAAAAAGAT TTGTTTTTTG TTTTGTTTAA CCTTTGTCTG GACCTGGAAG TTCTCCACAA GTTCCTAAAT AGAAAAAGAA AAACAAGTTG TAAAACTGAC CTTTTTTCTA
C protein YF17D
• T A H L K R L W K M L D P R Q G L A V L R K V K R V V A S L M R G 301 CACAGCCCAC CTAAAGAGGT TGTGGAAAAT GCTGGACCCA AGACAAGGCT TGGCTGTTCT AAGGAAAGTC AAGAGAGTGG TGGCCAGTTT GATGAGAGGA GTGTCGGGTG GATTTCTCCA ACACCTTTTA CGACCTGGGT TCTGTTCCGA ACCGACAAGA TTCCTTTCAG TTCTCTCACC ACCGGTCAAA CTACTCTCCT
C protein YF17D TBEV partial signal
YF 17D partial signal prM protein Langat E5
L S S R K R R S H D V L T V Q F L I L G M L G M T I A A T V R R E R
401 TTGTCCTCAA GGAAACGCCG TTCCCATGAT GTTCTGACTG TGCAATTCCT AATTTTGGGC ATGCTGGGGA TGACGATCGC AGCTACTGTG CGAAGGGAGA AACAGGAGTT CCTTTGCGGC AAGGGTACTA CAAGACTGAC ACGTTAAGGA TTAAAACCCG TACGACCCCT ACTGCTAGCG TCGATGACAC GCTTCCCTCT
prM protein Langat E5
• D G S M V I R A E G R D A A T Q V R V E N G T C V I L A T D M G S 501 GAGACGGCTC TATGGTGATC AGAGCCGAAG GTAGGGACGC TGCGACCCAG GTGAGGGTCG AAAATGGCAC CTGTGTTATT CTGGCGACCG ACATGGGCTC CTCTGCCGAG ATACCACTAG TCTCGGCTTC CATCCCTGCG ACGCTGGGTC CACTCCCAGC TTTTACCGTG GACACAATAA GACCGCTGGC TGTACCCGAG
prM protein Langat E5
• W C D D S L A Y E C V T I D Q G E E P V D V D C F C R G V E K V T 601 CTGGTGTGAT GATTCTCTGG CTTATGAATG TGTTACTATT GATCAGGGTG AAGAGCCTGT GGACGTGGAC TGTTTCTGTA GAGGCGTCGA GAAAGTGACC GACCACACTA CTAAGAGACC GAATACTTAC ACAATGATAA CTAGTCCCAC TTCTCGGACA CCTGCACCTG ACAAAGACAT CTCCGCAGCT CTTTCACTGG
pr protein Langat E5
L E Y G R C G R R E G S R S R R S V L I P S H A Q R D L T G R G H Q
701 CTGGAATATG GACGATGTGG CCGGCGAGAA GGCTCCAGGA GTCGGAGATC CGTGTTGATC CCTTCACATG CGCAGCGCGA TCTGACAGGG AGGGGTCACC GACCTTATAC CTGCTACACC GGCCGCTCTT CCGAGGTCCT CAGCCTCTAG GCACAACTAG GGAAGTGTAC GCGTCGCGCT AGACTGTCCC TCCCCAGTGG
prM protein Langat E5
• L E G E A V A H L T R V E G W V W K H K L F T L S L V M V A W 801 AGTGGCTCGA AGGCGAAGCA GTCAAGGCCC ATCTGACTCG CGTTGAAGGC TGGGTGTGGA AAAACAAACT CTTTACCCTT AGCCTGGTGA TGGTCGCGTG TCACCGAGCT TCCGCTTCGT CAGTTCCGGG TAGACTGAGC GCAACTTCCG ACCCACACCT TTTTGTTTGA GAAATGGGAA TCGGACCACT ACCAGCGCAC
prM protein Langat E5
E protein Langat E5
• L M V D G L L P R I L I V V V A L A L A P A Y A S R C T H L E N R 901 GCTGATGGTA GACGGACTCC TTCCCCGCAT TCTCATTGTT GTGGTGGCTC TCGCGCTCGC CCCTGCATAC GCGTCCAGGT GTACGCACCT CGAAAATCGA CGACTACCAT CTGCCTGAGG AAGGGGCGTA AGAGTAACAA CACCACCGAG AGCGCGAGCG GGGACGTATG CGCAGGTCCA CATGCGTGGA GCTTTTAGCT
E protein Langat E5
D F V T G V Q G T T R L T L V L E L G G C V T V T A D G K P S L D V
1001 GATTTCGTCA CAGGCGTCCA AGGTACTACC CGGCTCACCC TCGTGCTGGA GCTGGGAGGC TGTGTCACTG TTACAGCCGA CGGAAAACCT AGTCTGGATG CTAAAGCAGT GTCCGCAGGT TCCATGATGG GCCGAGTGGG AGCACGACCT CGACCCTCCG ACACAGTGAC AATGTCGGCT GCCTTTTGGA TCAGACCTAC
E protein Langat E5
■ W L D S I Y Q E S P A Q T R E Y C L H A K L T G T K V A A R C P T 1101 TGTGGCTGGA CTCCATCTAT CAGGAGAGCC CGGCACAGAC CAGGGAGTAC TGCCTCCACG CTAAGCTGAC TGGGACAAAG GTAGCCGCAA GATGTCCCAC ACACCGACCT GAGGTAGATA GTCCTCTCGG GCCGTGTCTG GTCCCTCATG ACGGAGGTGC GATTCGACTG ACCCTGTTTC CATCGGCGTT CTACAGGGTG
E protein Langat E5
• M G P A T L P E E H Q S G T V C K R D Q S D R G G N H C G L F G 1201 AATGGGGCCT GCCACCTTGC CCGAGGAACA CCAATCCGGT ACGGTATGCA AGCGAGATCA GTCTGATCGC GGATGGGGGA ATCATTGCGG CCTCTTCGGT TTACCCCGGA CGGTGGAACG GGCTCCTTGT GGTTAGGCCA TGCCATACGT TCGCTCTAGT CAGACTAGCG CCTACCCCCT TAGTAACGCC GGAGAAGCCA
E protein Langat E5
K G S I V T C V K V T C E D K K K A T G H V Y D V N K I T Y T I K V
1301 AAAGGCAGCA TTGTCACTTG CGTGAAGGTG ACATGCGAGG ACAAGAAGAA GGCCACAGGT CATGTATATG ATGTGAACAA AATCACATAT ACCATTAAGG TTTCCGTCGT AACAGTGAAC GCACTTCCAC TGTACGCTCC TGTTCTTCTT CCGGTGTCCA GTACATATAC TACACTTGTT TTAGTGTATA TGGTAATTCC
E protein Langat E5
• E P H T G E F V A A N E T H S G R K S A S F T V S S E K T I L T L
1401 TAGAACCACA TACAGGGGAA TTCGTGGCAG CAAACGAGAC TCATAGCGGA CGAAAGTCCG CCTCCTTCAC CGTCTCCTCC GAGAAAACAA TCCTGACCCT
ATCTTGGTGT ATGTCCCCTT AAGCACCGTC GTTTGCTCTG AGTATCGCCT GCTTTCAGGC GGAGGAAGTG GCAGAGGAGG CTCTTTTGTT AGGACTGGGA
E protein Langat E5
• G D Y G D V S L L C R V A S G V D L A Q T V V L A L D K T H E H L 1501 CGGAGACTAC GGCGACGTAT CTTTGCTGTG CAGGGTGGCC AGCGGCGTGG ACCTTGCTCA GACAGTCGTG TTGGCCCTGG ACAAGACACA TGAGCACTTG GCCTCTGATG CCGCTGCATA GAAACGACAC GTCCCACCGG TCGCCGCACC TGGAACGAGT CTGTCAGCAC AACCGGGACC TGTTCTGTGT ACTCGTGAAC
E protein Langat E5
P T A W Q V H R D W F N D L A L P K H D G A E W N E A G R L V E
1601 CCAACAGCCT GGCAGGTGCA CAGGGACTGG TTTAACGACC TGGCGCTCCC GTGGAAACAT GACGGCGCTG AAGCATGGAA TGAGGCAGGG AGACTGGTGG
GGTTGTCGGA CCGTCCACGT GTCCCTGACC AAATTGCTGG ACCGCGAGGG CACCTTTGTA CTGCCGCGAC TTCGTACCTT ACTCCGTCCC TCTGACCACC
E protein Langat E5
■ F G T P H A V K M D V F N L G D Q T G V L L K S L A G V P V A S I 1701 AATTTGGAAC CCCACACGCC GTAAAGATGG ACGTTTTCAA TCTTGGTGAC CAGACAGGGG TGCTCCTGAA ATCACTGGCG GGCGTGCCTG TAGCCAGCAT TTAAACCTTG GGGTGTGCGG CATTTCTACC TGCAAAAGTT AGAACCACTG GTCTGTCCCC ACGAGGACTT TAGTGACCGC CCGCACGGAC ATCGGTCGTA
E protein Langat E5
■ E G T K Y H L K S G H V T C E V G L E K L K M K G L T Y T V C D K 1801 CGAGGGCACA AAGTATCACC TGAAGTCTGG GCATGTAACC TGCGAAGTGG GCCTGGAAAA GCTGAAGATG AAAGGACTTA CGTACACTGT TTGTGATAAG GCTCCCGTGT TTCATAGTGG ACTTCAGACC CGTACATTGG ACGCTTCACC CGGACCTTTT CGACTTCTAC TTTCCTGAAT GCATGTGACA AACACTATTC
E protein Langat E5
T K F T R A P T D S G H D T V V M E V G F S G T R P C R I P V R
1901 ACCAAGTTTA CATGGAAGCG AGCCCCAACG GATTCCGGCC ATGATACCGT CGTGATGGAG GTTGGTTTCT CCGGCACCAG ACCATGTAGA ATACCAGTGA TGGTTCAAAT GTACCTTCGC TCGGGGTTGC CTAAGGCCGG TACTATGGCA GCACTACCTC CAACCAAAGA GGCCGTGGTC TGGTACATCT TATGGTCACT
E protein Langat E5
■ A V A H G V P E V N V A M L I T P N P T M E N N G G G F I E M Q L 2001 GAGCTGTCGC CCACGGTGTA CCCGAGGTAA ACGTGGCCAT GCTGATTACA CCGAATCCCA CTATGGAGAA CAATGGCGGA GGGTTCATCG AAATGCAGCT CTCGACAGCG GGTGCCACAT GGGCTCCATT TGCACCGGTA CGACTAATGT GGCTTAGGGT GATACCTCTT GTTACCGCCT CCCAAGTAGC TTTACGTCGA
E protein Langat E5
• P P G D N I I Y V G D L D H Q W F Q K G S S I G R V L Q K T R K G
2101 GCCGCCTGGA GACAACATCA TTTATGTCGG CGACCTCGAT CATCAATGGT TCCAGAAAGG GTCTTCCATC GGCCGCGTCC TTCAGAAGAC ACGAAAAGGC
CGGCGGACCT CTGTTGTAGT AAATACAGCC GCTGGAGCTA GTAGTTACCA AGGTCTTTCC CAGAAGGTAG CCGGCGCAGG AAGTCTTCTG TGCTTTTCCG
E protein Langat E5
I E R L T V L G E H A W D F G S V G G V M T S I G R A M H T V L G G
2201 ATTGAAAGAC TTACAGTCCT GGGCGAACAT GCCTGGGACT TCGGGTCAGT TGGCGGGGTA ATGACAAGCA TAGGCAGAGC TATGCACACC GTTCTCGGTG
TAACTTTCTG AATGTCAGGA CCCGCTTGTA CGGACCCTGA AGCCCAGTCA ACCGCCCCAT TACTGTTCGT ATCCGTCTCG ATACGTGTGG CAAGAGCCAC
E protein Langat E5
• A F N T L L G G V G F L P K I L L G V A M A W L G L N M R N P T L 2301 GGGCATTTAA TACTCTGTTG GGTGGCGTGG GTTTTCTTCC GAAAATCCTG CTCGGTGTCG CAATGGCCTG GCTTGGACTG AATATGCGCA ATCCTACACT CCCGTAAATT ATGAGACAAC CCACCGCACC CAAAAGAAGG CTTTTAGGAC GAGCCACAGC GTTACCGGAC CGAACCTGAC TTATACGCGT TAGGATGTGA
E protein Langat E5
NS1 gene of YF17D
• S M G F L L S G G L V L A M T L G V G A D Q G C A I N F G K R E L 2401 GAGTATGGGG TTTCTTCTGT CAGGAGGCCT GGTCCTGGCA ATGACTCTGG GAGTGGGCGC CGATCAAGGA TGCGCCATCA ACTTTGGCAA GAGAGAGCTC CTCATACCCC AAAGAAGACA GTCCTCCGGA CCAGGACCGT TACTGAGACC CTCACCCGCG GCTAGTTCCT ACGCGGTAGT TGAAACCGTT CTCTCTCGAG
CV-TBEV Hypr with YFV/TBEV chimeric signal and dC2 deletion in C protein (p59; SEQ ID NOs:49-51)
5' DTR
1 AGTAAATCCT GTGTGCTAAT TGAGGTGCAT TGGTCTGCAA ATCGAGTTGC TAGGCAATAA ACACATTTGG ATTAATTTTA ATCGTTCGTT GAGCGATTAG TCATTTAGGA CACACGATTA ACTCCACGTA ACCAGACGTT TAGCTCAACG ATCCGTTATT TGTGTAAACC TAATTAAAAT TAGCAAGCAA CTCGCTAATC
5' UTR
C protein
M S G R A Q G T L G V N M V R R G V R S L S N K I K
101 CAGAGAACTG ACCAGAACAT GTCTGGTCGT AAAGCTCAGG GAAAAACCCT GGGCGTCAAT ATGGTACGAC GAGGAGTTCG CTCCTTGTCA AACAAAATAA GTCTCTTGAC TGGTCTTGTA CAGACCAGCA TTTCGAGTCC CTTTTTGGGA CCCGCAGTTA TACCATGCTG CTCCTCAAGC GAGGAACAGT TTGTTTTATT
dC2 deletion (PSR)
C protein
• Q K T K Q I G N R P G G V Q G F I F F F L F H I L T G K I T A H · 201 AACAAAAAAC AAAACAAATT GGAAACAGAC CTGGAGGTGT TCAAGGATTT ATCTTTTTCT TTTTGTTCAA CATTTTGACT GGAAAAAAGA TCACAGCCCA TTGTTTTTTG TTTTGTTTAA CCTTTGTCTG GACCTCCACA AGTTCCTAAA TAGAAAAAGA AAAACAAGTT GTAAAACTGA CCTTTTTTCT AGTGTCGGGT
C protein
■ L K R L W K M L D P R Q G L A V L R K V K R V V A S L M R G L S S 301 CCTAAAGAGG TTGTGGAAAA TGCTGGACCC AAGACAAGGC TTGGCTGTTC TAAGGAAAGT CAAGAGAGTG GTGGCCAGTT TGATGAGAGG ATTGTCCTCA GGATTTCTCC AACACCTTTT ACGACCTGGG TTCTGTTCCG AACCGACAAG ATTCCTTTCA GTTCTCTCAC CACCGGTCAA ACTACTCTCC TAACAGGAGT YF17D partial signal
TBEV partial signal
C protein Hypr prM protein
R K R R S H D V L T V Q F L I L G M L G M T I A A T V R K E R D G S
401 AGGAAACGCC GTTCCCATGA TGTTCTGACT GTGCAATTCC TAATTTTGGG CATGCTGGGC ATGACAATCG CAGCTACGGT TCGCAAGGAA AGAGACGGCA TCCTTTGCGG CAAGGGTACT ACAAGACTGA CACGTTAAGG ATTAAAACCC GTACGACCCG TACTGTTAGC GTCGATGCCA AGCGTTCCTT TCTCTGCCGT
Hypr prM protein
• T V I R A E G K D A A T Q V R V E N G T C V I L A T D M G S C D 501 GTACGGTCAT ACGCGCGGAA GGTAAGGATG CCGCTACCCA AGTGAGAGTG GAAAATGGTA CCTGCGTCAT TCTGGCCACC GACATGGGCT CTTGGTGTGA CATGCCAGTA TGCGCGCCTT CCATTCCTAC GGCGATGGGT TCACTCTCAC CTTTTACCAT GGACGCAGTA AGACCGGTGG CTGTACCCGA GAACCACACT
Hypr prM protein
• D S L S Y E C V T I D Q G E E P V D V D C F C R N V D G V Y L E Y 601 TGATAGCCTT TCTTATGAGT GCGTAACCAT AGATCAAGGT GAGGAACCTG TTGACGTTGA TTGCTTCTGC CGAAACGTGG ATGGGGTGTA TCTCGAATAT ACTATCGGAA AGAATACTCA CGCATTGGTA TCTAGTTCCA CTCCTTGGAC AACTGCAACT AACGAAGACG GCTTTGCACC TACCCCACAT AGAGCTTATA
Hypr prM protein
G R C G K Q E G S R T R R S V L 1 Ρ Ξ Η A Q G E L T G R G H K L E 701 GGACGGTGTG GTAAACAAGA AGGAAGCAGA ACCAGACGCT CAGTGCTTAT ACCCTCCCAC GCTCAAGGAG AGCTGACCGG ACGGGGACAT AAATGGTTGG CCTGCCACAC CATTTGTTCT TCCTTCGTCT TGGTCTGCGA GTCACGAATA TGGGAGGGTG CGAGTTCCTC TCGACTGGCC TGCCCCTGTA TTTACCAACC
Hypr prM protein
• G D S L R T H L T R V E G W V W K N R L L A L A M V T V V W L T L 801 AGGGCGACTC ACTCCGAACA CATTTGACCC GCGTCGAGGG CTGGGTCTGG AAAAATCGGC TGTTGGCCCT CGCTATGGTG ACAGTCGTTT GGCTCACGCT TCCCGCTGAG TGAGGCTTGT GTAAACTGGG CGCAGCTCCC GACCCAGACC TTTTTAGCCG ACAACCGGGA GCGATACCAC TGTCAGCAAA CCGAGTGCGA
Hypr E protein
Hypr prM protein
■ E S V V T R V A V L V V L L C L A P V Y A S R C T H L E N R D F V 901 GGAGTCTGTG GTTACTCGCG TGGCAGTGCT GGTGGTGCTC CTCTGTCTTG CCCCTGTCTA CGCGTCCAGG TGTACTCATT TGGAAAACAG AGATTTTGTC CCTCAGACAC CAATGAGCGC ACCGTCACGA CCACCACGAG GAGACAGAAC GGGGACAGAT GCGCAGGTCC ACATGAGTAA ACCTTTTGTC TCTAAAACAG
Hypr E protein
T G T Q G T T R V T L V L E L G G C V T I T A E G K P S M D V W L D
1001 ACCGGCACCC AGGGGACGAC TCGGGTAACC CTGGTGCTTG AACTGGGTGG TTGCGTTACT ATTACCGCTG AGGGCAAACC CTCTATGGAT GTGTGGCTGG TGGCCGTGGG TCCCCTGCTG AGCCCATTGG GACCACGAAC TTGACCCACC AACGCAATGA TAATGGCGAC TCCCGTTTGG GAGATACCTA CACACCGACC
Hypr E protein
• A I Y Q E N P A Q T R E Y C L H A K L S D T K V A A R C P T M G P 1101 ATGCAATCTA TCAGGAGAAT CCCGCACAAA CCAGGGAATA TTGCCTTCAC GCAAAGCTGT CCGATACAAA GGTCGCGGCT AGGTGCCCAA CAATGGGACC TACGTTAGAT AGTCCTCTTA GGGCGTGTTT GGTCCCTTAT AACGGAAGTG CGTTTCGACA GGCTATGTTT CCAGCGCCGA TCCACGGGTT GTTACCCTGG
Hypr E protein
■ A T L A E E H Q G G T V C K R D Q S D R G W G N H C G L F G K G S 1201 GGCCACCCTG GCGGAGGAAC ATCAGGGAGG TACAGTGTGC AAACGGGACC AGAGTGATAG AGGCTGGGGT AATCACTGCG GCCTGTTCGG CAAAGGAAGT CCGGTGGGAC CGCCTCCTTG TAGTCCCTCC ATGTCACACG TTTGCCCTGG TCTCACTATC TCCGACCCCA TTAGTGACGC CGGACAAGCC GTTTCCTTCA
Hypr E protein
I V A C V K A A C E A K A T G H V Y D A N K I V Y T V K V E P H
1301 ATTGTCGCTT GCGTCAAGGC AGCCTGTGAG GCCAAAAAGA AGGCTACTGG GCACGTCTAT GACGCCAACA AGATCGTTTA TACAGTGAAA GTGGAACCAC TAACAGCGAA CGCAGTTCCG TCGGACACTC CGGTTTTTCT TCCGATGACC CGTGCAGATA CTGCGGTTGT TCTAGCAAAT ATGTCACTTT CACCTTGGTG
Hypr E protein
• T G D Y V A A N E T H S G R K T A S F T V S S E K T I L T M G E Y 1401 ACACAGGGGA TTACGTGGCG GCCAACGAGA CTCATTCCGG TCGCAAAACG GCCAGCTTCA CCGTGTCATC CGAAAAGACC ATCCTCACTA TGGGGGAGTA TGTGTCCCCT AATGCACCGC CGGTTGCTCT GAGTAAGGCC AGCGTTTTGC CGGTCGAAGT GGCACAGTAG GCTTTTCTGG TAGGAGTGAT ACCCCCTCAT
Hypr E protein
• G D V S L L C R V A S G V D L A Q T V I L E L D K T V E H L P T A
1501 TGGCGACGTT TCTCTGCTCT GCCGGGTGGC TAGCGGAGTC GACCTGGCCC AGACAGTCAT CCTGGAACTG GATAAAACAG TTGAGCATCT GCCTACCGCT
ACCGCTGCAA AGAGACGAGA CGGCCCACCG ATCGCCTCAG CTGGACCGGG TCTGTCAGTA GGACCTTGAC CTATTTTGTC AACTCGTAGA CGGATGGCGA
Hypr E protein
Q V H R D W F N D L A L P W K H E G A R N W N N A E R L V E F G A
1601 TGGCAGGTGC ACAGGGATTG GTTTAACGAC CTTGCCCTGC CATGGAAACA TGAAGGAGCG AGAAACTGGA ATAATGCAGA GCGACTCGTA GAATTCGGTG ACCGTCCACG TGTCCCTAAC CAAATTGCTG GAACGGGACG GTACCTTTGT ACTTCCTCGC TCTTTGACCT TATTACGTCT CGCTGAGCAT CTTAAGCCAC
Hypr E protein
• P H A V K M D V Y N L G D Q T G V L L K A L A G V P V A H I E G T 1701 CCCCTCATGC CGTGAAGATG GACGTCTACA ATCTGGGTGA TCAGACCGGC GTTCTCCTTA AAGCTCTCGC TGGCGTACCA GTTGCCCACA TCGAAGGAAC GGGGAGTACG GCACTTCTAC CTGCAGATGT TAGACCCACT AGTCTGGCCG CAAGAGGAAT TTCGAGAGCG ACCGCATGGT CAACGGGTGT AGCTTCCTTG
Hypr E protein
• K Y H L K S G H V T C E V G L E K L K M K G L T Y T M C D K T K F 1801 GAAGTACCAC CTGAAGTCAG GCCATGTAAC TTGCGAGGTG GGCCTGGAGA AGTTGAAAAT GAAAGGTCTT ACGTACACAA TGTGTGACAA GACCAAGTTC CTTCATGGTG GACTTCAGTC CGGTACATTG AACGCTCCAC CCGGACCTCT TCAACTTTTA CTTTCCAGAA TGCATGTGTT ACACACTGTT CTGGTTCAAG
Hypr E protein
T W K R A P T D S G H D T V V M E V T F S G T K P C R I P V R A V A
1901 ACATGGAAGA GGGCCCCCAC AGATAGCGGC CACGATACTG TGGTGATGGA GGTGACCTTT TCTGGAACAA AACCCTGCAG AATACCCGTG CGGGCTGTAG TGTACCTTCT CCCGGGGGTG TCTATCGCCG GTGCTATGAC ACCACTACCT CCACTGGAAA AGACCTTGTT TTGGGACGTC TTATGGGCAC GCCCGACATC
Hypr E protein
■ H G S P D V N V A M L I T P N P T I E N N G G G F I E M Q L P P G 2001 CTCACGGATC TCCCGATGTC AATGTTGCTA TGCTGATTAC ACCTAACCCT ACCATCGAGA ATAACGGTGG TGGTTTTATT GAGATGCAGC TTCCGCCAGG GAGTGCCTAG AGGGCTACAG TTACAACGAT ACGACTAATG TGGATTGGGA TGGTAGCTCT TATTGCCACC ACCAAAATAA CTCTACGTCG AAGGCGGTCC
Hypr E protein
• D N I I Y V G E L S Y Q W F Q K G S S I G R V F Q K T K K G I E R 2101 CGATAftCATC ATCTACGTGG GCGAACTCTC TTACCAGTGG TTTCAGAAAG GGAGTTCAAT TGGGCGGGTC TTCCAAAAAA CGAAGAAGGG AATCGAACGA GCTATTGTAG TAGATGCACC CGCTTGAGAG AATGGTCACC AAAGTCTTTC CCTCAAGTTA ACCCGCCCAG AAGGTTTTTT GCTTCTTCCC TTAGCTTGCT
Hypr E protein
L T V I G E H A W D F G S A G G F L S S I G K A L H T V L G G A F N
2201 TTGACGGTTA TCGGCGAGCA CGCATGGGAT TTTGGTTCCG CAGGGGGATT CCTGTCTTCT ATTGGTAAGG CACTGCATAC CGTGCTGGGG GGCGCATTCA AACTGCCAAT AGCCGCTCGT GCGTACCCTA AAACCAAGGC GTCCCCCTAA GGACAGAAGA TAACCATTCC GTGACGTATG GCACGACCCC CCGCGTAAGT
Hypr E protein
• S I F G G V G F L P K L L L G V A L A W L G L N M R N P T M S M S
2301 ATTCTATTTT CGGGGGCGTG GGGTTCCTGC CTAAACTCCT GCTGGGAGTA GCCCTGGCCT GGTTGGGACT GAATATGCGG AATCCGACGA TGTCCATGTC
TAAGATAAAA GCCCCCGCAC CCCAAGGACG GATTTGAGGA CGACCCTCAT CGGGACCGGA CCAACCCTGA CTTATACGCC TTAGGCTGCT ACAGGTACAG
Hypr E protein
NS1 gene of YF17D
• F L L A G V L V L A M T L G V G A D Q G C A I N F G K R E L
ATTCCTCTTG GCCGGCGTGC TTGTACTGGC CATGACACTG GGCGTTGGCG CCGATCAAGG ATGCGCCATC AACTTTGGCA AGAGAGAGCT C TAAGGAGAAC CGGCCGCACG AACATGACCG GTACTGTGAC CCGCAACCGC GGCTAGTTCC TACGCGGTAG TTGAAACCGT TCTCTCTCGA G
Sequence Appendix 3
PIV-WNTBEV Hypr with TBEV signal (p39; SEQ ID NOs:52-54)
deleted C UTR
M S
1 AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA NV deleted C protein
• K K P G G P G K S R A V Y L L K R G M P R V L S L I G L K R S S 101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGC GGAGCTCCAA GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCG CCTCGAGGTT
TBEV signal
deleted C prM Hypr
■ Q K R G G T D W M S W L L V 1 G M L G M T I A A T V R K E R D G 201 ACAAAAGAAA CGGGGGGGAA CAGACTGGAT GAGCTGGCTG CTCGTAATCG GCATGCTGGG CATGACAATC GCAGCTACGG TTCGCAAGGA AAGAGACGGC TGTTTTCTTT GCCCCCCCTT GTCTGACCTA CTCGACCGAC GAGCATTAGC CGTACGACCC GTACTGTTAG CGTCGATGCC AAGCGTTCCT TTCTCTGCCG
prM Hypr
S T V I R A E G K D A A T Q V R V E N G T C V I L A T D M G S C D
301 AGTACGGTCA TACGCGCGGA AGGTAAGGAT GCCGCTACCC AAGTGAGAGT GGAAAATGGT ACCTGCGTCA TTCTGGCCAC CGACATGGGC TCTTGGTGTG
TCATGCCAGT ATGCGCGCCT TCCATTCCTA CGGCGATGGG TTCACTCTCA CCTTTTACCA TGGACGCAGT AAGACCGGTG GCTGTACCCG AGAACCACAC prM Hypr
■ D S L S Y E C V T I D Q G E E P V D V D C F C R N V D G V Y L E Y 401 ATGATAGCCT TTCTTATGAG TGCGTAACCA TAGATCAAGG TGAGGAACCT GTTGACGTTG ATTGCTTCTG CCGAAACGTG GATGGGGTGT ATCTCGAATA TACTATCGGA AAGAATACTC ACGCATTGGT ATCTAGTTCC ACTCCTTGGA CAACTGCAAC TAACGAAGAC GGCTTTGCAC CTACCCCACA TAGAGCTTAT
prM Hypr
• G R C G Q E G S R T R R S V L I P S H A Q G E L T G R G H K L 501 TGGACGGTGT GGTAAACAAG AAGGAAGCAG AACCAGACGC TCAGTGCTTA TACCCTCCCA CGCTCAAGGA GAGCTGACCG GACGGGGACA TAAATGGTTG ACCTGCCACA CCATTTGTTC TTCCTTCGTC TTGGTCTGCG AGTCACGAAT ATGGGAGGGT GCGAGTTCCT CTCGACTGGC CTGCCCCTGT ATTTACCAAC
prM Hypr
E G D S L R T H L T R V E G W V W K N R L L A L A M V T V V W L T L
601 GAGGGCGACT CACTCCGAAC ACATTTGACC CGCGTCGAGG GCTGGGTCTG GAAAAATCGG CTGTTGGCCC TCGCTATGGT GACAGTCGTT TGGCTCACGC CTCCCGCTGA GTGAGGCTTG TGTAAACTGG GCGCAGCTCC CGACCCAGAC CTTTTTAGCC GACAACCGGG AGCGATACCA CTGTCAGCAA ACCGAGTGCG
E Hypr
prM Hypr
• E S V V T R V A V L V V L L C L A P V Y A S R C T H L E N R D F V · 701 TGGAGTCTGT GGTTACTCGC GTGGCAGTGC TGGTGGTGCT CCTCTGTCTT GCCCCTGTCT ACGCGTCCAG GTGTACTCAT TTGGAAAACA GAGATTTTGT ACCTCAGACA CCAATGAGCG CACCGTCACG ACCACCACGA GGAGACAGAA CGGGGACAGA TGCGCAGGTC CACATGAGTA AACCTTTTGT CTCTAAAACA
E Hypr
• T G T Q G T T R V T L V L E L G G C V T I T A E G K P S M D V W L 801 CACCGGCACC CAGGGGACGA CTCGGGTAAC CCTGGTGCTT GAACTGGGTG GTTGCGTTAC TATTACCGCT GAGGGCAAAC CCTCTATGGA TGTGTGGCTG GTGGCCGTGG GTCCCCTGCT GAGCCCATTG GGACCACGAA CTTGACCCAC CAACGCAATG ATAATGGCGA CTCCCGTTTG GGAGATACCT ACACACCGAC
E Hypr
D A I Y Q E N P A Q T R E Y C L H A K L S D T K V A A R C P T M G P
901 GATGCAATCT ATCAGGAGAA TCCCGCACAA ACCAGGGAAT ATTGCCTTCA CGCAAAGCTG TCCGATACAA AGGTCGCGGC TAGGTGCCCA ACAATGGGAC CTACGTTAGA TAGTCCTCTT AGGGCGTGTT TGGTCCCTTA TAACGGAAGT GCGTTTCGAC AGGCTATGTT TCCAGCGCCG ATCCACGGGT TGTTACCCTG
E Hypr
• A T L A E E H Q G G T V C K R D Q S D R G W G N H C G L F G K G S
1001 CGGCCACCCT GGCGGAGGAA CATCAGGGAG GTACAGTGTG CAAACGGGAC CAGAGTGATA GAGGCTGGGG TAATCACTGC GGCCTGTTCG GCAAAGGAAG
GCCGGTGGGA CCGCCTCCTT GTAGTCCCTC CATGTCACAC GTTTGCCCTG GTCTCACTAT CTCCGACCCC ATTAGTGACG CCGGACAAGC CGTTTCCTTC
E Hypr
• I V A C V K A A C E A K K K A T G H V Y D A N K I V Y T V K V E P 1101 TATTGTCGCT TGCGTCAAGG CAGCCTGTGA GGCCAAAAAG AAGGCTACTG GGCACGTCTA TGACGCCAAC AAGATCGTTT ATACAGTGAA AGTGGAACCA ATAACAGCGA ACGCAGTTCC GTCGGACACT CCGGTTTTTC TTCCGATGAC CCGTGCAGAT ACTGCGGTTG TTCTAGCAAA TATGTCACTT TCACCTTGGT
E Hypr
H T G D Y V A A N E T H S G R T A S F T V S S E K T I L T M G E Y
1201 CACACAGGGG ATTACGTGGC GGCCAACGAG ACTCATTCCG GTCGCAAAAC GGCCAGCTTC ACCGTGTCAT CCGAAAAGAC CATCCTCACT ATGGGGGAGT GTGTGTCCCC TAATGCACCG CCGGTTGCTC TGAGTAAGGC CAGCGTTTTG CCGGTCGAAG TGGCACAGTA GGCTTTTCTG GTAGGAGTGA TACCCCCTCA
E Hypr
• G D V S L L C R V A S G V D L A Q T V I L E L D T V E H L P T A 1301 ATGGCGACGT TTCTCTGCTC TGCCGGGTGG CTAGCGGAGT CGACCTGGCC CAGACAGTCA TCCTGGAACT GGATAAAACA GTTGAGCATC TGCCTACCGC TACCGCTGCA AAGAGACGAG ACGGCCCACC GATCGCCTCA GCTGGACCGG GTCTGTCAGT AGGACCTTGA CCTATTTTGT CAACTCGTAG ACGGATGGCG
E Hypr
' W Q V H R D F N D L A L P W K H E G A R N W N N A E R L V E F G 1401 TTGGCAGGTG CACAGGGATT GGTTTAACGA CCTTGCCCTG CCATGGAAAC ATGAAGGAGC GAGAAACTGG AATAATGCAG AGCGACTCGT AGAATTCGGT AACCGTCCAC GTGTCCCTAA CCAAATTGCT GGAACGGGAC GGTACCTTTG TACTTCCTCG CTCTTTGACC TTATTACGTC TCGCTGAGCA TCTTAAGCCA
E Hypr
A P H A V M D V Y N L G D Q T G V L L A L A G V P V A H I E G T
1501 GCCCCTCATG CCGTGAAGAT GGACGTCTAC AATCTGGGTG ATCAGACCGG CGTTCTCCTT AAAGCTCTCG CTGGCGTACC AGTTGCCCAC ATCGAAGGAA CGGGGAGTAC GGCACTTCTA CCTGCAGATG TTAGACCCAC TAGTCTGGCC GCAAGAGGAA TTTCGAGAGC GACCGCATGG TCAACGGGTG TAGCTTCCTT
E Hypr
• K Y H L K S G H V T C E V G L E K L K M K G L T Y T M C D K T K F 1601 CGAAGTACCA CCTGAAGTCA GGCCATGTAA CTTGCGAGGT GGGCCTGGAG AAGTTGAAAA TGAAAGGTCT TACGTACACA ATGTGTGACA AGACCAAGTT GCTTCATGGT GGACTTCAGT CCGGTACATT GAACGCTCCA CCCGGACCTC TTCAACTTTT ACTTTCCAGA ATGCATGTGT TACACACTGT TCTGGTTCAA
E Hypr
• T W K R A P T D S G H D T V V M E V T F S G T K P C R I P V R A V 1701 CACATGGAAG AGGGCCCCCA CAGATAGCGG CCACGATACT GTGGTGATGG AGGTGACCTT TTCTGGAACA AAACCCTGCA GAATACCCGT GCGGGCTGTA GTGTACCTTC TCCCGGGGGT GTCTATCGCC GGTGCTATGA CACCACTACC TCCACTGGAA AAGACCTTGT TTTGGGACGT CTTATGGGCA CGCCCGACAT
E Hypr
A H G S P D V N V A M L I T P N P T I E N N G G G F I E M Q L P P G
1801 GCTCACGGAT CTCCCGATGT CAATGTTGCT ATGCTGATTA CACCTAACCC TACCATCGAG AATAACGGTG GTGGTTTTAT TGAGATGCAG CTTCCGCCAG CGAGTGCCTA GAGGGCTACA GTTACAACGA TACGACTAAT GTGGATTGGG ATGGTAGCTC TTATTGCCAC CACCAAAATA ACTCTACGTC GAAGGCGGTC
E Hypr
• D N I I Y V G E L S Y Q W F Q K G S S I G R V F Q K T K K G I E R 1901 GCGATAACAT CATCTACGTG GGCGAACTCT CTTACCAGTG GTTTCAGAAA GGGAGTTCAA TTGGGCGGGT CTTCCAAAAA ACGAAGAAGG GAATCGAACG CGCTATTGTA GTAGATGCAC CCGCTTGAGA GAATGGTCAC CAAAGTCTTT CCCTCAAGTT AACCCGCCCA GAAGGTTTTT TGCTTCTTCC CTTAGCTTGC
E Hypr
■ L T V I G E H A W D F G S A G G F L S S I G K A L H T V L G G A F
2001 ATTGACGGTT ATCGGCGAGC ACGCATGGGA TTTTGGTTCC GCAGGGGGAT TCCTGTCTTC TATTGGTAAG GCACTGCATA CCGTGCTGGG GGGCGCATTC
TAACTGCCAA TAGCCGCTCG TGCGTACCCT AAAACCAAGG CGTCCCCCTA AGGACAGAAG ATAACCATTC CGTGACGTAT GGCACGACCC CCCGCGTAAG
E Hypr
N S I F G G V G F L P K L L L G V A L A L G L N M R N P T M S M S
2101 AATTCTATTT TCGGGGGCGT GGGGTTCCTG CCTAAACTCC TGCTGGGAGT AGCCCTGGCC TGGTTGGGAC TGAATATGCG GAATCCGACG ATGTCCATGT
TTAAGATAAA AGCCCCCGCA CCCCAAGGAC GGATTTGAGG ACGACCCTCA TCGGGACCGG ACCAACCCTG ACTTATACGC CTTAGGCTGC TACAGGTACA
E Hypr
WNV KS1 protein
■ F L L A G V L V L A M T L G V G A D T G C A I D I S R Q
2201 CATTCCTCTT GGCCGGCGTG CTTGTACTGG CCATGACACT GGGCGTTGGC GCCGACACTG GGTGTGCCAT AGACATCAGC CGGCAA
GTAAGGAGAA CCGGCCGCAC GAACATGACC GGTACTGTGA CCCGCAACCG CGGCTGTGAC CCACACGGTA TCTGTAGTCG GCCGTT
PIV-WNTBEV Hypr with WNV signal {p40; SEQ ID NOs:55-57)
deleted C
UTR
M S
AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
WNV deleted C
• K K P G G P G K S R A V Y L L K R G M P R V L S L I G L K R S S K
101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGC GGAGCTCCAA GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCG CCTCGAGGTT
WNV signal
WNV deleted C pr Hypr
• Q K K R G G K T G I A V M I G M L A C V G A A T V R K E R D G S T
201 GCAAAAGAAA CGCGGGGGAA AGACAGGCAT AGCTGTGATG ATAGGCATGC TGGCTTGTGT CGGAGCAGCT ACCGTGCGAA AAGAACGCGA CGGAAGCACC CGTTTTCTTT GCGCCCCCTT TCTGTCCGTA TCGACACTAC TATCCGTACG ACCGAACACA GCCTCGTCGA TGGCACGCTT TTCTTGCGCT GCCTTCGTGG
prM Hypr
V I R A E G K D A A T Q V R V E N G T C V I L A T D M G S W C D D S
301 GTGATAAGGG CTGAGGGTAA GGATGCGGCT ACGCAGGTGA GAGTAGAGAA TGGCACTTGC GTAATACTCG CGACTGATAT GGGATCCTGG TGTGACGATA
CACTATTCCC GACTCCCATT CCTACGCCGA TGCGTCCACT CTCATCTCTT ACCGTGAACG CATTATGAGC GCTGACTATA CCCTAGGACC ACACTGCTAT
prM Hypr
• L S Y E C V T I D Q G E E P V D V D C F C R H V D G V Y L E Y G R 401 GCCTCAGTTA TGAATGCGTA ACAATAGACC AGGGCGAAGA ACCTGTGGAC GTTGACTGTT TCTGTAGAAA TGTGGATGGC GTTTATCTGG AGTACGGCCG CGGAGTCAAT ACTTACGCAT TGTTATCTGG TCCCGCTTCT TGGACACCTG CAACTGACAA AGACATCTTT ACACCTACCG CAAATAGACC TCATGCCGGC
prM Hypr
• C G K Q E G S R T R R S V L I P S H A Q G E L T G R G H K W L E G 501 CTGTGGAAAA CAGGAGGGCT CACGAACTCG AAGATCTGTG CTGATTCCAA GTCACGCGCA AGGAGAGTTG ACCGGTAGAG GCCACAAGTG GCTTGAAGGG GACACCTTTT GTCCTCCCGA GTGCTTGAGC TTCTAGACAC GACTAAGGTT CAGTGCGCGT TCCTCTCAAC TGGCCATCTC CGGTGTTCAC CGAACTTCCC
prM Hypr
D S L R T H L T R V E G W V W K N R L L A L A M V T V V W L T L E S
601 GACTCATTGA GGACCCACCT GACTAGGGTG GAGGGTTGGG TTTGGAAGAA TCGGTTGCTC GCGCTCGCTA TGGTCACCGT CGTGTGGCTG ACACTGGAGA CTGAGTAACT CCTGGGTGGA CTGATCCCAC CTCCCAACCC AAACCTTCTT AGCCAACGAG CGCGAGCGAT ACCAGTGGCA GCACACCGAC TGTGACCTCT
E Hypr
prM Hypr
• V V T R V A V L V V L L C L A P V Y A S R C T H L E N R D F V T G
701 GTGTCGTGAC TCGGGTTGCT GTGTTGGTTG TCCTCCTCTG TTTGGCCCCA GTGTACGCGT CCAGGTGTAC TCATTTGGAA AACAGAGATT TTGTCACCGG
CACAGCACTG AGCCCAACGA CACAACCAAC AGGAGGAGAC AAACCGGGGT CACATGCGCA GGTCCACATG AGTAAACCTT TTGTCTCTAA AACAGTGGCC
E Hypr
• T Q G T T R V T L V L E L G G C V T I T A E G P S M D V W L D A
801 CACCCAGGGG ACGACTCGGG TAACCCTGGT GCTTGAACTG GGTGGTTGCG TTACTATTAC CGCTGAGGGC AAACCCTCTA TGGATGTGTG GCTGGATGCA
GTGGGTCCCC TGCTGAGCCC ATTGGGACCA CGAACTTGAC CCACCAACGC AATGATAATG GCGACTCCCG TTTGGGAGAT ACCTACACAC CGACCTACGT
E Hypr
I Y Q E N P A Q T R E Y C L H A K L S D T K V A A R C P T G P A T
901 ATCTATCAGG AGAATCCCGC ACAAACCAGG GAATATTGCC TTCACGCAAA GCTGTCCGAT ACAAAGGTCG CGGCTAGGTG CCCAACAATG GGACCGGCCA TAGATAGTCC TCTTAGGGCG TGTTTGGTCC CTTATAACGG AAGTGCGTTT CGACAGGCTA TGTTTCCAGC GCCGATCCAC GGGTTGTTAC CCTGGCCGGT
E Hypr
• L A E E H Q G G T V C K R D Q S D R G W G N H C G L F G K G S I V 1001 CCCTGGCGGA GGAACATCAG GGAGGTACAG TGTGCAAACG GGACCAGAGT GATAGAGGCT GGGGTAATCA CTGCGGCCTG TTCGGCAAAG GAAGTATTGT GGGACCGCCT CCTTGTAGTC CCTCCATGTC ACACGTTTGC CCTGGTCTCA CTATCTCCGA CCCCATTAGT GACGCCGGAC AAGCCGTTTC CTTCATAACA
E Hypr
• A C V K A A C E A K K K A T G H V Y D A N K I V Y T V K V E P H T 1101 CGCTTGCGTC AAGGCAGCCT GTGAGGCCAA AAAGAAGGCT ACTGGGCACG TCTATGACGC CAACAAGATC GTTTATACAG TGAAAGTGGA ACCACACACA GCGAACGCAG TTCCGTCGGA CACTCCGGTT TTTCTTCCGA TGACCCGTGC AGATACTGCG GTTGTTCTAG CAAATATGTC ACTTTCACCT TGGTGTGTGT
E Hypr
G D Y V A A N E T H S G R T A S F T V S S E K T I L T M G E Y G D
1201 GGGGATTACG TGGCGGCCAA CGAGACTCAT TCCGGTCGCA AAACGGCCAG CTTCACCGTG TCATCCGAAA AGACCATCCT CACTATGGGG GAGTATGGCG CCCCTAATGC ACCGCCGGTT GCTCTGAGTA AGGCCAGCGT TTTGCCGGTC GAAGTGGCAC AGTAGGCTTT TCTGGTAGGA GTGATACCCC CTCATACCGC
E Hypr
• V S L L C R V A S G V D L A Q T V I L E L D K T V E H L P T A W Q 1301 ACGTTTCTCT GCTCTGCCGG GTGGCTAGCG GAGTCGACCT GGCCCAGACA GTCATCCTGG AACTGGATAA AACAGTTGAG CATCTGCCTA CCGCTTGGCA TGCAAAGAGA CGAGACGGCC CACCGATCGC CTCAGCTGGA CCGGGTCTGT CAGTAGGACC TTGACCTATT TTGTCAACTC GTAGACGGAT GGCGAACCGT
E Hypr
• V H R D F N D L A L P W K H E G A R N N N A E R L V E F G A P
1401 GGTGCACAGG GATTGGTTTA ACGACCTTGC CCTGCCATGG AAACATGAAG GAGCGAGAAA CTGGAATAAT GCAGAGCGAC TCGTAGAATT CGGTGCCCCT
CCACGTGTCC CTAACCAAAT TGCTGGAACG GGACGGTACC TTTGTACTTC CTCGCTCTTT GACCTTATTA CGTCTCGCTG AGCATCTTAA GCCACGGGGA
E Hypr
H A V M D V Y N L G D Q T G V L L K A L A G V P V A H I E G T K Y
1501 CATGCCGTGA AGATGGACGT CTACAATCTG GGTGATCAGA CCGGCGTTCT CCTTAAAGCT CTCGCTGGCG TACCAGTTGC CCACATCGAA GGAACGAAGT GTACGGCACT TCTACCTGCA GATGTTAGAC CCACTAGTCT GGCCGCAAGA GGAATTTCGA GAGCGACCGC ATGGTCAACG GGTGTAGCTT CCTTGCTTCA
E Hypr
• H L K S G H V T C E V G L E K L M G L T Y T M C D K T K F T W 1601 ACCACCTGAA GTCAGGCCAT GTAACTTGCG AGGTGGGCCT GGAGAAGTTG AAAATGAAAG GTCTTACGTA CACAATGTGT GACAAGACCA AGTTCACATG TGGTGGACTT CAGTCCGGTA CATTGAACGC TCCACCCGGA CCTCTTCAAC TTTTACTTTC CAGAATGCAT GTGTTACACA CTGTTCTGGT TCAAGTGTAC
E Hypr
■ K R A P T D S G H D T V V M E V T F S G T K P C R I P V R A V A H 1701 GAAGAGGGCC CCCACAGATA GCGGCCACGA TACTGTGGTG ATGGAGGTGA CCTTTTCTGG AACAAAACCC TGCAGAATAC CCGTGCGGGC TGTAGCTCAC CTTCTCCCGG GGGTGTCTAT CGCCGGTGCT ATGACACCAC TACCTCCACT GGAAAAGACC TTGTTTTGGG ACGTCTTATG GGCACGCCCG ACATCGAGTG
E Hypr
G S P D V N V A M L I T P N P T I E N N G G G F I E M Q L P P G D N
1801 GGATCTCCCG ATGTCAATGT TGCTATGCTG ATTACACCTA ACCCTACCAT CGAGAATAAC GGTGGTGGTT TTATTGAGAT GCAGCTTCCG CCAGGCGATA CCTAGAGGGC TACAGTTACA ACGATACGAC TAATGTGGAT TGGGATGGTA GCTCTTATTG CCACCACCAA AATAACTCTA CGTCGAAGGC GGTCCGCTAT
E Hypr
■ I I Y V G E L S Y Q F Q K G S S I G R V F Q K T K K G I E R L T
ACATCATCTA CGTGGGCGAA CTCTCTTACC AGTGGTTTCA GAAAGGGAGT TCAATTGGGC GGGTCTTCCA AAAAACGAAG AAGGGAATCG AACGATTGAC
TGTAGTAGAT GCACCCGCTT GAGAGAATGG TCACCAAAGT CTTTCCCTCA AGTTAACCCG CCCAGAAGGT TTTTTGCTTC TTCCCTTAGC TTGCTAACTG
E Hypr
• V I G E H A W D F G S A G G F L S S I G A L H T V L G G A F N S
GGTTATCGGC GAGCACGCAT GGGATTTTGG TTCCGCAGGG GGATTCCTGT CTTCTATTGG TAAGGCACTG CATACCGTGC TGGGGGGCGC ATTCAATTCT
CCAATAGCCG CTCGTGCGTA CCCTAAAACC AAGGCGTCCC CCTAAGGACA GAAGATAACC ATTCCGTGAC GTATGGCACG ACCCCCCGCG TAAGTTAAGA
E Hypr
I F G G V G F L P K L L L G V A L A W L G L N M R N P T M S M S F L ATTTTCGGGG GCGTGGGGTT CCTGCCTAAA CTCCTGCTGG GAGTAGCCCT GGCCTGGTTG GGACTGAATA TGCGGAATCC GACGATGTCC ATGTCATTCC TAAAAGCCCC CGCACCCCAA GGACGGATTT GAGGACGACC CTCATCGGGA CCGGACCAAC CCTGACTTAT ACGCCTTAGG CTGCTACAGG TACAGTAAGG E Hypr
WNV NSl protein
• L A G V L V L A M T L G V G A D T G C A I D I S R Q
TCTTGGCCGG CGTGCTTGTA CTGGCCATGA CACTGGGCGT TGGCGCCGAC ACTGGGTGTG CCATAGACAT CAGCCGGCAA AGAACCGGCC GCACGAACAT GACCGGTACT GTGACCCGCA ACCGCGGCTG TGACCCACAC GGTATCTGTA GTCGGCCGTT
Sequence Appendix 4. WN PIV constructs expressing rabies virus G protein.
WN (ACprME)-Rabies PIV sequence (partial; SEQ ID NOs:58-60)
N-terminus of C
5' UTR
M S
1 AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCA CACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
N-terminus of C
■ K K P G G P G K S R A V Y L L K R G M ! P R V L S L I G L K Q K K R
101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGC AAAAGAAGCG GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCG TTTTCTTCGC
N-terminus of C Rabies-G signal
partial C signal Rabies-G protein
■ G G K T G I A V I V P Q A L L F V P L L V F P L C F G K F P I Y T
201 AGGGGGCAAG ACTGGTATAG CTGTGATCGT TCCTCAGGCT CTTTTGTTTG TACCCTTGCT GGTATTTCCC CTTTGCTTTG GTAAATTTCC TATCTATACC TCCCCCGTTC TGACCATATC GACACTAGCA AGGAGTCCGA GAAAACAAAC ATGGGAACGA CCATAAAGGG GAAACGAAAC CATTTAAAGG ATAGATATGG
Rabies-G protein
I P D K L G P W S P I D I H H L S C P N N L V V E D E G C T N L S G
301 ATCCCTGATA AGCTCGGGCC TTGGAGTCCC ATTGATATTC ACCATTTGAG CTGCCCAAAC AACCTCGTCG TTGAGGATGA AGGGTGCACT AATCTTTCTG TAGGGACTAT TCGAGCCCGG AACCTCAGGG TAACTATAAG TGGTAAACTC GACGGGTTTG TTGGAGCAGC AACTCCTACT TCCCACGTGA TTAGAAAGAC
Rabies-G protein
• F S Y M E L K V G Y I S A I K M N G F T C T G V V T E A E 1 Y T N
401 GATTTTCCTA CATGGAGTTG AAAGTGGGCT ATATTTCAGC CATTAAGATG AACGGCTTTA CTTGTACAGG AGTCGTGACC GAAGCCGAGA CATATACAAA CTAAAAGGAT GTACCTCAAC TTTCACCCGA TATAAAGTCG GTAATTCTAC TTGCCGAAAT GAACATGTCC TCAGCACTGG CTTCGGCTCT GTATATGTTT
Rabies-G protein
■ F V G Y V T T T F K R K H F R P T P D A C R A A Y N W K M A G
501 TTTCGTGGGA TACGTCACCA CCACCTTCAA GAGAAAACAC TTCCGCCCAA CGCCTGACGC TTGTCGGGCC GCTTACAACT GGAAGATGGC AGGAGATCCT AAAGCACCCT ATGCAGTGGT GGTGGAAGTT CTCTTTTGTG AAGGCGGGTT GCGGACTGCG AACAGCCCGG CGAATGTTGA CCTTCTACCG TCCTCTAGGA
Rabies-G protein
R Y E E S L H N P Y P D Y H W L R T V K T T K E S L V I I S P S V A
601 CGATATGAAG AATCTCTGCA CAACCCGTAT CCTGATTACC ATTOGCTGCG GACAGTCAAG ACTACCAAGG AGAGTCTGGT CATTATATCA CCAAGCGTGG GCTATACTTC TTAGAGACGT GTTGGGCATA GGACTAATGG TAACCGACGC CTGTCAGTTC TGATGGTTCC TCTCAGACCA GTAATATAGT GGTTCGCACC
Rabies-G protein
• D L D P Y D R S L H S R V P G G N C S G V A V S S T Y C S T H H
701 CCGATCTTGA TCCTTATGAT AGATCCCTGC ACAGTAGGGT TTTTCCTGGC GGGAATTGTA GCGGTGTTGC AGTATCAAGT ACCTACTGCT CCACTAACCA GGCTAGAACT AGGAATACTA TCTAGGGACG TGTCATCCCA AAAAGGACCG CCCTTAACAT CGCCACAACG TCATAGTTCA TGGATGACGA GGTGATTGGT
Rabies-G protein
• D Y T I W P E N P R L G M S C D I F T N S R G K R A S K G S E T
801 CGACTACACT ATATGGATGC CTGAGAACCC TCGACTCGGT ATGAGTTGCG ACAT TTAC GAACTCACGG GGCAAGCGGG CATCTAAGGG GTCTGAAACA GCTGATGTGA TATACCTACG GACTCTTGGG AGCTGAGCCA TACTCAACGC TGTAAAAATG CTTGAGTGCC CCGTTCGCCC GTAGATTCCC CAGACTTTGT
Rabies-G protein
C G F V D E R G L Y K S L K G A C K L K L C G V L G L R L M D G T W
901 TGCGGGTTTG TTGATGAGCG GGGGTTGTAT AAATCTCTTA AAGGCGCCTG TAAGCTGAAA CTCTGTGGCG TACTGGGGCT GCGCCTGATG GACGGCACAT ACGCCCAAAC AACTACTCGC CCCCAACATA TTTAGAGAAT TTCCGCGGAC ATTCGACTTT GAGACACCGC ATGACCCCGA CGCGGACTAC CTGCCGTGTA
Rabies-G protein
• V A M Q T S N E T K W C P P G Q L V N L H D F R S D E I E ¥. [ L V V
1001 GGGTGGCTAT GCAGACAAGC AATGAAACAA AGTGGTGTCC CCCTGGTCAG CTGGTTAATC TGCACGACTT TAGGTCTGAC GAAATCGAGC ACCTTGTGGT CCCACCGATA CGTCTGTTCG TTACTTTGTT TCACCACAGG GGGACCAGTC GACCAATTAG ACGTGCTGAA ATCCAGACTG CTTTAGCTCG TGGAACACCA
Rabies-G protein • E E L V K R E E C L D A L E S I M T T K S V S F R R L S H L R K
1101 GGAGGAACTG GTGAAGAAAC GCGAAGAGTG CCTGGACGCA CTTGAGAGTA TTATGACCAC CAAATCCGTT TCCTTCAGAA GACTGAGCCA CCTGCGAAAG CCTCCTTGAC CACTTCTTTG CGCTTCTCAC GGACCTGCGT GAACTCTCAT AATACTGGTG GTTTAGGCAA AGGAAGTCTT CTGACTCGGT GGACGCTTTC
Rabies-G protein
L V P G F G K A Y T I F N K T L M E A D A H Y K S V R T W N E I I 1
1201 CTGGTGCCAG GGTTCGGGAA GGCTTATACT ATTTTCAACA AGACTCTTAT GGAGGCGGAT GCCCATTATA AGTCAGTTAG GACTTGGAAT GAGATAATTC GACCACGGTC CCAAGCCCTT CCGAATATGA TAAAAGTTGT TCTGAGAATA CCTCCGCCTA CGGGTAATAT TCAGTCAATC CTGAACCTTA CTCTATTAAG
Rabies-G protein
• S K G C L R V G G R C H P H V N G V F F N G I I L G P D G N V L I
1301 CCTCCAAAGG ATGTCTGAGA GTCGGTGGGA GATGCCACCC CCATGTCAAT GGGGTGTTCT TTAACGGAAT CATCCTGGGA CCTGACGGGA ACGTGCTGAT GGAGGTTTCC TACAGACTCT CAGCCACCCT CTACGGTGGG GGTACAGTTA CCCCACAAGA AATTGCCTTA GTAGGACCCT GGACTGCCCT TGCACGACTA
Rabies-G protein
• P E M Q S S L L Q Q H M E L L V S S V I P L M H P L A D P S T V F
1401 TCCCGAGATG CAATCTTCCC TTCTGCAGCA ACACATGGAA CTCCTGGTGT CTTCAGTGAT ACCCCTGATG CACCCACTGG CCGACCCCAG CACTGTGTTC AGGGCTCTAC GTTAGAAGGG AAGACGTCGT TGTGTACCTT GAGGACCACA GAAGTCACTA TGGGGACTAC GTGGGTGACC GGCTGGGGTC GTGACACAAG
Rabies-G protein
K N G D E A E D F V E V H L P D V H E R I S G V D L G L P N W G K '
1501 AAAAATGGCG ATGAGGCCGA AGACTTTGTG GAAGTTCACC TGCCCGATGT ACACGAAAGG ATATCTGGAG TAGACCTGGG CCTTCCTAAT TGGGGTAAGT TTTTTACCGC TACTCCGGCT TCTGAAACAC CTTCAAGTGG ACGGGCTACA TGTGCTTTCC TATAGACCTC ATCTGGACCC GGAAGGATTA ACCCCATTCA
Rabies-G protein
■ V L L S A G A L T A L M L I I F L M T C R R V N R S E P T Q H N
1601 ACGTGCTCCT GAGTGCGGGT GCCTTGACCG CTTTGATGCT GATCATTTTT CTGATGACCT GCTGGCGGAG GGTGAATCGC TCCGAGCCGA CACAGCACAA TGCACGAGGA CTCACGCCCA CGGAACTGGC GAAACTACGA CTAGTAAAAA GACTACTGGA CGACCGCCTC CCACTTAGCG AGGCTCGGCT GTGTCGTGTT
Rabies-G protein
FMEK V 2A
■ L R G T G R E V S V T P Q S G K I I S S W E S Y K S G G E G L
1701 TCTCAGAGGG ACAGGCCGGG AAGTAAGTGT GACTCCGCAA TCTGGCAAGA TTATTAGTAG TTGGGAGAGT TACAAGTCTG GAGGAGAGAC TGGGTTGAAT AGAGTCTCCC TGTCCGGCCC TTCATTCACA CTGAGGCGTT AGACCGTTCT AATAATCATC AACCCTCTCA ATGTTCAGAC CTCCTCTCTG ACCCAACTTA
preNSl signal
FMDV 2A NSl signal
F D L L K L A G D V E S N P G P A R D R S I A L T F L A V G G V L 1
1801 TTTGATCTGC TCAAACTTGC AGGCGATGTA GAATCAAATC CTGGACCCGC CCGGGACAGG TCCATAGCTC TCACGTTTCT CGCAGTTGGA GGAGTTCTGC AAACTAGACG AGTTTGAACG TCCGCTACAT CTTAGTTTAG GACCTGGGCG GGCCCTGTCC AGGTATCGAG AGTGCAAAGA GCGTCAACCT CCTCAAGACG NSl signal
NSl
■ F L S V M V H A D T G C A I D I S R Q E L R C G S G V F I H N D V
1901 TCTTCCTCTC CGTGAACGTG CACGCTGACA CTGGGTGTGC CATAGACATC AGCCGGCAAG AGCTGAGATG TGGAAGTGGA GTGTTCATAC ACAATGATGT AGAAGGAGAG GCACTTGCAC GTGCGACTGT GACCCACACG GTATCTGTAG TCGGCCGTTC TCGACTCTAC ACCTTCACCT CACAAGTATG TGTTACTACA
(AC)-Rabies G PIV sequence (partial; SEQ ID NOs:61-63).
5'UTR
1 AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
N-terminus of C
• K K P G G P G K S R V N M L K R G M ] P R V L S L I G L K ζ 2 K K R
101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCAA TATGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGC AAAAGAAGCG GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGTT ATACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCG TTTTCTTCGC
N-terminus of C Rabies-G protein partial C signal RAbies-G signal
• G G K T G I A V I V P Q A L L F V P L L V F P L C F G K F P I Y T
201 AGGGGGCAAG ACTGGTATAG CTGTGATCGT TCCTCAGGCT CTTTTGTTTG TACCCTTGCT GGTATTTCCC CTTTGCTTTG GTAAATTTCC TATCTATACC TCCCCCGTTC TGACCATATC GACACTAGCA AGGAGTCCGA GAAAACAAAC ATGGGAACGA CCATAAAGGG GAAACGAAAC CATTTAAAGG ATAGATATGG
Rabies-G protein
I P D K L G P W S P I D I H H L S C P N N L V V E D E G C T N L S C
301 ATCCCTGATA AGCTCGGGCC TTGGAGTCCC ATTGATATTC ACCATTTGAG CTGCCCAAAC AACCTCGTCG TTGAGGATGA AGGGTGCACT AATCTTTCTG TAGGGACTAT TCGAGCCCGG AACCTCAGGG TAACTATAAG TGGTAAACTC GACGGGTTTG TTGGAGCAGC AACTCCTACT TCCCACGTGA TTAGAAAGAC
Rabies-G protein
• F S Y M E L K V G Y I S A I K M N G F T C T G V V T E A E T Y T N
401 GATTTTCCTA CATGGAGTTG AAAGTGGGCT ATATTTCAGC CATTAAGATG AACGGCTTTA CTTGTACAGG AGTCGTGACC GAAGCCGAGA CATATACAAA CTAAAAGGAT GTACCTCAAC TTTCACCCGA TATAAAGTCG GTAATTCTAC TTGCCGAAAT GAACATGTCC TCAGCACTGG CTTCGGCTCT GTATATGTTT
Rabies-G protein
• F V G Y V T T T F K R K H F R P T P D A C R A A Y N K M A G D P
501 TTTCGTGGGA TACGTCACCA CCACCTTCAA GAGAAAACAC TTCCGCCCAA CGCCTGACGC TTGTCGGGCC GCTTACAACT GGAAGATGGC AGGAGATCCT AAAGCACCCT ATGCAGTGGT GGTGGAAGTT CTCTTTTGTG AAGGCGGGTT GCGGACTGCG AACAGCCCGG CGAATGTTGA CCTTCTACCG TCCTCTAGGA
Rabies-G protein
R Y E E S L H N P Y P D Y H W L R T V K T T K E S L V I I S P S V A
601 CGATATGAAG AATCTCTGCA CAACCCGTAT CCTGATTACC ATTGGCTGCG GACAGTCAAG ACTACCAAGG AGAGTCTGGT CATTATATCA CCAAGCGTGG GCTATACTTC TTAGAGACGT GTTGGGCATA GGACTAATGG TAACCGACGC CTGTCAGTTC TGATGGTTCC TCTCAGACCA GTAATATAGT GGTTCGCACC
Rabies-G protein
■ D L D P Y D R S L H S R V F P G G N C S G V A V S S T Y C S T N H
701 CCGATCTTGA TCCTTATGAT AGATCCCTGC ACAGTAGGGT TTTTCCTGGC GGGAATTGTA GCGGTGTTGC AGTATCAAGT ACCTACTGCT CCACTAACCA GGCTAGAACT AGGAATACTA TCTAGGGACG TGTCATCCCA AAAAGGACCG CCCTTAACAT CGCCACAACG TCATAGTTCA TGGATGACGA GG GATTGGT
Rabies-G protein
■ D Y T I W M P E N P R L G M S C D I F T N S R G K R A S K G S E T
801 CGACTACACT ATATGGATGC CTGAGAACCC TCGACTCGGT ATGAGTTGCG ACATTTTTAC GAACTCACGG GGCAAGCGGG CATCTAAGGG GTCTGAAACA GCTGATGTGA TATACCTACG GACTCTTGGG AGCTGAGCCA TACTCAACGC TGTAAAAATG CTTGAGTGCC CCGTTCGCCC GTAGATTCCC CAGACTTTGT
Rabies-G protein
C G F V D E R G L Y K S L K G A C K L K L C G V L G L R L M D G T W
901 TGCGGGTTTG TTGATGAGCG GGGGTTGTAT AAATCTCTTA AAGGCGCCTG TAAGCTGAAA CTCTGTGGCG TACTGGGGCT GCGCCTGATG GACGGCACAT ACGCCCAAAC AACTACTCGC CCCCAACATA TTTAGAGAAT TTCCGCGGAC ATTCGACTTT GAGACACCGC ATGACCCCGA CGCGGACTAC CTGCCGTGTA
Rabies-G protein
• V A M Q T S N E T K W C P P G Q L V M L H D F R S D E I E H L V V
1001 GGGTGGCTAT GCAGACAAGC AATGAAACAA AGTGGTGTCC CCCTGGTCAG CTGGTTAATC TGCACGACTT TAGGTCTGAC GAAATCGAGC ACCTTGTGGT CCCACCGATA CGTCTGTTCG TTACTTTGTT TCACCACAGG GGGACCAGTC GACCAATTAG ACGTGCTGAA ATCCAGACTG CTTTAGCTCG TGGAACACCA
Rabies-G protein
■ E E L V K K R E E C L D A L E S I M T T K S V S F R R L S H L R K
1101 GGAGGAACTG GTGAAGAAAC GCGAAGAGTG CCTGGACGCA CTTGAGAGTA TTATGACCAC CAAATCCGTT TCCTTCAGAA GACTGAGCCA CCTGCGAAAG CCTCCTTGAC CACTTCTTTG CGCTTCTCAC GGACCTGCGT GAACTCTCAT AATACTGGTG GTTTAGGCAA AGGAAGTCTT CTGACTCGGT GGACGCTTTC
Rabies-G protein L V P G F G K A Y T I F N K L M E A D A H Y K S V R T W N E I I ]
1201 CTGGTGCCAG GGTTCGGGAA GGCTTATACT ATTTTCAACA AGACTCTTAT GGAGGCGGAT GCCCATTATA AGTCAGTTAG GACTTGGAAT GAGATAATTC
GACCACGGTC CCAAGCCCTT CCGAATATGA TAAAAGTTGT TCTGAGAATA CCTCCGCCTA CGGGTAATAT TCAGTCAATC CTGAACCTTA CTCTATTAAG
Rabies-G protein
■ S K G C L R V G G R C H P H V N G V F ] F N G I I L G P D G N V L I
1301 CCTCCAAAGG ATGTCTGAGA GTCGGTGGGA GATGCCACCC CCA GTCAAT GGGGTGTTCT TTAACGGAAT CATCCTGGGA CCTGACGGGA ACGTGCTGAT
GGAGGTTTCC TACAGACTCT CAGCCACCCT CTACGGTGGG GGTACAGTTA CCCCACAAGA AATTGCCTTA GTAGGACCCT GGACTGCCCT TGCACGACTA
Rabies-G protein
■ P E M Q S S L L Q Q H M E L L V S S V I P L M H P L A D P S T V F
1401 TCCCGAGATG CAATCTTCCC TTCTGCAGCA ACACATGGAA CTCCTGGTGT CTTCAGTGAT ACCCCTGATG CACCCACTGG CCGACCCCAG CACTGTGTTC
AGGGCTCTAC GTTAGAAGGG AAGACGTCGT TGTGTACCTT GAGGACCACA GAAGTCACTA TGGGGACTAC GTGGGTGACC GGCTGGGGTC GTGACACAAG
Rabies-G protein
K N G D E A E D F V E V H L P D V H E R I S G V D L G L P N W G K '
1501 AAAAATGGCG ATGAGGCCGA AGACTTTGTG GAAGTTCACC TGCCCGATGT ACACGAAAGG ATATCTGGAG TAGACCTGGG CCTTCCTAAT TGGGGTAAGT
TTTTTACCGC TACTCCGGCT TCTGAAACAC CTTCAAGTGG ACGGGCTACA TGTGCTTTCC TATAGACCTC ATCTGGACCC GGAAGGATTA ACCCCATTCA
Rabies-G protein
• V L L S A G A L T A L M L I I F L M T C W R R V N R S E P Q H N
1601 ACGTGCTCCT GAGTGCGGGT GCCTTGACCG CTTTGATGCT GATCATTTTT CTGATGACCT GCTGGCGGAG GGTGAATCGC TCCGAGCCGA CACAGCACAA
TGCACGAGGA CTCACGCCCA CGGAACTGGC GAAACTACGA CTAGTAAAAA GACTACTGGA CGACCGCCTC CCACTTAGCG AGGCTCGGCT GTGTCGTGTT FMD^
Rabies-G protein
• L R G T G R E V S V T P Q S G K I I S S W E S Y K S G G E G L N
1701 TCTCAGAGGG ACAGGCCGGG AAGTAAGTGT GACTCCGCAA TCTGGCAAGA TTATTAGTAG TTGGGAGAGT TACAAGTCTG GAGGAGAGAC TGGGTTGAAT
AGAGTCTCCC TGTCCGGCCC TTCATTCACA CTGAGGCGTT AGACCGTTCT AATAATCATC AACCCTCTCA ATGTTCAGAC CTCCTCTCTG ACCCAACTTA
C/prM signal
FMDV 2A
F D L L K L A G D V E S N ! P G P G G K T G I A V M I G L I A C V G i
1801 TTTGATCTGC TCAAACTTGC AGGCGATGTA GAATCAAATC CTGGACCCGG AGGAAAGACC GGTATTGCAG TCATGATTGG CCTGATCGCC TGCGTAGGAG AAACTAGACG AGTTTGAACG TCCGCTACAT CTTAGTTTAG GACCTGGGCC TCCTTTCTGG CCATAACGTC AGTACTAACC GGACTAGCGG ACGCATCCTC
C/prM signal
prM
• V T L S N F Q G V M M T V N A T D V T D V I I P T A A G K N L
1901 CAGTTACCCT CTCTAACTTC CAAGGGAAGG TGATGATGAC GGTAAATGCT ACTGACGTCA CAGATGTCAT CACGATTCCA ACAGCTGCTG GAAAGAACCT G CAA GGGA GAGATTGAAG GTTCCCTTCC ACTACTACTG CCATTTACGA TGACTGCAGT GTCTACAGTA GTGCTAAGGT TGTCGACGAC CTTTCTTGGA
prM
• C I R A M D V G Y M C D D T I T Y E C P V L S A G N D P E D I D
2001 ATGCATTGTC AGAGCAATGG ATGTGGGATA CATGTGCGAT GATACTATCA CTTATGAATG CCCAGTGCTG TCGGCTGGTA ATGATCCAGA AGACATCGAC TACGTAACAG TCTCGTTACC TACACCCTAT GTACACGCTA CTATGATAGT GAATACTTAC GGGTCACGAC AGCCGACCAT TACTAGGTCT TCTGTAGCTG
prM
C W C T K S A V Y V R Y G R C T K T R H S R R 1 3 R R S L T Q T H !
2101 TGTTGGTGCA CAAAGTCAGC AGTCTACGTC AGGTATGGAA GATGCACCAA GACACGCCAC TCAAGACGCA GTCGGAGGTC ACTGACAGTG CAGACACACG ACAACCACGT GTTTCAGTCG CAGATGCAG TCCATACCTT CTACGTGGTT CTGTGCGGTG AGTTCTGCGT CAGCCTCCAG TGAC G CAC GTCTGTGTGC
prM
• E S T L A N K K G A W M D S T K A T R Y L V K T E S W I L R N P G
2201 GAGAAAGCAC TCTAGCGAAC AAGAAGGGGG CTTGGATGGA CAGCACCAAG GCCACAAGGT ATTTGGTAAA AACAGAATCA TGGATCTTGA GGAACCCTGG CTCTTTCGTG AGATCGCTTG TTCTTCCCCC GAACCTACCT GTCGTGGTTC CGGTGTTCCA TAAACCATTT TTGTCTTAGT ACCTAGAACT CCTTGGGACC
prM
■ Y A L V A A V I G W M L G S N T M Q R V V F V V L L L L V A P A Y
2301 ATATGCCCTG GTGGCAGCCG TCATTGGTTG GATGCTTGGG AGCAACACCA TGCAGAGAGT TGTGTTTGTC GTGCTATTGC TTTTGGTGGC CCCAGCTTAC TATACGGGAC CACCGTCGGC AGTAACCAAC CTACGAACCC TCGTTGTGGT ACGTCTCTCA ACACAAACAG CACGATAACG AAAACCACCG GGGTCGAATG
E
p M
S F N C L G M S N R D F L E G V S G A W V D L V L E G D S C V T :
2401 AGCTTTAACT GCCTTGGAAT GAGCAACAGA GACTTCTTGG AAGGAGTGTC TGGAGCAACA TGGGTGGATT TGGTTCTCGA AGGCGACAGC TGCGTGACTA TCGAAATTGA CGGAACCTTA CTCGTTGTCT CTGAAGAACC TTCCTCACAG ACCTCGTTGT ACCCACCTAA ACCAAGAGCT TCCGCTGTCG ACGCACTGAT M S K D K P T I D V K M M N E A A N L A E V R S Y C Y L A T V S
2501 TCATGTCTAA GGACAAGCCT ACCATCGATG TGAAGATGAT GAATATGGAG GCGGCCAACC TGGCAGAGGT CCGCAGTTAT TGCTATTTGG CTACCGTCAG AGTACAGATT CCTGTTCGGA TGGTAGCTAC ACTTCTACTA CTTATACCTC CGCCGGTTGG ACCGTCTCCA GGCGTCAATA ACGATAAACC GATGGCAGTC
E
■ D L S T A A C P A M G E A H N D K R A D P A F V C R Q G V V D R
2601 CGATCTCTCC ACCAAAGCTG CGTGCCCGGC CATGGGAGAA GCTCACAATG ACAAACGTGC TGACCCAGCT TTTGTGTGCA GACAAGGAGT GGTGGACAGG GCTAGAGAGG TGGTTTCGAC GCACGGGCCG GTACCCTCTT CGAGTGTTAC TGTTTGCACG ACTGGGTCGA AAACACACGT CTGTTCCTCA CCACCTGTCC E
G W G N G C G L F G K G S Γ D T C A K F A C S T K A I G R T I L K I
2701 GGCTGGGGCA ACGGCTGCGG ACTATTTGGC AAAGGAAGCA TTGACACATG CGCCAAATTT GCCTGCTCTA CCAAGGCAAT AGGAAGAACC ATTTTGAAAG CCGACCCCGT TGCCGACGCC TGATAAACCG TTTCCTTCGT AACTGTGTAC GCGGTTTAAA CGGACGAGAT GGTTCCGTTA TCCTTCTTGG TAAAACTTTC
E
■ I K Y E V A I F V H G P T T V E S H G N Y S T Q V G A T Q A G R
2801 AGAATATCAA GTACGAAGTG GCCATTTTTG TCCATGGACC AACTACTGTG GAGTCGCACG GAAACTACTC CACACAGGTT GGAGCCACTC AGGCAGGGAG TCTTATAGTT CATGCTTCAC CGGTAAAAAC AGGTACCTGG TTGATGACAC CTCAGCGTGC CTTTGATGAG GTGTGTCCAA CCTCGGTGAG TCCGTCCCTC
E
■ F S I T P A A P S Y T L K L G E Y G E V T V D C E P R S G I D T N
2901 ATTCAGCATC ACTCCTGCGG CGCCTTCATA CACACTAAAG CTTGGAGAAT ATGGAGAGGT GACAGTGGAC TGTGAACCAC GGTCAGGGAT TGACACCAAT TAAGTCGTAG TGAGGACGCC GCGGAAGTAT GTGTGATTTC GAACCTCTTA TACCTCTCCA CTGTCACCTG ACACTTGGTG CCAGTCCCTA ACTGTGGTTA E
A Y Y V M T V G T K T F L V H R E W F M D L N L P W S S A G S T V 5
3001 GCATACTACG TGATGACTGT TGGAACAAAG ACGTTCTTGG TCCATCGTGA GTGGTTCATG GACCTCAACC TCCCTTGGAG CAGTGCTGGA AGTACTGTGT CGTATGATGC ACTACTGACA ACCTTGTTTC TGCAAGAACC AGGTAGCACT CACCAAGTAC CTGGAGTTGG AGGGAACCTC GTCACGACCT TCATGACACA
E
■ R N R E T L M E F E E P H A T K Q S V ] [ A L G S Q E G A L H Q A L
3101 GGAGGAACAG AGAGACGTTA ATGGAGTTTG AGGAACCACA CGCCACGAAG CAGTCTGTGA TAGCATTGGG CTCACAAGAG GGAGCTCTGC ATCAAGCTTT CCTCCTTGTC TCTCTGCAAT TACCTCAAAC TCCTTGGTGT GCGGTGCTTC GTCAGACACT ATCGTAACCC GAGTGTTCTC CCTCGAGACG TAGTTCGAAA
E
■ A G A I P V E F S S N T V K T S G H L K C R V K M E K L Q L K G
3201 GGCTGGAGCC ATTCCTGTGG AATTTTCAAG CAACACTGTC AAGTTGACGT CGGGTCATTT GAAGTGTAGA GTGAAGATGG AAAAATTGCA GTTGAAGGGA CCGACCTCGG TAAGGACACC TTAAAAGTTC GTTGTGACAG TTCAACTGCA GCCCAGTAAA CTTCACATCT CACTTCTACC TTTTTAACGT CAACTTCCCT E
T T Y G V C S K A F K F L G T P A D T G H G T V V L E L Q Y T G T I
3301 ACAACCTATG GCGTCTGTTC AAAGGCTTTC AAGTTTCTTG GGACTCCCGC AGACACAGGT CACGGCACTG TGGTGTTGGA ATTGCAGTAC ACTGGCACGG TGTTGGATAC CGCAGACAAG TTTCCGAAAG TTCAAAGAAC CCTGAGGGCG TCTGTGTCCA GTGCCGTGAC ACCACAACCT TAACGTCATG TGACCGTGCC
E
• G P C K V P I S S V A S L N D L T P V G R L V T V N P F V S V A T
3401 ATGGACCTTG CAAAGTTCCT ATCTCGTCAG TGGCTTCATT GAACGACCTA ACGCCAGTGG GCAGATTGGT CACTGTCAAC CCTTTTGTTT CAGTGGCCAC TACCTGGAAC GTTTCAAGGA TAGAGCAGTC ACCGAAGTAA CTTGCTGGAT TGCGGTCACC CGTCTAACCA GTGACAGTTG GGAAAACAAA GTCACCGGTG
E
• A N A K V L ] [ E L E P P F G D S Y I V V G R G E Q Q ] [ N H H W H K
3501 GGCCAACGCT AAGGTCCTGA TTGAATTGGA ACCACCCTTT GGAGACTCAT ACATAGTGGT GGGCAGAGGA GAACAACAGA TCAATCACCA CTGGCACAAG CCGGTTGCGA TTCCAGGACT AACTTAACCT TGGTGGGAAA CCTCTGAGTA TGTATCACCA CCCGTCTCCT CTTGTTGTCT AGTTAGTGGT GACCGTGTTC E
S G S S I G K A F T T T L K G A Q R L A A L G D A W D F G S V G C
3601 TCTGGAAGCA GCATTGGCAA AGCCTTTACA ACCACCCTCA AAGGAGCGCA GAGACTAGCC GCTCTAGGAG ACACAGCTTG GGACTTTGGA TCAGTTGGAG AGACCTTCGT CGTAACCGTT TCGGAAATGT TGGTGGGAGT TTCCTCGCGT CTCTGATCGG CGAGATCCTC TGTGTCGAAC CCTGAAACCT AGTCAACCTC
E
• V F T S V G K A V H Q V F G G A F R S L F G G M S W I T Q G L L G
3701 GGGTGTTCAC CTCAGTTGGG AAGGCTGTCC ATCAAGTGTT CGGAGGAGCA TTCCGCTCAC TGTTCGGAGG CATGTCCTGG ATAACGCAAG GATTGCTGGG CCCACAAGTG GAGTCAACCC TTCCGACAGG TAGTTCACAA GCCTCCTCGT AAGGCGAGTG ACAAGCCTCC GTACAGGACC TATTGCGTTC CTAACGACCC
E
• A L L L W M G I N A R D R S I A L T F L A V G G V L L F L S V N V
3801 GGCTCTCCTG TTGTGGATGG GCATCAATGC TCGTGACAGG TCCATAGCTC TCACGTTTCT CGCAGTTGGA GGAGTTCTGC TCTTCCTCTC CGTGAACGTG CCGAGAGGAC AACACCTACC CGTAGTTACG AGCACTGTCC AGGTATCGAG AGTGCAAAGA GCGTCAACCT CCTCAAGACG AGAAGGAGAG GCACTTGCAC
E H A D T G C A I D I S R Q E L R C G S G V F I H N D V E A
3901 CACGCTGACA CTGGGTGTGC CATAGAC TC AGCCGGCAAG AGCTGAGATG TGGAAGTGGA GTGTTCATAC ACAATGATGT GGAGGCTTGG ATGGACCGGT GTGCGACTGT GACCCACACG GTATCTGTAG TCGGCCGTTC TCGACTCTAC ACCTTCACCT CACAAGTATG TGTTACTACA CCTCCGAACC TACCTGGCCA
(AprME)-Rabies G PIV sequence (partial; SEQ ID NOs:64-66)
C protein
5' UTR
M S
1 AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT
TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
C protein
■ K K P G G P G K S R A V Y L L K R G M E R V L S L I G L K R A M L
CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGA GGGCTATGTT GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCT CCCGATACAA
C protein
■ S L I D G K G P I R F V L A L L A F F R F T A I A P 1 R A V L D R
GAGCCTGATC GACGGCAAGG GGCCAATACG ATTTGTGTTG GCTCTCTTGG CGTTCTTCAG GTTCACAGCA ATTGCTCCGA CCCGAGCAGT GCTGGATCGA CTCGGACTAG CTGCCGTTCC CCGGTTATGC TAAACACAAC CGAGAGAACC GCAAGAAGTC CAAGTGTCGT TAACGAGGCT GGGCTCGTCA CGACCTAGCT
C protein
W R G V N K Q T A M K H L L S F K K E L G T L T S A I N R R S S K (
TGGAGAGGTG TGAACAAACA AACAGCGATG AAACACCTTC TGAGTTTCAA GAAGGAACTA GGGACCTTGA CCAGTGCTAT CAATCGGCGG AGCTCAAAGC ACCTCTCCAC ACTTGTTTGT G CGC AC TTTGTGGAAG ACTCAAAGTT CTTCCTTGAT CCCTGGAACT GGTCACGATA GTTAGCCGCC TCGAGTTTCG
Rabies-G signal
C protein partial C signal RAbies-G protein
K K R G G K T G I A V I V P Q A L L F V P L L V F P L C F G K F P '
401 AAAAGAAGCG AGGGGGCAAG ACTGGTATAG CTGTGATCGT TCCTCAGGCT CTTTTGTTTG TACCCTTGCT GGTATTTCCC CTTTGCTTTG GTAAATTTCC TTTTCTTCGC TCCCCCGTTC TGACCATATC GACACTAGCA AGGAGTCCGA GAAAACAAAC ATGGGAACGA CCATAAAGGG GAAACGAAAC CATTTAAAGG RAbies-G protein
■ I Y T I P D K L G P S P I D I H H L S C P N N L V V E D E G C T
501 TATCTATACC ATCCCTGATA AGCTCGGGCC TTGGAGTCCC A TGATAT C ACCATTTGAG CTGCCCAAAC AACCTCGTCG TTGAGGATGA AGGGTGCACT ATAGATATGG TAGGGACTAT TCGAGCCCGG AACCTCAGGG TAACTATAAG TGGTAAACTC GACGGGTTTG TTGGAGCAGC AACTCCTACT TCCCACGTGA RAbies-G protein
N L S G F S Y M E L K G I S A I K M N G F T C T G V V T E A E '
601 AATCTTTCTG GATTTTCCTA CATGGAGTTG AAAGTGGGCT ATATTTCAGC CATTAAGATG AACGGCTTTA CTTGTACAGG AGTCGTGACC GAAGCCGAGA TTAGAAAGAC CTAAAAGGAT GTACCTCAAC TTTCACCCGA TATAAAGTCG GTAA CTAC TTGCCGAAAT GAACATGTCC TCAGCACTGG CTTCGGCTCT RAbies-G protein
■ Y T N F V G Y V T T T F K R K H F R P T P D A C R A A Y N W K M A
701 CATATACAAA TTTCGTGGGA TACGTCACCA CCACCTTCAA GAGAAAACAC TTCCGCCCAA CGCCTGACGC TTGTCGGGCC GCTTACAACT GGAAGATGGC GTATATGTTT AAAGCACCCT ATGCAGTGGT GGTGGAAGTT CTCTTTTGTG AAGGCGGGTT GCGGACTGCG AACAGCCCGG CGAATGTTGA CCTTCTACCG RAbies-G protein
■ G D P R Y E Ξ S L H N P Y P D Y H W L R T V K T T K E S L V I I S
801 AGGAGATCCT CGATATGAAG AATCTCTGCA CAACCCGTAT CCTGATTACC ATTGGCTGCG GACAGTCAAG ACTACCAAGG AGAGTCTGGT CATTATATCA TCCTCTAGGA GCTATACTTC TTAGAGACGT GTTGGGCATA GGACTAATGG TAACCGACGC CTGTCAGTTC TGATGGTTCC TCTCAGACCA GTAATATAGT RAbies-G protein
P S V A D L D P Y D R S L H S R V F P G G N C £ I G V A V S S T Y C i
901 CCAAGCGTGG CCGATCTTGA TCCTTATGAT AGATCCCTGC ACAGTAGGGT TTTTCCTGGC GGGAATTGTA GCGGTGTTGC AGTATCAAGT ACCTACTGCT GGTTCGCACC GGCTAGAACT AGGAATACTA TCTAGGGACG TGTCATCCCA AAAAGGACCG CCCTTAACAT CGCCACAACG TCATAGTTCA TGGATGACGA RAbies-G protein
■ T N H D Y T I W M P E N P R L G M S C D I F T N S R G K R A S K G 1001 CCACTAACCA CGACTACACT ATATGGATGC CTGAGAACCC TCGACTCGGT ATGAGTTGCG ACATTTTTAC GAACTCACGG GGCAAGCGGG CATCTAAGGG GGTGATTGGT GCTGATGTGA TATACCTACG GACTCTTGGG AGCTGAGCCA TACTCAACGC TGTAAAAATG CTTGAGTGCC CCGTTCGCCC GTAGATTCCC
RAbies-G protein
■ S E T C G F V D E R G L Y K S L K G A C K L K L C G V L G L R L M
HOI GTCTGAAACA TGCGGGTTTG TTGATGAGCG GGGGTTGTAT AAATCTCTTA AAGGCGCCTG TAAGCTGAAA CTCTGTGGCG TACTGGGGCT GCGCCTGATG CAGACTTTGT ACGCCCAAAC AACTACTCGC CCCCAACATA TTTAGAGAAT TTCCGCGGAC ATTCGACTTT GAGACACCGC ATGACCCCGA CGCGGACTAC RAbies-G protein
D G T W V A M Q T S N E T K W C P P G Q L V N L H D F R S D E I E I
1201 GACGGCACAT GGGTGGCTAT GCAGACAAGC AATGAAACAA AGTGGTGTCC CCCTGGTCAG CTGGTTAATC TGCACGACTT TAGGTCTGAC GAAATCGAGC CTGCCGTGTA CCCACCGATA CGTCTGTTCG TTACTTTGTT TCACCACAGG GGGACCAGTC GACCAATTAG ACGTGCTGAA ATCCAGACTG CTTTAGCTCG RAbies-G protein
• L V V E E L V K K R E E C L D A L E S I M T T K S V S F R R L S H
1301 ACCTTGTGGT GGAGGAACTG GTGAAGAAAC GCGAAGAGTG CCTGGACGCA CTTGAGAGTA TTATGACCAC CAAATCCGTT TCCTTCAGAA GACTGAGCCA TGGAACACCA CCTCCTTGAC CACTTCTTTG CGCTTCTCAC GGACCTGCGT GAACTCTCAT AATACTGGTG GTTTAGGCAA AGGAAGTCTT CTGACTCGGT RAbies-G protein
• L R K L V P G F G K A Y T I F N K T L M E A D A H Y K S V R T W N
1401 CCTGCGAAAG CTGGTGCCAG GGTTCGGGAA GGCTTATACT ATTTTCAACA AGACTCTTAT GGAGGCGGAT GCCCATTATA AGTCAGTTAG GACTTGGAAT GGACGCTTTC GACCACGGTC CCAAGCCCTT CCGAATATGA TAAAAGTTGT TCTGAGAATA CCTCCGCCTA CGGGTAATAT TCAGTCAATC CTGAACCTTA RAbies-G protein
E I I P S K G C L R V G G R C H P H V N G V F ] N G I I L G P D G I
1501 GAGATAATTC CCTCCAAAGG ATGTCTGAGA GTCGGTGGGA GATGCCACCC CCATGTCAAT GGGGTGTTCT TTAACGGAAT CATCCTGGGA CCTGACGGGA CTCTATTAAG GGAGGTTTCC TACAGACTCT CAGCCACCCT CTACGGTGGG GGTACAGTTA CCCCACAAGA AATTGCCTTA GTAGGACCCT GGACTGCCCT RAbies-G protein
• V L I P E M Q S S L L Q Q H E L L V S S V I P L M H P L A D P S
1601 ACGTGCTGAT TCCCGAGATG CAATCTTCCC TTCTGCAGCA ACACATGGAA CTCCTGGTGT CTTCAGTGAT ACCCCTGATG CACCCACTGG CCGACCCCAG TGCACGACTA AGGGCTCTAC GTTAGAAGGG AAGACGTCGT TGTGTACCTT GAGGACCACA GAAGTCACTA TGGGGACTAC GTGGGTGACC GGCTGGGGTC RAbies-G protein
■ T V F K N G D E A E D F V E V H L P D V H E R I S G V D L G L P N
1701 CACTGTGTTC AAAAATGGCG ATGAGGCCGA AGACTTTGTG GAAGTTCACC TGCCCGATGT ACACGAAAGG ATATCTGGAG TAGACCTGGG CCTTCCTAAT GTGACACAAG TTTTTACCGC TACTCCGGCT TCTGAAACAC CTTCAAGTGG ACGGGCTACA TGTGCTTTCC TATAGACCTC ATCTGGACCC GGAAGGATTA RAbies-G protein
W G K Y V L L S A G A L T A L M L I I F L M T C W R R V N R S E P
1801 TGGGGTAAGT ACGTGCTCCT GAGTGCGGGT GCCTTGACCG CTTTGATGCT GATCATTTTT CTGATGACCT GCTGGCGGAG GGTGAATCGC TCCGAGCCGA ACCCCATTCA TGCACGAGGA CTCACGCCCA CGGAACTGGC GAAACTACGA CTAGTAAAAA GACTACTGGA CGACCGCCTC CCACTTAGCG AGGCTCGGCT RAbies-G protein
• Q H N L R G T G R E V S V T P Q S G K ] : I s s W E S Y K S G G E T
1901 CACAGCACAA TCTCAGAGGG ACAGGCCGGG AAGTAAGTGT GACTCCGCAA TCTGGCAAGA TTATTAGTAG TTGGGAGAGT TACAAGTCTG GAGGAGAGAC GTGTCGTGTT AGAGTCTCCC TGTCCGGCCC TTCATTCACA CTGAGGCGTT AGACCGTTCT AATAATCATC AACCCTCTCA ATGTTCAGAC CTCCTCTCTG FMDV 2A NS1 signal
RAbies-G protein preNSl signal
■ G L N F D L L K L A G D V E S N P G P A R D R S I A L T F L A V G
2001 TGGGTTGAAT TTTGATCTGC TCAAACTTGC AGGCGATGTA GAATCAAATC CTGGACCCGC CCGGGACAGG TCCATAGCTC TCACGTTTCT CGCAGTTGGA ACCCAACTTA AAACTAGACG AGTTTGAACG TCCGCTACAT CTTAGTTTAG GACCTGGGCG GGCCCTGTCC AGGTATCGAG AGTGCAAAGA GCGTCAACCT NS1
NS1 signal
G V L L F L S V N V H A D T G C A I D I S R Q E L R C G S G V F I I
2101 GGAGTTCTGC TCTTCCTCTC CGTGAACGTG CACGCTGACA CTGGGTGTGC CATAGACATC AGCCGGCAAG AGCTGAGATG TGGAAGTGGA GTGTTCATAC CCTCAAGACG AGAAGGAGAG GCACTTGCAC GTGCGACTGT GACCCACACG GTATCTGTAG TCGGCCGTTC TCGACTCTAC ACCTTCACCT CACAAGTATG Sequence Appendix 4 (continued)
PIV-WNV helper ANSI (SEQ ID NOs:67-69)
UTR
M S
1 AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
C
• K K P G G P G K S R A V N M L K R G M P R V L S L I G L K R A M L 101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCAA TATGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGA GGGCTATGTT GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGTT ATACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCT CCCGATACAA
C
• S L I D G K G P I R F V L A L L A F F R F T A I A P T R A V L D R 201 GAGCCTGATC GACGGCAAGG GGCCAATACG ATTTGTGTTG GCTCTCTTGG CGTTCTTCAG GTTCACAGCA ATTGCTCCGA CCCGAGCAGT GCTGGATCGA CTCGGACTAG CTGCCGTTCC CCGGTTATGC TAAACACAAC CGAGAGAACC GCAAGAAGTC CAAGTGTCGT TAACGAGGCT GGGCTCGTCA CGACCTAGCT
C R G V N K Q T A M K H L L S F K K E L G T L T S A I N R R S S K Q
301 TGGAGAGGTG TGAACAAACA AACAGCGATG AAACACCTTC TGAGTTTCAA GAAGGAACTA GGGACCTTGA CCAGTGCTAT CAATCGGCGG AGCTCAAAAC ACCTCTCCAC ACTTGTTTGT TTGTCGCTAC TTTGTGGAAG ACTCAAAGTT CTTCCTTGAT CCCTGGAACT GGTCACGATA GTTAGCCGCC TCGAGTTTTG
Signal peptide
prM
• K K R G G T G I A V M I G L I A S V G A V T L S N F Q G V M M 401 AAAAGAAAAG AGGAGGAAAG ACCGGAATTG CAGTCATGAT TGGCCTGATC GCCAGCGTAG GAGCAGTTAC CCTCTCTAAC TTCCAAGGGA AGGTGATGAT TTTTCTTTTC TCCTCCTTTC TGGCCTTAAC GTCAGTACTA ACCGGACTAG CGGTCGCATC CTCGTCAATG GGAGAGATTG AAGGTTCCCT TCCACTACTA
prM
• T V N A T D V T D V I T I P T A A G K N L C I V R A M D V G Y M C 501 GACGGTAAAT GCTACTGACG TCACAGATGT CATCACGATT CCAACAGCTG CTGGAAAGAA CCTATGCATT GTCAGAGCAA TGGATGTGGG ATACATGTGC CTGCCATTTA CGATGACTGC AGTGTCTACA GTAGTGCTAA GGTTGTCGAC GACCTTTCTT GGATACGTAA CAGTCTCGTT ACCTACACCC TATGTACACG
prM
D D T I T Y E C P V L S A G N D P E D I D C W C T S A V Y V R Y G
601 GATGATACTA TCACTTATGA ATGCCCAGTG CTGTCGGCTG GTAATGATCC AGAAGACATC GACTGTTGGT GCACAAAGTC AGCAGTCTAC GTCAGGTATG CTACTATGAT AGTGAATACT TACGGGTCAC GACAGCCGAC CATTACTAGG TCTTCTGTAG CTGACAACCA CGTGTTTCAG TCGTCAGATG CAGTCCATAC
prM
• R C T K T R H S R R S R R S L T V Q T H G E S T L A N K K G A W M 701 GAAGATGCAC CAAGACACGC CACTCAAGAC GCAGTCGGAG GTCACTGACA GTGCAGACAC ACGGAGAAAG CACTCTAGCG AACAAGAAGG GGGCTTGGAT CTTCTACGTG GTTCTGTGCG GTGAGTTCTG CGTCAGCCTC CAGTGACTGT CACGTCTGTG TGCCTCTTTC GTGAGATCGC TTGTTCTTCC CCCGAACCTA
prM
■ D S T K A T R Y L V K T E S W I L R N P G Y A L V A A V I G W M L 801 GGACAGCACC AAGGCCACAA GGTATTTGGT AAAAACAGAA TCATGGATCT TGAGGAACCC TGGATATGCC CTGGTGGCAG CCGTCATTGG TTGGATGCTT CCTGTCGTGG TTCCGGTGTT CCATAAACCA TTTTTGTCTT AGTACCTAGA ACTCCTTGGG ACCTATACGG GACCACCGTC GGCAGTAACC AACCTACGAA
E
prM
G S N T M Q R V V F V V L L L L V A P A Y S F N C L G M S N R D F L
901 GGGAGCAACA CCATGCAGAG AGTTGTGTTT GTCGTGCTAT TGCTTTTGGT GGCCCCAGCT TACAGCTTTA ACTGCCTTGG AATGAGCAAC AGAGACTTCT CCCTCGTTGT GGTACGTCTC TCAACACAAA CAGCACGATA ACGAAAACCA CCGGGGTCGA ATGTCGAAAT TGACGGAACC TTACTCGTTG TCTCTGAAGA
• E G V S G A T W V D L V L E G D S C V T I M S K D K P T I D V K M 1001 TGGAAGGAGT GTCTGGAGCA ACATGGGTGG ATTTGGTTCT CGAAGGCGAC AGCTGCGTGA CTATCATGTC TAAGGACAAG CCTACCATCG ATGTGAAGAT ACCTTCCTCA CAGACCTCGT TGTACCCACC TAAACCAAGA GCTTCCGCTG TCGACGCACT GATAGTACAG ATTCCTGTTC GGATGGTAGC TACACTTCTA
E
• M N M E A A N L A E V R S Y C Y L A T V S D L S T K A A C P A M G 1101 GATGAATATG GAGGCGGCCA ACCTGGCAGA GGTCCGCAGT TATTGCTATT TGGCTACCGT CAGCGATCTC TCCACCAAAG CTGCGTGCCC GGCCATGGGA CTACTTATAC CTCCGCCGGT TGGACCGTCT CCAGGCGTCA ATAACGATAA ACCGATGGCA GTCGCTAGAG AGGTGGTTTC GACGCACGGG CCGGTACCCT
E
E A H N D R A D P A F V C R Q G V V D R G G N G C G L F G K G S
1201 GAAGCTCACA ATGACAAACG TGCTGACCCA GCTTTTGTGT GCAGACAAGG AGTGGTGGAC AGGGGCTGGG GCAACGGCTG CGGACTATTT GGCAAAGGAA CTTCGAGTGT TACTGTTTGC ACGACTGGGT CGAAAACACA CGTCTGTTCC TCACCACCTG TCCCCGACCC CGTTGCCGAC GCCTGATAAA CCGTTTCCTT
• I D T C A K F A C S T K A I G R T I L K E N I Y E V A I F V H G 1301 GCATTGACAC ATGCGCCAAA TTTGCCTGCT CTACCAAGGC AATAGGAAGA ACCATTTTGA AAGAGAATAT CAAGTACGAA GTGGCCATTT TTGTCCATGG CGTAACTGTG TACGCGGTTT AAACGGACGA GATGGTTCCG TTATCCTTCT TGGTAAAACT TTCTCTTATA GTTCATGCTT CACCGGTAAA AACAGGTACC
E
• P T T V E S H G N Y S T Q V G A T Q A G R F S I T P A A P S Y T L 1401 ACCAACTACT GTGGAGTCGC ACGGAAACTA CTCCACACAG GTTGGAGCCA CTCAGGCAGG GAGATTCAGC ATCACTCCTG CGGCGCCTTC ATACACACTA TGGTTGATGA CACCTCAGCG TGCCTTTGAT GAGGTGTGTC CAACCTCGGT GAGTCCGTCC CTCTAAGTCG TAGTGAGGAC GCCGCGGAAG TATGTGTGAT
E
K L G E Y G E V T V D C E P R S G I D T N A Y Y V M T V G T K T F L
1501 AAGCTTGGAG AATATGGAGA GGTGACAGTG GACTGTGAAC CACGGTCAGG GATTGACACC AATGCATACT ACGTGATGAC TGTTGGAACA AAGACGTTCT TTCGAACCTC TTATACCTCT CCACTGTCAC CTGACACTTG GTGCCAGTCC CTAACTGTGG TTACGTATGA TGCACTACTG ACAACCTTGT TTCTGCAAGA
• V H R E W F M D L N L P S S A G S T V W R N R E T L M E F E E P - 1601 TGGTCCATCG TGAGTGGTTC ATGGACCTCA ACCTCCCTTG GAGCAGTGCT GGAAGTACTG TGTGGAGGAA CAGAGAGACG TTAATGGAGT TTGAGGAACC ACCAGGTAGC ACTCACCAAG TACCTGGAGT TGGAGGGAAC CTCGTCACGA CCTTCATGAC ACACCTCCTT GTCTCTCTGC AATTACCTCA AACTCCTTGG
■ H A T K Q S V I A L G S Q E G A L H Q A L A G A I P V E F S S N T 1701 ACACGCCACG AAGCAGTCTG TGATAGCATT GGGCTCACAA GAGGGAGCTC TGCATCAAGC TTTGGCTGGA GCCATTCCTG TGGAATTTTC AAGCAACACT TGTGCGGTGC TTCGTCAGAC ACTATCGTAA CCCGAGTGTT CTCCCTCGAG ACGTAGTTCG AAACCGACCT CGGTAAGGAC ACCTTAAAAG TTCGTTGTGA
E
V K L T S G H L C R V K M E K L Q L K G T T Y G V C S K A F K F L
1801 GTCAAGTTGA CGTCGGGTCA TTTGAAGTGT AGAGTGAAGA TGGAAAAATT GCAGTTGAAG GGAACAACCT ATGGCGTCTG TTCAAAGGCT TTCAAGTTTC
CAGTTCAACT GCAGCCCAGT AAACTTCACA TCTCACTTCT ACCTTTTTAA CGTCAACTTC CCTTGTTGGA TACCGCAGAC AAGTTTCCGA AAGTTCAAAG
E
• G T P A D T G H G T V V L E L Q Y T G T D G P C K V P I S S V A S
1901 TTGGGACTCC CGCAGACACA GGTCACGGCA CTGTGGTGTT GGAATTGCAG TACACTGGCA CGGATGGACC TTGCAAAGTT CCTATCTCGT CAGTGGCTTC
AACCCTGAGG GCGTCTGTGT CCAGTGCCGT GACACCACAA CCTTAACGTC ATGTGACCGT GCCTACCTGG AACGTTTCAA GGATAGAGCA GTCACCGAAG
E
■ L N D L T P V G R L V T V N P F V S V A T A N A K V L I E L E P P
2001 ATTGAACGAC CTAACGCCAG TGGGCAGATT GGTCACTGTC AACCCTTTTG TTTCAGTGGC CACGGCCAAC GCTAAGGTCC TGATTGAATT GGAACCACCC
TAACTTGCTG GATTGCGGTC ACCCGTCTAA CCAGTGACAG TTGGGAAAAC AAAGTCACCG GTGCCGGTTG CGATTCCAGG ACTAACTTAA CCTTGGTGGG
E
F G D S Y I V V G R G E Q Q I N H H H K S G S S I G K A F T T T L
2101 TTTGGAGACT CATACATAGT GGTGGGCAGA GGAGAACAAC AGATCAATCA CCACTGGCAC AAGTCTGGAA GCAGCATTGG CAAAGCCTTT ACAACCACCC AAACCTCTGA GTATGTATCA CCACCCGTCT CCTCTTGTTG TCTAGTTAGT GGTGACCGTG TTCAGACCTT CGTCGTAACC GTTTCGGAAA TGTTGGTGGG
■ K G A Q R L A A L G D T A W D F G S V G G V F T S V G K A V H Q V 2201 TCAAAGGAGC GCAGAGACTA GCCGCTCTAG GAGACACAGC TTGGGACTTT GGATCAGTTG GAGGGGTGTT CACCTCAGTT GGGAAGGCTG TCCATCAAGT AGTTTCCTCG CGTCTCTGAT CGGCGAGATC CTCTGTGTCG AACCCTGAAA CCTAGTCAAC CTCCCCACAA GTGGAGTCAA CCCTTCCGAC AGGTAGTTCA
E
• F G G A F R S L F G G S W I T Q G L L G A L L L W M G I N A R D 2301 GTTCGGAGGA GCATTCCGCT CACTGTTCGG AGGCATGTCC TGGATAACGC AAGGATTGCT GGGGGCTCTC CTGTTGTGGA TGGGCATCAA TGCTCGTGAC CAAGCCTCCT CGTAAGGCGA GTGACAAGCC TCCGTACAGG ACCTATTGCG TTCCTAACGA CCCCCGAGAG GACAACACCT ACCCGTAGTT ACGAGCACTG
deleted NS1
E
R S I A L T F L A V G G V L L F L S V N V H A D T G I H R G P A T R
2401 AGGTCCATAG CTCTCACGTT TCTCGCAGTT GGAGGAGTTC TGCTCTTCCT CTCCGTGAAC GTGCACGCTG ACACTGGGAT CCACCGTGGA CCTGCCACTC TCCAGGTATC GAGAGTGCAA AGAGCGTCAA CCTCCTCAAG ACGAGAAGGA GAGGCACTTG CACGTGCGAC TGTGACCCTA GGTGGCACCT GGACGGTGAG
deleted NS1
• T T T E S G L I T D C C R S C T L P P L R Y Q T D S G C W Y G
2501 GCACCACCAC AGAGAGCGGA AAGTTGATAA CAGATTGGTG CTGCAGGAGC TGCACCTTAC CACCACTGCG CTACCAAACT GACAGCGGCT GTTGGTATGG
CGTGGTGGTG TCTCTCGCCT TTCAACTATT GTCTAACCAC GACGTCCTCG ACGTGGAATG GTGGTGACGC GATGGTTTGA CTGTCGCCGA CAACCATACC
deleted NS1
NS2A
• M E I R P Q R H D E K T L V Q S Q V N A Y N A D M I D P F Q L G L 2601 TATGGAGATC AGACCACAGA GACATGATGA AAAGACCCTC GTGCAGTCAC AAGTGAATGC TTATAATGCT GATATGATTG ACCCTTTTCA GTTGGGCCTT ATACCTCTAG TCTGGTGTCT CTGTACTACT TTTCTGGGAG CACGTCAGTG TTCACTTACG AATATTACGA CTATACTAAC TGGGAAAAGT CAACCCGGAA
Sequence Appendix 5
PIV-WNV(AprME)/RSV-F (SEQ ID NO:70-72)
C protein
OTR
M S
AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
C protein
■ K K P G G P G K S R A V Y L L R G M P R V L S L I G L K R A M L
101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGA GGGCTATGTT
GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCT CCCGATACAA
C protein
■ S L I D G K G P I R F V L A L L A F F R F T A I A P T R A V L D R
201 GAGCCTGATC GACGGCAAGG GGCCAATACG ATTTGTGTTG GCTCTCTTGG CGTTCTTCAG GTTCACAGCA ATTGCTCCGA CCCGAGCAGT GCTGGATCGA
CTCGGACTAG CTGCCGTTCC CCGGTTATGC TAAACACAAC CGAGAGAACC GCAAGAAGTC CAAGTGTCGT TAACGAGGCT GGGCTCGTCA CGACCTAGCT
NS3 cleavage
C protein
W R G V N K Q T A M K H L L S F K E L G T L T S A I N R R S S Q
301 TGGAGAGGTG TGAACAAACA AACAGCGATG AAACACCTTC TGAGTTTCAA GAAGGAACTA GGGACCTTGA CCAGTGCTAT CAATCGGCGG AGCTCAAAGC
ACCTCTCCAC ACTTGTTTGT TTGTCGCTAC TTTGTGGAAG ACTCAAAGTT CTTCCTTGAT CCCTGGAACT GGTCACGATA GTTAGCCGCC TCGAGTTTCG
F signal
NS3 cleavage Fl
• K K R G G E L L I L K A N A I T T I L T A V T F C F A S G Q N I T 401 AAAAGAAGCG AGGGGGCGAG TTGCTAATCC TCAAAGCAAA TGCAATTACC ACAATCCTCA CTGCAGTCAC ATTTTGTTTT GCTTCTGGTC AAAACATCAC
TTTTCTTCGC TCCCCCGCTC AACGATTAGG AGTTTCGTTT ACGTTAATGG TGTTAGGAGT GACGTCAGTG TAAAACAAAA CGAAGACCAG TTTTGTAGTG
Fl
• E E F Y Q S T C S A V S K G Y L S A L R T G W Y T S V I T I E L S 501 TGAAGAATTT TATCAATCAA CATGCAGTGC AGTTAGCAAA GGCTATCTTA GTGCTCTGAG AACTGGTTGG TATACCAGTG TTATAACTAT AGAATTAAGT
ACTTCTTAAA ATAGTTAGTT GTACGTCACG TCAATCGTTT CCGATAGAAT CACGAGACTC TTGACCAACC ATATGGTCAC AATATTGATA TCTTAATTCA
Fl
N I K E N K C N G T D A K V K L I K Q E L D K Y K N A V T E L Q L L - 501 AATATCAAGG AAAATAAGTG TAATGGAACA GATGCTAAGG -TAAAATTGAT AAAACAAGAA TTAGATAAAT ATAAAAATGC TGTAACAGAA TTGCAGTTGC
TTATAGTTCC TTTTATTCAC ATTACCTTGT CTACGATTCC ATTTTAACTA TTTTGTTCTT AATCTATTTA TATTTTTACG ACATTGTCTT AACGTCAACG
Fi
~~ M Q S T P P T N N R A R R E L P R F M N Y ~T L N N A K K T~ N V T L
701 TCATGCAAAG CACACCACCA ACAAACAATC GAGCCAGAAG AGAACTACCA AGGTTTATGA ATTATACACT CAACAATGCC AAAAAAACCA ATGTAACATT
AGTACGTTTC GTGTGGTGGT TGTTTGTTAG CTCGGTCTTC TCTTGATGGT TCCAAATACT TAATATGTGA GTTGTTACGG TTTTTTTGGT TACATTGTAA
Fl
F2
• S K K R K R R F L G F L L G V G S A I A S G V A V S' K V L H L E G
801 AAGCAAGAAA AGGAAAAGAA GATTTCTTGG TTTTTTGTTA GGTGTTGGAT CTGCAATCGC CAGTGGCGTT GCTGTATCTA AGGTCCTGCA CCTAGAAGGG
TTCGTTCTTT TCCTTTTCTT CTAAAGAACC AAAAAACAAT CCACAACCTA GACGTTAGCG GTCACCGCAA CGACATAGAT TCCAGGACGT GGATCTTCCC
F2
E V N K I K S A L L S T N K A V V S L S N G V S V L T S K V L D L K
901 GAAGTGAACA AGATCAAAAG TGCTCTACTA TCCACAAACA AGGCTGTAGT CAGCTTATCA AATGGAGTTA GTGTCTTAAC CAGCAAAGTG TTAGACCTCA CTTCACTTGT TCTAGTTTTC ACGAGATGAT AGGTGTTTGT TCCGACATCA GTCGAATAGT TTACCTCAAT CACAGAATTG GTCGTTTCAC AATCTGGAGT
F2
• N Y I D K Q L L P I V N K Q S C S I S K I E T V I E F Q Q N N R 1001 AAAACTATAT AGATAAACAA TTGTTACCTA TTGTGAACAA GCAAAGCTGC AGCATATCAA ATATAGAAAC TGTGATAGAG TTCCAACAAA AGAACAACAG TTTTGATATA TCTATTTGTT AACAATGGAT AACACTTGTT CGTTTCGACG TCGTATAGTT TATATCTTTG ACACTATCTC AAGGTTGTTT TCTTGTTGTC
F2
• L L E I T R E F S V N A G V T T P V S T Y M L T N S E L L S L I N 1101 ACTACTAGAG ATTACCAGGG AATTTAGTGT TAATGCAGGT GTAACTACAC CTGTAAGCAC TTACATGTTA ACTAATAGTG AATTATTGTC ATTAATCAAT TGATGATCTC TAATGGTCCC TTAAATCACA ATTACGTCCA CATTGATGTG GACATTCGTG AATGTACAAT TGATTATCAC TTAATAACAG TAATTAGTTA
F2
D M P I T N D Q K K L M S N N V Q I V R Q Q S Y S I M S I I K E E V
1201 GATATGCCTA TAACAAATGA TCAGAAAAAG TTAATGTCCA ACAATGTTCA AATAGTTAGA CAGCAAAGTT ACTCTATCAT GTCCATAATA AAAGAGGAAG CTATACGGAT ATTGTTTACT AGTCTTTTTC AATTACAGGT TGTTACAAGT TTATCAATCT GTCGTTTCAA TGAGATAGTA CAGGTATTAT TTTCTCCTTC
F2
■ L A Y V V Q L P L Y G V I D T P C W K L H T S P L C T T N T K E G 1301 TCTTAGCATA TGTAGTACAA TTACCACTAT ATGGTGTTAT AGATACACCC TGTTGGAAAC CAC CATC CCCTCTATGT ACAACCAACA CAAAAGAAGG AGAATCGTAT ACATCATGTT AATGGTGATA TACCACAATA TCTATGTGGG ACAACCTTTG ATGTGTGTAG GGGAGATACA TGTTGGTTGT GTTTTCTTCC
F2
■ S N I C L T R T D R G W Y C D N A G S V S F F P Q A E T C K V Q S
1401 GTCCAACATC TGTTTAACAA GAACTGACAG AGGATGGTAC TGTGACAATG CAGGATCAGT ATCTTTCTTC CCACAAGCTG AAACATGTAA AGTTCAATCA
CAGGTTGTAG ACAAATTGTT CTTGACTGTC TCCTACCATG ACACTGTTAC GTCCTAGTCA TAGAAAGAAG GGTGTTCGAC TTTGTACATT TCAAGTTAGT
F2
N R V F C D T M N S L T L P S E I N L C N V D I F N P K Y D C K I M
1501 AATCGAGTAT TTTGTGACAC AATGAACAGT TTAACATTAC CAAGTGAAAT AAATCTCTGC AATGTTGACA TATTCAACCC CAAATATGAT TGTAAAATTA TTAGCTCATA AAACACTGTG TTACTTGTCA AATTGTAATG GTTCACTTTA TTTAGAGACG TTACAACTGT ATAAGTTGGG GTTTATACTA ACATTTTAAT
F2
■ T S K T D V S S S V I T S L G A I V S C Y G K T K C T A S N K N R 1601 TGACTTCAAA AACAGATGTA AGCAGCTCCG TTATCACATC TCTAGGAGCC ATTGTGTCAT GCTATGGCAA AACTAAATGT ACAGCATCCA ATAAAAATCG ACTGAAGTTT TTGTCTACAT TCGTCGAGGC AATAGTGTAG AGATCCTCGG TAACACAGTA CGATACCGTT TTGATTTACA TGTCGTAGGT TATTTTTAGC
F2
• G I I K T F S N G C D Y V S N K G M D T V S V G N T L Y Y V N K Q 1701 TGGAATCATA AAGACATTTT CTAACGGGTG CGATTATGTA TCAAATAAAG GGATGGACAC TGTGTCTGTA GGTAACACAT TATATTATGT AAATAAGCAA ACCTTAGTAT TTCTGTAAAA GATTGCCCAC GCTAATACAT AGTTTATTTC CCTACCTGTG ACACAGACAT CCATTGTGTA ATATAATACA TTTATTCGTT
F2
E G K S L Y V K G E P I I N F Y D P L V F P S D E F D A S I S Q V N
1801 GAAGGTAAAA GTCTCTATGT AAAAGGTGAA CCAATAATAA ATTTCTATGA CCCATTAGTA TTCCCCTCTG ATGAATTTGA TGCATCAATA TCTCAAGTCA CTTCCATTTT CAGAGATACA TTTTCCACTT GGTTATTATT TAAAGATACT GGGTAATCAT AAGGGGAGAC TACTTAAACT ACGTAGTTAT AGAGTTCAGT
F2
M Domain
• E K I N Q S L A F I R K S D E L L H N V N A G S T T N I M I T T
1901 ACGAGAAGAT TAACCAGAGC CTAGCATTTA TTCGTAAATC CGATGAATTA TTACATAATG TAAATGCTGG TAAATCCACC ACAAATATCA TGATAACTAC
TGCTCTTCTA ATTGGTCTCG GATCGTAAAT AAGCATTTAG GCTACTTAAT AATGTATTAC ATTTACGACC ATTTAGGTGG TGTTTATAGT ACTATTGATG
TM Domain
Cytoplasmic Tail
• I I I V I I V I L L S L I A V G L L L Y C K A R S T P V T L S K D
2001 TATAATTATA GTGATTATAG TAATATTGTT ATCATTAATT GCTGTTGGAC TGCTCTTATA CTGTAAGGCC AGAAGCACAC CAGTCACACT AAGCAAAGAT
ATATTAATAT CACTAATATC ATTATAACAA TAGTAATTAA CGACAACCTG ACGAGAATAT GACATTCCGG TCTTCGTGTG GTCAGTGTGA TTCGTTTCTA
FMDV 2A
Transmembrane domain of WNV E (split)
Cytoplasmic Tail pre E/NS1 signal
Q L S G I N N I A F S N N F D L L K L A G D V E S N P G P A R D R S
2101 CAACTGAGTG GTATAAATAA TATTGCATTT AGTAACAATT TTGATCTGCT CAAACTTGCA GGCGATGTAG AATCAAATCC TGGACCCGCC CGGGACAGGT
GTTGACTCAC CATATTTATT ATAACGTAAA TCATTGTTAA AACTAGACGA GTTTGAACGT CCGCTACATC TTAGTTTAGG ACCTGGGCGG GCCCTGTCCA
NS1
Transmembrane domain of WNV E (split)
• I A L T F L A V G G V L L F L S V N V H A D T G C A I D I S R Q
2201 CCATAGCTCT CACGTTTCTC GCAGTTGGAG GAGTTCTGCT CTTCCTCTCC GTGAACGTGC ACGCTGACAC TGGGTGTGCC ATAGACATCA GCCGGCAA
GGTATCGAGA GTGCAAAGAG CGTCAACCTC CTCAAGACGA GAAGGAGAGG CACTTGCACG TGCGACTGTG ACCCACACGG TATCTGTAGT CGGCCGTT
PIV-WNV(ACpr E)/RSV-F (SEQ ID NOs:73-75)
deleted C protein
M S
1 AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT
TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
deleted C protein
NS3 cleavage
• K P G G P G S R A V N M L K R G M P R V L S L I G L K Q K K R
CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCAA TATGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGC AAAAGAAGCG
GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGTT ATACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCG TTTTCTTCGC
F signal
NS3 cleavage Fl
• G G E L L I L K A N A I T T I L T A V T F C F A S G Q N I T E E F 201 AGGGGGCGAG TTGCTAATCC TCAAAGCAAA TGCAATTACC ACAATCCTCA CTGCAGTCAC ATTTTGTTTT GCTTCTGGTC AAAACATCAC TGAAGAATTT TCCCCCGCTC AACGATTAGG AGTTTCGTTT ACGTTAATGG TGTTAGGAGT GACGTCAGTG TAAAACAAAA CGAAGACCAG TTTTGTAGTG ACTTCTTAAA
Fl
Y Q S T C S A V S G Y L S A L R T G Y T S V I T I E L S N I K E
301 TATCAATCAA CATGCAGTGC AGTTAGCAAA GGCTATCTTA GTGCTCTGAG AACTGGTTGG TATACCAGTG TTATAACTAT AGAATTAAGT AATATCAAGG ATAGTTAGTT GTACGTCACG TCAATCGTTT CCGATAGAAT CACGAGACTC TTGACCAACC ATATGGTCAC AATATTGATA TCTTAATTCA TTATAGTTCC
Fl
• N K C N G T D A K V K L I K Q E L D K Y K N A V T E L Q L L M Q S 401 AAAATAAGTG TAATGGAACA GATGCTAAGG TAAAATTGAT AAAACAAGAA TTAGATAAAT ATAAAAATGC TGTAACAGAA TTGCAGTTGC TCATGCAAAG TTTTATTCAC ATTACCTTGT CTACGATTCC ATTTTAACTA TTTTGTTCTT AATCTATTTA TATTTTTACG ACATTGTCTT AACGTCAACG AGTACGTTTC
Fl
• T P P T N N R A R R E L P R F M N Y T L N N A K K T N V T L S K K 501 CACACCACCA ACAAACAATC GAGCCAGAAG AGAACTACCA AGGTTTATGA ATTATACACT CAACAATGCC AAAAAAACCA ATGTAACATT AAGCAAGAAA GTGTGGTGGT TGTTTGTTAG CTCGGTCTTC TCTTGATGGT TCCAAATACT TAATATGTGA GTTGTTACGG TTTTTTTGGT TACATTGTAA TTCGTTCTTT
Fl
F2
R K R R F L G F L L G V G S A I A S G V A V S K V L H L E G E V N K
601 AGGAAAAGAA GATTTCTTGG TTTTTTGTTA GGTGTTGGAT CTGCAATCGC CAGTGGCGTT GCTGTATCTA AGGTCCTGCA CCTAGAAGGG GAAGTGAACA TCCTTTTCTT CTAAAGAACC AAAAAACAAT CCACAACCTA GACGTTAGCG GTCACCGCAA CGACATAGAT TCCAGGACGT GGATCTTCCC CTTCACTTGT
F2
■ I K S A L L S T N K A V V S L S N G V S V L T S K V L D L K N Y I 701 AGATCAAAAG TGCTCTACTA TCCACAAACA AGGCTGTAGT CAGCTTATCA AATGGAGTTA GTGTCTTAAC CAGCAAAGTG TTAGACCTCA AAAACTATAT TCTAGTTTTC ACGAGATGAT AGGTGTTTGT TCCGACATCA GTCGAATAGT TTACCTCAAT CACAGAATTG GTCGTTTCAC AATCTGGAGT TTTTGATATA
F2
- D K Q L L P I V N K Q S C S I S N I E T V I E F Q Q K N N R L L E 801 AGATAAACAA TTGTTACCTA TTGTGAACAA GCAAAGCTGC AGCATATCAA ATATAGAAAC TGTGATAGAG TTCCAACAAA AGAACAACAG ACTACTAGAG TCTATTTGTT AACAATGGAT AACACTTGTT CGTTTCGACG TCGTATAGTT TATATCTTTG ACACTATCTC AAGGTTGTTT TCTTGTTGTC TGATGATCTC
F2
I T R E F S V N A G V T T P V S T Y M L T N S E L L S L I N D M P I
901 ATTACCAGGG AATTTAGTGT TAATGCAGGT GTAACTACAC CTGTAAGCAC TTACATGTTA ACTAATAGTG AATTATTGTC ATTAATCAAT GATATGCCTA TAATGGTCCC TTAAATCACA ATTACGTCCA CATTGATGTG GACATTCGTG AATGTACAAT TGATTATCAC TTAATAACAG TAATTAGTTA CTATACGGAT
F2
• T N D Q K K L M S N N V Q I V R Q Q S Y S I M S I I K E E V L A Y - 1001 TAACAAATGA TCAGAAAAAG TTAATGTCCA ACAATGTTCA AATAGTTAGA CAGCAAAGTT ACTCTATCAT GTCCATAATA AAAGAGGAAG TCTTAGCATA ATTGTTTACT AGTCTTTTTC AATTACAGGT TGTTACAAGT TTATCAATCT GTCGTTTCAA TGAGATAGTA CAGGTATTAT TTTCTCCTTC AGAATCGTAT
F2
• V V Q L P L Y G V I D T P C W K L H T S P L C T T N T K E G S N I 1101 TGTAGTACAA TTACCACTAT ATGGTGTTAT AGATACACCC TGTTGGAAAC TACACACATC CCCTCTATGT ACAACCAACA CAAAAGAAGG GTCCAACATC ACATCATGTT AATGGTGATA TACCACAATA TCTATGTGGG ACAACCTTTG ATGTGTGTAG GGGAGATACA TGTTGGTTGT GTTTTCTTCC CAGGTTGTAG
F2
C L T R T D R G W Y C D N A G S V S F F P Q A E T C K V Q S N R V F
1201 TGTTTAACAA GAACTGACAG AGGATGGTAC TGTGACAATG CAGGATCAGT ATCTTTCTTC CCACAAGCTG AAACATGTAA AGTTCAATCA AATCGAGTAT
ACAAATTGTT CTTGACTGTC TCCTACCATG ACACTGTTAC GTCCTAGTCA TAGAAAGAAG GGTGTTCGAC TTTGTACATT TCAAGTTAGT TTAGCTCATA
F2
■ C D T M M S L T L P S E I N L C N V D I F N P K Y D C I M T S
1301 TTTGTGACAC AATGAACAGT TTAACATTAC CAAGTGAAAT AAATCTCTGC AATGTTGACA TATTCAACCC CAAATATGAT TGTAAAATTA TGACTTCAAA
AAACACTGTG TTACTTGTCA AATTGTAATG GTTCACTTTA TTTAGAGACG TTACAACTGT ATAAGTTGGG GTTTATACTA ACATTTTAAT ACTGAAGTTT
F2
■ T D V S S S V I T S L G A I V S C Y G K T K C T A S N K N R G i l
1 01 AACAGATGTA AGCAGCTCCG TTATCACATC TCTAGGAGCC ATTGTGTCAT GCTATGGCAA AACTAAATGT ACAGCATCCA ATAAAAATCG TGGAATCATA
TTGTCTACAT TCGTCGAGGC AATAGTGTAG AGATCCTCGG TAACACAGTA CGATACCGTT TTGATTTACA TGTCGTAGGT TATTTTTAGC ACCTTAGTAT
F2
K T F S N G C D Y V S N K G D T V S V G N T L Y Y V N K Q E G K S
1501 AAGACATTTT CTAACGGGTG CGATTATGTA TCAAATAAAG GGATGGACAC TGTGTCTGTA GGTAACACAT TATATTATGT AAATAAGCAA GAAGGTAAAA
TTCTGTAAAA GATTGCCCAC GCTAATACAT AGTTTATTTC CCTACCTGTG ACACAGACAT CCATTGTGTA ATATAATACA TTTATTCGTT CTTCCATTTT
F2
• L Y V K G E P I I N F Y D P L V F P S D E F D A S I S Q V N E K I
1601 GTCTCTATGT AAAAGGTGAA CCAATAATAA ATTTCTATGA CCCATTAGTA TTCCCCTCTG ATGAATTTGA TGCATCAATA TCTCAAGTCA ACGAGAAGAT
CAGAGATACA TTTTCCACTT GGTTATTATT TAAAGATACT GGGTAATCAT AAGGGGAGAC TACTTAAACT ACGTAGTTAT AGAGTTCAGT TGCTCTTCTA
F2
TM Domain
- N Q S L A F I R K S D E L L H N V N A G K S T T N I I T T I I I
1701 TAACCAGAGC CTAGCATTTA TTCGTAAATC CGATGAATTA TTACATAATG TAAATGCTGG TAAATCCACC ACAAATATCA TGATAACTAC TATAATTATA
ATTGGTCTCG GATCGTAAAT AAGCATTTAG GCTACTTAAT AATGTATTAC ATTTACGACC ATTTAGGTGG TGTTTATAGT ACTATTGATG ATATTAATAT TM Domain
Cytoplasmic Tail
V I I V I L L S L I A V G L L L Y C A R S T P V T L S K D Q L S G
1801 GTGATTATAG TAATATTGTT ATCATTAATT GCTGTTGGAC TGCTCTTATA CTGTAAGGCC AGAAGCACAC CAGTCACACT AAGCAAAGAT CAACTGAGTG
CACTAATATC ATTATAACAA TAGTAATTAA CGACAACCTG ACGAGAATAT GACATTCCGG TCTTCGTGTG GTCAGTGTGA TTCGTTTCTA GTTGACTCAC
FMDV 2A
membrane domain of WNV E (split)
Cytoplasmic Tail pre E/NSl signal
N N K H D L K L A D V E N P A R D R S A L
1901 GTATAAATAA TATTGCATTT AGTAACAATT TTGATCTGCT CAAACTTGCA GGCGATGTAG AATCAAATCC TGGACCCGCC CGGGACAGGT CCATAGCTCT
CATATTTATT ATAACGTAAA TCATTGTTAA AACTAGACGA GTTTGAACGT CCGCTACATC TTAGTTTAGG ACCTGGGCGG GCCCTGTCCA GGTATCGAGA
Transmembrane domain of WNV E (split)
NS1
• T F L A V G G V L L F L S V N V H A D T G C A I D I S R Q E L R
2001 CACGTTTCTC GCAGTTGGAG GAGTTCTGCT CTTCCTCTCC GTGAACGTGC ACGCTGACAC TGGGTGTGCC ATAGACATCA GCCGGCAAGA GCTGAGA
GTGCAAAGAG CGTCAACCTC CTCAAGACGA GAAGGAGAGG CACTTGCACG TGCGACTGTG ACCCACACGG TATCTGTAGT CGGCCGTTCT CGACTCT
PIV-WNV(AC)/RSV-F (SEQ ID NOs:76-78)
1 GATCCTAATA CGACTCACTA TAGAGTAGTT CGCCTGTGTG AGCTGACAAA CTTAGTAGTG TTTGTGAGGA TTAACAACAA TTAACACAGT GCGAGCTGTT CTAGGATTAT GCTGAGTGAT ATCTCATCAA GCGGACACAC TCGACTGTTT GAATCATCAC AAACACTCCT AATTGTTGTT AATTGTGTCA CGCTCGACAA
N-terminus of C
M S K K P G G P G K S R A V N M L K R G M P R V L S L
101 TCTTAGCACG AAGATCTCGA TGTCTAAGAA ACCAGGAGGG CCCGGCAAGA GCCGGGCTGT CAATATGCTA AAACGCGGAA TGCCCCGCGT GTTGTCCTTG
AGAATCGTGC TTCTAGAGCT ACAGATTCTT TGGTCCTCCC GGGCCGTTCT CGGCCCGACA GTTATACGAT TTTGCGCCTT ACGGGGCGCA CAACAGGAAC
N-terminus of C F signal
NS3 cleavage Fl
I G L K Q K R G G E L L I L K A N A I T T I L T A V T F C F A S G
201 ATTGGACTTA AGCAAAAGAA GCGAGGGGGC GAGTTGCTAA TCCTCAAAGC AAATGCAATT ACCACAATCC TCACTGCAGT CACATTTTGT TTTGCTTCTG
TAACCTGAAT TCGTTTTCTT CGCTCCCCCG CTCAACGATT AGGAGTTTCG TTTACGTTAA TGGTGTTAGG AGTGACGTCA GTGTAAAACA AAACGAAGAC
Fl
■ Q N I T E E F Y Q S T C S A V S K G Y L S A L R T G Y T S V I T 301 GTCAAAACAT CACTGAAGAA TTTTATCAAT CAACATGCAG TGCAGTTAGC AAAGGCTATC TTAGTGCTCT GAGAACTGGT TGGTATACCA GTGTTATAAC CAGTTTTGTA GTGACTTCTT AAAATAGTTA GTTGTACGTC ACGTCAATCG TTTCCGATAG AATCACGAGA CTCTTGACCA ACCATATGGT CACAATATTG
Fl
• I E L S N I K E N K C N G T D A K V K L I R Q E L D K Y K N A V T 401 TATAGAATTA AGTAATATCA AGGAAAATAA GTGTAATGGA ACAGATGCTA AGGTAAAATT GATAAAACAA GAATTAGATA AATATAAAAA TGCTGTAACA ATATCTTAAT TCATTATAGT TCCTTTTATT CACATTACCT TGTCTACGAT TCCATTTTAA CTATTTTGTT CTTAATCTAT TTATATTTTT ACGACATTGT
Fl
E L Q L L M Q S T P P T H N R A R R E L P R F M N Y T L N N A K K T '
501 GAATTGCAGT TGCTCATGCA AAGCACACCA CCAACAAACA ATCGAGCCAG AAGAGAACTA CCAAGGTTTA TGAATTATAC ACTCAACAAT GCCAAAAAAA
CTTAACGTCA ACGAGTACGT TTCGTGTGGT GGTTGTTTGT TAGCTCGGTC TTCTCTTGAT GGTTCCAAAT ACTTAATATG TGAGTTGTTA CGGTTTTTTT
F2
Fl
• N V T L S K K R K R R F L G F L L G V G S A I A S G V A V S K V L
601 CCAATGTAAC ATTAAGCAAG AAAAGGAAAA GAAGATTTCT TGGTTTTTTG TTAGGTGTTG GATCTGCAAT CGCCAGTGGC GTTGCTGTAT CTAAGGTCCT
GGTTACATTG TAATTCGTTC TTTTCCTTTT CTTCTAAAGA ACCAAAAAAC AATCCACAAC CTAGACGTTA GCGGTCACCG CAACGACATA GATTCCAGGA
F2
• H L E G E V N K I K S A L L S T N K A V V S L S N G V S V L T S K 701 GCACCTAGAA GGGGAAGTGA ACAAGATCAA AAGTGCTCTA CTATCCACAA ACAAGGCTGT AGTCAGCTTA TCAAATGGAG TTAGTGTCTT AACCAGCAAA CGTGGATCTT CCCCTTCACT TGTTCTAGTT TTCACGAGAT GATAGGTGTT TGTTCCGACA TCAGTCGAAT AGTTTACCTC AATCACAGAA TTGGTCGTTT
F2
V L D L K N Y I D K Q L L P I V N K Q S C S I S N I E T V I E F Q Q
801 GTGTTAGACC TCAAAAACTA TATAGATAAA CAATTGTTAC CTATTGTGAA CAAGCAAAGC TGCAGCATAT CAAATATAGA AACTGTGATA GAGTTCCAAC CACAATCTGG AGTTTTTGAT ATATCTATTT GTTAACAATG GATAACACTT GTTCGTTTCG ACGTCGTATA GTTTATATCT TTGACACTAT CTCAAGGTTG
F2
• N N R L L E I T R E F S V N A G V T T P V S T Y M L T N S E L L
901 AAAAGAACAA CAGACTACTA GAGATTACCA GGGAATTTAG TGTTAATGCA GGTGTAACTA CACCTGTAAG CACTTACATG TTAACTAATA GTGAATTATT TTTTCTTGTT GTCTGATGAT CTCTAATGGT CCCTTAAATC ACAATTACGT CCACATTGAT GTGGACATTC GTGAATGTAC AATTGATTAT CACTTAATAA
F2
• S L I N D M P I T N D Q K K L S N N V Q I V R Q Q S Y S I M S I 1001 GTCATTAATC AATGATATGC CTATAACAAA TGATCAGAAA AAGTTAATGT CCAACAATGT TCAAATAGTT AGACAGCAAA GTTACTCTAT CATGTCCATA
CAGTAATTAG TTACTATACG GATATTGTTT ACTAGTCTTT TTCAATTACA GGTTGTTACA AGTTTATCAA TCTGTCGTTT CAATGAGATA GTACAGGTAT
F2
I K E E V L A Y V V Q L P L Y G V I D T P C W K L H T S P L C T T N
1101 ATAAAAGAGG AAGTCTTAGC ATATGTAGTA CAATTACCAC TATATGGTGT TATAGATACA CCCTGTTGGA AACTACACAC ATCCCCTCTA TGTACAACCA
TATTTTCTCC TTCAGAATCG TATACATCAT GTTAATGGTG ATATACCACA ATATCTATGT GGGACAACCT TTGATGTGTG TAGGGGAGAT ACATGTTGGT
F2
• T K E G S N I C L T T D R G W Y C D N A G S V S F F P Q A E T C
1201 ACACAAAAGA AGGGTCCAAC ATCTGTTTAA CAAGAACTGA CAGAGGATGG TACTGTGACA ATGCAGGATC AGTATCTTTC TTCCCACAAG CTGAAACATG
TGTGTTTTCT TCCCAGGTTG TAGACAAATT GTTCTTGACT GTCTCCTACC ATGACACTGT TACGTCCTAG TCATAGAAAG AAGGGTGTTC GACTTTGTAC
F2
• K V Q S N R V F C D T M N S L T L P S E I N L C N V D I F N P K Y
1301 TAAAGTTCAA TCAAATCGAG TATTTTGTGA CACAATGAAC AGTTTAACAT TACCAAGTGA AATAAATCTC TGCAATGTTG ACATATTCAA CCCCAAATAT
ATTTCAAGTT AGTTTAGCTC ATAAAACACT GTGTTACTTG TCAAATTGTA ATGGTTCACT TTATTTAGAG ACGTTACAAC TGTATAAGTT GGGGTTTATA
F2
D C I M T S K T D V S S S V I T S L G A I V S C Y G K T K C T A S - 1401 GATTGTAAAA TTATGACTTC AAAAACAGAT GTAAGCAGCT CCGTTATCAC ATCTCTAGGA GCCATTGTGT CATGCTATGG CAAAACTAAA TGTACAGCAT
CTAACATTTT AATACTGAAG TTTTTGTCTA CATTCGTCGA GGCAATAGTG TAGAGATCCT CGGTAACACA GTACGATACC GTTTTGATTT ACATGTCGTA
F2
■ N K N R G I I K T F S N G C D Y V S N K G M D T V S V G N T L Y Y 1501 CCAATAAAAA TCGTGGAATC ATAAAGACAT TTTCTAACGG GTGCGATTAT GTATCAAATA AAGGGATGGA CACTGTGTCT GTAGGTAACA CATTATATTA
GGTTATTTTT AGCACCTTAG TATTTCTGTA AAAGATTGCC CACGCTAATA CATAGTTTAT TTCCCTACCT GTGACACAGA CATCCATTGT GTAATATAAT
F2
■ V N K Q E G S L Y V K G E P I I N F Y D P L V F P S D E F D A S 1601 TGTAAATAAG CAAGAAGGTA AAAGTCTCTA TGTAAAAGGT GAACCAATAA TAAATTTCTA TGACCCATTA GTATTCCCCT CTGATGAATT TGATGCATCA
ACATTTATTC GTTCTTCCAT TTTCAGAGAT ACATTTTCCA CTTGGTTATT ATTTAAAGAT ACTGGGTAAT CATAAGGGGA GACTACTTAA ACTACGTAGT
F2
TM Domain
I S Q V N E K I N Q S L A F I R K S D E L L H N V N A G K S T T N I - 1701 ATATCTCAAG TCAACGAGAA GATTAACCAG AGCCTAGCAT TTATTCGTAA ATCCGATGAA TTATTACATA ATGTAAATGC TGGTAAATCC ACCACAAATA
TATAGAGTTC AGTTGCTCTT CTAATTGGTC TCGGATCGTA AATAAGCATT TAGGCTACTT AATAATGTAT TACATTTACG ACCATTTAGG TGGTGTTTAT
TM Domain
Cytoplasmic Tail
■ M I T T I I I V I I V I L L S L I A V G L L L Y C K A R S T P V T
1801 TCATGATAAC TACTATAATT ATAGTGATTA TAGTAATATT GTTATCATTA ATTGCTGTTG GACTGCTCTT ATACTGTAAG GCCAGAAGCA CACCAGTCAC
AGTACTATTG ATGATATTAA TATCACTAAT ATCATTATAA CAATAGTAAT TAACGACAAC CTGACGAGAA TATGACATTC CGGTCTTCGT GTGGTCAGTG
FMDV 2A
Cytoplasmic Tail
• L S K D Q L S G I N N I A F S N N F D L L K L A G D V E S N P G P
1901 ACTAAGCAAA GATCAACTGA GTGGTATAAA TAATATTGCA TTTAGTAACA ATTTTGATCT GCTCAAACTT GCAGGCGATG TAGAATCAAA TCCTGGACCC
TGATTCGTTT CTAGTTGACT CACCATATTT ATTATAACGT AAATCATTGT TAAAACTAGA CGAGTTTGAA CGTCCGCTAC ATCTTAGTTT AGGACCTGGG
rM
C/prM signal
G G K T G I A V M I G L I A C V G A V T L S N F Q G K V M M T V N A
2001 GGAGGAAAGA CCGGTATTGC AGTCATGATT GGCCTGATCG CCTGCGTAGG AGCAGTTACC CTCTCTAACT TCCAAGGGAA GGTGATGATG ACGGTAAATG CCTCCTTTCT GGCCATAACG TCAGTACTAA CCGGACTAGC GGACGCATCC TCGTCAATGG GAGAGATTGA AGGTTCCCTT CCACTACTAC TGCCATTTAC
prM
■ T D V T D V I T I P T A A G N L C I V R A M D V G Y M C D D T I 2101 CTACTGACGT CACAGATGTC ATCACGATTC CAACAGCTGC TGGAAAGAAC CTATGCATTG TCAGAGCAAT GGATGTGGGA TACATGTGCG ATGATACTAT GATGACTGCA GTGTCTACAG TAGTGCTAAG GTTGTCGACG ACCTTTCTTG GATACGTAAC AGTCTCGTTA CCTACACCCT ATGTACACGC TACTATGATA
prM
• T Y E C P V L S A G N D P E D I D C C T K S A V Y V R Y G R C T 2201 CACTTATGAA TGCCCAGTGC TGTCGGCTGG TAATGATCCA GAAGACATCG ACTGTTGGTG CACAAAGTCA GCAGTCTACG TCAGGTATGG AAGATGCACC GTGAATACTT ACGGGTCACG ACAGCCGACC ATTACTAGGT CTTCTGTAGC TGACAACCAC GTGTTTCAGT CGTCAGATGC AGTCCATACC TTCTACGTGG
prM
K T R H S R R S R R S L T V Q T H G E S T L A N K K G A W M D S T K
2301 AAGACACGCC ACTCAAGACG CAGTCGGAGG TCACTGACAG TGCAGACACA CGGAGAAAGC ACTCTAGCGA ACAAGAAGGG GGCTTGGATG GACAGCACCA TTCTGTGCGG TGAGTTCTGC GTCAGCCTCC AGTGACTGTC ACGTCTGTGT GCCTCTTTCG TGAGATCGCT TGTTCTTCCC CCGAACCTAC CTGTCGTGGT
rM
■ A T R Y L V K T E S W I L R N P G Y A L V A A V I G M L G S H T 2 01 AGGCCACAAG GTATTTGGTA AAAACAGAAT CATGGATCTT GAGGAACCCT GGATATGCCC TGGTGGCAGC CGTCATTGGT TGGATGCTTG GGAGCAACAC TCCGGTGTTC CATAAACCAT TTTTGTCTTA GTACCTAGAA CTCCTTGGGA CCTATACGGG ACCACCGTCG GCAGTAACCA ACCTACGAAC CCTCGTTGTG
prM
■ M Q R V V F V V L L L L V A P A Y S F N C L G M S N R D F L E G V 2501 CATGCAGAGA GTTGTGTTTG TCGTGCTATT GCTTTTGGTG GCCCCAGCTT ACAGCTTTAA CTGCCTTGGA ATGAGCAACA GAGACTTCTT GGAAGGAGTG GTACGTCTCT CAACACAAAC AGCACGATAA CGAAAACCAC CGGGGTCGAA TGTCGAAATT GACGGAACCT TACTCGTTGT CTCTGAAGAA CCTTCCTCAC
prM
E
S G A T W V D L V L E G D S C V T I M S K D K P T I D V K M M N M E
2601 TCTGGAGCAA CATGGGTGGA TTTGGTTCTC GAAGGCGACA GCTGCGTGAC TATCATGTCT AAGGACAAGC CTACCATCGA TGTGAAGATG ATGAATATGG AGACCTCGTT GTACCCACCT AAACCAAGAG CTTCCGCTGT CGACGCACTG ATAGTACAGA TTCCTGTTCG GATGGTAGCT ACACTTCTAC TACTTATACC
• A A N L A E V R S Y C Y L A T V S D L S T K A A C P A M G E A H K 2701 AGGCGGCCAA CCTGGCAGAG GTCCGCAGTT ATTGCTATTT GGCTACCGTC AGCGATCTCT CCACCAAAGC TGCGTGCCCG GCCATGGGAG AAGCTCACAA TCCGCCGGTT GGACCGTCTC CAGGCGTCAA TAACGATAAA CCGATGGCAG TCGCTAGAGA GGTGGTTTCG ACGCACGGGC CGGTACCCTC TTCGAGTGTT
E
• D K R A D P A F V C R Q G V V D R G G N G C G L F G K G S I D T 2801 TGACAAACGT GCTGACCCAG CTTTTGTGTG CAGACAAGGA GTGGTGGACA GGGGCTGGGG CAACGGCTGC GGACTATTTG GCAAAGGAAG CATTGACACA ACTGTTTGCA CGACTGGGTC GAAAACACAC GTCTGTTCCT CACCACCTGT CCCCGACCCC GTTGCCGACG CCTGATAAAC CGTTTCCTTC GTAACTGTGT
E
C A K F A C S T K A I G R T I L K E N I Y E V A I F V H G P T T V
2901 TGCGCCAAAT TTGCCTGCTC TACCAAGGCA ATAGGAAGAA CCATTTTGAA AGAGAATATC AAGTACGAAG TGGCCATTTT TGTCCATGGA CCAACTACTG ACGCGGTTTA AACGGACGAG ATGGTTCCGT TATCCTTCTT GGTAAAACTT TCTCTTATAG TTCATGCTTC ACCGGTAAAA ACAGGTACCT GGTTGATGAC
E
• E S H G N Y S T Q V G A T Q A G R F S I T P A A P S Y T L K L G E 3001 TGGAGTCGCA CGGAAACTAC TCCACACAGG TTGGAGCCAC TCAGGCAGGG AGATTCAGCA TCACTCCTGC GGCGCCTTCA TACACACTAA AGCTTGGAGA ACCTCAGCGT GCCTTTGATG AGGTGTGTCC AACCTCGGTG AGTCCGTCCC TCTAAGTCGT AGTGAGGACG CCGCGGAAGT ATGTGTGATT TCGAACCTCT
E
• Y G E V T V D C E P R S G I D T N A Y Y V T V G T K T F L V H R 3101 ATATGGAGAG GTGACAGTGG ACTGTGAACC ACGGTCAGGG ATTGACACCA ATGCATACTA CGTGATGACT GTTGGAACAA AGACGTTCTT GGTCCATCGT TATACCTCTC CACTGTCACC TGACACTTGG TGCCAGTCCC TAACTGTGGT TACGTATGAT GCACTACTGA CAACCTTGTT TCTGCAAGAA CCAGGTAGCA
E
E W F M D N L P W S S A G S T V W R N R E T L M E F E E P H A T K
3201 GAGTGGTTCA TGGACCTCAA CCTCCCTTGG AGCAGTGCTG GAAGTACTGT GTGGAGGAAC AGAGAGACGT TAATGGAGTT TGAGGAACCA CACGCCACGA CTCACCAAGT ACCTGGAGTT GGAGGGAACC TCGTCACGAC CTTCATGACA CACCTCCTTG TCTCTCTGCA ATTACCTCAA ACTCCTTGGT GTGCGGTGCT
• Q S V I A L G S Q E G A L H Q A L A G A I P V E F S S N T V K L T 3301 AGCAGTCTGT GATAGCATTG GGCTCACAAG AGGGAGCTCT GCATCAAGCT TTGGCTGGAG CCATTCCTGT GGAATTTTCA AGCAACACTG TCAAGTTGAC TCGTCAGACA CTATCGTAAC CCGAGTGTTC TCCCTCGAGA CGTAGTTCGA AACCGACCTC GGTAAGGACA CCTTAAAAGT TCGTTGTGAC AGTTCAACTG
E
• S G H L K C R V M E K L Q L K G T T Y G V C S K A F K F L G T P 3401 GTCGGGTCAT TTGAAGTGTA GAGTGAAGAT GGAAAAATTG CAGTTGAAGG GAACAACCTA TGGCGTCTGT TCAAAGGCTT TCAAGTTTCT TGGGACTCCC CAGCCCAGTA AACTTCACAT CTCACTTCTA CCTTTTTAAC GTCAACTTCC CTTGTTGGAT ACCGCAGACA AGTTTCCGAA AGTTCAAAGA ACCCTGAGGG
E
A D T G H G T V V L E L Q Y T G T D G P C K V P I S S V A S L N D L
3501 GCAGACACAG GTCACGGCAC TGTGGTGTTG GAATTGCAGT ACACTGGCAC GGATGGACCT TGCAAAGTTC CTATCTCGTC AGTGGCTTCA TTGAACGACC CGTCTGTGTC CAGTGCCGTG ACACCACAAC CTTAACGTCA TGTGACCGTG CCTACCTGGA ACGTTTCAAG GATAGAGCAG TCACCGAAGT AACTTGCTGG
■ T P V G R L V T V N P F V S V A T A N A K V L I E L E P P F G D S 3601 TAACGCCAGT GGGCAGATTG GTCACTGTCA ACCCTTTTGT TTCAGTGGCC ACGGCCAACG CTAAGGTCCT GATTGAATTG GAACCACCCT TTGGAGACTC ATTGCGGTCA CCCGTCTAAC CAGTGACAGT TGGGAAAACA AAGTCACCGG TGCCGGTTGC GATTCCAGGA CTAACTTAAC CTTGGTGGGA AACCTCTGAG
E
• Y I V V G R G E Q Q I N H H W H K S G S S I G K A F T T T L K G A
3701 ATACATAGTG GTGGGCAGAG GAGAACAACA GATCAATCAC CACTGGCACA AGTCTGGAAG CAGCATTGGC AAAGCCTTTA CAACCACCCT CAAAGGAGCG
TATGTATCAC CACCCGTCTC CTCTTGTTGT CTAGTTAGTG GTGACCGTGT TCAGACCTTC GTCGTAACCG TTTCGGAAAT GTTGGTGGGA GTTTCCTCGC
E
Q R L A A L G D T A D F G S V G G V F T S V G K A V H Q V F G G A
3801 CAGAGACTAG CCGCTCTAGG AGACACAGCT TGGGACTTTG GATCAGTTGG AGGGGTGTTC ACCTCAGTTG GGAAGGCTGT CCATCAAGTG TTCGGAGGAG GTCTCTGATC GGCGAGATCC TCTGTGTCGA ACCCTGAAAC CTAGTCAACC TCCCCACAAG TGGAGTCAAC CCTTCCGACA GGTAGTTCAC AAGCCTCCTC
E
' F R S L F G G M S I T Q G L L G A L L L W G I N A R D R S I A 3901 CATTCCGCTC ACTGTTCGGA GGCATGTCCT GGATAACGCA AGGATTGCTG GGGGCTCTCC TGTTGTGGAT GGGCATCAAT GCTCGTGACA GGTCCATAGC GTAAGGCGAG TGACAAGCCT CCGTACAGGA CCTATTGCGT TCCTAACGAC CCCCGAGAGG ACAACACCTA CCCGTAGTTA CGAGCACTGT CCAGGTATCG
NSl
E
■ L T F L A V G G V L L F L S V N V H A D T G C A I D I S R Q E L R
TCTCACGTTT CTCGCAGTTG GAGGAGTTCT GCTCTTCCTC TCCGTGAACG TGCACGCTGA CACTGGGTGT GCCATAGACA TCAGCCGGCA AGAGCTGAGA AGAGTGCAAA GAGCGTCAAC CTCCTCAAGA CGAGAAGGAG AGGCACTTGC ACGTGCGACT GTGACCCACA CGGTATCTGT AGTCGGCCGT TCTCGACTCT
Sequence Appendix 6
Construct 1
1. PIV-WN (ACprME)-SIV 9AA FMD Gag
Figure imgf000117_0001
RV2309M-FMD-Gag
2. Sequence of PIV-WN (ACprME)-SIV 9AA FMD Gag (partial).
UTR
M S
AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA CATCACAAA CACTCCTAAT GTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
C
• K K P G G P G K S R A V Y L L K R G M P R V L S L I G L K R A M L
101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGA GGGCTATGTT
GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCT CCCGATACAA
C
• S L I D G K G P I R F V L A L L A F F R F T A I A P T R A V L D R
201 GAGCCTGATC GACGGCAAGG GGCCAATACG ATTTGTGTTG GCTCTCTTGG CGTTCTTCAG GTTCACAGCA ATTGCTCCGA CCCGAGCAGT GCTGGATCGA
CTCGGACTAG CTGCCGTTCC CCGGTTATGC TAAACACAAC CGAGAGAACC GCAAGAAGTC CAAGTGTCGT TAACGAGGCT GGGCTCGTCA CGACCTAGCT
C
Cleavage
W R G V N K Q T A K H L L S F K K E L G T L T S A I N R R S S Q · 301 TGGAGAGGTG TGAACAAACA AACAGCGATG AAACACCTTC TGAGTTTCAA GAAGGAACTA GGGACCTTGA CCAGTGCTAT CAATCGGCGG AGCTCAAAGC
ACCTCTCCAC ACTTGTTTGT TTGTCGCTAC TTTGTGGAAG ACTCAAAGTT CTTCCTTGAT CCCTGGAACT GGTCACGATA GTTAGCCGCC TCGAGTTTCG FMDV2A
9AA C Signal
NS3 Cleavage Gag
• K K R G G K T G I A V I N F D L L L A G D V E S N P G P M G A R 401 AGAAAAAGCG GGGCGGAAAG ACAGGTATTG CTGTGATCAA TTTTGACCTG TTAAAACTGG CCGGGGACGT CGAAAGCAAC CCCGGTCCGA TGGGCGCTAG TCTTTTTCGC CCCGCCTTTC TGTCCATAAC GACACTAGTT AAAACTGGAC AATTTTGACC GGCCCCTGCA GCTTTCGTTG GGGCCAGGCT ACCCGCGATC
Gag
- N S V L S G K K A D E L E K I R L R P G G K K K Y M L K H V V W A
501 GAATAGCGTG CTTAGTGGCA AAAAGGCTGA TGAACTTGAG AAGATCCGGC TCCGTCCGGG CGGGAAGAAG AAGTATATGT TGAAACATGT CGTGTGGGCC
CTTATCGCAC GAATCACCGT TTTTCCGACT ACTTGAACTC TTCTAGGCCG AGGCAGGCCC GCCCTTCTTC TTCATATACA ACTTTGTACA GCACACCCGG
Gag
A N E L D R F G L A E S L L E N K E G C Q K I L S V L A P L V P T G
601 GCCAACGAGT TAGATAGGTT TGGGCTAGCA GAGTCATTGC TCGAAAACAA GGAAGGATGT CAGAAGATAC TAAGTGTCCT GGCACCTTTG GTACCCACGG CGGTTGCTCA ATCTATCCAA ACCCGATCGT CTCAGTAACG AGCTTTTGTT CCTTCCTACA GTCTTCTATG ATTCACAGGA CCGTGGAAAC CATGGGTGCC
Gag
• S E N L K S L Y N T V C V I W C I H A E E K V K H T E E A K Q I V 701 GGTCTGAGAA CTTAAAGAGT CTGTATAACA CTGTGTGCGT GATCTGGTGC ATTCACGCCG AAGAGAAAGT GAAGCACACC GAAGAAGCTA AGCAAATAGT CCAGACTCTT GAATTTCTCA GACATATTGT GACACACGCA CTAGACCACG TAAGTGCGGC TTCTCTTTCA CTTCGTGTGG CTTCTTCGAT TCGTTTATCA
Gag
• Q R H L V V E T G T A E T M P K T S R P T A P S S G R G G N Y P V 801 GCAGAGACAT TTGGTCGTGG AAACCGGGAC CGCCGAGACT ATGCCCAAAA CATCCCGTCC AACCGCTCCA AGTAGTGGAA GAGGAGGTAA CTACCCCGTT CGTCTCTGTA AACCAGCACC TTTGGCCCTG GCGGCTCTGA TACGGGTTTT GTAGGGCAGG TTGGCGAGGT TCATCACCTT CTCCTCCATT GATGGGGCAA
Gag
Q Q I G G N Y V H L P L S P R T L N A W V K L I E E K K F G A E V V
901 CAGCAAATCG GGGGGAATTA CGTGCATCTC CCTTTGTCAC CAAGGACCCT CAATGCATGG GTCAAACTCA TCGAGGAAAA GAAGTTCGGA GCGGAAGTGG GTCGTTTAGC CCCCCTTAAT GCACGTAGAG GGAAACAGTG GTTCCTGGGA GTTACGTACC CAGTTTGAGT AGCTCCTTTT CTTCAAGCCT CGCCTTCACC
Gag
■ P G F Q A L S E G C T P Y D I N Q M L H C V G D H Q A A M Q I I R 1001 TCCCAGGGTT CCAGGCACTG AGTGAAGGGT GCACTCCCTA TGACATCAAC CAGATGCTTA ACTGCGTCGG CGACCATCAG GCCGCGATGC AGATTATTCG AGGGTCCCAA GGTCCGTGAC TCACTTCCCA CGTGAGGGAT ACTGTAGTTG GTCTACGAAT TGACGCAGCC GCTGGTAGTC CGGCGCTACG TCTAATAAGC
Gag
• D I I N E E A A D W D L Q H P Q P A P Q Q G Q L R E P S G S D I A 1101 GGACATAATC AACGAGGAGG CTGCAGACTG GGATTTGCAG CACCCCCAAC CCGCCCCTCA GCAAGGGCAG CTAAGGGAGC CTTCCGGCAG CGACATAGCT CCTGTATTAG TTGCTCCTCC GACGTCTGAC CCTAAACGTC GTGGGGGTTG GGCGGGGAGT CGTTCCCGTC GATTCCCTCG GAAGGCCGTC GCTGTATCGA
Gag
G T T S S V D E Q I Q W M Y R Q Q N P I P V G N I Y R R W I Q L G L
1201 GGGACTACTA GCTCCGTGGA TGAACAGATT CAATGGATGT ACAGACAGCA GAATCCGATC CCCGTTGGCA ACATCTACCG GCGCTGGATT CAACTCGGAC CCCTGATGAT CGAGGCACCT ACTTGTCTAA GTTACCTACA TGTCTGTCGT CTTAGGCTAG GGGCAACCGT TGTAGATGGC CGCGACCTAA GTTGAGCCTG
Gag
• Q C V R M Y N P T N I L D V K Q G P K E P F Q S Y V D R F Y S 1301 TTCAGAAGTG CGTCAGAATG TACAACCCCA CCAATATTCT GGATGTGAAA CAGGGGCCGA AAGAGCCCTT TCAATCCTAC GTCGACCGTT TCTACAAAAG AAGTCTTCAC GCAGTCTTAC ATGTTGGGGT GGTTATAAGA CCTACACTTT GTCCCCGGCT TTCTCGGGAA AGTTAGGATG CAGCTGGCAA AGATGTTTTC
Gag
■ L R A E Q T D A A V K N W M T Q T L L I Q N A N P D C K L V L K G
1401 TCTACGCGCC GAGCAGACCG ATGCCGCAGT GAAGAACTGG ATGACACAGA CGCTCCTGAT ACAGAATGCT AACCCTGATT GTAAACTCGT GCTGAAGGGC
AGATGCGCGG CTCGTCTGGC TACGGCGTCA CTTCTTGACC TACTGTGTCT GCGAGGACTA TGTCTTACGA TTGGGACTAA CATTTGAGCA CGACTTCCCG
Gag
L G V N P T L E E M L T A C Q G V G G P G Q K A R L M A E A L K E A
1501 TTAGGGGTAA ACCCAACGCT GGAAGAAATG TTAACCGCCT GCCAGGGAGT TGGTGGACCC GGACAGAAGG CCCGGCTAAT GGCCGAGGCG CTGAAAGAAG AATCCCCATT TGGGTTGCGA CCTTCTTTAC AATTGGCGGA CGGTCCCTCA ACCACCTGGG CCTGTCTTCC GGGCCGATTA CCGGCTCCGC GACTTTCTTC
Gag
• L A P V P I P F A A A Q Q R G P R K P I K C W N C G K E G H S A K
1601 CATTGGCTCC AGTACCCATT CCTTTTGCTG CCGCACAACA GAGAGGTCCC CGTAAACCGA TCAAATGCTG GAACTGTGGG AAGGAGGGGC ACTCCGCTAA
GTAACCGAGG TCATGGGTAA GGAAAACGAC GGCGTGTTGT CTCTCCAGGG GCATTTGGCT AGTTTACGAC CTTGACACCC TTCCTCCCCG TGAGGCGATT
Gag
■ Q C R A P R R Q G C W K C G K D H V M A K C P D R Q A G F L G L 1701 ACAATGTCGA GCGCCTAGAC GTCAGGGGTG TTGGAAGTGT GGTAAAATGG ACCACGTTAT GGCCAAATGC CCCGACAGAC AAGCCGGGTT CCTCGGGTTA TGTTACAGCT CGCGGATCTG CAGTCCCCAC AACCTTCACA CCATTTTACC TGGTGCAATA CCGGTTTACG GGGCTGTCTG TTCGGCCCAA GGAGCCCAAT
Gag
G P G K K P R N F P M A Q V H Q G L T P T A P P E D P A V D L L
1801 GGGCCTTGGG GAAAAAAGCC CAGAAACTTC CCAATGGCGC AAGTACACCA GGGCCTGACC CCGACCGCCC CCCCAGAGGA CCCAGCCGTA GACCTCTTGA CCCGGAACCC CTTTTTTCGG GTCTTTGAAG GGTTACCGCG TTCATGTGGT CCCGGACTGG GGCTGGCGGG GGGGTCTCCT GGGTCGGCAT CTGGAGAACT
Gag
• N Y M Q L G Q Q R E S R E K P Y K E V T E D L L H L N S L F G G
1901 AAAACTATAT GCAGCTGGGG AAGCAGCAGC GCGAGAGTAG AGAGAAGCCC TACAAGGAGG TTACGGAAGA TCTGTTACAC CTTAATTCGT TATTTGGTGG
TTTTGATATA CGTCGACCCC TTCGTCGTCG CGCTCTCATC TCTCTTCGGG ATGTTCCTCC AATGCCTTCT AGACAATGTG GAATTAAGCA ATAAACCACC
FMDV2A TM Domain WN E (split)
Gag prE/NSl Sig
• D Q N F D L L K L A G D V E S N P G P A R D R S I A L T F L A V G 2001 TGATCAGAAT TTCGACCTGC TTAAACTTGC TGGCGACGTT GAGTCAAATC CGGGCCCTGC CCGGGACAGG TCCATAGCTC TCACGTTTCT CGCAGTTGGA ACTAGTCTTA AAGCTGGACG AATTTGAACG ACCGCTGCAA CTCAGTTTAG GCCCGGGACG GGCCCTGTCC AGGTATCGAG AGTGCAAAGA GCGTCAACCT TM Domain WN E (split)
NSl
G V L L F L S V N V H A D T G C A I D I S R Q E L R C G S G V F I H
2101 GGAGTTCTGC TCTTCCTCTC CGTGAACGTG CACGCTGACA CTGGGTGTGC CATAGACATC AGCCGGCAAG AGCTGAGATG TGGAAGTGGA GTGTTCATAC CCTCAAGACG AGAAGGAGAG GCACTTGCAC GTGCGACTGT GACCCACACG GTATCTGTAG TCGGCCGTTC TCGACTCTAC ACCTTCACCT CACAAGTATG
NSl
• N D V E A W M D R Y K Y Y P E T P Q G L A K I I Q A H K E G V C 2201 ACAATGATGT GGAGGCTTGG ATGGACCGGT ACAAGTATTA CCCTGAAACG CCACAAGGCC TAGCCAAGAT CATTCAGAAA GCTCATAAGG AAGGAGTGTG TGTTACTACA CCTCCGAACC TACCTGGCCA TGTTCATAAT GGGACTTTGC GGTGTTCCGG ATCGGTTCTA GTAAGTCTTT CGAGTATTCC TTCCTCACAC
NSl
• G L R S V S R L E H Q M W E A V K D E L N T L L K
2301 CGGTCTACGA TCAGTTTCCA GACTGGAGCA TCAAATGTGG GAAGCAGTGA AGGACGAGCT GAACACTCTT TTGAAG
GCCAGATGCT AGTCAAAGGT CTGACCTCGT AGTTTACACC CTTCGTCACT TCCTGCTCGA CTTGTGAGAA AACTTC
Construct 2
1. PIV-WN (ACprME)-SIV 9AA FMD Gag & Pr
Figure imgf000120_0001
RV2309AA-FMD-GagPro
2. Sequence of PIV-WN (ACprME)-SIV 9AA FMD Gag & Pr (partial).
OTR
M S
1 AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
C
• K P G G P G K S R A V Y L L K R G M P R V L S L I G L K R A M L · 101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGA GGGCTATGTT GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCT CCCGATACAA
• S L I D G K G P I R F V L A L L A F F R F T A I A P T R A V L D R 201 GAGCCTGATC GACGGCAAGG GGCCAATACG ATTTGTGTTG GCTCTCTTGG CGTTCTTCAG GTTCACAGCA ATTGCTCCGA CCCGAGCAGT GCTGGATCGA CTCGGACTAG CTGCCGTTCC CCGGTTATGC TAAACACAAC CGAGAGAACC GCAAGAAGTC CAAGTGTCGT TAACGAGGCT GGGCTCGTCA CGACCTAGCT
C
NS3 CI
W R G V N K Q T A M K H L L S F K K E L G T L T S A I N R R S S Q
301 TGGAGAGGTG TGAACAAACA AACAGCGATG AAACACCTTC TGAGTTTCAA GAAGGAACTA GGGACCTTGA CCAGTGCTAT CAATCGGCGG AGCTCAAAGC ACCTCTCCAC ACTTGTTTGT TTGTCGCTAC TTTGTGGAAG ACTCAAAGTT CTTCCTTGAT CCCTGGAACT GGTCACGATA GTTAGCCGCC TCGAGTTTCG FMDV2A
9AA C Signal
NS3 Cleavage Gag
■ K K R G G K T G I A V I N F D L L K L A G D V E S N P G P M G A R 401 AGAAAAAGCG GGGCGGAAAG ACAGGTATTG CTGTGATCAA TTTTGACCTG TTAAAACTGG CCGGGGACGT CGAAAGCAAC CCCGGTCCGA TGGGCGCTAG TCTTTTTCGC CCCGCCTTTC TGTCCATAAC GACACTAGTT AAAACTGGAC AATTTTGACC GGCCCCTGCA GCTTTCGTTG GGGCCAGGCT ACCCGCGATC
Gag
• N S V L S G K K A D E L E K I R L R P G G K K K Y M L K H V V W A 501 GAATAGCGTG CTTAGTGGCA AAAAGGCTGA TGAACTTGAG AAGATCCGGC TCCGTCCGGG CGGGAAGAAG AAGTATATGT TGAAACATGT CGTGTGGGCC CTTATCGCAC GAATCACCGT TTTTCCGACT ACTTGAACTC TTCTAGGCCG AGGCAGGCCC GCCCTTCTTC TTCATATACA ACTTTGTACA GCACACCCGG
Gag
A N E L D R F G L A E S L L E N K E G C Q K I L S V L A P L V P T G
601 GCCAACGAGT TAGATAGGTT TGGGCTAGCA GAGTCATTGC TCGAAAACAA GGAAGGATGT CAGAAGATAC TAAGTGTCCT GGCACCTTTG GTACCCACGG CGGTTGCTCA ATCTATCCAA ACCCGATCGT CTCAGTAACG AGCTTTTGTT CCTTCCTACA GTCTTCTATG ATTCACAGGA CCGTGGAAAC CATGGGTGCC
Gag
• S E N L K S L Y N T V C V I W C I H A E E K V K H T E E A K Q I V · 701 GGTCTGAGAA CTTAAAGAGT CTGTATAACA CTGTGTGCGT GATCTGGTGC ATTCACGCCG AAGAGAAAGT GAAGCACACC GAAGAAGCTA AGCAAATAGT CCAGACTCTT GAATTTCTCA GACATATTGT GACACACGCA CTAGACCACG TAAGTGCGGC TTCTCTTTCA CTTCGTGTGG CTTCTTCGAT TCGTTTATCA
Gag
■ Q R H L V V E T G T A E T M P K T S R P T A P S S G R G G N Y P V 801 GCAGAGACAT TTGGTCGTGG AAACCGGGAC CGCCGAGACT ATGCCCAAAA CATCCCGTCC AACCGCTCCA AGTAGTGGAA GAGGAGGTAA CTACCCCGTT CGTCTCTGTA AACCAGCACC TTTGGCCCTG GCGGCTCTGA TACGGGTTTT GTAGGGCAGG TTGGCGAGGT TCATCACCTT CTCCTCCATT GATGGGGCAA
Gag
Q Q I G G N Y V H L P L S P R T L N A V K L I E E K K F G A E V V
901 CAGCAAATCG GGGGGAATTA CGTGCATCTC CCTTTGTCAC CAAGGACCCT CAATGCATGG GTCAAACTCA TCGAGGAAAA GAAGTTCGGA GCGGAAGTGG GTCGTTTAGC CCCCCTTAAT GCACGTAGAG GGAAACAGTG GTTCCTGGGA GTTACGTACC CAGTTTGAGT AGCTCCTTTT CTTCAAGCCT CGCCTTCACC
Gag
• P G F Q A L S E G C T P Y D I N Q M L N C V G D H Q A A M Q I I R 1001 TCCCAGGGTT CCAGGCACTG AGTGAAGGGT GCACTCCCTA TGACATCAAC CAGATGCTTA ACTGCGTCGG CGACCATCAG GCCGCGATGC AGATTATTCG AGGGTCCCAA GGTCCGTGAC TCACTTCCCA CGTGAGGGAT ACTGTAGTTG GTCTACGAAT TGACGCAGCC GCTGGTAGTC CGGCGCTACG TCTAATAAGC
Gag
■ D I I H E E A A D W D L Q H P Q P A P Q Q G Q L R E P S G S D I A
1101 GGACATAATC AACGAGGAGG CTGCAGACTG GGATTTGCAG CACCCCCAAC CCGCCCCTCA GCAAGGGCAG CTAAGGGAGC CTTCCGGCAG CGACATAGCT
CCTGTATTAG TTGCTCCTCC GACGTCTGAC CCTAAACGTC GTGGGGGTTG GGCGGGGAGT CGTTCCCGTC GATTCCCTCG GAAGGCCGTC GCTGTATCGA
Gag
G T T S S V D E Q I Q W M Y R Q Q N P I P V G N I Y R R I Q L G L
1201 GGGACTACTA GCTCCGTGGA TGAACAGATT CAATGGATGT ACAGACAGCA GAATCCGATC CCCGTTGGCA ACATCTACCG GCGCTGGATT CAACTCGGAC CCCTGATGAT CGAGGCACCT ACTTGTCTAA GTTACCTACA TGTCTGTCGT CTTAGGCTAG GGGCAACCGT TGTAGATGGC CGCGACCTAA GTTGAGCCTG
Gag
• Q K C V R M Y N P T N I L D V K Q G P K E P F Q S Y V D R F Y S 1301 TTCAGAAGTG CGTCAGAATG TACAACCCCA CCAATATTCT GGATGTGAAA CAGGGGCCGA AAGAGCCCTT TCAATCCTAC GTCGACCGTT TCTACAAAAG AAGTCTTCAC GCAGTCTTAC ATGTTGGGGT GGTTATAAGA CCTACACTTT GTCCCCGGCT TTCTCGGGAA AGTTAGGATG CAGCTGGCAA AGATGTTTTC
Gag
• L R A E Q T D A A V K N W M T Q T L L I Q N A N P D C K L V L K G 1401 TCTACGCGCC GAGCAGACCG ATGCCGCAGT GAAGAACTGG ATGACACAGA CGCTCCTGAT ACAGAATGCT AACCCTGATT GTAAACTCGT GCTGAAGGGC AGATGCGCGG CTCGTCTGGC TACGGCGTCA CTTCTTGACC TACTGTGTCT GCGAGGACTA TGTCTTACGA TTGGGACTAA CATTTGAGCA CGACTTCCCG
Gag
L G V N P T L E E M L T A C Q G V G G P G Q K A R L M A E A L K E A
1501 TTAGGGGTAA ACCCAACGCT GGAAGAAATG TTAACCGCCT GCCAGGGAGT TGGTGGACCC GGACAGAAGG CCCGGCTAAT GGCCGAGGCG CTGAAAGAAG
AATCCCCATT TGGGTTGCGA CCTTCTTTAC AATTGGCGGA CGGTCCCTCA ACCACCTGGG CCTGTCTTCC GGGCCGATTA CCGGCTCCGC GACTTTCTTC
Gag
• L A P V P I P F A A A Q Q R G P R K P I K C W N C G K E G H S A K 1601 CATTGGCTCC AGTACCCATT CCTTTTGCTG CCGCACAACA GAGAGGTCCC CGTAAACCGA TCAAATGCTG GAACTGTGGG AAGGAGGGGC ACTCCGCTAA GTAACCGAGG TCATGGGTAA GGAAAACGAC GGCGTGTTGT CTCTCCAGGG GCATTTGGCT AGTTTACGAC CTTGACACCC TTCCTCCCCG TGAGGCGATT
Gag
• Q C R A P R R Q G C K C G M D H V M A R C P D R Q A G F L G L 1701 ACAATGTCGA GCGCCTAGAC GTCAGGGGTG TTGGAAGTGT GGTAAAATGG ACCACGTTAT GGCCAAATGC CCCGACAGAC AAGCCGGGTT CCTCGGGTTA TGTTACAGCT CGCGGATCTG CAGTCCCCAC AACCTTCACA CCATTTTACC TGGTGCAATA CCGGTTTACG GGGCTGTCTG TTCGGCCCAA GGAGCCCAAT
Gag
G P G K K P R N F P A Q V H Q G L T P T A P P E D P A V D L L
1801 GGGCCTTGGG GAAAAAAGCC CAGAAACTTC CCAATGGCGC AAGTACACCA GGGCCTGACC CCGACCGCCC CCCCAGAGGA CCCAGCCGTA GACCTCTTGA CCCGGAACCC CTTTTTTCGG GTCTTTGAAG GGTTACCGCG TTCATGTGGT CCCGGACTGG GGCTGGCGGG GGGGTCTCCT GGGTCGGCAT CTGGAGAACT
Gag
• N Y Q L G K Q Q R E S R E K P Y K E V T E D L L H L N S L F G G 1901 AAAACTATAT GCAGCTGGGG AAGCAGCAGC GCGAGAGTAG AGAGAAGCCC TACAAGGAGG TTACGGAAGA TCTGTTACAC CTTAATTCGT TATTTGGTGG TTTTGATATA CGTCGACCCC TTCGTCGTCG CGCTCTCATC TCTCTTCGGG ATGTTCCTCC AATGCCTTCT AGACAATGTG GAATTAAGCA ATAAACCACC
FMDV2A
Gag Pro
• D Q N F D L L L A G D V E S N P G P V L E L R Q R G P Q R Q A V 2001 TGATCAGAAT TTCGACCTGC TTAAACTTGC TGGCGACGTT GAGTCAAATC CGGGCCCTGT GCTGGAGTTG AGACAGCGCG GGCCCCAGCG GCAGGCTGTT ACTAGTCTTA AAGCTGGACG AATTTGAACG ACCGCTGCAA CTCAGTTTAG GCCCGGGACA CGACCTCAAC TCTGTCGCGC CCGGGGTCGC CGTCCGACAA
Pro
Q S P S E T G L L E V W Q D G P R D G Q M P R Q T G G F F R P W S
2101 CAGAGCCCAT CAGAGACGGG TCTACTTGAG GTGTGGCAGG ATGGCCCCCG TGATGGACAG ATGCCTCGCC AGACGGGAGG GTTCTTCCGA CCCTGGAGTA GTCTCGGGTA GTCTCTGCCC AGATGAACTC CACACCGTCC TACCGGGGGC ACTACCTGTC TACGGAGCGG TCTGCCCTCC CAAGAAGGCT GGGACCTCAT
Pro
• G E A P Q F P H G S S A S G A D A N C S P R G P S C G S A E L 2201 TGGGAAAGGA GGCCCCGCAG TTCCCTCATG GCTCTTCTGC CTCTGGCGCG GATGCCAATT GTAGCCCCCG AGGCCCTTCT TGCGGCTCAG CCAAGGAGCT ACCCTTTCCT CCGGGGCGTC AAGGGAGTAC CGAGAAGACG GAGACCGCGC CTACGGTTAA CATCGGGGGC TCCGGGAAGA ACGCCGAGTC GGTTCCTCGA
Pro
• H A V G Q A A E R K Q R E A L Q G G D R G F A A P Q F S L W R R P 2301 GCACGCAGTG GGCCAGGCAG CAGAGCGCAA ACAGCGAGAA GCACTGCAGG GCGGTGACCG TGGTTTTGCC GCCCCACAAT TCAGTCTGTG GCGCCGACCT CGTGCGTCAC CCGGTCCGTC GTCTCGCGTT TGTCGCTCTT CGTGACGTCC CGCCACTGGC ACCAAAACGG CGGGGTGTTA AGTCAGACAC CGCGGCTGGA
Pro
V V T A H I E G Q P V E V L L D T G A D D S I V T G I E L G P H Y T GTCGTGACTG CTCATATCGA GGGTCAGCCC GTGGAGGTTT TACTGGACAC TGGCGCAGAC GATTCTATTG TGACTGGCAT TGAACTAGGC CCCCATTACA CAGCACTGAC GAGTATAGCT CCCAGTCGGG CACCTCCAAA ATGACCTGTG ACCGCGTCTG CTAAGATAAC ACTGACCGTA ACTTGATCCG GGGGTAATGT
Pro
• P K I V G G I G G F I N T K E Y K N V E I E V L G K R I K G T I M 2501 CTCCAAAAAT CGTAGGGGGG ATAGGAGGAT TTATCAACAC GAAGGAGTAT AAGAATGTGG AGATCGAGGT TCTCGGAAAA CGCATTAAGG GAACGATTAT GAGGTTTTTA GCATCCCCCC TATCCTCCTA AATAGTTGTG CTTCCTCATA TTCTTACACC TCTAGCTCCA AGAGCCTTTT GCGTAATTCC CTTGCTAATA
FMDV2A
Pro
• T G D T P I N I F G R N L L T A L G M S L N L N F D L L K L A G D 2601 GACAGGCGAT ACACCCATTA ACATCTTTGG ACGCAATCTA CTTACGGCCC TCGGAATGAG CCTTAACCTC AACTTCGACT TACTCAAGCT CGCCGGAGAC CTGTCCGCTA TGTGGGTAAT TGTAGAAACC TGCGTTAGAT GAATGCCGGG AGCCTTACTC GGAATTGGAG TTGAAGCTGA ATGAGTTCGA GCGGCCTCTG
TM domain WN E (split)
prE/NSl Sig
FMDV2A NSl
V E S N P G P A R D R S I A L T F L A V G G V L L F L S V N V H A D -
2701 GTGGAGTCCA ATCCCGGCCC AGCCCGGGAC AGGTCCATAG CTCTCACGTT TCTCGCAGTT GGAGGAGTTC TGCTCTTCCT CTCCGTGAAC GTGCACGCTG
CACCTCAGGT TAGGGCCGGG TCGGGCCCTG TCCAGGTATC GAGAGTGCAA AGAGCGTCAA CCTCCTCAAG ACGAGAAGGA GAGGCACTTG CACGTGCGAC
NSl
• T G C A I D I S R Q E L R C G S G V F I H N D V E A W M D R Y K Y " ACACTGGGTG TGCCATAGAC ATCAGCCGGC AAGAGCTGAG ATGTGGAAGT GGAGTGTTCA TACACAATGA TGTGGAGGCT TGGATGGACC GGTACAAGTA TGTGACCCAC ACGGTATCTG TAGTCGGCCG TTCTCGACTC TACACCTTCA CCTCACAAGT ATGTGTTACT ACACCTCCGA ACCTACCTGG CCATGTTCAT
NSl
■ Y P E T P Q G L A K I I Q K A H K E G V C G L R S V S R L E H Q M 2901 TTACCCTGAA ACGCCACAAG GCCTAGCCAA GATCATTCAG AAAGCTCATA AGGAAGGAGT GTGCGGTCTA CGATCAGTTT CCAGACTGGA GCATCAAATG AATGGGACTT TGCGGTGTTC CGGATCGGTT CTAGTAAGTC TTTCGAGTAT TCCTTCCTCA CACGCCAGAT GCTAGTCAAA GGTCTGACCT CGTAGTTTAC
NSl
W E A V K D E L N T L L K TGGGAAGCAG TGAAGGACGA GCTGAACACT CTTTTGAAG ACCCTTCGTC ACTTCCTGCT CGACTTGTGA GAAAACTTC
Construct 3
1. PIV-WN (ACprME)-SIV Anch Gag
Figure imgf000124_0001
DeleteC230AnchGag
13Η3Φ
2. Sequence of PIV-WN (ACprME)-SIV Anch Gag (partial).
UTR
M S
1 AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
C
NS3 cleavage
• P G G P G K S R A V Y L L K R G M P R V L S L I G L K Q K K R
1 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGC AAAAGAAGCG
GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCG TTTTCTTCGC
C Anchor
NS3 cleavage FMDV2A
■ G G K T G I A V M I G L I A S V G A N F D L L K L A G D V E S N P 1 AGGCGGAAAG ACAGGTATTG CTGTGATGAT TGGCCTGATC GCCAGCGTAG GAGCAAATTT TGACCTGTTA AAACTGGCCG GGGACGTCGA AAGCAACCCC TCCGCCTTTC TGTCCATAAC GACACTACTA ACCGGACTAG CGGTCGCATC CTCGTTTAAA ACTGGACAAT TTTGACCGGC CCCTGCAGCT TTCGTTGGGG FMDV2A
Gag
G P M G A R N S V L S G K K A D E L E I R L R P G G K K Y M L K 301 GGTCCGATGG GCGCTAGGAA TAGCGTGCTT AGTGGCAAAA AGGCTGATGA ACTTGAGAAG ATCCGGCTCC GTCCGGGCGG GAAGAAGAAG TATATGTTGA CCAGGCTACC CGCGATCCTT ATCGCACGAA TCACCGTTTT TCCGACTACT TGAACTCTTC TAGGCCGAGG CAGGCCCGCC CTTCTTCTTC ATATACAACT
Gag
• H V V W A A N E L D R F G L A E S L L E N K E G C Q K I L S V L A 401 AACATGTCGT GTGGGCCGCC AACGAGTTAG ATAGGTTTGG GCTAGCAGAG TCATTGCTCG AAAACAAGGA AGGATGTCAG AAGATACTAA GTGTCCTGGC TTGTACAGCA CACCCGGCGG TTGCTCAATC TATCCAAACC CGATCGTCTC AGTAACGAGC TTTTGTTCCT TCCTACAGTC TTCTATGATT CACAGGACCG
Gag
• P L V P T G S E N L K S L Y N T V C V I W C I H A E E K V K H T E 501 ACCTTTGGTA CCCACGGGGT CTGAGAACTT AAAGAGTCTG TATAACACTG TGTGCGTGAT CTGGTGCATT CACGCCGAAG AGAAAGTGAA GCACACCGAA TGGAAACCAT GGGTGCCCCA GACTCTTGAA TTTCTCAGAC ATATTGTGAC ACACGCACTA GACCACGTAA GTGCGGCTTC TCTTTCACTT CGTGTGGCTT
Gag
E A K Q I V Q R H L V V E T G T A E T M P K T S R P T A P S S G R G
601 GAAGCTAAGC AAATAGTGCA GAGACATTTG GTCGTGGAAA CCGGGACCGC CGAGACTATG CCCAAAACAT CCCGTCCAAC CGCTCCAAGT AGTGGAAGAG CTTCGATTCG TTTATCACGT CTCTGTAAAC CAGCACCTTT GGCCCTGGCG GCTCTGATAC GGGTTTTGTA GGGCAGGTTG GCGAGGTTCA TCACCTTCTC
Gag
• G N Y P V Q Q I G G N Y V H L P L S P R T L N A W V K L I E E K K 701 GAGGTAACTA CCCCGTTCAG CAAATCGGGG GGAATTACGT GCATCTCCCT TTGTCACCAA GGACCCTCAA TGCATGGGTC AAACTCATCG AGGAAAAGAA CTCCATTGAT GGGGCAAGTC GTTTAGCCCC CCTTAATGCA CGTAGAGGGA AACAGTGGTT CCTGGGAGTT ACGTACCCAG TTTGAGTAGC TCCTTTTCTT
Gag
• F G A E V V P G F Q A L S E G C T P Y D I N Q M L N C V G D H Q A 801 GTTCGGAGCG GAAGTGGTCC CAGGGTTCCA GGCACTGAGT GAAGGGTGCA CTCCCTATGA CATCAACCAG ATGCTTAACT GCGTCGGCGA CCATCAGGCC CAAGCCTCGC CTTCACCAGG GTCCCAAGGT CCGTGACTCA CTTCCCACGT GAGGGATACT GTAGTTGGTC TACGAATTGA CGCAGCCGCT GGTAGTCCGG
Gag
A M Q I I R D I I N E E A A D D L Q H P Q P A P Q Q G Q L R E P S
901 GCGATGCAGA TTATTCGGGA CATAATCAAC GAGGAGGCTG CAGACTGGGA TTTGCAGCAC CCCCAACCCG CCCCTCAGCA AGGGCAGCTA AGGGAGCCTT CGCTACGTCT AATAAGCCCT GTATTAGTTG CTCCTCCGAC GTCTGACCCT AAACGTCGTG GGGGTTGGGC GGGGAGTCGT TCCCGTCGAT TCCCTCGGAA
Gag
■ G S D I A G T T S S V D E Q I Q W M Y R Q Q N P I P V G N I Y R R 1001 CCGGCAGCGA CATAGCTGGG ACTACTAGCT CCGTGGATGA ACAGATTCAA TGGATGTACA GACAGCAGAA TCCGATCCCC GTTGGCAACA TCTACCGGCG GGCCGTCGCT GTATCGACCC TGATGATCGA GGCACCTACT TGTCTAAGTT ACCTACATGT CTGTCGTCTT AGGCTAGGGG CAACCGTTGT AGATGGCCGC
Gag
• W I Q L G L Q K C V R M Y N P T N I L D V K Q G P K E P F Q S Y V 1101 CTGGATTCAA CTCGGACTTC AGAAGTGCGT CAGAATGTAC AACCCCACCA ATATTCTGGA TGTGAAACAG GGGCCGAAAG AGCCCTTTCA ATCCTACGTC GACCTAAGTT GAGCCTGAAG TCTTCACGCA GTCTTACATG TTGGGGTGGT TATAAGACCT ACACTTTGTC CCCGGCTTTC TCGGGAAAGT TAGGATGCAG
Gag
D R F Y K S L R A E Q T D A A V K N W M T Q T L L I Q N A N P D C K
1201 GACCGTTTCT ACAAAAGTCT ACGCGCCGAG CAGACCGATG CCGCAGTGAA GAACTGGATG ACACAGACGC TCCTGATACA GAATGCTAAC CCTGATTGTA CTGGCAAAGA TGTTTTCAGA TGCGCGGCTC GTCTGGCTAC GGCGTCACTT CTTGACCTAC TGTGTCTGCG AGGACTATGT CTTACGATTG GGACTAACAT
Gag
• L V L K G L G V N P T L E E L T A C Q G V G G P G Q K A R L M A 1301 AACTCGTGCT GAAGGGCTTA GGGGTAAACC CAACGCTGGA AGAAATGTTA ACCGCCTGCC AGGGAGTTGG TGGACCCGGA CAGAAGGCCC GGCTAATGGC
TTGAGCACGA CTTCCCGAAT CCCCATTTGG GTTGCGACCT TCTTTACAAT TGGCGGACGG TCCCTCAACC ACCTGGGCCT GTCTTCCGGG CCGATTACCG
Gag
- E A L R E A L A P V P I P F A A A Q Q G P R K P I K C W N C G K
1401 CGAGGCGCTG AAAGAAGCAT TGGCTCCAGT ACCCATTCCT TTTGCTGCCG CACAACAGAG AGGTCCCCGT AAACCGATCA AATGCTGGAA CTGTGGGAAG
GCTCCGCGAC TTTCTTCGTA ACCGAGGTCA TGGGTAAGGA AAACGACGGC GTGTTGTCTC TCCAGGGGCA TTTGGCTAGT TTACGACCTT GACACCCTTC
Gag
E G H S A K Q C R A P R R Q G C W K C G K M D H V M A K C P D R Q A
1501 GAGGGGCACT CCGCTAAACA ATGTCGAGCG CCTAGACGTC AGGGGTGTTG GAAGTGTGGT AAAATGGACC ACGTTATGGC CAAATGCCCC GACAGACAAG
CTCCCCGTGA GGCGATTTGT TACAGCTCGC GGATCTGCAG TCCCCACAAC CTTCACACCA TTTTACCTGG TGCAATACCG GTTTACGGGG CTGTCTGTTC
Gag
• G F L G L G P W G K K P R N F P M A Q V H Q G L T P T A P P E D P
1601 CCGGGTTCCT CGGGTTAGGG CCTTGGGGAA AAAAGCCCAG AAACTTCCCA ATGGCGCAAG TACACCAGGG CCTGACCCCG ACCGCCCCCC CAGAGGACCC
GGCCCAAGGA GCCCAATCCC GGAACCCCTT TTTTCGGGTC TTTGAAGGGT TACCGCGTTC ATGTGGTCCC GGACTGGGGC TGGCGGGGGG GTCTCCTGGG
Gag
■ A V D L L K N Y M Q L G K Q Q R E S R E K P Y K E V T E D L L H L
1701 AGCCGTAGAC CTCTTGAAAA ACTATATGCA GCTGGGGAAG CAGCAGCGCG AGAGTAGAGA GAAGCCCTAC AAGGAGGTTA CGGAAGATCT GTTACACCTT
TCGGCATCTG GAGAACTTTT TGATATACGT CGACCCCTTC GTCGTCGCGC TCTCATCTCT CTTCGGGATG TTCCTCCAAT GCCTTCTAGA CAATGTGGAA
FMDV2A pre E/NS1 signal
Gag Transmembrane domain of WNV E
(split)
N S G G D Q N F K L A G D V E S N P A R D R S A L T
1801 AATTCGTTAT TTGGTGGTGA TCAGAATTTC GACCTGCTTA AACTTGCTGG CGACGTTGAG TCAAATCCGG GCCCTGCCCG GGACAGGTCC ATAGCTCTCA
TTAAGCAATA AACCACCACT AGTCTTAAAG CTGGACGAAT TTGAACGACC GCTGCAACTC AGTTTAGGCC CGGGACGGGC CCTGTCCAGG TATCGAGAGT
Transmembrane domain of WNV E (split)
NSl
• F L A V G G V L L F L S V N V H A D T G C A I D I S R Q E L R C G
1901 CGTTTCTCGC AGTTGGAGGA GTTCTGCTCT TCCTCTCCGT GAACGTGCAC GCTGACACTG GGTGTGCCAT AGACATCAGC CGGCAAGAGC TGAGATGTGG
GCAAAGAGCG TCAACCTCCT CAAGACGAGA AGGAGAGGCA CTTGCACGTG CGACTGTGAC CCACACGGTA TCTGTAGTCG GCCGTTCTCG ACTCTACACC
NSl
• S G V F I H N D V E A W M D R Y K Y Y P E T P Q G L A K I I Q K A
2001 AAGTGGAGTG TTCATACACA ATGATGTGGA GGCTTGGATG GACCGGTACA AGTATTACCC TGAAACGCCA CAAGGCCTAG CCAAGATCAT TCAGAAAGCT
TTCACCTCAC AAGTATGTGT TACTACACCT CCGAACCTAC CTGGCCATGT TCATAATGGG ACTTTGCGGT GTTCCGGATC GGTTCTAGTA AGTCTTTCGA
NSl
H E G V C G L R S V S R L E H Q M W E A V K D E L N T L L K
2101 CATAAGGAAG GAGTGTGCGG TCTACGATCA GTTTCCAGAC TGGAGCATCA AATGTGGGAA GCAGTGAAGG ACGAGCTGAA CACTCTTTTG AAG
GTATTCCTTC CTCACACGCC AGATGCTAGT CAAAGGTCTG ACCTCGTAGT TTACACCCTT CGTCACTTCC TGCTCGACTT GTGAGAAAAC TTC
Construct 4
1. PIV-WN (ACprME)-SIV Anc Gag &Pro
Figure imgf000127_0001
DeleteC23DAnchGag&Pr
2. Sequence of PIV-WN (ACprME)-SIV Anch Gag & Pro (partial).
5' UTR
M S
1 AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
NS3 cleavage
C
- K K P G G P G K S R A V Y L L K R G P R V L S L I G L K Q K K R 101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGC AAAAGAAGCG GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCG TTTTCTTCGC
C Anchor
KS3 cleavage FMDV2A
• G G K T G I A V M I G L I A S V G A N F D L L K L A G D V E S N P 201 AGGCGGAAAG ACAGGTATTG CTGTGATGAT TGGCCTGATC GCCAGCGTAG GAGCAAATTT TGACCTGTTA AAACTGGCCG GGGACGTCGA AAGCAACCCC TCCGCCTTTC TGTCCATAAC GACACTACTA ACCGGACTAG CGGTCGCATC CTCGTTTAAA ACTGGACAAT TTTGACCGGC CCCTGCAGCT TTCGTTGGGG F DV2A
Gag
G P M G A R N S V L S G K K A D E L E K I R L R P G G K K Y L K
301 GGTCCGATGG GCGCTAGGAA TAGCGTGCTT AGTGGCAAAA AGGCTGATGA ACTTGAGAAG ATCCGGCTCC GTCCGGGCGG GAAGAAGAAG TATATGTTGA CCAGGCTACC CGCGATCCTT ATCGCACGAA TCACCGTTTT TCCGACTACT TGAACTCTTC TAGGCCGAGG CAGGCCCGCC CTTCTTCTTC ATATACAACT
Gag
■ H V V W A A N E L D R F G L A E S L L E N K E G C Q K I L S V L A 401 AACATGTCGT GTGGGCCGCC AACGAGTTAG ATAGGTTTGG GCTAGCAGAG TCATTGCTCG AAAACAAGGA AGGATGTCAG AAGATACTAA GTGTCCTGGC TTGTACAGCA CACCCGGCGG TTGCTCAATC TATCCAAACC CGATCGTCTC AGTAACGAGC TTTTGTTCCT TCCTACAGTC TTCTATGATT CACAGGACCG
Gag
■ P L V P T G S E N L K S L Y N T V C V I W C I H A E E K V K H T E
501 ACCTTTGGTA CCCACGGGGT CTGAGAACTT AAAGAGTCTG TATAACACTG TGTGCGTGAT CTGGTGCATT CACGCCGAAG AGAAAGTGAA GCACACCGAA
TGGAAACCAT GGGTGCCCCA GACTCTTGAA TTTCTCAGAC ATATTGTGAC ACACGCACTA GACCACGTAA GTGCGGCTTC TCTTTCACTT CGTGTGGCTT
Gag
E A K Q I V Q R H L V V E T G T A E T M P K T S R P T A P S S. G R G 601 GAAGCTAAGC AAATAGTGCA GAGACATTTG GTCGTGGAAA CCGGGACCGC CGAGACTATG CCCAAAACAT CCCGTCCAAC CGCTCCAAGT AGTGGAAGAG CTTCGATTCG TTTATCACGT CTCTGTAAAC CAGCACCTTT GGCCCTGGCG GCTCTGATAC GGGTTTTGTA GGGCAGGTTG GCGAGGTTCA TCACCTTCTC
Gag
• G N Y P V Q Q I G G N Y V H L P L S P R T L N A W V L I E E K K · 701 GAGGTAACTA CCCCGTTCAG CAAATCGGGG GGAATTACGT GCATCTCCCT TTGTCACCAA GGACCCTCAA TGCATGGGTC AAACTCATCG AGGAAAAGAA CTCCATTGAT GGGGCAAGTC GTTTAGCCCC CCTTAATGCA CGTAGAGGGA AACAGTGGTT CCTGGGAGTT ACGTACCCAG TTTGAGTAGC TCCTTTTCTT
Gag
■ F G A E V V P G F Q A L S E G C T P Y D I N Q M L N C V G D H Q A
801 GTTCGGAGCG GAAGTGGTCC CAGGGTTCCA GGCACTGAGT GAAGGGTGCA CTCCCTATGA CATCAACCAG ATGCTTAACT GCGTCGGCGA CCATCAGGCC
CAAGCCTCGC CTTCACCAGG GTCCCAAGGT CCGTGACTCA CTTCCCACGT GAGGGATACT GTAGTTGGTC TACGAATTGA CGCAGCCGCT GGTAGTCCGG
Gag
A M Q I I R D I I N E E A A D W D L Q H P Q P A P Q Q G Q L R E P S
901 GCGATGCAGA TTATTCGGGA CATAATCAAC GAGGAGGCTG CAGACTGGGA TTTGCAGCAC CCCCAACCCG CCCCTCAGCA AGGGCAGCTA AGGGAGCCTT CGCTACGTCT AATAAGCCCT GTATTAGTTG CTCCTCCGAC GTCTGACCCT AAACGTCGTG GGGGTTGGGC GGGGAGTCGT TCCCGTCGAT TCCCTCGGAA
Gag
• G S D I A G T T S S V D E Q I Q W M Y R Q Q N P I P V G N I Y R R 1001 CCGGCAGCGA CATAGCTGGG ACTACTAGCT CCGTGGATGA ACAGATTCAA TGGATGTACA GACAGCAGAA TCCGATCCCC GTTGGCAACA TCTACCGGCG GGCCGTCGCT GTATCGACCC TGATGATCGA GGCACCTACT TGTCTAAGTT ACCTACATGT CTGTCGTCTT AGGCTAGGGG CAACCGTTGT AGATGGCCGC
Gag
• I Q L G L Q K C V R M Y N P T N I L D V K Q G P K E P F Q S Y V 1101 CTGGATTCAA CTCGGACTTC AGAAGTGCGT CAGAATGTAC AACCCCACCA ATATTCTGGA TGTGAAACAG GGGCCGAAAG AGCCCTTTCA ATCCTACGTC GACCTAAGTT GAGCCTGAAG TCTTCACGCA GTCTTACATG TTGGGGTGGT TATAAGACCT ACACTTTGTC CCCGGCTTTC TCGGGAAAGT TAGGATGCAG
Gag
D R F Y K S L R A E Q T D A A V K N M T Q T L L I Q N A N P D C K
1201 GACCGTTTCT ACAAAAGTCT ACGCGCCGAG CAGACCGATG CCGCAGTGAA GAACTGGATG ACACAGACGC TCCTGATACA GAATGCTAAC CCTGATTGTA CTGGCAAAGA TGTTTTCAGA TGCGCGGCTC GTCTGGCTAC GGCGTCACTT CTTGACCTAC TGTGTCTGCG AGGACTATGT CTTACGATTG GGACTAACAT
Gag
• L V L K G L G V N P T L E E M L T A C Q G V G G P G Q K A R L M A 1301 AACTCGTGCT GAAGGGCTTA GGGGTAAACC CAACGCTGGA AGAAATGTTA ACCGCCTGCC AGGGAGTTGG TGGACCCGGA CAGAAGGCCC GGCTAATGGC TTGAGCACGA CTTCCCGAAT CCCCATTTGG GTTGCGACCT TCTTTACAAT TGGCGGACGG TCCCTCAACC ACCTGGGCCT GTCTTCCGGG CCGATTACCG
Gag
• E A L K E A L A P V P I P F A A A Q Q R G P R K P I K C N C G 1401 CGAGGCGCTG AAAGAAGCAT TGGCTCCAGT ACCCATTCCT TTTGCTGCCG CACAACAGAG AGGTCCCCGT AAACCGATCA AATGCTGGAA CTGTGGGAAG GCTCCGCGAC TTTCTTCGTA ACCGAGGTCA TGGGTAAGGA AAACGACGGC GTGTTGTCTC TCCAGGGGCA TTTGGCTAGT TTACGACCTT GACACCCTTC
Gag
E G H S A K Q C R A P R R Q G C C G M D H V M A K C P D R Q A
1501 GAGGGGCACT CCGCTAAACA ATGTCGAGCG CCTAGACGTC AGGGGTGTTG GAAGTGTGGT AAAATGGACC ACGTTATGGC CAAATGCCCC GACAGACAAG CTCCCCGTGA GGCGATTTGT TACAGCTCGC GGATCTGCAG TCCCCACAAC CTTCACACCA TTTTACCTGG TGCAATACCG GTTTACGGGG CTGTCTGTTC
Gag
• G F L G L G P W G K K P R H F P M A Q V H Q G L T P T A P P E D P 1601 CCGGGTTCCT CGGGTTAGGG CCTTGGGGAA AAAAGCCCAG AAACTTCCCA ATGGCGCAAG TACACCAGGG CCTGACCCCG ACCGCCCCCC CAGAGGACCC GGCCCAAGGA GCCCAATCCC GGAACCCCTT TTTTCGGGTC TTTGAAGGGT TACCGCGTTC ATGTGGTCCC GGACTGGGGC TGGCGGGGGG GTCTCCTGGG
Gag
■ A V D L L K N Y M Q L G K Q Q R E S R E K P Y K E V T E D L L H L 1701 AGCCGTAGAC CTCTTGAAAA ACTATATGCA GCTGGGGAAG CAGCAGCGCG AGAGTAGAGA GAAGCCCTAC AAGGAGGTTA CGGAAGATCT GTTACACCTT TCGGCATCTG GAGAACTTTT TGATATACGT CGACCCCTTC GTCGTCGCGC TCTCATCTCT CTTCGGGATG TTCCTCCAAT GCCTTCTAGA CAATGTGGAA
FMDV2A
Gag Pro
N S L F G G D Q N F D L L K L A G D V E S N P G P V L E L R Q R G P
1801 AATTCGTTAT TTGGTGGTGA TCAGAATTTC GACCTGCTTA AACTTGCTGG CGACGTTGAG TCAAATCCGG GCCCTGTGCT GGAGTTGAGA CAGCGCGGGC TTAAGCAATA AACCACCACT AGTCTTAAAG CTGGACGAAT TTGAACGACC GCTGCAACTC AGTTTAGGCC CGGGACACGA CCTCAACTCT GTCGCGCCCG
Pro
• Q R Q A V Q S P S E T G L L E V Q D G P R D G Q M P R Q T G G F 1901 CCCAGCGGCA GGCTGTTCAG AGCCCATCAG AGACGGGTCT ACTTGAGGTG TGGCAGGATG GCCCCCGTGA TGGACAGATG CCTCGCCAGA CGGGAGGGTT GGGTCGCCGT CCGACAAGTC TCGGGTAGTC TCTGCCCAGA TGAACTCCAC ACCGTCCTAC CGGGGGCACT ACCTGTCTAC GGAGCGGTCT GCCCTCCCAA
Pro
• F R P W S M G K E A P Q F P H G S S A S G A D A N C S P R G P S C
2001 CTTCCGACCC TGGAGTATGG GAAAGGAGGC CCCGCAGTTC CCTCATGGCT CTTCTGCCTC TGGCGCGGAT GCCAATTGTA GCCCCCGAGG CCCTTCTTGC
GAAGGCTGGG ACCTCATACC CTTTCCTCCG GGGCGTCAAG GGAGTACCGA GAAGACGGAG ACCGCGCCTA CGGTTAACAT CGGGGGCTCC GGGAAGAACG
Pro
G S A K E L H A V G Q A A E R K Q R E A L Q G G D R G F A A P Q F S
2101 GGCTCAGCCA AGGAGCTGCA CGCAGTGGGC CAGGCAGCAG AGCGCAAACA GCGAGAAGCA CTGCAGGGCG GTGACCGTGG TTTTGCCGCC CCACAATTCA
CCGAGTCGGT TCCTCGACGT GCGTCACCCG GTCCGTCGTC TCGCGTTTGT CGCTCTTCGT GACGTCCCGC CACTGGCACC AAAACGGCGG GGTGTTAAGT
Pro
• L W R R P V V T A H I E G Q P V E V L L D T G A D D S I V T G I E
2201 GTCTGTGGCG CCGACCTGTC GTGACTGCTC ATATCGAGGG TCAGCCCGTG GAGGTTTTAC TGGACACTGG CGCAGACGAT TCTATTGTGA CTGGCATTGA
CAGACACCGC GGCTGGACAG CACTGACGAG TATAGCTCCC AGTCGGGCAC CTCCAAAATG ACCTGTGACC GCGTCTGCTA AGATAACACT GACCGTAACT
Pro
• L G P H Y T P K I V G G I G G F I N T K E Y K N V E I E V L G K R
2301 ACTAGGCCCC CATTACACTC CAAAAATCGT AGGGGGGATA GGAGGATTTA TCAACACGAA GGAGTATAAG AATGTGGAGA TCGAGGTTCT CGGAAAACGC TGATCCGGGG GTAATGTGAG GTTTTTAGCA TCCCCCCTAT CCTCCTAAAT AGTTGTGCTT CCTCATATTC TTACACCTCT AGCTCCAAGA GCCTTTTGCG
FMDV2A
Pro
I K G T I M T G D T P I N I F G R N L L T A L G M S L N L N F D L L
2401 ATTAAGGGAA CGATTATGAC AGGCGATACA CCCATTAACA TCTTTGGACG CAATCTACTT ACGGCCCTCG GAATGAGCCT TAACCTCAAC TTCGACTTAC TAATTCCCTT GCTAATACTG TCCGCTATGT GGGTAATTGT AGAAACCTGC GTTAGATGAA TGCCGGGAGC CTTACTCGGA ATTGGAGTTG AAGCTGAATG
pre E/NS1 signal
FMDV2A Transmembrane domain of WNV E (split)
• K L A G D V E S N P G P A R D R S I A L T F L A V G G V L L F L S 2501 TCAAGCTCGC CGGAGACGTG GAGTCCAATC CCGGCCCAGC CCGGGACAGG TCCATAGCTC TCACGTTTCT CGCAGTTGGA GGAGTTCTGC TCTTCCTCTC AGTTCGAGCG GCCTCTGCAC CTCAGGTTAG GGCCGGGTCG GGCCCTGTCC AGGTATCGAG AGTGCAAAGA GCGTCAACCT CCTCAAGACG AGAAGGAGAG
Transmembrane domain of WNV E (split)
HS1
• V N V H A D T G C A I D I S R Q E L R C G S G V F I H O V E A W
2601 CGTGAACGTG CACGCTGACA CTGGGTGTGC CATAGACATC AGCCGGCAAG AGCTGAGATG TGGAAGTGGA GTGTTCATAC ACAATGATGT GGAGGCTTGG
GCACTTGCAC GTGCGACTGT GACCCACACG GTATCTGTAG TCGGCCGTTC TCGACTCTAC ACCTTCACCT CACAAGTATG TGTTACTACA CCTCCGAACC
NS1
M D R Y K Y Y P E T P Q G L A K I I Q K A H K E G V C G L R S V S R
2701 ATGGACCGGT ACAAGTATTA CCCTGAAACG CCACAAGGCC TAGCCAAGAT CATTCAGAAA GCTCATAAGG AAGGAGTGTG CGGTCTACGA TCAGTTTCCA TACCTGGCCA TGTTCATAAT GGGACTTTGC GGTGTTCCGG ATCGGTTCTA GTAAGTCTTT CGAGTATTCC TTCCTCACAC GCCAGATGCT AGTCAAAGGT
NS1
- L E H Q M W E A V K D E L N T L L K
2801 GACTGGAGCA TCAAATGTGG GAAGCAGTGA AGGACGAGCT GAACACTCTT TTGAAG
CTGACCTCGT AGTTTACACC CTTCGTCACT TCCTGCTCGA CTTGTGAGAA AACTTC
Construct 5
1. PIV-WN (ACprME)-SIV FMD2a Gag
Figure imgf000131_0001
De!eteC230FMD2AGag uence of PIV-WN (ACprME)-SIV FMD2a Gag
5' DTR
M S
1 AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
C
NS3 cleavage
• K K P G G P G K S R A V Y L L K R G M P R V L S L I G L Q K K R
101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGC AAAAGAAGCG
GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCG TTTTCTTCGC
FMDV2A
NS3 cleavage Gag
■ N F D L L K L A G D V E S N P G P M G A R N S V L S G K K A D E L 201 AAATTTTGAC CTGTTAAAAC TGGCCGGGGA CGTCGAAAGC AACCCCGGTC CGATGGGCGC TAGGAATAGC GTGCTTAGTG GCAAAAAGGC TGATGAACTT TTTAAAACTG GACAATTTTG ACCGGCCCCT GCAGCTTTCG TTGGGGCCAG GCTACCCGCG ATCCTTATCG CACGAATCAC CGTTTTTCCG ACTACTTGAA
Gag
E K I R L R P G G K K K Y M L K H V V W A A N E L D R F G L A E S L
301 GAGAAGATCC GGCTCCGTCC GGGCGGGAAG AAGAAGTATA TGTTGAAACA TGTCGTGTGG GCCGCCAACG AGTTAGATAG GTTTGGGCTA GCAGAGTCAT CTCTTCTAGG CCGAGGCAGG CCCGCCCTTC TTCTTCATAT ACAACTTTGT ACAGCACACC CGGCGGTTGC TCAATCTATC CAAACCCGAT CGTCTCAGTA
Gag
■ L E N K E G C Q K I L S V L A P L V P T G S E N L K S L Y N T V C
401 TGCTCGAAAA CAAGGAAGGA TGTCAGAAGA TACTAAGTGT CCTGGCACCT TTGGTACCCA CGGGGTCTGA GAACTTAAAG AGTCTGTATA ACACTGTGTG
ACGAGCTTTT GTTCCTTCCT ACAGTCTTCT ATGATTCACA GGACCGTGGA AACCATGGGT GCCCCAGACT CTTGAATTTC TCAGACATAT TGTGACACAC
Gag
• V I W C I H A E E K V K H T E E A K Q I V Q R H L V V E T G T A E 501 CGTGATCTGG TGCATTCACG CCGAAGAGAA AGTGAAGCAC ACCGAAGAAG CTAAGCAAAT AGTGCAGAGA CATTTGGTCG TGGAAACCGG GACCGCCGAG GCACTAGACC ACGTAAGTGC GGCTTCTCTT TCACTTCGTG TGGCTTCTTC GATTCGTTTA TCACGTCTCT GTAAACCAGC ACCTTTGGCC CTGGCGGCTC
Gag
T M P T S R P T A P S S G R G G N Y P V Q Q I G G N Y V H L P L S
601 ACTATGCCCA AAACATCCCG TCCAACCGCT CCAAGTAGTG GAAGAGGAGG TAACTACCCC GTTCAGCAAA TCGGGGGGAA TTACGTGCAT CTCCCTTTGT TGATACGGGT TTTGTAGGGC AGGTTGGCGA GGTTCATCAC CTTCTCCTCC ATTGATGGGG CAAGTCGTTT AGCCCCCCTT AATGCACGTA GAGGGAAACA
Gag
■ P R T L N A V K L I E E K K F G A E V V P G F Q A L S E G C T P
701 CACCAAGGAC CCTCAATGCA TGGGTCAAAC TCATCGAGGA AAAGAAGTTC GGAGCGGAAG TGGTCCCAGG GTTCCAGGCA CTGAGTGAAG GGTGCACTCC
GTGGTTCCTG GGAGTTACGT ACCCAGTTTG AGTAGCTCCT TTTCTTCAAG CCTCGCCTTC ACCAGGGTCC CAAGGTCCGT GACTCACTTC CCACGTGAGG
Gag
■ Y D I N Q M L N C V G D H Q A A M Q I I R D I I N E E A A D W D L 801 CTATGACATC AACCAGATGC TTAACTGCGT CGGCGACCAT CAGGCCGCGA TGCAGATTAT TCGGGACATA ATCAACGAGG AGGCTGCAGA CTGGGATTTG GATACTGTAG TTGGTCTACG AATTGACGCA GCCGCTGGTA GTCCGGCGCT ACGTCTAATA AGCCCTGTAT TAGTTGCTCC TCCGACGTCT GACCCTAAAC
Gag
Q H P Q P A P Q Q G Q L R E P S G S D I A G T T S S V D E Q I Q W M
901 CAGCACCCCC AACCCGCCCC TCAGCAAGGG CAGCTAAGGG AGCCTTCCGG CAGCGACATA GCTGGGACTA CTAGCTCCGT GGATGAACAG ATTCAATGGA GTCGTGGGGG TTGGGCGGGG AGTCGTTCCC GTCGATTCCC TCGGAAGGCC GTCGCTGTAT CGACCCTGAT GATCGAGGCA CCTACTTGTC TAAGTTACCT
Gag
• Y R Q Q N P I P V G N I Y R R I Q L G L Q K C V R M Y N P T N I 1001 TGTACAGACA GCAGAATCCG ATCCCCGTTG GCAACATCTA CCGGCGCTGG ATTCAACTCG GACTTCAGAA GTGCGTCAGA ATGTACAACC CCACCAATAT ACATGTCTGT CGTCTTAGGC TAGGGGCAAC CGTTGTAGAT GGCCGCGACC TAAGTTGAGC CTGAAGTCTT CACGCAGTCT TACATGTTGG GGTGGTTATA
Gag
• L D V K Q G P K E P F Q S Y V D R F Y K S L R A E Q T D A A V K N 1101 TCTGGATGTG AAACAGGGGC CGAAAGAGCC CTTTCAATCC TACGTCGACC GTTTCTACAA AAGTCTACGC GCCGAGCAGA CCGATGCCGC AGTGAAGAAC AGACCTACAC TTTGTCCCCG GCTTTCTCGG GAAAGTTAGG ATGCAGCTGG CAAAGATGTT TTCAGATGCG CGGCTCGTCT GGCTACGGCG TCACTTCTTG
Gag
M T Q T L L I Q N A N P D C K L V L K G L G V N P T L E E M L T A
1201 TGGATGACAC AGACGCTCCT GATACAGAAT GCTAACCCTG ATTGTAAACT CGTGCTGAAG GGCTTAGGGG TAAACCCAAC GCTGGAAGAA ATGTTAACCG ACCTACTGTG TCTGCGAGGA CTATGTCTTA CGATTGGGAC TAACATTTGA GCACGACTTC CCGAATCCCC ATTTGGGTTG CGACCTTCTT TACAATTGGC
Gag
■ C Q G V G G P G Q K A R L M A E A L K E A L A P V P I P F A A A Q
1301 CCTGCCAGGG AGTTGGTGGA CCCGGACAGA AGGCCCGGCT AATGGCCGAG GCGCTGAAAG AAGCATTGGC TCCAGTACCC ATTCCTTTTG CTGCCGCACA
GGACGGTCCC TCAACCACCT GGGCCTGTCT TCCGGGCCGA TTACCGGCTC CGCGACTTTC TTCGTAACCG AGGTCATGGG TAAGGAAAAC GACGGCGTGT
Gag
• Q R G P R K P I C N C G K E G H S A K Q C R A P R R Q G C W K 1401 ACAGAGAGGT CCCCGTAAAC CGA C G CTGGAACTGT GGGAAGGAGG GGCACTCCGC TAAACAATGT CGAGCGCCTA GACGTCAGGG GTGTTGGAAG TGTCTCTCCA GGGGCATTTG GCTAGTTTAC GACCTTGACA CCCTTCCTCC CCGTGAGGCG ATTTGTTACA GCTCGCGGAT CTGCAGTCCC CACAACCTTC
Gag
C G K M D H V M A K C P D R Q A G F L G L G P G K K P R N F P M A
1501 TGTGGTAAAA TGGACCACGT TATGGCCAAA TGCCCCGACA GACAAGCCGG GTTCCTCGGG TTAGGGCCTT GGGGAAAAAA GCCCAGAAAC TTCCCAATGG ACACCATTTT ACCTGGTGCA ATACCGGTTT ACGGGGCTGT CTGTTCGGCC CAAGGAGCCC AATCCCGGAA CCCCTTTTTT CGGGTCTTTG AAGGGTTACC
Gag
• Q V H Q G L T P T A P P E D P A V D L L H Y M Q L G K Q Q R E S 1601 CGCAAGTACA CCAGGGCCTG ACCCCGACCG CCCCCCCAGA GGACCCAGCC GTAGACCTCT TGAAAAACTA TATGCAGCTG GGGAAGCAGC AGCGCGAGAG GCGTTCATGT GGTCCCGGAC TGGGGCTGGC GGGGGGGTCT CCTGGGTCGG CATCTGGAGA ACTTTTTGAT ATACGTCGAC CCCTTCGTCG TCGCGCTCTC
FMDV2A
Gag
■ R E K P Y K E V T E D L L H L N S L F G G D Q H F D L L L A G D 1701 TAGAGAGAAG CCCTACAAGG AGGTTACGGA AGATCTGTTA CACCTTAATT CGTTATTTGG TGGTGATCAG AATTTCGACC TGCTTAAACT TGCTGGCGAC ATCTCTCTTC GGGATGTTCC TCCAATGCCT TCTAGACAAT GTGGAATTAA GCAATAAACC ACCACTAGTC TTAAAGCTGG ACGAATTTGA ACGACCGCTG
Transmembrane domain of WNV E (split)
pre E/NS1 signal
FMDV2A NSl
V E S N P G P A R D R S I A L T F L A V G G V L L F L S V N V H A D ·
1801 GTTGAGTCAA ATCCGGGCCC TGCCCGGGAC AGGTCCATAG CTCTCACGTT TCTCGCAGTT GGAGGAGTTC TGCTCTTCCT CTCCGTGAAC GTGCACGCTG
CAACTCAGTT TAGGCCCGGG ACGGGCCCTG TCCAGGTATC GAGAGTGCAA AGAGCGTCAA CCTCCTCAAG ACGAGAAGGA GAGGCACTTG CACGTGCGAC
NSl
• T G C A I D I S R Q E L R C G S G V F I H N D V E A W M D R Y K Y ■ 1901 ACACTGGGTG TGCCATAGAC ATCAGCCGGC AAGAGCTGAG ATGTGGAAGT GGAGTGTTCA TACACAATGA TGTGGAGGCT TGGATGGACC GGTACAAGTA TGTGACCCAC ACGGTATCTG TAGTCGGCCG TTCTCGACTC TACACCTTCA CCTCACAAGT ATGTGTTACT ACACCTCCGA ACCTACCTGG CCATGTTCAT
NSl
• Y P E T P Q G L A K I I Q K A H K E G V C G L R S V S R L E H Q M
2001 TTACCCTGAA ACGCCACAAG GCCTAGCCAA GATCATTCAG AAAGCTCATA AGGAAGGAGT GTGCGGTCTA CGATCAGTTT CCAGACTGGA GCATCAAATG
AATGGGACTT TGCGGTGTTC CGGATCGGTT CTAGTAAGTC TTTCGAGTAT TCCTTCCTCA CACGCCAGAT GCTAGTCAAA GGTCTGACCT CGTAGTTTAC
NSl
W E A V K D E L N T L L K
2101 TGGGAAGCAG TGAAGGACGA GCTGAACACT CTTTTGAAG
ACCCTTCGTC ACTTCCTGCT CGACTTGTGA GAAAACTTC
Construct 6
1. PIV-WN (ACprME)-SIV FMD2a Gag & Pr
Figure imgf000134_0001
DeleteC230FMD2AGag&Pr
2. Sequence of PIV-WN (ACpr E)-SIV fmd2A Gag & Pr (partial).
UTR
M S
1 AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
NS3 cleavage
C
■ K K P G G P G K S R A V Y L L K R G P R V L S L I G L K Q K K R 101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGC AAAAGAAGCG GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCG TTTTCTTCGC
F DV2A
HS3 cleavage Gag
■ N F D L L K L A G D V E S N P G P M G A R N S V L S G K K A D E L 201 AAATTTTGAC CTGTTAAAAC TGGCCGGGGA CGTCGAAAGC AACCCCGGTC CGATGGGCGC TAGGAATAGC GTGCTTAGTG GCAAAAAGGC TGATGAACTT TTTAAAACTG GACAATTTTG ACCGGCCCCT GCAGCTTTCG TTGGGGCCAG GCTACCCGCG ATCCTTATCG CACGAATCAC CGTTTTTCCG ACTACTTGAA
Gag
E K I R L R P G G K K K Y M L K H V V A A N E L D R F G L A E S L
301 GAGAAGATCC GGCTCCGTCC GGGCGGGAAG AAGAAGTATA TGTTGAAACA TGTCGTGTGG GCCGCCAACG AGTTAGATAG GTTTGGGCTA GCAGAGTCAT CTCTTCTAGG CCGAGGCAGG CCCGCCCTTC TTCTTCATAT ACAACTTTGT ACAGCACACC CGGCGGTTGC TCAATCTATC CAAACCCGAT CGTCTCAGTA
Gag
■ L E N K E G C Q K I L S V L A P L V P T G S E N L K S L Y N T V C 401 TGCTCGAAAA CAAGGAAGGA TGTCAGAAGA TACTAAGTGT CCTGGCACCT TTGGTACCCA CGGGGTCTGA GAACTTAAAG AGTCTGTATA ACACTGTGTG ACGAGCTTTT GTTCCTTCCT ACAGTCTTCT ATGATTCACA GGACCGTGGA AACCATGGGT GCCCCAGACT CTTGAATTTC TCAGACATAT TGTGACACAC
Gag
■ V I W C I H A E E K V K H T E E A K Q I V Q R H L V V E T G T A E
501 CGTGATCTGG TGCATTCACG CCGAAGAGAA AGTGAAGCAC ACCGAAGAAG CTAAGCAAAT AGTGCAGAGA CATTTGGTCG TGGAAACCGG GACCGCCGAG
GCACTAGACC ACGTAAGTGC GGCTTCTCTT TCACTTCGTG TGGCTTCTTC GATTCGTTTA TCACGTCTCT GTAAACCAGC ACCTTTGGCC CTGGCGGCTC
Gag
T M P K T S R P T A P S S G R G G N Y P V Q Q I G G N Y V H L P L S
601 ACTATGCCCA AAACATCCCG TCCAACCGCT CCAAGTAGTG GAAGAGGAGG TAACTACCCC GTTCAGCAAA TCGGGGGGAA TTACGTGCAT CTCCCTTTGT
TGATACGGGT TTTGTAGGGC AGGTTGGCGA GGTTCATCAC CTTCTCCTCC ATTGATGGGG CAAGTCGTTT AGCCCCCCTT AATGCACGTA GAGGGAAACA
Gag
• P R T L N A V K L I E E K K F G A E V V P G F Q A L S E G C T P " 701 CACCAAGGAC CCTCAATGCA TGGGTCAAAC TCATCGAGGA AAAGAAGTTC GGAGCGGAAG TGGTCCCAGG GTTCCAGGCA CTGAGTGAAG GGTGCACTCC GTGGTTCCTG GGAGTTACGT ACCCAGTTTG AGTAGCTCCT TTTCTTCAAG CCTCGCCTTC ACCAGGGTCC CAAGGTCCGT GACTCACTTC CCACGTGAGG
Gag
■ Y D I N Q M L N C V G D H Q A A Q I I R D I I N E E A A D W D L
801 CTATGACATC AACCAGATGC TTAACTGCGT CGGCGACCAT CAGGCCGCGA TGCAGAT AT TCGGGACATA ATCAACGAGG AGGCTGCAGA CTGGGATTTG
GATACTGTAG TTGGTCTACG AATTGACGCA GCCGCTGGTA GTCCGGCGCT ACGTCTAATA AGCCCTGTAT TAGTTGCTCC TCCGACGTCT GACCCTAAAC
Gag
Q H P Q P A P Q Q G Q L R E P S G S D I A G T T S S V D E Q I Q W M
901 CAGCACCCCC AACCCGCCCC TCAGCAAGGG CAGCTAAGGG AGCCTTCCGG CAGCGACATA GCTGGGACTA CTAGCTCCGT GGATGAACAG ATTCAATGGA GTCGTGGGGG TTGGGCGGGG AGTCGTTCCC GTCGATTCCC TCGGAAGGCC GTCGCTGTAT CGACCCTGAT GATCGAGGCA CCTACTTGTC TAAGTTACCT
Gag
• Y R Q Q N P I P V G N I Y R R W I Q L G L Q K C V R M Y N P T N I 1001 TGTACAGACA GCAGAATCCG ATCCCCGTTG GCAACATCTA CCGGCGCTGG ATTCAACTCG GACTTCAGAA GTGCGTCAGA ATGTACAACC CCACCAATAT ACATGTCTGT CGTCTTAGGC TAGGGGCAAC CGTTGTAGAT GGCCGCGACC TAAGTTGAGC CTGAAGTCTT CACGCAGTCT TACATGTTGG GGTGGTTATA
Gag
• L D V K Q G P K E P F Q S Y V D R F Y K S L R A E Q T D A A V K N 1101 TCTGGATGTG AAACAGGGGC CGAAAGAGCC CTTTCAATCC TACGTCGACC GTTTCTACAA AAGTCTACGC GCCGAGCAGA CCGATGCCGC AGTGAAGAAC AGACCTACAC TTTGTCCCCG GCTTTCTCGG GAAAGTTAGG ATGCAGCTGG CAAAGATGTT TTCAGATGCG CGGCTCGTCT GGCTACGGCG TCACTTCTTG
Gag
M T Q T L L I Q N A N P D C K L V L K G L G V N P T L E E M L T A
1201 TGGATGACAC AGACGCTCCT GATACAGAAT GCTAACCCTG ATTGTAAACT CGTGCTGAAG GGCTTAGGGG TAAACCCAAC GCTGGAAGAA ATGTTAACCG ACCTACTGTG TCTGCGAGGA CTATGTCTTA CGATTGGGAC TAACATTTGA GCACGACTTC CCGAATCCCC ATTTGGGTTG CGACCTTCTT TACAATTGGC
Gag
• C Q G V G G P G Q K A R L M A E A L K E A L A P V P I P F A A A Q 1301 CCTGCCAGGG AGTTGGTGGA CCCGGACAGA AGGCCCGGCT AATGGCCGAG GCGCTGAAAG AAGCATTGGC TCCAGTACCC ATTCCTTTTG CTGCCGCACA GGACGGTCCC TCAACCACCT GGGCCTGTCT TCCGGGCCGA TTACCGGCTC CGCGACTTTC TTCGTAACCG AGGTCATGGG TAAGGAAAAC GACGGCGTGT
Gag
■ Q R G P R K P I K C W N C G K E G H S A K Q C R A P R R Q G C W K 1401 ACAGAGAGGT CCCCGTAAAC CGATCAAATG CTGGAACTGT GGGAAGGAGG GGCACTCCGC TAAACAATGT CGAGCGCCTA GACGTCAGGG GTGTTGGAAG TGTCTCTCCA GGGGCATTTG GCTAGTTTAC GACCTTGACA CCCTTCCTCC CCGTGAGGCG ATTTGTTACA GCTCGCGGAT CTGCAGTCCC CACAACCTTC
Gag
C G K M D H V M A K C P D R Q A G F L G L G P W G K K P R N F P M A
1501 TGTGGTAAAA TGGACCACGT TATGGCCAAA TGCCCCGACA GACAAGCCGG GTTCCTCGGG TTAGGGCCTT GGGGAAAAAA GCCCAGAAAC TTCCCAATGG ACACCATTTT ACCTGGTGCA ATACCGGTTT ACGGGGCTGT CTGTTCGGCC CAAGGAGCCC AATCCCGGAA CCCCTTTTTT CGGGTCTTTG AAGGGTTACC
Gag
• Q V H Q G L T P T A P P E D P A V D L L K N Y M Q L G K Q Q R E S 1601 CGCAAGTACA CCAGGGCCTG ACCCCGACCG CCCCCCCAGA GGACCCAGCC GTAGACCTCT TGAAAAACTA TATGCAGCTG GGGAAGCAGC AGCGCGAGAG GCGTTCATGT GGTCCCGGAC TGGGGCTGGC GGGGGGGTCT CCTGGGTCGG CATCTGGAGA ACTTTTTGAT ATACGTCGAC CCCTTCGTCG TCGCGCTCTC
FMDV2A
Gag
■ R E K P Y K E V T E D L L H L N S L F G G D Q N F D L L L A G D 1701 TAGAGAGAAG CCCTACAAGG AGGTTACGGA AGATCTGTTA CACCTTAATT CGTTATTTGG TGGTGATCAG AATTTCGACC TGCTTAAACT TGCTGGCGAC ATCTCTCTTC GGGATGTTCC TCCAATGCCT TCTAGACAAT GTGGAATTAA GCAATAAACC ACCACTAGTC TTAAAGCTGG ACGAATTTGA ACGACCGCTG
Pro
FMDV2A
V E S N P G P V L E L R Q R G P Q R Q A V Q S P S E T G L L E V W Q
1801 GTTGAGTCAA ATCCGGGCCC TGTGCTGGAG TTGAGACAGC GCGGGCCCCA GCGGCAGGCT GTTCAGAGCC CATCAGAGAC GGGTCTACTT GAGGTGTGGC CAACTCAGTT TAGGCCCGGG ACACGACCTC AACTCTGTCG CGCCCGGGGT CGCCGTCCGA CAAGTCTCGG GTAGTCTCTG CCCAGATGAA CTCCACACCG
Pro
■ D G P R D G Q M P R Q T G G F F R P W S M G K E A P Q F P H G S S
1901 AGGATGGCCC CCGTGATGGA CAGATGCCTC GCCAGACGGG AGGGTTCTTC CGACCCTGGA GTATGGGAAA GGAGGCCCCG CAGTTCCCTC ATGGCTCTTC
TCCTACCGGG GGCACTACCT GTCTACGGAG CGGTCTGCCC TCCCAAGAAG GCTGGGACCT CATACCCTTT CCTCCGGGGC GTCAAGGGAG TACCGAGAAG
Pro
■ A S G A D A N C S P R G P S C G S A K E L H A V G Q A A E R K Q R
2001 TGCCTCTGGC GCGGATGCCA ATTGTAGCCC CCGAGGCCCT TCTTGCGGCT CAGCCAAGGA GCTGCACGCA GTGGGCCAGG CAGCAGAGCG CAAACAGCGA
ACGGAGACCG CGCCTACGGT TAACATCGGG GGCTCCGGGA AGAACGCCGA GTCGGTTCCT CGACGTGCGT CACCCGGTCC GTCGTCTCGC GTTTGTCGCT
Pro
E A L Q G G D R G F A A P Q F S L W R R P V V T A H I E G Q P V E V
2101 GAAGCACTGC AGGGCGGTGA CCGTGGTTTT GCCGCCCCAC AATTCAGTCT GTGGCGCCGA CCTGTCGTGA CTGCTCATAT CGAGGGTCAG CCCGTGGAGG CTTCGTGACG TCCCGCCACT GGCACCAAAA CGGCGGGGTG TTAAGTCAGA CACCGCGGCT GGACAGCACT GACGAGTATA GCTCCCAGTC GGGCACCTCC
Pro
• L L D T G A D D S I V T G I E L G P H Y T P K I V G G I G G F I N .2201 TTTTACTGGA CACTGGCGCA GACGATTCTA TTGTGACTGG CATTGAACTA GGCCCCCATT ACACTCCAAA AATCGTAGGG GGGATAGGAG GATTTATCAA AAAATGACCT GTGACCGCGT CTGCTAAGAT AACACTGACC GTAACTTGAT CCGGGGGTAA TGTGAGGTTT TTAGCATCCC CCCTATCCTC CTAAATAGTT
Pro
■ T K E Y N V E I E V L G K R I K G T I M T G D T P I N I F G R N 2301 CACGAAGGAG TATAAGAATG TGGAGATCGA GGTTCTCGGA AAACGCATTA AGGGAACGAT TATGACAGGC GATACACCCA TTAACATCTT TGGACGCAAT GTGCTTCCTC ATATTCTTAC ACCTCTAGCT CCAAGAGCCT TTTGCGTAAT TCCCTTGCTA ATACTGTCCG CTATGTGGGT AATTGTAGAA ACCTGCGTTA
pre E/NS1 signal
F DV2A
Pro Transmembrane domain of NV E (split)
L L T A L G M S L N L N F D L L K L A G D V E S N P G P A R D R S I
2401 CTACTTACGG CCCTCGGAAT GAGCCTTAAC CTCAACTTCG ACTTACTCAA GCTCGCCGGA GACGTGGAGT CCAATCCCGG CCCAGCCCGG GACAGGTCCA
GATGAATGCC GGGAGCCTTA CTCGGAATTG GAGTTGAAGC TGAATGAGTT CGAGCGGCCT CTGCACCTCA GGTTAGGGCC GGGTCGGGCC CTGTCCAGGT
Transmembrane domain of WNV E (split)
NSl
■ A L T F L A V G G V L L F L S V N V H A D T G C A I D I S R Q E L
2501 TAGCTCTCAC GTTTCTCGCA GTTGGAGGAG TTCTGCTCTT CCTCTCCGTG AACGTGCACG CTGACACTGG GTGTGCCATA GACATCAGCC GGCAAGAGCT
ATCGAGAGTG CAAAGAGCGT CAACCTCCTC AAGACGAGAA GGAGAGGCAC TTGCACGTGC GACTGTGACC CACACGGTAT CTGTAGTCGG CCGTTCTCGA
NSl
- R C G S G V F I H N D V E A W M D R Y Y Y P E T P Q G L A K I I
2601 GAGATGTGGA AGTGGAGTGT TCATACACAA TGATGTGGAG GCTTGGATGG ACCGGTACAA GTATTACCCT GAAACGCCAC AAGGCCTAGC CAAGATCATT
CTCTACACCT TCACCTCACA AGTATGTGTT ACTACACCTC CGAACCTACC TGGCCATGTT CATAATGGGA CTTTGCGGTG TTCCGGATCG GTTCTAGTAA
NSl
Q K A H K E G V C G L R S V S R L E H Q M W E A V K D E L N T L L K
2701 CAGAAAGCTC ATAAGGAAGG AGTGTGCGGT CTACGATCAG TTTCCAGACT GGAGCATCAA ATGTGGGAAG CAGTGAAGGA CGAGCTGAAC ACTCTTTTGA
GTCTTTCGAG TATTCCTTCC TCACACGCCA GATGCTAGTC AAAGGTCTGA CCTCGTAGTT TACACCCTTC GTCACTTCCT GCTCGACTTG TGAGAAAACT
NSl
• K
2801 AG
TC
Construct 7
1. PIV-WN (ACprME)-SlV Env
Figure imgf000138_0001
DeieteC230ENV
2. Sequence of PIV-WN (ACprME)-SIV Env (partial).
UTR
M S
1 AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
C
NS3 cleavage
• K K P G G P G K S R A V Y L L K R G M P R V L S L I G L K Q K K R CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGC AAAAGAAGCG GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCG TTTTCTTCGC NS3 cleavage tpa
partial C signal
• G G T G I A V I M D A M K R G L C C V L L L C G A V F V T T T E
01 AGGGGGCAAG ACTGGTATAG CTGTGATCAT GGACGCCATG AAGAGGGGAC TTTGTTGTGT GCTCCTGCTG TGCGGAGCTG TGTTCGTTAC AACAACGGAG
TCCCCCGTTC TGACCATATC GACACTAGTA CCTGCGGTAC TTCTCCCCTG AAACAACACA CGAGGACGAC ACGCCTCGAC ACAAGCAATG T GTTGCCTC
Env
tpa
A I Y C T Q Y V T V F Y G V P A W R N A T I P L F C A T K N R D T W
301 GCGATTTACT GCACCCAGTA TGTCACCGTG TTTTACGGTG TCCCCGCCTG GCGGAACGCC ACCATCCCTC TGTTTTGTGC CACCAAGAAT AGAGATACGT CGCTAAATGA CGTGGGTCAT ACAGTGGCAC AAAATGCCAC AGGGGCGGAC CGCCTTGCGG TGGTAGGGAG ACAAAACACG GTGGTTCTTA TCTCTATGCA
Env
■ G T T Q C L P D N G D Y S E L A L N V T E S F D A W E N T V T E Q 401 GGGGCACCAC ACAATGCCTT CCCGATAATG GCGATTACTC TGAATTAGCC CTGAACGTCA CGGAAAGTTT TGATGCTTGG GAAAATACGG TTACCGAACA CCCCGTGGTG TGTTACGGAA GGGCTATTAC CGCTAATGAG ACTTAATCGG GACTTGCAGT GCCTTTCAAA ACTACGAACC CTTTTATGCC AATGGCTTGT
Env
■ A I E D V W Q L F E T S I K P C V L S P L C I T M R C N K S E T 501 GGCCATCGAA GATGTCTGGC AGTTATTCGA AACTAGTATC AAACCTTGCG TTAAGCTGAG TCCTTTGTGC ATAACGATGC GGTGCAACAA GAGCGAAACG CCGGTAGCTT CTACAGACCG TCAATAAGCT TTGATCATAG TTTGGAACGC AATTCGACTC AGGAAACACG TATTGCTACG CCACGTTGTT CTCGCTTTGC
Env
D K W G L T K S S T T T A S T T T T T A P A K I D V N E T S S C I
601 GACAAATGGG GCTTAACCAA ATCTTCAACC ACCACCGCCT CCACCAC AC GACAACCGCA CCTGCCAAGA TCGACATGGT TAACGAAACC TCTAGTTGCA CTGTTTACCC CGAATTGGTT TAGAAGTTGG TGGTGGCGGA GGTGGTGATG CTGTTGGCGT GGACGGTTCT AGCTGTACCA ATTGCTTTGG AGATCAACGT
Env
• T H D N C T G L E Q E Q M I G C K F N M T G L R D K T K E Y N E 701 TTACCCATGA CAACTGCACA GGCCTCGAAC AAGAACAAAT GATCGGCTGT AAATTCAATA TGACCGGACT GAAGAGAGAC AAGACAAAAG AGTACAACGA AATGGGTACT GTTGACGTGT CCGGAGCTTG TTCTTGTTTA CTAGCCGACA TTTAAGTTAT ACTGGCCTGA CTTCTCTCTG TTCTGTTTTC TCATGTTGCT
Env
• T W Y S T D L V C E Q G N S T D N E S R C Y M N H C N T S I I Q E 801 GACTTGGTAC AGCACCGACT TAGTGTGTGA GCAGGGGAAC TCAACCGATA ACGAGTCCCG CTGTTATATG AACCACTGCA ATACGAGCAT CATCCAAGAG CTGAACCATG TCGTGGCTGA ATCACACACT CGTCCCCTTG AGTTGGCTAT TGCTCAGGGC GACAATATAC TTGGTGACGT TATGCTCGTA GTAGGTTCTC
Env
S C D K H Y W D T I R F R Y C A P P G Y A L L R C N D T N Y S G F M
901 TCGTGCGACA AACACTATTG GGACACTATC CGATTTAGGT ACTGTGCCCC GCCGGGCTAT GCGCTTCTGC GTTGTAATGA TACCAATTAC AGTGGGTTCA AGCACGCTGT TTGTGATAAC CCTGTGATAG GCTAAATCCA TGACACGGGG CGGCCCGATA CGCGAAGACG CAACATTACT ATGGTTAATG TCACCCAAGT
Env
• P K C S V V V Ξ S C T R M M E T Q T S T W F G F N G T R A E N R 1001 TGCCGAAGTG TAGCAAAGTC GTGGTGTCCT CTTGTACCCG CATGATGGAG ACGCAGACTT CCACCTGGTT TGGCTTTAAC GGAACTCGAG CTGAAAACCG ACGGCTTCAC ATCGTTTCAG CACCACAGGA GAACATGGGC GTACTACCTC TGCGTCTGAA GGTGGACCAA ACCGAAATTG CCTTGAGCTC GACTTTTGGC
Env
■ T Y I Y W H G R D N R T I I S L N K Y Y K L T M K C R R P G N K T
1101 GACGTATATC TACTGGCACG GACGAGATAA CCGAACGATC ATCTCACTGA ACAAGTACTA CAATCTGACC ATGAAATGCC GGCGCCCAGG CAATAAGACG
CTGCATATAG ATGACCGTGC CTGCTCTATT GGCTTGCTAG TAGAGTGACT TGTTCATGAT GTTAGACTGG TACTTTACGG CCGCGGGTCC GTTATTCTGC
Env
V L P V T I M S G L V F H S Q P V N E R P N Q A W C W F G G N W K D
1201 GTACTTCCTG TCACTATTAT GAGCGGACTT GTATTTCACT CGCAGCCGGT CAATGAGCGC CCGAACCAAG CCTGGTGCTG GTTTGGAGGC AACTGGAAAG CATGAAGGAC AGTGATAATA CTCGCCTGAA CATAAAGTGA GCGTCGGCCA GTTACTCGCG GGCTTGGTTC GGACCACGAC CAAACCTCCG TTGACCTTTC
Env
• A I K E V K Q T I V K H P Y T G T N N T D K I N L T A P R G G D
1301 ATGCGATTAA GGAAGTTAAA CAAACCATCG TAAAGCATCC CCGCTACACC GGCACCAACA ATACGGATAA GATCAACCTC ACAGCCCCTC GTGGCGGCGA
TACGCTAATT CCTTCAATTT GTTTGGTAGC ATTTCGTAGG GGCGATGTGG CCGTGGTTGT TATGCCTATT CTAGTTGGAG TGTCGGGGAG CACCGCCGCT
Env
• P E V T F M W T N C R G E F L Y C K M N W F L N V E D R D L T T 1401 TCCAGAGGTG ACCTTCATGT GGACTAACTG TCGCGGTGAA TTTCTGTACT GTAAGATGAA TTGGTTTCTG AACTGGGTCG AGGATAGGGA TCTGACAACA AGGTCTCCAC TGGAAGTACA CCTGATTGAC AGCGCCACTT AAAGACATGA CATTCTACTT AACCAAAGAC TTGACCCAGC TCCTATCCCT AGACTGTTGT
Env
Q R P K E R H R R N Y V P C H I R Q I I N T W H K V G K N V Y L P P
1501 CAACGGCCTA AGGAGAGGCA CCGCCGTAAC TATGTGCCTT GTCATATCAG ACAGATCATC AATACATGGC ATAAGGTGGG TAAAAACGTA TACCTCCCTC GTTGCCGGAT TCCTCTCCGT GGCGGCATTG ATACACGGAA CAGTATAGTC TGTCTAGTAG TTATGTACCG TATTCCACCC ATTTTTGCAT ATGGAGGGAG
Env
• R E G D L T C N S T V T S L I A N I D W T D G N Q T N I T- M Ξ Α Ε 1601 CCCGCGAGGG CGACCTGACA TGTAATAGTA CAGTAACCAG CCTCATCGCT AACATAGACT GGACTGATGG AAATCAGACC AACATCACTA TGTCAGCCGA GGGCGCTCCC GCTGGACTGT ACATTATCAT GTCATTGGTC GGAGTAGCGA TTGTATCTGA CCTGACTACC TTTAGTCTGG TTGTAGTGAT ACAGTCGGCT
Env
■ V A E L Y R L E L G D Y K L V E I T P I G L A P T D V K R Y T T G 1701 GGTAGCCGAA CTGTATAGGC TAGAACTCGG TGACTATAAG CTCGTCGAGA TCACCCCGAT AGGGCTCGCC CCTACAGACG TGAAACGTTA TACCACCGGC CCATCGGCTT GACATATCCG ATCTTGAGCC ACTGATATTC GAGCAGCTCT AGTGGGGCTA TCCCGAGCGG GGATGTCTGC ACTTTGCAAT ATGGTGGCCG
Env
TM
G T S R N K R Y G I Y I V V G V I L L R I V I Y I V Q M L N R V R Q
1801 GGTACATCAA GGAACAAACG CTACGGCATC TACATCGTGG TAGGGGTCAT CCTCTTACGG ATTGTCATCT ATATCGTTCA GATGCTGAAT AGGGTGAGGC CCATGTAGTT CCTTGTTTGC GATGCCGTAG ATGTAGCACC ATCCCCAGTA GGAGAATGCC TAACAGTAGA TATAGCAAGT CTACGACTTA TCCCACTCCG TM pre E/NS1 signal
Env
FMDV2A Transmembrane domain of WNV E (split)
• G N F D L L K L A G D V E S N P G P A R D R S I A L T F L A V G G AGGGCAATTT TGACCTGTTA AAACTGGCCG GGGACGTCGA AAGCAACCCC GGTCCGGCCC GGGACAGGTC CATAGCTCTC ACGTTTCTCG CAGTTGGAGG TCCCGTTAAA ACTGGACAAT TTTGACCGGC CCCTGCAGCT TTCGTTGGGG CCAGGCCGGG CCCTGTCCAG GTATCGAGAG TGCAAAGAGC GTCAACCTCC
Transmembrane domain of WNV E (split)
NS1
■ V L L F L S V N V H A D T G C A I D I S R Q E L R C G S G V F I H
2001 AGTTCTGCTC TTCCTCTCCG TGAACGTGCA CGCTGACACT GGGTGTGCCA TAGACATCAG CCGGCAAGAG CTGAGATGTG GAAGTGGAGT GTTCATACAC
TCAAGACGAG AAGGAGAGGC ACTTGCACGT GCGACTGTGA CCCACACGGT ATCTGTAGTC GGCCGTTCTC GACTCTACAC CTTCACCTCA CAAGTATGTG
NS1
N D V E A W M D R Y K Y Y P E T P Q G L A K I I Q K A H K E G V C G
2101 AATGATGTGG AGGCTTGGAT GGACCGGTAC AAGTATTACC CTGAAACGCC ACAAGGCCTA GCCAAGATCA TTCAGAAAGC TCATAAGGAA GGAGTGTGCG TTACTACACC TCCGAACCTA CCTGGCCATG TTCATAATGG GACTTTGCGG TGTTCCGGAT CGGTTCTAGT AAGTCTTTCG AGTATTCCTT CCTCACACGC
NS1
• L R S V S R L E H Q M W E A V K D E L N T L L K
01 GTCTACGATC AGTTTCCAGA CTGGAGCATC AAATGTGGGA AGCAGTGAAG GACGAGCTGA ACACTCTTTT GAAG
CAGATGCTAG TCAAAGGTCT GACCTCGTAG TTTACACCCT TCGTCACTTC CTGCTCGACT TGTGAGAAAA CTTC
Construct 8
1. PIV-WN (ACprME)-SIV Env No Transmembrane (TM)
Figure imgf000141_0001
dC RVC230 ENV No TM
uence of PIV-WN (ACprME)-SIV Env No Transmembrane (partial).
UTR
M S
1 AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
NS3 cleavage
C
• K P G G P G S R A V Y L L K R G M P R V L S L I G L K Q K R
101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGC AAAAGAAGCG
GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCG TTTTCTTCGC NS3 cleavage tpa
partial C signal
• G G K T G I A V I M D A M K R G L C C V L L L C G A V F V T T T E 201 AGGGGGCAAG ACTGGTATAG CTGTGATCAT GGACGCCATG AAGAGGGGAC TTTGTTGTGT GCTCCTGCTG TGCGGAGCTG TGTTCGTTAC AACAACGGAG TCCCCCGTTC TGACCATATC GACACTAGTA CCTGCGGTAC TTCTCCCCTG AAACAACACA CGAGGACGAC ACGCCTCGAC ACAAGCAATG TTGTTGCCTC
tpa
Env
A I Y C T Q Y V T V F Y G V P A W R N A T I P L F C A T K N R D T W
301 GCGATTTACT GCACCCAGTA TGTCACCGTG TTTTACGGTG TCCCCGCCTG GCGGAACGCC ACCATCCCTC TGTTTTGTGC CACCAAGAAT AGAGATACGT CGCTAAATGA CGTGGGTCAT ACAGTGGCAC AAAATGCCAC AGGGGCGGAC CGCCTTGCGG TGGTAGGGAG ACAAAACACG GTGGTTCTTA TCTCTATGCA
Env
• G T T Q C L P D N G D Y S E L A L N V T E S F D A W E N T V T E Q
401 GGGGCACCAC ACAATGCCTT CCCGATAATG GCGATTACTC TGAATTAGCC CTGAACGTCA CGGAAAGTTT TGATGCTTGG GAAAATACGG TTACCGAACA
CCCCGTGGTG TGTTACGGAA GGGCTATTAC CGCTAATGAG ACTTAATCGG GACTTGCAGT GCCTTTCAAA ACTACGAACC CTTTTATGCC AATGGCTTGT
Env
■ A I E D V W Q L F E T S I K P C V K L S P L C I T M R C N S E T 501 GGCCATCGAA GATGTCTGGC AGTTATTCGA AACTAGTATC AAACCTTGCG TTAAGCTGAG TCCTTTGTGC ATAACGATGC GGTGCAACAA GAGCGAAACG CCGGTAGCTT CTACAGACCG TCAATAAGCT TTGATCATAG TTTGGAACGC AATTCGACTC AGGAAACACG TATTGCTACG CCACGTTGTT CTCGCTTTGC
Env
D K W G L T K S S T T T A S T T T T T A P A I D M V N E T S S C I
601 GACAAATGGG GCTTAACCAA ATCTTCAACC ACCACCGCCT CCACCACTAC GACAACCGCA CCTGCCAAGA TCGACATGGT TAACGAAACC TCTAGTTGCA CTGTTTACCC CGAATTGGTT TAGAAGTTGG TGGTGGCGGA GGTGGTGATG CTGTTGGCGT GGACGGTTCT AGCTGTACCA ATTGCTTTGG AGATCAACGT
Env
• T H D N C T G L E Q E Q M I G C K F N M T G L K R D K T K E Y H E 701 TTACCCATGA CAACTGCACA GGCCTCGAAC AAGAACAAAT GATCGGCTGT AAATTCAATA TGACCGGACT GAAGAGAGAC AAGACAAAAG AGTACAACGA AATGGGTACT GTTGACGTGT CCGGAGCTTG TTCTTGTTTA CTAGCCGACA TTTAAGTTAT ACTGGCCTGA CTTCTCTCTG TTCTGTTTTC TCATGTTGCT
Env
• T W Y S T D L V C E Q G N S T D N E S R C Y M N H C N T S I I Q E 801 GACTTGGTAC AGCACCGACT TAGTGTGTGA GCAGGGGAAC TCAACCGATA ACGAGTCCCG CTGTTATATG AACCACTGCA ATACGAGCAT CATCCAAGAG CTGAACCATG TCGTGGCTGA ATCACACACT CGTCCCCTTG AGTTGGCTAT TGCTCAGGGC GACAATATAC TTGGTGACGT TATGCTCGTA GTAGGTTCTC
Env
S C D K H Y W D T I R F R Y C A P P G Y A L L R C N D T N Y S G F M
901 TCGTGCGACA AACACTATTG GGACACTATC CGATTTAGGT ACTGTGCCCC GCCGGGCTAT GCGCTTCTGC GTTGTAATGA TACCAATTAC AGTGGGTTCA AGCACGCTGT TTGTGATAAC CCTGTGATAG GCTAAATCCA TGACACGGGG CGGCCCGATA CGCGAAGACG CAACATTACT ATGGTTAATG TCACCCAAGT
Env
• P K C S K V V V S S C T R M M E T Q T S T W F G F N G T R A E N R 1001 TGCCGAAGTG TAGCAAAGTC GTGGTGTCCT CTTGTACCCG CATGATGGAG ACGCAGACTT CCACCTGGTT TGGCTTTAAC GGAACTCGAG CTGAAAACCG ACGGCTTCAC ATCGTTTCAG CACCACAGGA GAACATGGGC GTACTACCTC TGCGTCTGAA GGTGGACCAA ACCGAAATTG CCTTGAGCTC GACTTTTGGC
Env
■ T Y I Y W H G R D N R T I I S L N K Y Y N L T M K C R R P G N K T 1101 GACGTATATC TACTGGCACG GACGAGATAA CCGAACGATC ATCTCACTGA ACAAGTACTA CAATCTGACC ATGAAATGCC GGCGCCCAGG CAATAAGACG CTGCATATAG ATGACCGTGC CTGCTCTATT GGCTTGCTAG TAGAGTGACT TGTTCATGAT GTTAGACTGG TACTTTACGG CCGCGGGTCC GTTATTCTGC
Env
V L P V T I M S G L V F H S Q P V N E R P N Q A W C F G G K K D
1201 GTACTTCCTG TCACTATTAT GAGCGGACTT GTATTTCACT CGCAGCCGGT CAATGAGCGC CCGAACCAAG CCTGGTGCTG GTTTGGAGGC AACTGGAAAG
CATGAAGGAC AGTGATAATA CTCGCCTGAA CATAAAGTGA GCGTCGGCCA GTTACTCGCG GGCTTGGTTC GGACCACGAC CAAACCTCCG TTGACCTTTC
Env
• A I K E V K Q T I V K H P R Y T G T N N T D K I N L T A P R G G D
1301 ATGCGATTAA GGAAGTTAAA CAAACCATCG TAAAGCATCC CCGCTACACC GGCACCAACA ATACGGATAA GATCAACCTC ACAGCCCCTC GTGGCGGCGA
TACGCTAATT CCTTCAATTT GTTTGGTAGC ATTTCGTAGG GGCGATGTGG CCGTGGTTGT TATGCCTATT CTAGTTGGAG TGTCGGGGAG CACCGCCGCT
Env
■ P E V T F M W T N C R G E F L Y C K M N W F L N W V E D R D L T T
1401 TCCAGAGGTG ACCTTCATGT GGACTAACTG TCGCGGTGAA TTTCTGTACT GTAAGATGAA TTGGTTTCTG AACTGGGTCG AGGATAGGGA TCTGACAACA
AGGTCTCCAC TGGAAGTACA CCTGATTGAC AGCGCCACTT AAAGACATGA CATTCTACTT AACCAAAGAC TTGACCCAGC TCCTATCCCT AGACTGTTGT
Env
Q R P K E R H R R N Y V P C H I R Q I I N T H K V G K N V Y L P P CAACGGCCTA AGGAGAGGCA CCGCCGTAAC TATGTGCCTT GTCATATCAG ACAGATCATC AATACATGGC ATAAGGTGGG TAAAAACGTA TACCTCCCTC GTTGCCGGAT TCCTCTCCGT GGCGGCATTG ATACACGGAA CAGTATAGTC TGTCTAGTAG TTATGTACCG TATTCCACCC ATTTTTGCAT ATGGAGGGAG
Env
• R E G D L T C N S T V T S L I A N I D W T D G N Q T N I T M S A E
1601 CCCGCGAGGG CGACCTGACA TGTAATAGTA CAGTAACCAG CCTCATCGCT AACATAGACT GGACTGATGG AAATCAGACC AACATCACTA TGTCAGCCGA
GGGCGCTCCC GCTGGACTGT ACATTATCAT GTCATTGGTC GGAGTAGCGA TTGTATCTGA CCTGACTACC TTTAGTCTGG TTGTAGTGAT ACAGTCGGCT
Env
• V A E L Y R L E L G D Y L V E I T P I G L A P T D V K R Y T T G
1701 GGTAGCCGAA CTGTATAGGC TAGAACTCGG TGACTATAAG CTCGTCGAGA TCACCCCGAT AGGGCTCGCC CCTACAGACG TGAAACGTTA TACCACCGGC
CCATCGGCTT GACATATCCG ATCTTGAGCC ACTGATATTC GAGCAGCTCT AGTGGGGCTA TCCCGAGCGG GGATGTCTGC ACTTTGCAAT ATGGTGGCCG
pre E/NS1 signal
FMDV2A
Env Transmembrane domain of NV E
(split)
G T R N K R N L K L A G D V E N P A R D R S L T
1801 GGTACATCAA GGAACAAACG CAATTTTGAC CTGTTAAAAC TGGCCGGGGA CGTCGAAAGC AACCCCGGTC CGGCCCGGGA CAGGTCCATA GCTCTCACGT
CCATGTAGTT CCTTGTTTGC GTTAAAACTG GACAATTTTG ACCGGCCCCT GCAGCTTTCG TTGGGGCCAG GCCGGGCCCT GTCCAGGTAT CGAGAGTGCA
Transmembrane domain of WNV E (split)
NS1
• L A V G G V L L F L S V N V H A D T G C A I D I S R Q E L R C G S
1901 TTCTCGCAGT TGGAGGAGTT CTGCTCTTCC TCTCCGTGAA CGTGCACGCT GACACTGGGT GTGCCATAGA CATCAGCCGG CAAGAGCTGA GATGTGGAAG
AAGAGCGTCA ACCTCCTCAA GACGAGAAGG AGAGGCACTT GCACGTGCGA CTGTGACCCA CACGGTATCT GTAGTCGGCC GTTCTCGACT CTACACCTTC
NS1
■ G V F I H N D V E A W M D R Y K Y Y P E T P Q G L A K I I Q A H
2001 TGGAGTGTTC ATACACAATG ATGTGGAGGC TTGGATGGAC CGGTACAAGT ATTACCCTGA AACGCCACAA GGCCTAGCCA AGATCATTCA GAAAGCTCAT
ACCTCACAAG TATGTGTTAC TACACCTCCG AACCTACCTG GCCATGTTCA TAATGGGACT TTGCGGTGTT CCGGATCGGT TCTAGTAAGT CTTTCGAGTA
NS1
K E G V C G L R S V S R L E H Q W E A V K D E L N T L L K
AAGGAAGGAG TGTGCGGTCT ACGATCAGTT TCCAGACTGG AGCATCAAAT GTGGGAAGCA GTGAAGGACG AGCTGAACAC TCTTTTGAAG TTCCTTCCTC ACACGCCAGA TGCTAGTCAA AGGTCTGACC TCGTAGTTTA CACCCTTCGT CACTTCCTGC TCGACTTGTG AGAAAACTTC
Construct 9
1. PIV-WN (ACprME)-SIV ENV Rab G Transmembrane TM)
Figure imgf000144_0001
RV230 dC Env Rab TM
2. Sequence of PIV-WN (ACpr E)- SIV ENV Rab G Transmembrane (TM) (partial).
5' UTR
M S
1 AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
NS3 Cleavage
c
• K K P G G P G K S R A V Y L L R G M P R V L S L I G L K Q K K R 101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGC AGAAAAAGCG GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCG TCTTTTTCGC NS3 Cleavage tpa
Partial C Signal
■ G G K T G I A V I M D A M K R G L C C V L L L C G A V F V T T T E 201 GGGCGGAAAG ACAGGTATTG CTGTGATCAT GGACGCCATG AAGAGGGGAC TTTGTTGTGT GCTCCTGCTG TGCGGAGCTG TGTTCGTTAC AACAACGGAG CCCGCCTTTC TGTCCATAAC GACACTAGTA CCTGCGGTAC TTCTCCCCTG AAACAACACA CGAGGACGAC ACGCCTCGAC ACAAGCAATG TTGTTGCCTC
Env
tpa
A I Y C T Q Y V T V F Y G V P A W R N A T I P L F C A T N R D T W
301 GCGATTTACT GCACCCAGTA TGTCACCGTG TTTTACGGTG TCCCCGCCTG GCGGAACGCC ACCATCCCTC TGTTTTGTGC CACCAAGAAT AGAGATACGT CGCTAAATGA CGTGGGTCAT ACAGTGGCAC AAAATGCCAC AGGGGCGGAC CGCCTTGCGG TGGTAGGGAG ACAAAACACG GTGGTTCTTA TCTCTATGCA
Env
• G T T Q C L P D N G D Y S E L A L N V T E S F D A W E N T V T E Q 401 GGGGCACCAC ACAATGCCTT CCCGATAATG GCGATTACTC TGAATTAGCC CTGAACGTCA CGGAAAGTTT TGATGCTTGG GAAAATACGG TTACCGAACA CCCCGTGGTG TGTTACGGAA GGGCTATTAC CGCTAATGAG ACTTAATCGG GACTTGCAGT GCCTTTCAAA ACTACGAACC CTTTTATGCC AATGGCTTGT
Env
• A I E D V W Q L F E T S I K P C V K L S P L C I T M R C H K S E T
501 GGCCATCGAA GATGTCTGGC AGTTATTCGA AACTAGTATC AAACCTTGCG TTAAGCTGAG TCCTTTGTGC ATAACGATGC GGTGCAACAA GAGCGAAACG
CCGGTAGCTT CTACAGACCG TCAATAAGCT TTGATCATAG TTTGGAACGC AATTCGACTC AGGAAACACG TATTGCTACG CCACGTTGTT CTCGCTTTGC
Env
D W G L T K S S T T T A S T T T T T A P A R I D M V N E T S S C I
601 GACAAATGGG GCTTAACCAA ATCTTCAACC ACCACCGCCT CCACCACTAC GACAACCGCA CCTGCCAAGA TCGACATGGT TAACGAAACC TCTAGTTGCA CTGTTTACCC CGAATTGGTT TAGAAGTTGG TGGTGGCGGA GGTGGTGATG CTGTTGGCGT GGACGGTTCT AGCTGTACCA ATTGCTTTGG AGATCAACGT
Env
• T H D N C T G L E Q E Q M I G C K F N M T G L K R D K T K E Y N E
701 TTACCCATGA CAACTGCACA GGCCTCGAAC AAGAACAAAT GATCGGCTGT AAATTCAATA TGACCGGACT GAAGAGAGAC AAGACAAAAG AGTACAACGA
AATGGGTACT GTTGACGTGT CCGGAGCTTG TTCTTGTTTA CTAGCCGACA TTTAAGTTAT ACTGGCCTGA CTTCTCTCTG TTCTGTTTTC TCATGTTGCT
Env
• T W Y S T D L V C E Q G N S T D N E S R C Y M N H C N T S I I Q E 801 GACTTGGTAC AGCACCGACT TAGTGTGTGA GCAGGGGAAC TCAACCGATA ACGAGTCCCG CTGTTATATG AACCACTGCA ATACGAGCAT CATCCAAGAG CTGAACCATG TCGTGGCTGA ATCACACACT CGTCCCCTTG AGTTGGCTAT TGCTCAGGGC GACAATATAC TTGGTGACGT TATGCTCGTA GTAGGTTCTC
Env
S C D H Y W D T I R F R Y C A P P G Y A L L R C N D T N Y S G F M
901 TCGTGCGACA AACACTATTG GGACACTATC CGATTTAGGT ACTGTGCCCC GCCGGGCTAT GCGCTTCTGC GTTGTAATGA TACCAATTAC AGTGGGTTCA AGCACGCTGT TTGTGATAAC CCTGTGATAG GCTAAATCCA TGACACGGGG CGGCCCGATA CGCGAAGACG CAACATTACT ATGGTTAATG TCACCCAAGT
Env
• P K C S V V V S S C T R M M E T Q T S T W F G F N G T R A E N R 1001 TGCCGAAGTG TAGCAAAGTC GTGGTGTCCT CTTGTACCCG CATGATGGAG ACGCAGACTT CCACCTGGTT TGGCTTTAAC GGAACTCGAG CTGAAAACCG
ACGGCTTCAC ATCGTTTCAG CACCACAGGA GAACATGGGC GTACTACCTC TGCGTCTGAA GGTGGACCAA ACCGAAATTG CCTTGAGCTC GACTTTTGGC
Env
• T Y I Y W H G R D N R T I I S L N Y Y N L T K C R R P G N K T 1101 GACGTATATC TACTGGCACG GACGAGATAA CCGAACGATC ATCTCACTGA ACAAGTACTA CAATCTGACC ATGAAATGCC GGCGCCCAGG CAATAAGACG CTGCATATAG ATGACCGTGC CTGCTCTATT GGCTTGCTAG TAGAGTGACT TGTTCATGAT GTTAGACTGG TACTTTACGG CCGCGGGTCC GTTATTCTGC
Env
V L P V T I M S G L V F H S Q P V N E R P N Q A W C W F G G N K D
1201 GTACTTCCTG TCAC ATTAT GAGCGGACTT GTATTTCACT CGCAGCCGGT CAATGAGCGC CCGAACCAAG CCTGGTGCTG GTTTGGAGGC AACTGGAAAG CATGAAGGAC AGTGATAATA CTCGCCTGAA CATAAAGTGA GCGTCGGCCA GTTACTCGCG GGCTTGGTTC GGACCACGAC CAAACCTCCG TTGACCTTTC
Env
■ A I K E V K Q T I V K H P R Y T G T N N T D K I N L T A P R G G D
1301 ATGCGATTAA GGAAGTTAAA CAAACCATCG TAAAGCATCC CCGCTACACC GGCACCAACA ATACGGATAA GATCAACCTC ACAGCCCCTC GTGGCGGCGA
TACGCTAATT CCTTCAATTT GTTTGGTAGC ATTTCGTAGG GGCGATGTGG CCGTGGTTGT TATGCCTATT CTAGTTGGAG TGTCGGGGAG CACCGCCGCT
Env
• P E V T F M T N C R G E F L Y C K M N F L N W V E D R D L T T
1401 TCCAGAGGTG ACCTTCATGT GGACTAACTG TCGCGGTGAA TTTCTGTACT GTAAGATGAA TTGGTTTCTG AACTGGGTCG AGGATAGGGA TCTGACAACA
AGGTCTCCAC TGGAAGTACA CCTGATTGAC AGCGCCACTT AAAGACATGA CATTCTACTT AACCAAAGAC TTGACCCAGC TCCTATCCCT AGACTGTTGT
Env
Q R P K E R H R R N Y V P C H I R Q I I K T W H K V G K N V Y L P P
1501 CAACGGCCTA AGGAGAGGCA CCGCCGTAAC TATGTGCCTT GTCATATCAG ACAGATCATC AATACATGGC ATAAGGTGGG TAAAAACGTA TACCTCCCTC GTTGCCGGAT TCCTCTCCGT GGCGGCATTG ATACACGGAA CAGTATAGTC TGTCTAGTAG TTATGTACCG TATTCCACCC ATTTTTGCAT ATGGAGGGAG
Env
• R E G D L T C N S T V T S L I A N I D W T D G N Q T N I T M S A E 1601 CCCGCGAGGG CGACCTGACA TGTAATAGTA CAGTAACCAG CCTCATCGCT AACATAGACT GGACTGATGG AAATCAGACC AACATCAC A TGTCAGCCGA GGGCGCTCCC GCTGGACTGT ACATTATCAT GTCATTGGTC GGAGTAGCGA TTGTATCTGA CCTGACTACC TTTAGTCTGG TTGTAGTGAT ACAGTCGGCT
Env
■ V A E L Y R L E L G D Y K L V E I T P I G L A P T D V K R Y T T G
1701 GGTAGCCGAA CTGTATAGGC TAGAACTCGG TGACTATAAG CTCGTCGAGA TCACCCCGAT AGGGCTCGCC CCTACAGACG TGAAACGTTA TACCACCGGC
CCATCGGCTT GACATATCCG ATCTTGAGCC ACTGATATTC GAGCAGCTCT AGTGGGGCTA TCCCGAGCGG GGATGTCTGC ACTTTGCAAT ATGGTGGCCG
Env
RabG TM S Cytoplasmic
G T S R N R Y V L L S A G A L T A L M L I I F L M T C W R R V N R
1801 GGTACATCAA GGAACAAACG CTACGTGCTC CTGAGTGCGG GTGCCTTGAC CGCTTTGATG CTGATCATTT TTCTGATGAC CTGCTGGCGG AGGGTGAATC
CCATGTAGTT CCTTGTTTGC GATGCACGAG GACTCACGCC CACGGAACTG GCGAAACTAC GACTAGTAAA AAGACTACTG GACGACCGCC TCCCACTTAG
RabG TM S Cytoplasmic
• S E P T Q H N L R G T G R E V S V T P Q S G K I I S S W E S Y K S 1901 GCTCCGAGCC GACACAGCAC AATCTCAGAG GGACAGGCCG GGAAGTAAGT GTGACTCCGC AATCTGGCAA GATTATTAGT AGTTGGGAGA GTTACAAGTC CGAGGCTCGG CTGTGTCGTG TTAGAGTCTC CCTGTCCGGC CCTTCATTCA CACTGAGGCG TTAGACCGTT CTAATAATCA TCAACCCTCT CAATGTTCAG
FMDV2A TM Domain HV E (split)
RabG TM S Cytoplasmic pre E/NS1 Signal
• G G E T G L N F D L L K L A G D V E S H P G P A R D R S I A L T F
2001 TGGAGGAGAG ACTGGGTTGA ATTTTGATCT GCTCAAACTT GCAGGCGATG TAGAATCAAA TCCTGGACCC GCCCGGGACA GGTCCATAGC TCTCACGTTT ACCTCCTCTC TGACCCAACT TAAAACTAGA CGAGTTTGAA CGTCCGCTAC ATCTTAGTTT AGGACCTGGG CGGGCCCTGT CCAGGTATCG AGAGTGCAAA
NSl
TM Domain WNV E (split)
L A V G G V L L F L S V N V H A D T G C A I D I S R Q E L R C G S G
2101 CTCGCAGTTG GAGGAGTTCT GCTCTTCCTC TCCGTGAACG TGCACGCTGA CACTGGGTGT GCCATAGACA TCAGCCGGCA AGAGCTGAGA TGTGGAAGTG
GAGCGTCAAC CTCCTCAAGA CGAGAAGGAG AGGCACTTGC ACGTGCGACT GTGACCCACA CGGTATCTGT AGTCGGCCGT TCTCGACTCT ACACCTTCAC
NSl
• V F I H N D V E A W M D R Y K Y Y P E T & Q G L A K I I Q K A H K ·
2201 GAGTGTTCAT ACACAATGAT GTGGAGGCTT GGATGGACCG GTACAAGTAT TACCCTGAAA CGCCACAAGG CCTAGCCAAG ATCATTCAGA AAGCTCATAA
CTCACAAGTA TGTGTTACTA CACCTCCGAA CCTACCTGGC CATGTTCATA ATGGGACTTT GCGGTGTTCC GGATCGGTTC TAGTAAGTCT TTCGAGTATT
NSl
■ E G V C G L R S V S R L E H Q M W E A V K D E L N T L L K
2301 GGAAGGAGTG TGCGGTCTAC GATCAGTTTC CAGACTGGAG CATCAAATGT GGGAAGCAGT GAAGGACGAG CTGAACACTC TTTTGAAG
CCTTCCTCAC ACGCCAGATG CTAGTCAAAG GTCTGACCTC GTAGTTTACA CCCTTCGTCA CTTCCTGCTC GACTTGTGAG AAAACTTC
Construct 10
1. PIV-WN (ACprME)-SIV Env RabG Chimera Signal Sequence and Transmembrane (TM)
Figure imgf000148_0001
dC RV230 ENV RabG Chimera
2. Sequence of PIV-WN (ACprME)-SIV Env RabG Chimera, Signal Sequence and Transmembrane (TM)
5' DTR
M S
1 AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
NS3 cleavage
C
• K K P G G P G K S R A V Y L L R G M P R V L S L I G L K Q K K R 101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGC AAAAGAAGCG GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCG TTTTCTTCGC NS3 cleavage Rab G Signal
partial C signal Env
■ G G K T G I A V I V P Q A L L F V P L L V F P L C F G C T Q Y V T 201 AGGGGGCAAG ACTGGTATAG CTGTGATCGT TCCTCAGGCT CTTTTGTTTG TACCCTTGCT GGTATTTCCC CTTTGCTTTG GTTGCACCCA GTATGTCACC TCCCCCGTTC TGACCATATC GACACTAGCA AGGAGTCCGA GAAAACAAAC ATGGGAACGA CCATAAAGGG GAAACGAAAC CAACGTGGGT CATACAGTGG
Env
V F Y G V P A W R N A T I P L F C A T K N R D T W G T T Q C L P D N
301 GTGTTTTACG GTGTCCCCGC CTGGCGGAAC GCCACCATCC CTCTGTTTTG TGCCACCAAG AATAGAGATA CGTGGGGCAC CACACAATGC CTTCCCGATA CACAAAATGC CACAGGGGCG GACCGCCTTG CGGTGGTAGG GAGACAAAAC ACGGTGGTTC TTATCTCTAT GCACCCCGTG GTGTGTTACG GAAGGGCTAT
Env
- G D Y S E L A L N V T E S F D A W E N T V T E Q A I E D V W Q L F 401 ATGGCGATTA CTCTGAATTA GCCCTGAACG TCACGGAAAG TTTTGATGCT TGGGAAAATA CGGTTACCGA ACAGGCCATC GAAGATGTCT GGCAGTTATT TACCGCTAAT GAGACTTAAT CGGGACTTGC AGTGCCTTTC AAAACTACGA ACCCTTTTAT GCCAATGGCT TGTCCGGTAG CTTCTACAGA CCGTCAATAA
Env
• E T S I P C V K L S P L C I T M R C N Κ Ξ Ε T D K W G L T K S S
501 CGAAACTAGT ATCAAACCTT GCGTTAAGCT GAGTCCTTTG TGCATAACGA TGCGGTGCAA CAAGAGCGAA ACGGACAAAT GGGGCTTAAC CAAATCTTCA
GCTTTGATCA TAGTTTGGAA CGCAATTCGA CTCAGGAAAC ACGTATTGCT ACGCCACGTT GTTCTCGCTT TGCCTGTTTA CCCCGAATTG GTTTAGAAGT
Env
T T T A S T T T T T A P A K I D M V N E T S S C I T H D N C T G L E
601 ACCACCACCG CCTCCACCAC TACGACAACC GCACCTGCCA AGATCGACAT GGTTAACGAA ACCTCTAGTT GCATTACCCA TGACAACTGC ACAGGCCTCG TGGTGGTGGC GGAGGTGGTG ATGCTGTTGG CGTGGACGGT TCTAGCTGTA CCAATTGCTT TGGAGATCAA CGTAATGGGT ACTGTTGACG TGTCCGGAGC
Env
- Q E Q M I G C K F N M T G L K R D K T K E Y N E T W Y S T D L V C
701 AACAAGAACA AATGATCGGC TGTAAATTCA ATATGACCGG ACTGAAGAGA GACAAGACAA AAGAGTACAA CGAGACTTGG TACAGCACCG ACTTAGTGTG
TTGTTCTTGT TTACTAGCCG ACATTTAAGT TATACTGGCC TGACTTCTCT CTGTTCTGTT TTCTCATGTT GCTCTGAACC ATGTCGTGGC TGAATCACAC
Env
• E Q G N S T D N E S R C Y M N H C N T S I I Q E S C D K H Y W D T 801 TGAGCAGGGG AACTCAACCG ATAACGAGTC CCGCTGTTAT ATGAACCACT GCAATACGAG CATCATCCAA GAGTCGTGCG ACAAACACTA TTGGGACACT ACTCGTCCCC TTGAGTTGGC TATTGCTCAG GGCGACAATA TACTTGGTGA CGTTATGCTC GTAGTAGGTT CTCAGCACGC TGTTTGTGAT AACCCTGTGA
Env
I R F R Y C A P P G Y A L L R C N D T N Y S G F M P K C S K V V V S
901 ATCCGATTTA GGTACTGTGC CCCGCCGGGC TATGCGCTTC TGCGTTGTAA TGATACCAAT TACAGTGGGT TCATGCCGAA GTGTAGCAAA GTCGTGGTGT TAGGCTAAAT CCATGACACG GGGCGGCCCG ATACGCGAAG ACGCAACATT ACTATGGTTA ATGTCACCCA AGTACGGCTT CACATCGTTT CAGCACCACA
Env
• S C T R M E T Q T S T W F G F N G T R A E N R T Y I Y W H G R D 1001 CCTCTTGTAC CCGCATGATG GAGACGCAGA CTTCCACCTG GTTTGGCTTT AACGGAACTC GAGCTGAAAA CCGGACGTAT ATCTACTGGC ACGGACGAGA GGAGAACATG GGCGTACTAC CTCTGCGTCT GAAGGTGGAC CAAACCGAAA TTGCCTTGAG CTCGACTTTT GGCCTGCATA TAGATGACCG TGCCTGCTCT
Env
• N R T I I S L N K Y Y N L T M K C R R P G N K T V L P V T I M S G 1101 TAACCGAACG ATCATCTCAC TGAACAAGTA CTACAATCTG ACCATGAAAT GCCGGCGCCC AGGCAATAAG ACGGTACTTC CTGTCACTAT TATGAGCGGA ATTGGCTTGC TAGTAGAGTG ACTTGTTCAT GATGTTAGAC TGGTACTTTA CGGCCGCGGG TCCGTTATTC TGCCATGAAG GACAGTGATA ATACTCGCCT
Env
L V F H S Q P V N E R P N Q A W C F G G N W D A I K E V K Q T I
1201 CTTGTATTTC ACTCGCAGCC GGTCAATGAG CGCCCGAACC AAGCCTGGTG CTGGTTTGGA GGCAACTGGA AAGATGCGAT TAAGGAAGTT AAACAAACCA GAACATAAAG TGAGCGTCGG CCAGTTACTC GCGGGCTTGG TTCGGACCAC GACCAAACCT CCGTTGACCT TTCTACGCTA ATTCCTTCAA TTTGTTTGGT
Env
• V K H P R Y T G T N N T D K I N L T A P R G G D P E V T F M W T N 1301 TCGTAAAGCA TCCCCGCTAC ACCGGCACCA ACAATACGGA TAAGATCAAC CTCACAGCCC CTCGTGGCGG CGATCCAGAG GTGACCTTCA TGTGGACTAA AGCATTTCGT AGGGGCGATG TGGCCGTGGT TGTTATGCCT ATTCTAGTTG GAGTGTCGGG GAGCACCGCC GCTAGGTCTC CACTGGAAGT ACACCTGATT
Env
■ C R G E F L Y C K M N F L N W V E D R D L T T Q R P K E R H R R
1401 CTGTCGCGGT GAATTTCTGT ACTGTAAGAT GAATTGGTTT CTGAACTGGG TCGAGGATAG GGATCTGACA ACACAACGGC CTAAGGAGAG GCACCGCCGT
GACAGCGCCA CTTAAAGACA TGACATTCTA CTTAACCAAA GACTTGACCC AGCTCCTATC CCTAGACTGT TGTGTTGCCG GATTCCTCTC CGTGGCGGCA
Env
N Y V P C H I R Q I I N T H K V G K N V Y L P P R E G D L T C N S ·
1501 AACTATGTGC CTTGTCATAT CAGACAGATC ATCAATACAT GGCATAAGGT GGGTAAAAAC GTATACCTCC CTCCCCGCGA GGGCGACCTG ACATGTAATA
TTGATACACG GAACAGTATA GTCTGTCTAG TAGTTATGTA CCGTATTCCA CCCATTTTTG CATATGGAGG GAGGGGCGCT CCCGCTGGAC TGTACATTAT
Env
■ T V T S L I A N I D T D G N Q T N I T M S A E V A E L Y R L E L "
GTACAGTAAC CAGCCTCATC GCTAACATAG ACTGGACTGA TGGAAATCAG ACCAACATCA CTATGTCAGC CGAGGTAGCC GAACTGTATA GGCTAGAACT CATGTCATTG GTCGGAGTAG CGATTGTATC TGACCTGACT ACCTTTAGTC TGGTTGTAGT GATACAGTCG GCTCCATCGG CTTGACATAT CCGATCTTGA
RabG TM & Cytoplasmii
Env
• G D Y K L V E I T P I G L A P T D V K R Y T T G G T S R N R Y V
1701 CGGTGACTAT AAGCTCGTCG AGATCACCCC GATAGGGCTC GCCCCTACAG ACGTGAAACG TTATACCACC GGCGGTACAT CAAGGAACAA ACGCTACGTG
GCCACTGATA TTCGAGCAGC TCTAGTGGGG CTATCCCGAG CGGGGATGTC TGCACTTTGC AATATGGTGG CCGCCATGTA GTTCCTTGTT TGCGATGCAC
RabG TM & Cytoplasmic
L L S A G A L T A L M L I I F L T C W R R V N R S E P T Q H N L R
1801 CTCCTGAGTG CGGGTGCCTT GACCGCTTTG ATGCTGATCA TTTTTCTGAT GACCTGCTGG CGGAGGGTGA ATCGCTCCGA GCCGACACAG CACAATCTCA
GAGGACTCAC GCCCACGGAA CTGGCGAAAC TACGACTAGT AAAAAGACTA CTGGACGACC GCCTCCCACT TAGCGAGGCT CGGCTGTGTC GTGTTAGAGT
FMDV2A
RabG TM s Cytoplasmic
• G T G R E V S V T P Q S G K I I S S W E S Y K S G G E T G L N F D
1901 GAGGGACAGG CCGGGAAGTA AGTGTGACTC CGCAATCTGG CAAGATTATT AGTAGTTGGG AGAGTTACAA GTCTGGAGGA GAGACTGGGT TGAATTTTGA
CTCCCTGTCC GGCCCTTCAT TCACACTGAG GCGTTAGACC GTTCTAATAA TCATCAACCC TCTCAATGTT CAGACCTCCT CTCTGACCCA ACTTAAAACT
Pre E/NS1 Signal
FMDV2A TM Domain of WNV E (split)
• L L K L A G D V E S N P G P A R D R S I A L T F L A V G G V L L F
2001 TCTGCTCAAA CTTGCAGGCG ATGTAGAATC AAATCCTGGA CCCGCCCGGG ACAGGTCCAT AGCTCTCACG TTTCTCGCAG TTGGAGGAGT TCTGCTCTTC
AGACGAGTTT GAACGTCCGC TACATCTTAG TTTAGGACCT GGGCGGGCCC TGTCCAGGTA TCGAGAGTGC AAAGAGCGTC AACCTCCTCA AGACGAGAAG
NS1
TM Domain of WNV E (split)
_
L S V N V H A D T G C A I D I S R Q E L R C G S G V F I H N D V E A '
2101 CTCTCCGTGA ACGTGCACGC TGACACTGGG TGTGCCATAG ACATCAGCCG GCAAGAGCTG AGATGTGGAA GTGGAGTGTT CATACACAAT GATGTGGAGG
GAGAGGCACT TGCACGTGCG ACTGTGACCC ACACGGTATC TGTAGTCGGC CGTTCTCGAC TCTACACCTT CACCTCACAA GTATGTGTTA CTACACCTCC
NS1
—,~— , , —,
• W M D R Y K Y Y P E T P Q G L A K I I Q A H K E G V C G L R S V ·
2201 CTTGGATGGA CCGGTACAAG TATTACCCTG AAACGCCACA AGGCCTAGCC AAGATCATTC AGAAAGCTCA TAAGGAAGGA GTGTGCGGTC TACGATCAGT
GAACCTACCT GGCCATGTTC ATAATGGGAC TTTGCGGTGT TCCGGATCGG TTCTAGTAAG TCTTTCGAGT ATTCCTTCCT CACACGCCAG ATGCTAGTCA
NS1
• S R L E H Q M W E A V K D E L N T L L E N G V D L S V V V E K Q 2301 TTCCAGACTG GAGCATCAAA TGTGGGAAGC AGTGAAGGAC GAGCTGAACA CTCTTTTGAA GGAGAATGGT GTGGACCTTA GTGTCGTGGT TGAGAAACAA AAGGTCTGAC CTCGTAGTTT ACACCCTTCG TCACTTCCTG CTCGACTTGT GAGAAAACTT CCTCTTACCA CACCTGGAAT CACAGCACCA ACTCTTTGTT
G G M Y K S A P K R L T A T T E K L E I G W K A W G K S I L F A P E
2401 GGGGGAATGT ACAAGTCAGC ACCTAAACGC CTCACCGCCA CCACGGAAAA ATTGGAAATT GGCTGGAAGG CCTGGGGAAA GAGTATTTTG TTTGCACCAG CCCCCTTACA TGTTCAGTCG TGGATTTGCG GAGTGGCGGT GGTGCCTTTT TAACCTTTAA CCGACCTTCC GGACCCCTTT CTCATAAAAC AAACGTGGTC
NSl
• L A N N T F V V D G P E T K E C P T Q N R A W N S L E V E D F G F
2501 AACTCGCCAA CAACACCTTT GTGGTTGATG GTCCGGAGAC CAAGGAATGT CCGACTCAGA ATCGCGCTTG GAATAGCTTA GAAGTGGAGG ATTTTGGATT
TTGAGCGGTT GTTGTGGAAA CACCAACTAC CAGGCCTCTG GTTCCTTACA GGCTGAGTCT TAGCGCGAAC CTTATCGAAT CTTCACCTCC TAAAACCTAA
NSl
- G L T S T R M F L K V R E S N T T E C D S K I I G T A V K N N L A 2601 TGGTCTCACC AGCACTCGGA TGTTCCTGAA GGTCAGAGAG AGCAACACAA CTGAATGTGA CTCGAAGATC ATTGGAACGG CTGTCAAGAA CAACTTGGCG ACCAGAGTGG TCGTGAGCCT ACAAGGACTT CCAGTCTCTC TCGTTGTGTT GACTTACACT GAGCTTCTAG TAACCTTGCC GACAGTTCTT GTTGAACCGC
NSl
I H S D L S Y W I E S R L N D T W K L E R A V L G E V K S C T W P E
2701 ATCCACAGTG ACCTGTCCTA TTGGATTGAA AGCAGGCTCA ATGATACGTG GAAGCTTGAA AGGGCAGTTC TGGGTGAAGT CAAATCATGT ACGTGGCCTG TAGGTGTCAC TGGACAGGAT AACCTAACTT TCGTCCGAGT TACTATGCAC CTTCGAACTT TCCCGTCAAG ACCCACTTCA GTTTAGTACA TGCACCGGAC
NSl
■ T H T L W G D G I L E S D L I I P V T L A G P R S N H N R R P G Y
2801 AGACGCATAC CTTGTGGGGC GATGGAATCC TTGAGAGTGA CTTGATAATA CCAGTCACAC TGGCGGGACC ACGAAGCAAT CACAATCGGA GACCTGGGTA
TCTGCGTATG GAACACCCCG CTACCTTAGG AACTCTCACT GAACTATTAT GGTCAGTGTG ACCGCCCTGG TGCTTCGTTA GTGTTAGCCT CTGGACCCAT
NSl
• K T Q N Q G P W D E G R V E I D F D Y C P G T T V T L S E S C G H 2901 TAAGACACAA AACCAGGGCC CATGGGACGA AGGCCGGGTA GAGATTGACT TCGATTACTG CCCAGGAACT ACGGTCACCC TGAGTGAGAG CTGCGGACAC ATTCTGTGTT TTGGTCCCGG GTACCCTGCT TCCGGCCCAT CTCTAACTGA AGCTAATGAC GGGTCCTTGA TGCCAGTGGG ACTCACTCTC GACGCCTGTG
NSl
R G P A T R T T T E S G K L I T D W C C R S C T L P P L R Y Q T D S
3001 CGTGGACCTG CCACTCGCAC CACCACAGAG AGCGGAAAGT TGATAACAGA TTGGTGCTGC AGGAGCTGCA CCTTACCACC ACTGCGCTAC CAAACTGACA GCACCTGGAC GGTGAGCGTG GTGGTGTCTC TCGCCTTTCA ACTATTGTCT AACCACGACG TCCTCGACGT GGAATGGTGG TGACGCGATG GTTTGACTGT
NSl
NS2A
• G C W Y G M E I R P Q R H D E K T L V Q S Q V N A Y N A D M I D P
3101 GCGGCTGTTG GTATGGTATG GAGATCAGAC CACAGAGACA TGATGAAAAG ACCCTCGTGC AGTCACAAGT GAATGCTTAT AATGCTGATA TGATTGACCC
CGCCGACAAC CATACCATAC CTCTAGTCTG GTGTCTCTGT ACTACTTTTC TGGGAGCACG TCAGTGTTCA CTTACGAATA TTACGACTAT ACTAACTGGG
NS2A
■ F Q L G L L V V F L A T Q E V L R K R W T A K I S M P A I L I A L 3201 TTTTCAGTTG GGCCTTCTGG TCGTGTTCTT GGCCACCCAG GAGGTCCTTC GCAAGAGGTG GACAGCCAAG ATCAGCATGC CAGCTATACT GATTGCTCTG AAAAGTCAAC CCGGAAGACC AGCACAAGAA CCGGTGGGTC CTCCAGGAAG CGTTCTCCAC CTGTCGGTTC TAGTCGTACG GTCGATATGA CTAACGAGAC
NS2A
L V L V F G G I T Y T D V L R Y V I L V G A A F A E S N S G G D V V
3301 CTAGTCCTGG TGTTTGGGGG CATTACTTAC ACTGATGTGT TACGCTATGT CATCTTGGTG GGGGCAGCTT TCGCAGAATC TAATTCGGGA GGAGACGTGG GATCAGGACC ACAAACCCCC GTAATGAATG TGACTACACA ATGCGATACA GTAGAACCAC CCCCGTCGAA AGCGTCTTAG ATTAAGCCCT CCTCTGCACC
NS2A
- H L A L M A T F K I Q P V F M V A S F L K A R W T N Q E H I L L M
3401 TACACTTGGC GCTCATGGCG ACCTTCAAGA TACAACCAGT GTTTATGGTG GCATCGTTTC TTAAAGCGAG ATGGACCAAC CAGGAGAACA TTTTGTTGAT
ATGTGAACCG CGAGTACCGC TGGAAGTTCT ATGTTGGTCA CAAATACCAC CGTAGCAAAG AATTTCGCTC TACCTGGTTG GTCCTCTTGT AAAACAACTA
NS2A
• L A A V F F Q M A Y H D A R Q I L L E I P D V L N S L A I A W M
3501 GTTGGCGGCT GTTTTCTTTC AAATGGCTTA TCACGATGCC CGCCAAATTC TGCTCTGGGA GATCCCTGAT GTGTTGAATT CACTGGCAAT AGCTTGGATG
CAACCGCCGA CAAAAGAAAG TTTACCGAAT AGTGCTACGG GCGGTTTAAG ACGAGACCCT CTAGGGACTA CACAACTTAA GTGACCGTTA TCGAACCTAC
NS2A
I L R A I T F T T T S N V V V P L L A L L T P G L R C L N L D V Y R
3601 ATACTGAGAG CCATAACATT CACAACGACA TCAAACGTGG TTGTTCCGCT GCTAGCCCTG CTAACACCCG GGCTGAGATG CTTGAATCTG GATGTGTACA TATGACTCTC GGTATTGTAA GTGTTGCTGT AGTTTGCACC AACAAGGCGA CGATCGGGAC GATTGTGGGC CCGACTCTAC GAACTTAGAC CTACACATGT
NS2A
• I L L L M V G I G S L I R E K R S A A A K K K G A S L L C L A L A 3701 GGATACTGCT GTTGATGGTC GGAATAGGCA GCTTGATCAG GGAGAAGAGG AGCGCAGCTG CAAAAAAGAA AGGAGCAAGT CTGCTATGCT TGGCTCTAGC CCTATGACGA CAACTACCAG CCTTATCCGT CGAACTAGTC CCTCTTCTCC TCGCGTCGAC GTTTTTTCTT TCCTCGTTCA GACGATACGA ACCGAGATCG
NS2B
NS2A
• S T G L F N P M I L A A G L I A C D P N R K R G W P A T E V M T A 3801 CTCAACAGGA CTCTTCAACC CCATGATCCT TGCTGCTGGA CTGATTGCAT GTGATCCCAA CCGTAAACGC GGGTGGCCCG CAACTGAAGT GATGACAGCT GAGTTGTCCT GAGAAGTTGG GGTACTAGGA ACGACGACCT GACTAACGTA CACTAGGGTT GGCATTTGCG CCCACCGGGC GTTGACTTCA CTACTGTCGA
NS2B
V G L M F A I V G G L A E L D I D Ξ Μ Α I P M T I A G L M F A A F V
3901 GTCGGCCTAA TGTTTGCCAT CGTCGGAGGG CTGGCAGAGC TTGACATTGA CTCCATGGCC ATTCCAATGA CTATCGCGGG GCTCATGTTT GCTGCTTTCG CAGCCGGATT ACAAACGGTA GCAGCCTCCC GACCGTCTCG AACTGTAACT GAGGTACCGG TAAGGTTACT GATAGCGCCC CGAGTACAAA CGACGAAAGC
NS2B
■ I S G K S T D M W I E R T A D I S W E S D A E I T G S S E R V D V 4001 TGATTTCTGG GAAATCAACA GATATGTGGA TTGAGAGAAC GGCGGACATT TCCTGGGAAA GTGATGCAGA GATTACAGGC TCGAGCGAAA GAGTTGATGT ACTAAAGACC CTTTAGTTGT CTATACACCT AACTCTCTTG CCGCCTGTAA AGGACCCTTT CACTACGTCT CTAATGTCCG AGCTCGCTTT CTCAACTACA
NS2B
• R L D D D G N F Q L M N D P G A P W K I W M L R M V C L A I S A Y 4101 GCGGCTTGAT GATGATGGAA ACTTCCAGCT CATGAATGAT CCAGGAGCAC CTTGGAAGAT ATGGATGCTC AGAATGGTCT GTCTCGCGAT TAGTGCGTAC CGCCGAACTA CTACTACCTT TGAAGGTCGA GTACTTACTA GGTCCTCGTG GAACCTTCTA TACCTACGAG TCTTACCAGA CAGAGCGCTA ATCACGCATG
NS3
ΝΞ2Β
T P W A I L P S V V G F W I T L Q Y T K R G G V L W D T P S P K E Y
4201 ACCCCCTGGG CAATCTTGCC CTCAGTAGTT GGATTTTGGA TAACTCTCCA ATACACAAAG AGAGGAGGCG TGTTGTGGGA CACTCCCTCA CCAAAGGAGT
TGGGGGACCC GTTAGAACGG GAGTCATCAA CCTAAAACCT ATTGAGAGGT TATGTGTTTC TCTCCTCCGC ACAACACCCT GTGAGGGAGT GGTTTCCTCA
NS3
• K K G D T T T G V Y R I M T R G L L G S Y Q A G A G V .M V E G V F 4301 ACAAAAAGGG GGACACGACC ACCGGCGTCT ACAGGATCAT GACTCGTGGG CTGCTCGGCA GTTATCAAGC AGGAGCAGGC GTGATGGTTG AAGGTGTTTT TGTTTTTCCC CCTGTGCTGG TGGCCGCAGA TGTCCTAGTA CTGAGCACCC GACGAGCCGT CAATAGTTCG TCCTCGTCCG CACTACCAAC TTCCACAAAA
NS3
• H T L W H T T K G A A L M S G E G R L D P Y W G S V K E D R L C Y 4401 CCACACCCTT TGGCATACAA CAAAAGGAGC CGCTTTGATG AGCGGAGAGG GCCGCCTGGA CCCATACTGG GGCAGTGTCA AGGAGGATCG ACTTTGTTAC GGTGTGGGAA ACCGTATGTT GTTTTCCTCG GCGAAACTAC TCGCCTCTCC CGGCGGACCT GGGTATGACC CCGTCACAGT TCCTCCTAGC TGAAACAATG
NS3
G G P W K L Q H K W H G Q D E V Q M I V V E P G K N V K N V Q T K P
4501 GGAGGACCCT GGAAATTGCA GCACAAGTGG AACGGGCAGG ATGAGGTGCA GATGATTGTG GTGGAACCTG GCAAGAACGT TAAGAACGTC CAGACGAAAC CCTCCTGGGA CCTTTAACGT CGTGTTCACC TTGCCCGTCC TACTCCACGT CTACTAACAC CACCTTGGAC CGTTCTTGCA ATTCTTGCAG GTCTGCTTTG
NS3
• G V F K T P E G E I G A V T L D F P T G T S G S P I V D N G D V 4601 CAGGGGTGTT CAAAACACCT GAAGGAGAAA TCGGGGCCGT GACTTTGGAC TTCCCCACTG GAACATCAGG CTCACCAATA GTGGACAAAA ACGGTGATGT GTCCCCACAA GTTTTGTGGA CTTCCTCTTT AGCCCCGGCA CTGAAACCTG AAGGGGTGAC CTTGTAGTCC GAGTGGTTAT CACCTGTTTT TGCCACTACA
NS3
■ I G L Y G N G V I M P N G S Y I S A I V Q G E R M D E P I P A G F 4701 GATTGGGCTT TATGGCAATG GAGTCATAAT GCCCAACGGC TCATACATAA GCGCGATAGT GCAGGGTGAA AGGATGGATG AGCCAATCCC AGCCGGATTC CTAACCCGAA ATACCGTTAC CTCAGTATTA CGGGTTGCCG AGTATGTATT CGCGCTATCA CGTCCCACTT TCCTACCTAC TCGGTTAGGG TCGGCCTAAG
NS3
E P E M L R K Q I T V L D L H P G A G K T R R I L P Q I I K E A I
801 GAACCTGAGA TGCTGAGGAA AAAACAGATC ACTGTACTGG ATCTCCATCC CGGCGCCGGT AAAACAAGGA GGATTCTGCC ACAGATCATC AAAGAGGCCA CTTGGACTCT ACGACTCCTT TTTTGTCTAG TGACATGACC TAGAGGTAGG GCCGCGGCCA TTTTGTTCCT CCTAAGACGG TGTCTAGTAG TTTCTCCGGT
NS3
• N R R L R T A V L A P T R V V A A E M A E A L R G L P I R Y Q T S 4901 TAAACAGAAG ACTGAGAACA GCCGTGCTAG CACCAACCAG GGTTGTGGCT GCTGAGATGG CTGAAGCACT GAGAGGACTG CCCATCCGGT ACCAGACATC ATTTGTCTTC TGACTCTTGT CGGCACGATC GTGGTTGGTC CCAACACCGA CGACTCTACC GACTTCGTGA CTCTCCTGAC GGGTAGGCCA TGGTCTGTAG
NS3
■ A V P R E H N G N E I V D V M C H A T L T H R L M S P H R V P N Y 5001 CGCAGTGCCC AGAGAACATA ATGGAAATGA GATTGTTGAT GTCATGTGTC ATGCTACCCT CACCCACAGG CTGATGTCTC CTCACAGGGT GCCGAACTAC GCGTCACGGG TCTCTTGTAT TACCTTTACT CTAACAACTA CAGTACACAG TACGATGGGA GTGGGTGTCC GACTACAGAG GAGTGTCCCA CGGCTTGATG
NS3
N L F V M D E A H F T D P A S I A A R G Y I S T K V E L G E A A A I
5101 AACCTGTTCG TGATGGATGA GGCTCATTTC ACCGACCCAG CTAGCATTGC AGCAAGAGGT TACATTTCCA CAAAGGTCGA GCTAGGGGAG GCGGCGGCAA TTGGACAAGC ACTACCTACT CCGAGTAAAG TGGCTGGGTC GATCGTAACG TCGTTCTCCA ATGTAAAGGT GTTTCCAGCT CGATCCCCTC CGCCGCCGTT
NS3
• F M T A T P P G T S D P F P E S N S P I S D L Q T E I P D R A W N 5201 TATTCATGAC AGCCACCCCA CCAGGCACTT CAGATCCATT CCCAGAGTCC AATTCACCAA TTTCCGACTT ACAGACTGAG ATCCCGGATC GAGCTTGGAA ATAAGTACTG TCGGTGGGGT GGTCCGTGAA GTCTAGGTAA GGGTCTCAGG TTAAGTGGTT AAAGGCTGAA TGTCTGACTC TAGGGCCTAG CTCGAACCTT
NS3
■ S G Y E W I T E Y T G K T V F V P S V M G N E I A L C L Q R A 5301 CTCTGGATAC GAATGGATCA CAGAATACAC CGGGAAGACG GTTTGGTTTG TGCCTAGTGT TAAGATGGGG AATGAGATTG CCCTTTGCCT ACAACGTGCT GAGACCTATG CTTACCTAGT GTCTTATGTG GCCCTTCTGC CAAACCAAAC ACGGATCACA ATTCTACCCC TTACTCTAAC GGGAAACGGA TGTTGCACGA
NS3
G K K V V Q L N R K S Y E T E Y P K C N D D W D F V I T T D I S E GGAAAGAAAG TAGTCCAATT GAACAGAAAG TCGTACGAGA CGGAGTACCC AAAATGTAAG AACGATGATT GGGACTTTGT TATCACAACA GACATATCTG CCTTTCTTTC ATCAGGTTAA CTTGTCTTTC AGCATGCTCT GCCTCATGGG TTTTACATTC TTGCTACTAA CCCTGAAACA ATAGTGTTGT CTGTATAGAC
NS3
■ M G A N F K A S R V I D S R K S V K P T I I T E G E G R V I L
5501 AAATGGGGGC TAACTTCAAG GCGAGCAGGG TGATTGACAG CCGGAAGAGT GTGAAACCAA CCATCATAAC AGAAGGAGAA GGGAGAGTGA TCCTGGG TTTACCCCCG ATTGAAGTTC CGCTCGTCCC ACTAACTGTC GGCCTTCTCA CACTTTGGTT GGTAGTATTG TCTTCCTCTT CCCTCTCACT AGGACCC
Construct 11
1. PIV-WN (AC)-SIV Env
Figure imgf000155_0001
RV909 Env
2. Sequence of PIV-WN (AC)-SIV Env (partial).
dC
UTR
S S S P V * A D K L S S V C E D * Q Q L T Q C E L F L S T K I S M S 1 AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
NS3 Signal dC
• K K P G G P G K S R A V N M L R G M P R V L S L I G L K Q K K R 101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCAA TATGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGC AAAAGAAGCG GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGTT ATACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCG TTTTCTTCGC
partial C signal
NS3 Signal Env
• G G K T G I A V I M D A M K R G L C C V L L L C G A V F V T T T E 201 AGGGGGCAAG ACTGGTATAG CTGTGATCAT GGACGCCATG AAGAGGGGAC TTTGTTGTGT GCTCCTGCTG TGCGGAGCTG TGTTCGTTAC AACAACGGAG TCCCCCGTTC TGACCATATC GACACTAGTA CCTGCGGTAC TTCTCCCCTG AAACAACACA CGAGGACGAC ACGCCTCGAC ACAAGCAATG TTGTTGCCTC
Env
A I Y C T Q Y V T V F Y G V P A W R N A T I P L F C A T K N R D T W 301 GCGATTTACT GCACCCAGTA TGTCACCGTG TTTTACGGTG TCCCCGCCTG GCGGAACGCC ACCATCCCTC TGTTTTGTGC CACCAAGAAT AGAGATACGT CGCTAAATGA CGTGGGTCAT ACAGTGGCAC AAAATGCCAC AGGGGCGGAC CGCCTTGCGG TGGTAGGGAG ACAAAACACG GTGGTTCTTA TCTCTATGCA
Env
■ G T T Q C L P D N G D Y S E L A L N V T E S F D A W E N T V T E Q 401 GGGGCACCAC ACAATGCCTT CCCGATAATG GCGATTACTC TGAATTAGCC CTGAACGTCA CGGAAAGTTT TGATGCTTGG GAAAATACGG TTACCGAACA CCCCGTGGTG TGTTACGGAA GGGCTATTAC CGCTAATGAG ACTTAATCGG GACTTGCAGT GCCTTTCAAA ACTACGAACC CTTTTATGCC AATGGCTTGT
Env
■ A I E D V W Q L F E T S I K P C V L S P L C I T M R C N K S E T 501 GGCCATCGAA GATGTCTGGC AGTTATTCGA AACTAGTATC AAACCTTGCG TTAAGCTGAG TCCTTTGTGC ATAACGATGC GGTGCAACAA GAGCGAAACG CCGGTAGCTT CTACAGACCG TCAATAAGCT TTGATCATAG TTTGGAACGC AATTCGACTC AGGAAACACG TATTGCTACG CCACGTTGTT CTCGCTTTGC
Env
D K W G L T K S S T T T A S T T T T T A P A K I D V N E T S S C I
601 GACAAATGGG GCTTAACCAA ATCTTCAACC ACCACCGCCT CCACCACTAC GACAACCGCA CCTGCCAAGA TCGACATGGT TAACGAAACC TCTAGTTGCA CTGTTTACCC CGAATTGGTT TAGAAGTTGG TGGTGGCGGA GGTGGTGATG CTGTTGGCGT GGACGGTTCT AGCTGTACCA ATTGCTTTGG AGATCAACGT
Env
■ T H D N C T G L E Q E Q M I G C K F N M T G L K R D K T K E Y N E 701 TTACCCATGA CAACTGCACA GGCCTCGAAC AAGAACAAAT GATCGGCTGT AAATTCAATA TGACCGGACT GAAGAGAGAC AAGACAAAAG AGTACAACGA AATGGGTACT GTTGACGTGT CCGGAGCTTG TTCTTGTTTA CTAGCCGACA TTTAAGTTAT ACTGGCCTGA CTTCTCTCTG TTCTGTTTTC TCATGTTGCT
Env
■ T W Y S T D L V C E Q G N S T D N E S R C Y M N H C N T S I I Q E 801 GACTTGGTAC AGCACCGACT TAGTGTGTGA GCAGGGGAAC TCAACCGATA ACGAGTCCCG CTGTTATATG AACCACTGCA ATACGAGCAT CATCCAAGAG CTGAACCATG TCGTGGCTGA ATCACACACT CGTCCCCTTG AGTTGGCTAT TGCTCAGGGC GACAATATAC TTGGTGACGT TATGCTCGTA GTAGGTTCTC
Env
S C D K H Y W D T I R F R Y C A P P G Y A L L R C N D T N Y S G F M
901 TCGTGCGACA AACACTATTG GGACACTATC CGATTTAGGT ACTGTGCCCC GCCGGGCTAT GCGCTTCTGC GTTGTAATGA TACCAATTAC AGTGGGTTCA AGCACGCTGT TTGTGATAAC CCTGTGATAG GCTAAATCCA TGACACGGGG CGGCCCGATA CGCGAAGACG CAACATTACT ATGGTTAATG TCACCCAAGT
Env
• P K C S K V V V S S C T R M M E T Q T S T W F G F N G T R A E N R 1001 TGCCGAAGTG TAGCAAAGTC GTGGTGTCCT CTTGTACCCG CATGATGGAG ACGCAGACTT CCACCTGGTT TGGCTTTAAC GGAACTCGAG CTGAAAACCG ACGGCTTCAC ATCGTTTCAG CACCACAGGA GAACATGGGC GTACTACCTC TGCGTCTGAA GGTGGACCAA ACCGAAATTG CCTTGAGCTC GACTTTTGGC
Env
■ T Y I Y W H G R D N R T I I S L H Y Y N L T M K C R R P G N T
1101 GACGTATATC TACTGGCACG GACGAGATAA CCGAACGATC ATCTCACTGA ACAAGTACTA CAATCTGACC ATGAAATGCC GGCGCCCAGG CAATAAGACG
CTGCATATAG ATGACCGTGC CTGCTCTATT GGCTTGCTAG TAGAGTGACT TGTTCATGAT GTTAGACTGG TACTTTACGG CCGCGGGTCC GTTATTCTGC
Env
V L P V T I M S G L V F H S Q P V N E R P N Q A W C W F G G N W K D
1201 GTACTTCCTG TCACTATTAT GAGCGGACTT GTATTTCACT CGCAGCCGGT CAATGAGCGC CCGAACCAAG CCTGGTGCTG GTTTGGAGGC AACTGGAAAG
CATGAAGGAC AGTGATAATA CTCGCCTGAA CATAAAGTGA GCGTCGGCCA GTTACTCGCG GGCTTGGTTC GGACCACGAC CAAACCTCCG TTGACCTTTC
Env
■ A I K E V K Q T I V K H P R Y T G T N N T D K I N L T A P R G G D 1301 ATGCGATTAA GGAAGTTAAA CAAACCATCG TAAAGCATCC CCGCTACACC GGCACCAACA ATACGGATAA GATCAACCTC ACAGCCCCTC GTGGCGGCGA
TACGCTAATT CCTTCAATTT GTTTGGTAGC ATTTCGTAGG GGCGATGTGG CCGTGGTTGT TATGCCTATT CTAGTTGGAG TGTCGGGGAG CACCGCCGCT
Env
■ P E V T F M W T N C R G E F L Y C K M N F L N W V E D R D L T T
1401 TCCAGAGGTG ACCTTCATGT GGACTAACTG TCGCGGTGAA TTTCTGTACT GTAAGATGAA TTGGTTTCTG AACTGGGTCG AGGATAGGGA TCTGACAACA
AGGTCTCCAC TGGAAGTACA CCTGATTGAC AGCGCCACTT AAAGACATGA CATTCTACTT AACCAAAGAC TTGACCCAGC TCCTATCCCT AGACTGTTGT
Env
Q R P E R H R R N Y V P C H I R Q I I N T H K V G E N V Y L P P
1501 CAACGGCCTA AGGAGAGGCA CCGCCGTAAC TATGTGCCTT GTCATATCAG ACAGATCATC AATACATGGC ATAAGGTGGG TAAAAACGTA TACCTCCCTC GTTGCCGGAT TCCTCTCCGT GGCGGCATTG ATACACGGAA CAGTATAGTC TGTCTAGTAG TTATGTACCG TATTCCACCC ATTTTTGCAT ATGGAGGGAG
Env
• R E G D L T C N S T V T S L I A N I D W T D G N Q T N I T M S A E · 1601 CCCGCGAGGG CGACCTGACA TGTAATAGTA CAGTAACCAG CCTCATCGCT AACATAGACT GGACTGATGG AAATCAGACC AACATCACTA TGTCAGCCGA GGGCGCTCCC GCTGGACTGT ACATTATCAT GTCATTGGTC GGAGTAGCGA TTGTATCTGA CCTGACTACC TTTAGTCTGG TTGTAGTGAT ACAGTCGGCT
Env
• V A E L Y R L E L G D Y K L V E I T P I G L A P T D V K R Y T T G
1701 GGTAGCCGAA CTGTATAGGC TAGAACTCGG TGACTATAAG CTCGTCGAGA TCACCCCGAT AGGGCTCGCC CCTACAGACG TGAAACGTTA TACCACCGGC
CCATCGGCTT GACATATCCG ATCTTGAGCC ACTGATATTC GAGCAGCTCT AGTGGGGCTA TCCCGAGCGG GGATGTCTGC ACTTTGCAAT ATGGTGGCCG
Env
G T S R N K R Y G I Y I V V G V I L L R I V I Y I V Q M L N R V R Q
1801 GGTACATCAA GGAACAAACG CTACGGCATC TACATCGTGG TAGGGGTCAT CCTCTTACGG ATTGTCATCT ATATCGTTCA GATGCTGAAT AGGGTGAGGC CCATGTAGTT CCTTGTTTGC GATGCCGTAG ATGTAGCACC ATCCCCAGTA GGAGAATGCC TAACAGTAGA TATAGCAAGT CTACGACTTA TCCCACTCCG
FMD2a
Env C Anchor
• G N F D L L K L A G D V E S K P G P G G K T G I A V M I G L I A C 1901 AGGGCAATTT TGACCTGTTA AAACTGGCCG GGGACGTCGA AAGCAACCCC GGTCCGGGAG GAAAGACCGG TATTGCAGTC ATGATTGGCC TGATCGCCTG TCCCGTTAAA ACTGGACAAT TTTGACCGGC CCCTGCAGCT TTCGTTGGGG CCAGGCCCTC CTTTCTGGCC ATAACGTCAG TACTAACCGG ACTAGCGGAC C Anchor
prM
• V G A V T L S N F Q G K V M M T V N A T D V T D V I T I P
2001 CGTAGGAGCA GTTACCCTCT CTAACTTCCA AGGGAAGGTG ATGATGACGG TAAATGCTAC TGACGTCACA GATGTCATCA CGATTCCA
GCATCCTCGT CAATGGGAGA GATTGAAGGT TCCCTTCCAC TACTACTGCC ATTTACGATG ACTGCAGTGT CTACAGTAGT GCTAAGGT
Appendix 7
1. PIV-WN (AprME)-HIV Gag
Figure imgf000158_0001
RV2309AA-FMD-AM96 Gag
2. Sequence of PIV-WN (AprME)-HIV Gag (partial).
DTR
M S
AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
C
• K K P G G P G K S R A V Y L L K R G M P R V L S L I G L K R A M L
101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGA GGGCTATGTT
GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCT CCCGATACAA
C
■ S L I D G K G P I R F V L A L L A F F R F T A I A P T R A V L D R
201 GAGCCTGATC GACGGCAAGG GGCCAATACG ATTTGTGTTG GCTCTCTTGG CGTTCTTCAG GTTCACAGCA ATTGCTCCGA CCCGAGCAGT GCTGGATCGA
CTCGGACTAG CTGCCGTTCC CCGGTTATGC TAAACACAAC CGAGAGAACC GCAAGAAGTC CAAGTGTCGT TAACGAGGCT GGGCTCGTCA CGACCTAGCT
C
NS3 Cleavage
W R G V N K Q T A M K H L L F K K E L G T L T S A N R R S S K Q ·
301 TGGAGAGGTG TGAACAAACA AACAGCGATG AAACACCTTC TGAGTTTCAA GAAGGAACTA GGGACCTTGA CCAGTGCTAT CAATCGGCGG AGCTCAAAGC ACCTCTCCAC ACTTGTTTGT TTGTCGCTAC TTTGTGGAAG ACTCAAAGTT CTTCCTTGAT CCCTGGAACT GGTCACGATA GTTAGCCGCC TCGAGTTTCG
9AA C Signal ZM96 Gag
NS3 Cleavage FMDV2A
■ K K R G G K T G I A V I N F D L L K L A G D V E S N P G P M G A R 401 AGAAAAAGCG GGGCGGAAAG ACAGGTATTG CTGTGATCAA TTTTGACCTG TTAAAACTGG CCGGGGACGT CGAAAGCAAC CCCGGTCCGA TGGGAGCCAG TCTTTTTCGC CCCGCCTTTC TGTCCATAAC GACACTAGTT AAAACTGGAC AATTTTGACC GGCCCCTGCA GCTTTCGTTG GGGCCAGGCT ACCCTCGGTC
ZM96 Gag
• A S I L R G G K L D K W E K I R L R P G G K K R Y I K H L V W A 501 AGCCAGCATC CTGAGAGGCG GAAAGCTGGA CAAGTGGGAG AAGATCCGGC TGAGACCTGG CGGAAAGAAA CGGTACATGA TCAAGCACCT GGTGTGGGCT TCGGTCGTAG GACTCTCCGC CTTTCGACCT GTTCACCCTC TTCTAGGCCG ACTCTGGACC GCCTTTCTTT GCCATGTACT AGTTCGTGGA CCACACCCGA
2M96 Gag
S R E L E R F A L N P G L L E T S E G C K Q 1 M K Q L Q P A L Q T G 601 TCTCGGGAGC TGGAAAGATT CGCCCTGAAT CCCGGCCTGC TGGAAACCAG CGAGGGCTGC AAGCAGATCA TGAAGCAGCT GCAGCCTGCC CTGCAGACCG AGAGCCCTCG ACCTTTCTAA GCGGGACTTA GGGCCGGACG ACCTTTGGTC GCTCCCGACG TTCGTCTAGT ACTTCGTCGA CGTCGGACGG GACGTCTGGC
ZM96 Gag
' T E E L R S L Y N T V A T L Y C V H E G V E V R D T K E A L D R I 701 GCACCGAGGA ACTGCGGAGC CTGTACAACA CCGTGGCCAC CCTGTACTGC GTGCACGAGG GCGTGGAAGT GCGGGACACC AAAGAGGCCC TGGACCGGAT CGTGGCTCCT TGACGCCTCG GACATGTTGT GGCACCGGTG GGACATGACG CACGTGCTCC CGCACCTTCA CGCCCTGTGG TTTCTCCGGG ACCTGGCCTA
2M96 Gag
• E E E Q N K I Q Q K I Q Q T Q Q A A D G K V S Q N Y P I V Q N L
801 CGAGGAAGAA CAGAACAAGA TCCAGCAGAA GATTCAGCAG AAAACCCAGC AGGCTGCCGA CGGCAAGGTG TCCCAGAACT ACCCCATCGT GCAGAACCTG
GCTCCTTCTT GTCTTGTTCT AGGTCGTCTT CTAAGTCGTC TTTTGGGTCG TCCGACGGCT GCCGTTCCAC AGGGTCTTGA TGGGGTAGCA CGTCTTGGAC
ZM96 Gag
Q G Q M V H Q K L S P R T L N A V K V I E E K A F S P E V I P M F
901 CAGGGCCAGA TGGTGCACCA GAAGCTGTCA CCTCGGACCC TGAACGCCTG GGTGAAAGTG ATCGAGGAAA AGGCCTTCAG CCCTGAAGTG ATCCCCATGT GTCCCGGTCT ACCACGTGGT CTTCGACAGT GGAGCCTGGG ACTTGCGGAC CCACTTTCAC TAGCTCCTTT TCCGGAAGTC GGGACTTCAC TAGGGGTACA
ZM96 Gag
• T A L S E G A T P Q D L N T M L N T V G G H Q A A M Q M L K D T I
1001 TCACAGCCCT GAGCGAGGGA GCCACACCCC AGGACCTGAA CACCATGCTG AACACCGTGG GAGGGCACCA GGCTGCCATG CAGATGCTGA AGGACACCAT
AGTGTCGGGA CTCGCTCCCT CGGTGTGGGG TCCTGGACTT GTGGTACGAC TTGTGGCACC CTCCCGTGGT CCGACGGTAC GTCTACGACT TCCTGTGGTA
ZM96 Gag
• N E E A A E W D R L H P V H A G P I A P G Q M R E P R G S D I A G 1101 CAACGAAGAG GCTGCCGAGT GGGACCGGCT GCACCCTGTC CATGCTGGAC CTATTGCCCC TGGCCAGATG CGGGAGCCCA GAGGCTCCGA TATTGCCGGC GTTGCTTCTC CGACGGCTCA CCCTGGCCGA CGTGGGACAG GTACGACCTG GATAACGGGG ACCGGTCTAC GCCCTCGGGT CTCCGAGGCT ATAACGGCCG
ZM96 Gag
T T S T L Q E Q I A W M T S N P P I P V G D I Y K R W I I L G L N K
1201 ACCACCTCCA CACTGCAAGA ACAGATCGCC TGGATGACCA GCAACCCTCC CATCCCCGTG GGCGACATCT ACAAGCGGTG GATCATCCTG GGCCTGAACA TGGTGGAGGT GTGACGTTCT TGTCTAGCGG ACCTACTGGT CGTTGGGAGG GTAGGGGCAC CCGCTGTAGA TGTTCGCCAC CTAGTAGGAC CCGGACTTGT
ZM96 Gag
• I V R M Y S P V S I L D I K Q G P K E P F R D Y V D R F F K T L R 1301 AGATCGTGCG GATGTACAGC CCTGTGTCCA TCCTGGACAT CAAGCAGGGA CCCAAAGAGC CCTTCCGGGA CTACGTGGAC CGGTTCTTCA AGACCCTGAG
TCTAGCACGC CTACATGTCG GGACACAGGT AGGACCTGTA GTTCGTCCCT GGGTTTCTCG GGAAGGCCCT GATGCACCTG GCCAAGAAGT TCTGGGACTC
ZM96 Gag
• A E Q A T Q E V K N W M T D T L L V Q N A N P D C K T I L K A L G
1401 AGCCGAGCAG GCCACCCAAG AGGTGAAGAA CTGGATGACC GACACCCTGC TGGTGCAGAA CGCCAACCCC GACTGCAAGA CCATCCTGAA GGCCCTGGGA
TCGGCTCGTC CGGTGGGTTC TCCACTTCTT GACCTACTGG CTGTGGGACG ACCACGTCTT GCGGTTGGGG CTGACGTTCT GGTAGGACTT CCGGGACCCT
ZM96 Gag
P G A T L E E M M T A C Q G V G G P S H K A R V L A E A M S Q T N S
1501 CCTGGAGCCA CCCTGGAAGA GATGATGACC GCCTGCCAGG GCGTGGGAGG ACCCAGCCAC AAGGCTCGGG TGCTGGCCGA GGCCATGAGC CAGACCAACA GGACCTCGGT GGGACCTTCT CTACTACTGG CGGACGGTCC CGCACCCTCC TGGGTCGGTG TTCCGAGCCC ACGACCGGCT CCGGTACTCG GTCTGGTTGT
ZM96 Gag
• V N I L M Q K S N F K G N K R M V K C F N C G K E G H I A R N C R - 1601 GCGTGAACAT CCTGATGCAG AAGTCCAACT TCAAGGGCAA CAAGCGGATG GTGAAGTGCT TCAACTGTGG AAAGGAGGGC CACATTGCCA GAAACTGCAG CGCACTTGTA GGACTACGTC TTCAGGTTGA AGTTCCCGTT GTTCGCCTAC CACTTCACGA AGTTGACACC TTTCCTCCCG GTGTAACGGT CTTTGACGTC
ZM96 Gag
• A P R K K G C K C G K E G H Q M K D C T E R Q A N F L G K I W P 1701 AGCCCCAAGA AAAAAGGGCT GCTGGAAGTG CGGCAAAGAG GGGCACCAGA TGAAGGACTG CACCGAGCGG CAGGCTAACT TCCTGGGCAA GATCTGGCCC TCGGGGTTCT TTTTTCCCGA CGACCTTCAC GCCGTTTCTC CCCGTGGTCT ACTTCCTGAC GTGGCTCGCC GTCCGATTGA AGGACCCGTT CTAGACCGGG
ZM96 Gag
S H K G R P G N F L Q N R P E P T A P P A E S F R F E E T T P A P K
1801 TCCCACAAGG GCAGACCAGG CAACTTCCTG CAGAACAGAC CCGAGCCAAC AGCCCCTCCT GCCGAGAGCT TCAGATTCGA GGAAACCACC CCTGCCCCAA AGGGTGTTCC CGTCTGGTCC GTTGAAGGAC GTCTTGTCTG GGCTCGGTTG TCGGGGAGGA CGGCTCTCGA AGTCTAAGCT CCTTTGGTGG GGACGGGGTT
ZM96 Gag
FMDV2A
■ Q E S K D R E A L T S L K S L F G S D P L S Q N F D L L K L A G D 1901 AGCAGGAAAG CAAGGACCGG GAGGCCCTGA CCTCCCTGAA GTCCCTGTTC GGCAGCGACC CCCTGAGCCA GAATTTCGAC CTGCTTAAAC TTGCTGGCGA TCGTCCTTTC GTTCCTGGCC CTCCGGGACT GGAGGGACTT CAGGGACAAG CCGTCGCTGG GGGACTCGGT CTTAAAGCTG GACGAATTTG AACGACCGCT
TM Domain WN E (split)
FMDV2A prE/NSl Sig
• V E S N P G P A R D R S I A L T F L A V G G V L L F L S V N V H A 2001 CGTTGAGTCA AATCCGGGCC CTGCCCGGGA CAGGTCCATA GCTCTCACGT TTCTCGCAGT TGGAGGAGTT CTGCTCTTCC TCTCCGTGAA CGTGCACGCT GCAACTCAGT TTAGGCCCGG GACGGGCCCT GTCCAGGTAT CGAGAGTGCA AAGAGCGTCA ACCTCCTCAA GACGAGAAGG AGAGGCACTT GCACGTGCGA
NSl
D T G C A I D I S R Q E L R C G S G V F I H N D V E A M D R Y K Y
2101 GACACTGGGT GTGCCATAGA CATCAGCCGG CAAGAGCTGA GATGTGGAAG TGGAGTGTTC ATACACAATG ATGTGGAGGC TTGGATGGAC CGGTACAAGT CTGTGACCCA CACGGTATCT GTAGTCGGCC GTTCTCGACT CTACACCTTC ACCTCACAAG TATGTGTTAC TACACCTCCG AACCTACCTG GCCATGTTCA
NSl
■ Y P E T P Q G L A K I I Q K A H K E G V C G L R S V S R L E H Q M 2201 ATTACCCTGA AACGCCACAA GGCCTAGCCA AGATCATTCA GAAAGCTCAT AAGGAAGGAG TGTGCGGTCT ACGATCAGTT TCCAGACTGG AGCATCAAAT TAATGGGACT TTGCGGTGTT CCGGATCGGT TCTAGTAAGT CTTTCGAGTA TTCCTTCCTC ACACGCCAGA TGCTAGTCAA AGGTCTGACC TCGTAGTTTA
KSl
• W E A V K D E L N T L L K
GTGGGAAGCA GTGAAGGACG AGCTGAACAC TCTTTTGAAG CACCCTTCGT CACTTCCTGC TCGACTTGTG AGAAAACTTC
1. PIV-WN (Apr E)-HIV Env Gp140
Figure imgf000161_0001
RV230 ZM96 EnvGP140
2. Sequence of PIV-WN (AprME)-HIV Env Gp140 (partial).
5' OTR
M S ·
1 AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT
TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
C
• K K P G G P G K S R A V Y L L K R G P R V L S L I G L K R A M L
101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGA GGGCTATGTT
GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCT CCCGATACAA
C
' S L I D G K G P I R F V L A L L A F F R F T A I A P T R A V L D R
201 GAGCCTGATC GACGGCAAGG GGCCAATACG ATTTGTGTTG GCTCTCTTGG CGTTCTTCAG GTTCACAGCA ATTGCTCCGA CCCGAGCAGT GCTGGATCGA
CTCGGACTAG CTGCCGTTCC CCGGTTATGC TAAACACAAC CGAGAGAACC GCAAGAAGTC CAAGTGTCGT TAACGAGGCT GGGCTCGTCA CGACCTAGCT
C
NS3 Cleavage
W R G V N K Q T A M K H L L S F K K E L G T L T S A I N R R S S K Q - 301 TGGAGAGGTG TGAACAAACA AACAGCGATG AAACACCTTC TGAGTTTCAA GAAGGAACTA GGGACCTTGA CCAGTGCTAT CAATCGGCGG AGCTCAAAGC
ACCTCTCCAC ACTTGTTTGT TTGTCGCTAC TTTGTGGAAG ACTCAAAGTT CTTCCTTGAT CCCTGGAACT GGTCACGATA GTTAGCCGCC TCGAGTTTCG
Partial C Signal
NS3 Cleavage gpl40
• K R G G K T G I A V I M G V R E I L R N Q R W W T W G I L G F
401 AGAAAAAGCG GGGCGGAAAG ACAGGTATTG CTGTGATCAT GGGAGTGCGG GAGATCCTGC GGAACTGGCA GCGGTGGTGG ACCTGGGGCA TCCTGGGCTT
TCTTTTTCGC CCCGCCTTTC TGTCCATAAC GACACTAGTA CCCTCACGCC CTCTAGGACG CCTTGACCGT CGCCACCACC TGGACCCCGT AGGACCCGAA
gpl40
• W M L M I C N V W G N L W V T V Y Y G V P V W K E A K T T L F C A
501 TTGGATGCTG ATGATCTGCA ACGTGTGGGG CAACCTGTGG GTGACCGTGT ACTACGGCGT GCCCGTGTGG AAAGAGGCCA AGACCACCCT GTTCTGCGCC
AACCTACGAC TACTAGACGT TGCACACCCC GTTGGACACC CACTGGCACA TGATGCCGCA CGGGCACACC TTTCTCCGGT TCTGGTGGGA CAAGACGCGG
gp!40
S D A K S Y E K E V H N V W A T H A C V P T D P N P Q E I V L G K V
601 AGCGACGCCA AGAGCTACGA GAAGGAAGTG CACAATGTGT GGGCCACCCA CGCCTGCGTG CCCACCGACC CCAACCCCCA GGAAATCGTC CTGGGCAACG TCGCTGCGGT TCTCGATGCT CTTCCTTCAC GTGTTACACA CCCGGTGGGT GCGGACGCAC GGGTGGCTGG GGTTGGGGGT CCTTTAGCAG GACCCGTTGC
gpl40
■ T E N F N M W K N D M V D Q M H E D I I S L W D Q S L K P C V K L 701 TGACCGAGAA CTTCAACATG TGGAAGAACG ACATGGTGGA CCAGATGCAC GAGGACATCA TCAGCCTGTG GGACCAGAGC CTGAAGCCCT GCGTGAAGCT ACTGGCTCTT GAAGTTGTAC ACCTTCTTGC TGTACCACCT GGTCTACGTG CTCCTGTAGT AGTCGGACAC CCTGGTCTCG GACTTCGGGA CGCACTTCGA
gpl40
■ T P L C V T L N C T E V N V T R N V N N S V V N N T T N V N Ν Ξ Μ 801 GACCCCCCTG TGCGTGACCC TGAACTGCAC CGAAGTGAAC GTGACCCGGA ACGTGAACAA CAGCGTGGTG AACAACACCA CCAACGTGAA TAACTCCATG CTGGGGGGAC ACGCACTGGG ACTTGACGTG GCTTCACTTG CACTGGGCCT TGCACTTGTT GTCGCACCAC TTGTTGTGGT GGTTGCACTT ATTGAGGTAC
gpl40
N G D M K N C S F N I T T E L K D K K K N V Y A L F Y K L D I V S L
901 AACGGCGACA TGAAGAACTG CAGCTTCAAC ATCACCACCG AGCTGAAGGA CAAGAAAAAG AACGTGTACG CCCTGTTCTA CAAGCTGGAC ATCGTGTCCC TTGCCGCTGT ACTTCTTGAC GTCGAAGTTG TAGTGGTGGC TCGACTTCCT GTTCTTTTTC TTGCACATGC GGGACAAGAT GTTCGACCTG TAGCACAGGG
gpl40
• N E T D D S E T G N S S K Y Y R L I N C N T S A L T Q A C P K V S 1001 TGAACGAGAC AGACGACAGC GAGACAGGCA ACAGCAGCAA GTACTACCGG CTGATCAACT GCAACACCAG CGCCCTGACC CAGGCCTGCC CCAAGGTGTC ACTTGCTCTG TCTGCTGTCG CTCTGTCCGT TGTCGTCGTT CATGATGGCC GACTAGTTGA CGTTGTGGTC GCGGGACTGG GTCCGGACGG GGTTCCACAG
gpl40
• F D P I P I H Y C A P A G Y A I L K C N N K T F N G T G P C H N V 1101 CTTCGACCCC ATCCCCATCC ACTACTGCGC CCCTGCCGGC TACGCCATCC TGAAGTGCAA CAACAAGACC TTCAACGGCA CCGGCCCCTG CCACAACGTG GAAGCTGGGG TAGGGGTAGG TGATGACGCG GGGACGGCCG ATGCGGTAGG ACTTCACGTT GTTGTTCTGG AAGTTGCCGT GGCCGGGGAC GGTGTTGCAC
gpl40
S T V Q C T H G I K P V V S T Q L L L N G S L A E E G I I I R S E N
1201 TCCACCGTGC AGTGCACCCA CGGCATCAAG CCCGTGGTGT CCACCCAGCT GCTGCTGAAC GGCAGCCTGG CCGAGGAAGG CATCATCATC AGAAGCGAGA AGGTGGCACG TCACGTGGGT GCCGTAGTTC GGGCACCACA GGTGGGTCGA CGACGACTTG CCGTCGGACC GGCTCCTTCC GTAGTAGTAG TCTTCGCTCT
gpl 0
■ L T N N V K T I I V H L N R S I E I V C V R P N N N . T R Q S I R I
1301 ACCTGACCAA CAACGTGAAA ACCATCATCG TGCACCTGAA CAGATCCATC GAGATCGTGT GCGTGCGGCC CAACAACAAC ACCCGGCAGA GCATCCGGAT
TGGACTGGTT GTTGCACTTT TGGTAGTAGC ACGTGGACTT GTCTAGGTAG CTCTAGCACA CGCACGCCGG GTTGTTGTTG TGGGCCGTCT CGTAGGCCTA
gpl40
• G P G Q T F Y A T G D I I G D I R Q A H C N I S R T H W T K T L R
1401 CGGCCCTGGC CAGACCTTTT ACGCCACCGG CGACATCATC GGCGACATCA GACAGGCCCA CTGCAACATC AGCCGGACCA ACTGGACCAA GACCCTGCGG
GCCGGGACCG GTCTGGAAAA TGCGGTGGCC GCTGTAGTAG CCGCTGTAGT CTGTCCGGGT GACGTTGTAG TCGGCCTGGT TGACCTGGTT CTGGGACGCC
gpl40
E V R N K L R E H F P N N I T F K P S S G G D L E I T T H S F N C
1501 GAAGTGCGGA ACAAGCTGCG GGAGCACTTC CCCAACAAGA ACATCACCTT CAAGCCCAGC TCTGGCGGCG ACCTGGAAAT CACCACCCAC AGCTTCAACT CTTCACGCCT TGTTCGACGC CCTCGTGAAG GGGTTGTTCT TGTAGTGGAA GTTCGGGTCG AGACCGCCGC TGGACCTTTA GTGGTGGGTG TCGAAGTTGA
gpl40
• R G E F F Y C U T S G L F S I N Y T E N N T D G T P I T L P C R I
1601 GCAGGGGCGA GTTCTTCTAC TGCAATACCT CCGGCCTGTT CAGCATCAAC TACACCGAGA ACAACACCGA CGGCACCCCC ATCACCCTGC CCTGCAGAAT
CGTCCCCGCT CAAGAAGATG ACGTTATGGA GGCCGGACAA GTCGTAGTTG ATGTGGCTCT TGTTGTGGCT GCCGTGGGGG TAGTGGGACG GGACGTCTTA
gpl40
• R Q I I N M H Q E V G R A M Y A P P I E G N I A C K S D I T G L L 1701 CCGGCAGATC ATCAATATGT GGCAGGAGGT GGGCAGGGCC ATGTACGCCC CTCCCATCGA GGGCAATATC GCCTGCAAGA GCGACATCAC CGGCCTGCTG GGCCGTCTAG TAGTTATACA CCGTCCTCCA CCCGTCCCGG TACATGCGGG GAGGGTAGCT CCCGTTATAG CGGACGTTCT CGCTGTAGTG GCCGGACGAC
gpl40
L V R D G G S T N D S T N N N T E I F R P A G G D M R D N W R S E L
1801 CTGGTGCGGG ACGGCGGCAG CACCAACGAC AGCACCAACA ACAATACCGA GATCTTCCGG CCTGCCGGCG GAGACATGCG GGACAACTGG CGGAGCGAGC GACCACGCCC TGCCGCCGTC GTGGTTGCTG TCGTGGTTGT TGTTATGGCT CTAGAAGGCC GGACGGCCGC CTCTGTACGC CCTGTTGACC GCCTCGCTCG
gp!40
■ Y K Y K V V E I K P L G I A P T E A R R V V E R E K S A V G I G 1901 TGTACAAGTA CAAGGTGGTG GAGATCAAGC CTCTGGGCAT TGCTCCCACC GAGGCCAAGC GGCGGGTGGT GGAGCGGGAG AAGAGCGCCG TGGGCATCGG ACATGTTCAT GTTCCACCAC CTCTAGTTCG GAGACCCGTA ACGAGGGTGG CTCCGGTTCG CCGCCCACCA CCTCGCCCTC TTCTCGCGGC ACCCGTAGCC
gpl 0
■ A V F L G F L G A A G S T M G A A S I T L T A Q A R Q V L S G I V 2001 CGCCGTGTTT CTGGGCTTCC TGGGAGCCGC CGGAAGCACA ATGGGAGCCG CCAGCATCAC CCTGACCGCC CAGGCCCGGC AGGTGCTGTC CGGCATCGTG GCGGCACAAA GACCCGAAGG ACCCTCGGCG GCCTTCGTGT TACCCTCGGC GGTCGTAGTG GGACTGGCGG GTCCGGGCCG TCCACGACAG GCCGTAGCAC
gpl40
Q Q Q S N L L R A I E A Q Q H L L Q L T V K G I K Q L Q T R V L A I
2101 CAGCAGCAGA GCAACCTGCT GAGAGCCATC GAGGCTCAGC AGCACCTGCT GCAGCTGACA GTGTGGGGCA TCAAGCAGCT GCAGACCCGG GTGCTGGCCA GTCGTCGTCT CGTTGGACGA CTCTCGGTAG CTCCGAGTCG TCGTGGACGA CGTCGACTGT CACACCCCGT AGTTCGTCGA CGTCTGGGCC CACGACCGGT
gpl40
■ E R Y L D Q Q L L G L G C S G L I C T T A V P W N I S W S H
2201 TCGAGAGATA CCTGAAGGAT CAGCAGCTCC TGGGCCTGTG GGGCTGCAGC GGCAAGCTGA TCTGCACCAC CGCCGTGCCC TGGAACATCA GCTGGTCCAA
AGCTCTCTAT GGACTTCCTA GTCGTCGAGG ACCCGGACAC CCCGACGTCG CCGTTCGACT AGACGTGGTG GCGGCACGGG ACCTTGTAGT CGACCAGGTT
gpl 0
■ K S K T D I W D N M T M Q W D R E I S N Y T N T I Y R L L E D S
2301 CAAGAGCAAG ACCGACATCT GGGACAACAT GACCTGGATG CAGTGGGACC GGGAGATCAG CAACTACACC AACACCATCT ACCGGCTGCT GGAAGATAGC
GTTCTCGTTC TGGCTGTAGA CCCTGTTGTA CTGGACCTAC GTCACCCTGG CCCTCTAGTC GTTGATGTGG TTGTGGTAGA TGGCCGACGA CCTTCTATCG
gpl40
FMDV2A
Q S Q Q E Q H E K D L L A L D S W N N N F D L L K L A G D V E S N P
2401 CAGAGCCAGC AGGAACAGAA CGAGAAGGAC CTGCTGGCCC TGGACAGCTG GAACAACAAT TTCGACCTGC TTAAACTTGC TGGCGACGTT GAGTCAAATC GTCTCGGTCG TCCTTGTCTT GCTCTTCCTG GACGACCGGG ACCTGTCGAC CTTGTTGTTA AAGCTGGACG AATTTGAACG ACCGCTGCAA CTCAGTTTAG
TM Domain WN E (split)
FMDV2A prE/NSl Sig NS1
• G P A R D R S I A L T F L A V G G V L L F L S V N V H A D T G C A
2501 CGGGCCCTGC CCGGGACAGG TCCATAGCTC TCACGTTTCT CGCAGTTGGA GGAGTTCTGC TCTTCCTCTC CGTGAACGTG CACGCTGACA CTGGGTGTGC GCCCGGGACG GGCCCTGTCC AGGTATCGAG AGTGCAAAGA GCGTCAACCT CCTCAAGACG AGAAGGAGAG GCACTTGCAC GTGCGACTGT GACCCACACG
NS1
• I D I S R Q E L R C G S G V F I H N D V E A W M D R Y K Y Y P E T
2601 CATAGACATC AGCCGGCAAG AGCTGAGATG TGGAAGTGGA GTGTTCATAC ACAATGATGT GGAGGCTTGG ATGGACCGGT ACAAGTATTA CCCTGAAACG GTATCTGTAG TCGGCCGTTC TCGACTCTAC ACCTTCACCT CACAAGTATG TGTTACTACA CCTCCGAACC TACCTGGCCA TGTTCATAAT GGGACTTTGC
NS1
P Q G L A K I I Q K A H K E G V C G L R S V S R L E H Q M W E A V K
2701 CCACAAGGCC TAGCCAAGAT CATTCAGAAA GCTCATAAGG AAGGAGTGTG CGGTCTACGA TCAGTTTCCA GACTGGAGCA TCAAATGTGG GAAGCAGTGA
GGTGTTCCGG ATCGGTTCTA GTAAGTCTTT CGAGTATTCC TTCCTCACAC GCCAGATGCT AGTCAAAGGT CTGACCTCGT AGTTTACACC CTTCGTCACT
NS1
• D E L N T L L K
2801 AGGACGAGCT GAACACTCTT TTGAAG
TCCTGCTCGA CTTGTGAGAA AACTTC
Appendix 8
Construct 1
1. PIV-WN (AprME)-HA New Caledonia
Figure imgf000165_0001
RV230 HA New Cal Sequence
2. Sequence of PIV-WN (AprME)-HA New Caledonia (partial).
UTR
M S
AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
C
• K K P G G P G K S R A V Y L L K R G P R V L S L I G L K R A M L " 101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGA GAGCCATGCT
GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCT CTCGGTACGA
■ S L I D G K G P I R F V L A L L A F F R F T A I A P T R A V L D R
201 TTCGCTCATT GACGGAAAGG GACCCATCCG ATTCGTACTG GCGTTGCTCG CATTCTTCCG GTTTACGGCT ATTGCGCCCA CGAGAGCGGT ACTCGACAGG
AAGCGAGTAA CTGCCTTTCC CTGGGTAGGC TAAGCATGAC CGCAACGAGC GTAAGAAGGC CAAATGCCGA TAACGCGGGT GCTCTCGCCA TGAGCTGTCC
NS3 Cleavage
W R G V N K Q T A M H L L S F K E L G T L T S A I N R R S S Q
301 TGGCGCGGAG TAAACAAGCA AACAGCCATG AAACACTTGT TGTCGTTCAA AAAGGAACTC GGGACCTTGA CCTCCGCCAT CAACCGACGG AGCTCAAAAC
ACCGCGCCTC ATTTGTTCGT TTGTCGGTAC TTTGTGAACA ACAGCAAGTT TTTCCTTGAG CCCTGGAACT GGAGGCGGTA GTTGGCTGCC TCGAGTTTTG Partial C signal New Cal HA
NS3 Cleavage HA signal
• K K R G G K T G I A V I K A K L L V L L C T F T A T Y A D T I C I 401 AGAAGAAGAG GGGAGGAAAG ACGGGAATCG CAGTCATTAA GGCAAAACTG TTGGTGTTGC TCTGTACATT CACTGCGACG TACGCGGATA CAATCTGTAT TCTTCTTCTC CCCTCCTTTC TGCCCTTAGC GTCAGTAATT CCGTTTTGAC AACCACAACG AGACATGTAA GTGACGCTGC ATGCGCCTAT GTTAGACATA
New Cal HA
- G Y H A N N S T D T V D T V L E K N V T V T H S V N L L E D S H N 501 CGGGTACCAT GCCAACAATT CGACCGACAC CGTGGATACC GTCTTGGAAA AGAATGTCAC AGTGACTCAT TCGGTAAACC TCCTTGAGGA TTCGCATAAC GCCCATGGTA CGGTTGTTAA GCTGGCTGTG GCACCTATGG CAGAACCTTT TCTTACAGTG TCACTGAGTA AGCCATTTGG AGGAACTCCT AAGCGTATTG
New Cal HA
G K L C L L K G I A P L Q L G N C S V A G W I L G N P E C E L L I S
601 GGGAAGTTGT GCCTTCTTAA AGGGATCGCA CCGCTGCAAC TGGGTAACTG TTCGGTCGCC GGCTGGATTC TCGGAAACCC CGAGTGTGAA CTGCTTATCT CCCTTCAACA CGGAAGAATT TCCCTAGCGT GGCGACGTTG ACCCATTGAC AAGCCAGCGG CCGACCTAAG AGCCTTTGGG GCTCACACTT GACGAATAGA
New Cal HA
- E S S Y I V E T P N P E N G T C Y P G Y F A D Y E E L R E Q L 701 CAAAGGAATC GTGGTCCTAT ATCGTGGAGA CACCGAACCC GGAGAATGGG ACGTGCTACC CTGGTTATTT CGCAGACTAC GAAGAACTTC GAGAACAACT GTTTCCTTAG CACCAGGATA TAGCACCTCT GTGGCTTGGG CCTCTTACCC TGCACGATGG GACCAATAAA GCGTCTGATG CTTCTTGAAG CTCTTGTTGA
New Cal HA
■ S S V S S F E R F E I F P K E S S W P N H T V T G V S A S C S H N 801 GTCCTCCGTC AGCTCGTTCG AGCGATTCGA AATCTTTCCG AAAGAGTCAT CGTGGCCGAA TCACACGGTA ACGGGTGTGT CCGCGTCATG TAGCCATAAT CAGGAGGCAG TCGAGCAAGC TCGCTAAGCT TTAGAAAGGC TTTCTCAGTA GCACCGGCTT AGTGTGCCAT TGCCCACACA GGCGCAGTAC ATCGGTATTA
New Cal HA
G K S S F Y R N L L W L T G K N G L Y P N L S K S Y V N N K E K E V
901 GGGAAGTCCT CGTTCTATCG CAACCTGTTG TGGCTTACTG GGAAAAACGG GTTGTACCCT AATCTCAGCA AGAGCTACGT CAATAACAAA GAAAAAGAGG CCCTTCAGGA GCAAGATAGC GTTGGACAAC ACCGAATGAC CCTTTTTGCC CAACATGGGA TTAGAGTCGT TCTCGATGCA GTTATTGTTT CTTTTTCTCC
New Cal HA
• L V L G V H H P P N I G N Q R A L Y H T E N A Y V S V V S S H Y 1001 TGCTGGTCTT GTGGGGTGTG CATCACCCAC CTAACATTGG GAATCAGAGG GCACTGTACC ACACTGAGAA TGCATACGTG AGCGTGGTGT CGAGCCACTA ACGACCAGAA CACCCCACAC GTAGTGGGTG GATTGTAACC CTTAGTCTCC CGTGACATGG TGTGACTCTT ACGTATGCAC TCGCACCACA GCTCGGTGAT
New Cal HA
• S R R F T P E I A K R P K V R D Q E G R I N Y Y W T L L E P G D T 1101 TAGCCGGAGA TTCACACCAG AGATTGCGAA GCGGCCCAAA GTCCGCGACC AGGAGGGGCG GATTAACTAC TACTGGACCC TCCTCGAGCC TGGCGATACG ATCGGCCTCT AAGTGTGGTC TCTAACGCTT CGCCGGGTTT CAGGCGCTGG TCCTCCCCGC CTAATTGATG ATGACCTGGG AGGAGCTCGG ACCGCTATGC
New Cal HA
I I F E A N G N L I A P Y A F A L S R G F G S G i l T S N A P M D 1201 ATCATCTTTG AAGCGAATGG TAATCTTATC GCCCCGTGGT ATGCTTTTGC GCTTTCAAGA GGATTTGGAT CAGGGATCAT CACATCAAAT GCGCCGATGG TAGTAGAAAC TTCGCTTACC ATTAGAATAG CGGGGCACCA TACGAAAACG CGAAAGTTCT CCTAAACCTA GTCCCTAGTA GTGTAGTTTA CGCGGCTACC
New Cal HA
• E C D A K C Q T P Q G A I N S S L P F Q N V H P V T I G E C P K Y 1301 ACGAGTGCGA TGCTAAGTGT CAGACTCCCC AAGGCGCTAT CAACTCGTCG TTGCCCTTTC AAAACGTGCA CCCCGTAACG ATCGGAGAGT GTCCCAAGTA
TGCTCACGCT ACGATTCACA GTCTGAGGGG TTCCGCGATA GTTGAGCAGC AACGGGAAAG TTTTGCACGT GGGGCATTGC TAGCCTCTCA CAGGGTTCAT
New Cal HA
■ V R S A K L R M V T G L R N I P S I Q S R G L F G A I A G F I E G 1401 TGTCAGATCG GCGAAACTTA GGATGGTGAC CGGACTCCGC AATATCCCCT CGATCCAGTC ACGGGGATTG TTTGGAGCCA TTGCGGGCTT CATCGAAGGG
ACAGTCTAGC CGCTTTGAAT CCTACCACTG GCCTGAGGCG TTATAGGGGA GCTAGGTCAG TGCCCCTAAC AAACCTCGGT AACGCCCGAA GTAGCTTCCC
New Cal HA
G T G M V D G W Y G Y H H Q N E Q G S G Y A A D Q K S T Q N A I N
1501 GGCTGGACTG GAATGGTCGA TGGGTGGTAC GGTTATCACC ACCAGAATGA GCAGGGTTCC GGGTATGCCG CGGATCAGAA ATCGACACAG AACGCAATCA
CCGACCTGAC CTTACCAGCT ACCCACCATG CCAATAGTGG TGGTCTTACT CGTCCCAAGG CCCATACGGC GCCTAGTCTT TAGCTGTGTC TTGCGTTAGT
New Cal HA
■ G I T N K V N S V I E K M N T Q F T A V G E F N K L E R R M E N 1601 ACGGGATTAC GAACAAGGTA AACAGCGTCA TTGAGAAGAT GAATACACAG TTTACAGCCG TGGGGAAAGA ATTCAACAAA CTCGAGCGCC GGATGGAGAA
TGCCCTAATG CTTGTTCCAT TTGTCGCAGT AACTCTTCTA CTTATGTGTC AAATGTCGGC ACCCCTTTCT TAAGTTGTTT GAGCTCGCGG CCTACCTCTT
New Cal HA
■ L N K V D D G F L D I W T Y N A E L L V L L E N E R T L D F H D
1701 TTTGAATAAG AAAGTGGACG ATGGTTTCCT CGATATCTGG ACGTACAATG CGGAGCTGCT TGTCCTGCTC GAAAATGAGA GGACGCTCGA CTTTCATGAC
AAACTTATTC TTTCACCTGC TACCAAAGGA GCTATAGACC TGCATGTTAC GCCTCGACGA ACAGGACGAG CTTTTACTCT CCTGCGAGCT GAAAGTACTG
New Cal HA
S N V K N L Y E K V K S Q L K N N A K E I G N G C F E F Y H K C N N
1801 TCCAATGTGA AGAACCTTTA CGAGAAGGTG AAGTCCCAAT TGAAGAATAA CGCCAAGGAA ATTGGAAACG GCTGCTTCGA ATTCTACCAC AAATGCAACA
AGGTTACACT TCTTGGAAAT GCTCTTCCAC TTCAGGGTTA ACTTCTTATT GCGGTTCCTT TAACCTTTGC CGACGAAGCT TAAGATGGTG TTTACGTTGT
New Cal HA
■ E C M E S V K N G T Y D Y P K Y S E E S K L N R E K I D G V K L E
1901 ATGAGTGCAT GGAATCGGTC AAAAATGGAA CATATGATTA TCCCAAATAC TCGGAGGAGT CAAAGCTTAA TAGGGAGAAA ATTGATGGGG TAAAACTTGA
TACTCACGTA CCTTAGCCAG TTTTTACCTT GTATACTAAT AGGGTTTATG AGCCTCCTCA GTTTCGAATT ATCCCTCTTT TAACTACCCC ATTTTGAACT
New Cal HA
• S M G V Y Q I L A I Y S T V A S S L V L L V S L G A I S F M C S
2001 GAGCATGGGT GTATATCAGA TCCTGGCAAT CTACTCAACC GTGGCGTCGT CACTGGTACT CCTCGTGTCC CTGGGCGCCA TTAGCTTTTG GATGTGTTCG
CTCGTACCCA CATATAGTCT AGGACCGTTA GATGAGTTGG CACCGCAGCA GTGACCATGA GGAGCACAGG GACCCGCGGT AATCGAAAAC CTACACAAGC
New Cal HA prE/NSl signal
FMD2A TM of WN E split
N G S L Q C R I C I N F D L L K L A G D V E S N P G P A R D R S I A - 2101 AATGGATCGC TCCAGTGCCG CATCTGCATC AACTTTGACC TGCTGAAGCT CGCGGGTGAC GTCGAATCCA ACCCAGGGCC AGCCCGGGAC AGAAGCATTG
TTACCTAGCG AGGTCACGGC GTAGACGTAG TTGAAACTGG ACGACTTCGA GCGCCCACTG CAGCTTAGGT TGGGTCCCGG TCGGGCCCTG TCTTCGTAAC
TM Of WN E split
~„—,~ ~~.—
NSl
~
• L T F L V G G V L L F L S V N V H A D T G C A I D I S R Q E L R -
2201 CGCTCACTTT TCTCGCGGTA GGAGGTGTGC TGTTGTTCCT GTCAGTGAAC GTCCACGCAG ACACGGGATG CGCGATTGAT ATCTCCAGAC AAGAATTGAG
GCGAGTGAAA AGAGCGCCAT CCTCCACACG ACAACAAGGA CAGTCACTTG CAGGTGCGTC TGTGCCCTAC GCGCTAACTA TAGAGGTCTG TTCTTAACTC
NSl
, , . ...
• C G S G V F I H N D V E A W M D R Y K Y Y P E T P Q G L A K I I Q
2301 GTGCGGGTCG GGGGTCTTTA TCCATAACGA CGTGGAGGCG TGGATGGACA GGTATAAGTA TTACCCTGAA ACGCCGCAGG GACTTGCGAA AATCATTCAG CACGCCCAGC CCCCAGAAAT AGGTATTGCT GCACCTCCGC ACCTACCTGT CCATATTCAT AATGGGACTT TGCGGCGTCC CTGAACGCTT TTAGTAAGTC
NS1
K A H K E G V C G L R S V S R L E H Q M W E A V K D E L N T L L K
2401 AAAGCCCATA AGGAAGGTGT GTGTGGATTG AGATCAGTCT CACGCCTTGA GCACCAGATG TGGGAGGCTG TCAAGGATGA ATTGAACACA CTTTTGAAGG TTTCGGGTAT TCCTTCCACA CACACCTAAC TCTAGTCAGA GTGCGGAACT CGTGGTCTAC ACCCTCCGAC AGTTCCTACT TAACTTGTGT GAAAACTTCC
Construct 2
1. PIV-WN (ACprME)-HA New Caledonia
Figure imgf000168_0001
DeleteC230 HA New Cal
2. Sequence of PIV-WN (ACprME)-HA New Caledonia (partial).
5' DTR
M S
1 AGTAGTTCGC CTGTGTGAGC TGACAAACTT AGTAGTGTTT GTGAGGATTA ACAACAATTA ACACAGTGCG AGCTGTTTCT TAGCACGAAG ATCTCGATGT TCATCAAGCG GACACACTCG ACTGTTTGAA TCATCACAAA CACTCCTAAT TGTTGTTAAT TGTGTCACGC TCGACAAAGA ATCGTGCTTC TAGAGCTACA
NS3 cleavage
c
• K K P G G P G K S R A V Y L L K R G M P R V L S L I G L K Q K K R 101 CTAAGAAACC AGGAGGGCCC GGCAAGAGCC GGGCTGTCTA TTTGCTAAAA CGCGGAATGC CCCGCGTGTT GTCCTTGATT GGACTTAAGC AGAAGAAGAG GATTCTTTGG TCCTCCCGGG CCGTTCTCGG CCCGACAGAT AAACGATTTT GCGCCTTACG GGGCGCACAA CAGGAACTAA CCTGAATTCG TCTTCTTCTC
Partial C signal New Cal HA
NS3 cleavage HA signal
- G G K T G I A V I K A K L L V L L C T F T A T Y A D T I C I G Y H 201 GGGAGGAAAG ACGGGAATCG CAGTCATTAA GGCAAAACTG TTGGTGTTGC TCTGTACATT CACTGCGACG TACGCGGATA CAATCTGTAT CGGGTACCAT CCCTCCTTTC TGCCCTTAGC GTCAGTAATT CCGTTTTGAC AACCACAACG AGACATGTAA GTGACGCTGC ATGCGCCTAT GTTAGACATA GCCCATGGTA
New Cal HA
A N N S T D T V D T V L E K N V T V T H S V N L L E D S H N G K L C
301 GCCAACAATT CGACCGACAC CGTGGATACC GTCTTGGAAA AGAATGTCAC AGTGACTCAT TCGGTAAACC TCCTTGAGGA TTCGCATAAC GGGAAGTTGT
CGGTTGTTAA GCTGGCTGTG GCACCTATGG CAGAACCTTT TCTTACAGTG TCACTGAGTA AGCCATTTGG AGGAACTCCT AAGCGTATTG CCCTTCAACA
New Cal HA
• L L K G I A P L Q L G N C S V A G W I L G N P E C E L L I S K E S
401 GCCTTCTTAA AGGGATCGCA CCGCTGCAAC TGGGTAACTG TTCGGTCGCC GGCTGGATTC TCGGAAACCC CGAGTGTGAA CTGCTTATCT CAAAGGAATC
CGGAAGAATT TCCCTAGCGT GGCGACGTTG ACCCATTGAC AAGCCAGCGG CCGACCTAAG AGCCTTTGGG GCTCACACTT GACGAATAGA GTTTCCTTAG
New Cal HA
• W S Y I V E T P N P E N G T C Y P G Y F A D Y E E L R E Q L S S V 501 GTGGTCCTAT ATCGTGGAGA CACCGAACCC GGAGAATGGG ACGTGCTACC CTGGTTATTT CGCAGACTAC GAAGAACTTC GAGAACAACT GTCCTCCGTC CACCAGGATA TAGCACCTCT GTGGCTTGGG CCTCTTACCC TGCACGATGG GACCAATAAA GCGTCTGATG CTTCTTGAAG CTCTTGTTGA CAGGAGGCAG
New Cal HA
S S F E R F E I F P K E S S W P N H T V T G V S A S C S H N G S S
601 AGCTCGTTCG AGCGATTCGA AATCTTTCCG AAAGAGTCAT CGTGGCCGAA TCACACGGTA ACGGGTGTGT CCGCGTCATG TAGCCATAAT GGGAAGTCCT
TCGAGCAAGC TCGCTAAGCT TTAGAAAGGC TTTCTCAGTA GCACCGGCTT AGTGTGCCAT TGCCCACACA GGCGCAGTAC ATCGGTATTA CCCTTCAGGA
New Cal HA
■ F Y R N L L W L T G K N G L Y P N L S K S Y V N N K E K E V L V L 701 CGTTCTATCG CAACCTGTTG TGGCTTACTG GGAAAAACGG GTTGTACCCT AATCTCAGCA AGAGCTACGT CAATAACAAA GAAAAAGAGG TGCTGGTCTT GCAAGATAGC GTTGGACAAC ACCGAATGAC CCTTTTTGCC CAACATGGGA TTAGAGTCGT TCTCGATGCA GTTATTGTTT CTTTTTCTCC ACGACCAGAA
New Cal HA
• W G V H H P P N I G N Q R A L Y H T E N A Y V S V V S S H Y S R R 801 GTGGGGTGTG CATCACCCAC CTAACATTGG GAATCAGAGG GCACTGTACC ACACTGAGAA TGCATACGTG AGCGTGGTGT CGAGCCACTA TAGCCGGAGA CACCCCACAC GTAGTGGGTG GATTGTAACC CTTAGTCTCC CGTGACATGG TGTGACTCTT ACGTATGCAC TCGCACCACA GCTCGGTGAT ATCGGCCTCT
New Cal HA
F T P E I A K R P K V R D Q E G R I N Y Y W T L L E P G D T I I F E
901 TTCACACCAG AGATTGCGAA GCGGCCCAAA GTCCGCGACC AGGAGGGGCG GATTAACTAC TACTGGACCC TCCTCGAGCC TGGCGATACG ATCATCTTTG AAGTGTGGTC TCTAACGCTT CGCCGGGTTT CAGGCGCTGG TCCTCCCCGC CTAATTGATG ATGACCTGGG AGGAGCTCGG ACCGCTATGC TAGTAGAAAC
New Cal HA
- A N G N L I A P W Y A F A L S R G F G S G i l T S N A P M D E C D 1001 AAGCGAATGG TAATCTTATC GCCCCGTGGT ATGCTTTTGC GCTTTCAAGA GGATTTGGAT CAGGGATCAT CACATCAAAT GCGCCGATGG ACGAGTGCGA
TTCGCTTACC ATTAGAATAG CGGGGCACCA TACGAAAACG CGAAAGTTCT CCTAAACCTA GTCCCTAGTA GTGTAGTTTA CGCGGCTACC TGCTCACGCT
New Cal HA
- A K C Q T P Q G A I N S S L P F Q N V H P V T I G E C P K Y V R S
1101 TGCTAAGTGT CAGACTCCCC AAGGCGCTAT CAACTCGTCG TTGCCCTTTC AAAACGTGCA CCCCGTAACG ATCGGAGAGT GTCCCAAGTA TGTCAGATCG
ACGATTCACA GTCTGAGGGG TTCCGCGATA GTTGAGCAGC AACGGGAAAG TTTTGCACGT GGGGCATTGC TAGCCTCTCA CAGGGTTCAT ACAGTCTAGC
New Cal HA
A K L R M V T G L R N I P S I 'Q S R G L F G A I A G F I E G G W T G
1201 GCGAAACTTA GGATGGTGAC CGGACTCCGC AATATCCCCT CGATCCAGTC ACGGGGATTG TTTGGAGCCA TTGCGGGCTT CATCGAAGGG GGCTGGACTG
CGCTTTGAAT CCTACCACTG GCCTGAGGCG TTATAGGGGA GCTAGGTCAG TGCCCCTAAC AAACCTCGGT AACGCCCGAA GTAGCTTCCC CCGACCTGAC
New Cal HA
• M V D G W Y G Y H H Q U E Q G S G Y A A D Q K S T Q N A I N G I T 1301 GAATGGTCGA TGGGTGGTAC GGTTATCACC ACCAGAATGA GCAGGGTTCC GGGTATGCCG CGGATCAGAA ATCGACACAG AACGCAATCA ACGGGATTAC CTTACCAGCT ACCCACCATG CCAATAGTGG TGGTCTTACT CGTCCCAAGG CCCATACGGC GCCTAGTCTT TAGCTGTGTC TTGCGTTAGT TGCCCTAATG
New Cal HA
• N K V N S V I E K M N T Q F T A V G K E F N K L E R R M E N L N K 1 01 GAACAAGGTA AACAGCGTCA TTGAGAAGAT GAATACACAG TTTACAGCCG TGGGGAAAGA ATTCAACAAA CTCGAGCGCC GGATGGAGAA TTTGAATAAG CTTGTTCCAT TTGTCGCAGT AACTCTTCTA CTTATGTGTC AAATGTCGGC ACCCCTTTCT TAAGTTGTTT GAGCTCGCGG CCTACCTCTT AAACTTATTC
New Cal HA
K V D D G F L D I T Y N A E L L V L L E N E R T L D F H D S N V K
1501 AAAGTGGACG ATGGTTTCCT CGATATCTGG ACGTACAATG CGGAGCTGCT TGTCCTGCTC GAAAATGAGA GGACGCTCGA CTTTCATGAC TCCAATGTGA TTTCACCTGC TACCAAAGGA GCTATAGACC TGCATGTTAC GCCTCGACGA ACAGGACGAG CTTTTACTCT CCTGCGAGCT GAAAGTACTG AGGTTACACT
New Cal HA
• N L Y E K V K S Q L K N N A K E I G N G C F E F Y H K C N N E C M 1601 AGAACCTTTA CGAGAAGGTG AAGTCCCAAT TGAAGAATAA CGCCAAGGAA ATTGGAAACG GCTGCTTCGA ATTCTACCAC AAATGCAACA ATGAGTGCAT TCTTGGAAAT GCTCTTCCAC TTCAGGGTTA ACTTCTTATT GCGGTTCCTT TAACCTTTGC CGACGAAGCT TAAGATGGTG TTTACGTTGT TACTCACGTA
New Cal HA
■ E S V K N G T Y D Y P K Y S E E S K L N R E K I D G V K L E S M G 1701 GGAATCGGTC AAAAATGGAA CATATGATTA TCCCAAATAC TCGGAGGAGT CAAAGCTTAA TAGGGAGAAA ATTGATGGGG TAAAACTTGA GAGCATGGGT CCTTAGCCAG TTTTTACCTT GTATACTAAT AGGGTTTATG AGCCTCCTCA GTTTCGAATT ATCCCTCTTT TAACTACCCC ATTTTGAACT CTCGTACCCA
New Cal HA
V Y Q I L A I Y S T V A S S L V L L V S L G A I S F W M C S N G S L
1801 GTATATCAGA TCCTGGCAAT CTACTCAACC GTGGCGTCGT CACTGGTACT CCTCGTGTCC CTGGGCGCCA TTAGCTTTTG GATGTGTTCG AATGGATCGC CATATAGTCT AGGACCGTTA GATGAGTTGG CACCGCAGCA GTGACCATGA GGAGCACAGG GACCCGCGGT AATCGAAAAC CTACACAAGC TTACCTAGCG
FMDV2A TM Of WN E split
New Cal HA prE/NSl Signal
• Q C R I C I N F D L L K L A G D V E S N P G P A R D R S I A L T F 1901 TCCAGTGCCG CATCTGCATC AACTTTGACC TGCTGAAGCT CGCGGGTGAC GTCGAATCCA ACCCAGGGCC AGCCCGGGAC AGAAGCATTG CGCTCACTTT AGGTCACGGC GTAGACGTAG TTGAAACTGG ACGACTTCGA GCGCCCACTG CAGCTTAGGT TGGGTCCCGG TCGGGCCCTG TCTTCGTAAC GCGAGTGAAA
NSl
TM of WN E split
Figure imgf000171_0001
Other Embodiments
All publications, patent applications, and patents mentioned in this specification are incorporated herein by reference in their entirety as if each individual publication, patent application, or patent were specifically and individually indicated to be
incorporated by reference. In particular, US 2011/0135686, WO/2009/114207, U.S. Serial No. 13/364,187, filed February 1, 2012, U.S. Serial No. 12/922,513, filed
September 14, 2012, U.S. Serial No. 61/069,451, filed March 14, 2008, and 61/092,814, filed August 29, 2008, are incorporated herein by reference in their entirety for all they disclose.
Various modifications and variations of the described viruses, vectors,
compositions, and methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the fields of medicine, pharmacology, or related fields are intended to be within the scope of the invention. Use of singular forms herein, such as "a" and "the," does not exclude indication of the corresponding plural form, unless the context indicates to the contrary. Similarly, use of plural terms does not exclude indication of a corresponding singular form. Other embodiments are within the scope of the following claims.

Claims

What is claimed is: CLAIMS
1. A replication-deficient pseudoinfectious flaviviras comprising a flavivirus genome comprising (i) one or more deletions or mutations in nucleotide sequences encoding one or more proteins selected from the group consisting of capsid (C), pre- membrane (prM), envelope (E), non-structural protein 1 (NSl), non-structural protein 3 (NS3), and non-structural protein 5 (NS5), and (ii) a sequence encoding one or more heterologous pathogen, cancer, or allergy-related immunogens.
2. The replication-deficient pseudoinfectious flaviviras of claim 1, wherein said one or more deletions or mutations is within capsid (C) sequences of the flavivirus genome.
3. The replication-deficient pseudoinfectious flavivirus of claim 1, wherein said one or more deletions or mutations is within pre-membrane (prM) and/or envelope (E) sequences of the flavivirus genome.
4. The replication-deficient pseudoinfectious flavivirus of claim 1 , wherein said one or more deletions or mutations is within capsid (C), pre-membrane (prM), and envelope (E) sequences of the flavivirus genome.
5. The replication-deficient pseudoinfectious flavivirus of claim 1, wherein said one or more deletions or mutations is within non-structural protein 1 (NSl) sequences of the flavivirus genome.
6. The replication-deficient pseudoinfectious flavivirus of any of claims 1-5, wherein said heterologous immunogen is from a pathogen selected from the group consisting of a human immunodeficiency virus, a simian immunodeficiency virus, a rabies virus, Borrelia burgdorferi, a tick, an influenza virus, a human papilloma virus, a respiratory syncytial virus, malaria parasite, and Mycobacterium tuberculosis.
7. The replication-deficient pseudoinfectious flavivirus of claim 6, wherein said heterologous immunogen comprises an optionally codon-optimized HIV or SIV gag, tat/nef, pol, pro, gp41, gpl20, gpl40, gpl45, or gpl60 protein, or an immunogenic fragment thereof, or a combination of 2 or more thereof.
8. The replication-deficient pseudoinfectious flavivirus of claim 6, wherein said heterologous immunogen comprises Borrelia burgdorferi OspA immunogen or an immunogenic fragment thereof.
9. The replication-deficient pseudoinfectious flavivirus of claim 6, wherein said heterologous immunogen comprises a tick saliva protein selected from the group consisting of 64TRP, Isac, and Salp20, or an immunogenic fragment thereof.
10. The replication-deficient pseudoinfectious flavivirus of claim 6, wherein said heterologous immunogen comprises an influenza virus M2, hemaglutinnin (HA), or neuraminidase (NA) epitope, or an immunogenic fragment thereof.
11. The replication-deficient pseudoinfectious flavivirus of claim 6, wherein said heterologous immunogen comprises a rabies virus G protein immunogen or an immunogenic fragment thereof.
12. The replication-deficient pseudoinfectious flavivirus of claim 6, wherein said heterologous immunogen comprises an HPV16 or HPV18 capsid protein LI or L2, or an immunogenic fragment thereof.
13. The replication-deficient pseudoinfectious flavivirus of claim 6, wherein said heterologous immunogen comprises a respiratory syncytial virus F or G glycoprotein.
14. The replication-deficient pseudoinfectious flavivirus of any of claims 1-13, wherein said flavivirus genome comprises sequences encoding a pre-membrane (prM) and/or envelope (E) protein.
15. The replication-deficient pseudoinfectious flavivirus of any of claims 1-14, wherein the flavivirus genome is selected from that of yellow fever virus, West Nile virus, tick-borne encephalitis virus, Langat virus, Japanese encephalitis virus, dengue virus, and St. Louis encephalitis virus sequences, and chimeras thereof.
16. The replication-deficient pseudoinfectious flavivirus of claim 15, wherein said chimera comprises pre-membrane (prM) and envelope (E) sequences of a first flavivirus, and capsid (C) and non-structural sequences of a second, different flavivirus.
17. The replication-deficient pseudoinfectious flavivirus of claim 16, wherein said first flavivirus is a tick-borne encephalitis virus or a Langat virus.
18. The replication-deficient pseudoinfectious flavivirus of claim 16 or 17, wherein said second, different flavivirus is a yellow fever virus or a West Nile virus or Langat virus.
19. The replication-deficient pseudoinfectious flavivirus of any of claims 1-18, wherein said genome is packaged in a particle comprising pre-membrane (prM) and envelope (E) sequences from a flavivirus that is the same or different from that of the genome.
20. The replication-deficient pseudoinfectious flavivirus of any of claims 1-19, wherein sequences encoding said heterologous immunogen are inserted in the place of or in combination with the one or more deletions or mutations of the one or more proteins.
21. The replication-deficient pseudoinfectious flavivirus of any of claims 1-20, wherein sequences encoding said heterologous immunogen are inserted in the flavivirus genome within sequences encoding the envelope (E) protein, within sequences encoding the non-structural 1 (NS1) protein, within sequences encoding the pre-membrane (prM) protein, intergenically between sequences encoding the envelope (E) protein and nonstructural protein 1 (NS1), intergenically between non-structural protein 2B (NS2B) and non-structural protein 3 (NS3), or as a bicistronic insertion in the 3' untranslated region of the flavivirus genome.
22. A composition comprising a first replication-deficient pseudoinfectious flavivirus of any of claims 1-21 and a second, different replication-deficient
pseudoinfectious flavivirus comprising a genome comprising one or more deletions or mutations in nucleotide sequences encoding one or more proteins selected from the group consisting of capsid (C), pre-membrane (prM), envelope (E), non-structural protein 1 (NS1), non-structural protein 3 (NS3), and non-structural protein 5 (NS5), wherein the one or more proteins encoded by the sequences in which the one or more deletion(s) or mutation(s) occur in the second, different replication-deficient pseudoinfectious flavivirus are different from the one or more proteins encoded by the sequences in which the one or more deletion(s) or mutation(s) occur in the first replication-deficient pseudoinfectious flavivirus.
23. A method of inducing an immune response to an immunogen in a subject, the method comprising administering to the subject one or more replication-deficient pseudoinfectious flaviviruses of any of claims 1-21 and/or a composition of claim 22 to the subject.
24. The method of claim 23, wherein the subject is at risk of but does not have an infection by said pathogen or a disease or condition associated with said cancer or allergy-related immunogen.
25. The method of claim 23, wherein the subject has an infection by said pathogen or a disease or condition associated with said cancer or allergy-related immunogen.
26. The method of any of claims 23-25, wherein the immunogen is from a pathogen selected from the group consisting of a rabies virus, Borrelia burgdorferi, a tick, an influenza virus, a human immunodeficiency virus, a simian immunodeficiency virus, a human papilloma virus, a respiratory syncytial virus, malaria parasite, and Mycobacterium tuberculosis.
27. The method of any of claims 23-26, wherein the method is for inducing an immune response against a protein encoded by the flavivirus genome, in addition to the source of the immunogen.
28. The method of claim 27, wherein the subject is at risk of but does not have an infection by the flavivirus corresponding to the genome of the pseudoinfectious flavivirus, which comprises sequences encoding a flavivirus pre-membrane and/or envelope protein.
29. The method of claim 27, wherein the subject has an infection by the flavivirus corresponding to the genome of the pseudoinfectious flavivirus which comprises sequences encoding a flavivirus pre-membrane and/or envelope protein.
30. A live, attenuated chimeric flavivirus comprising a yellow fever virus in which sequences encoding pre-membrane and envelope proteins are replaced with sequences encoding pre-membrane and envelope proteins of a tick-borne encephalitis virus or a Langat virus, and the signal sequence between the capsid and pre-membrane proteins of the chimeric flavivirus comprises a hybrid of yellow fever virus and tick- bome encephalitis or Langat virus capsid/pre-membrane signal sequences, or a variant thereof.
31. The live, attenuated chimeric flavivirus of claim 30, wherein the capsid/pre- membrane signal sequence of the chimeric flavivirus comprises yellow fever virus sequences in the amino terminal region and tick-borne encephalitis or Langat virus sequences in the carboxy terminal region.
32. A live, attenuated chimeric flavivirus comprising a West Nile virus in which sequences encoding pre-membrane and envelope proteins are replaced with sequences encoding pre-membrane and envelope proteins of a tick-borne encephalitis or a Langat virus, and the signal sequence between the capsid and pre-membrane proteins of the chimeric flavivirus comprises a tick-borne encephalitis or a Langat virus capsid/pre- membrane signal sequence, or a variant thereof.
33. A pharmaceutical composition comprising a pseudoinfectious flavivirus of any of claims 1-21, the composition of claim 22, or the live, attenuated flavivirus of any of claims 30-32 and a pharmaceutically acceptable carrier or diluent.
34. The pharmaceutical composition of claim 33, further comprising an adjuvant.
35. A replication-deficient pseudoinfectious flavivirus comprising a flavivirus genome comprising one or more deletions or mutations in nucleotide sequences encoding non-structural protein 1 (NSl), non-structural protein 3 (NS3), or non-structural protein 5 (NS5).
36. A nucleic acid molecule corresponding to the genome of a pseudoinfectious flavivirus of any of claims 1-21 or 35, or the genome of the live, attenuated flavivirus of any of claims 30-32.
37. A method of making a replication-deficient pseudoinfectious flavivirus of any of claims 1-21 or 35, the method comprising introducing a nucleic acid molecule of claim 36 into a cell that expresses the protein corresponding to any sequences deleted from the flavivirus genome of the replication-deficient pseudoinfectious flavivirus.
38. The method of claim 37, wherein the protein is expressed in the cell from the genome of a second, different, replication-deficient pseudoinfectious flavivirus.
39. The method of claim 37, wherein the protein is expressed from a replicon.
40. The method of claim 39, wherein the replicon is an alphavirus replicon.
41. The method of claim 40, wherein the alphavirus is a Venezuelan Equine Encephalitis virus.
42. A composition comprising two or more replication-deficient pseudoinfectious flaviviruses, wherein two of the replication-deficient pseudoinfectious flaviviruses are selected from the groups consisting of:
(a) a replication-deficient pseudoinfectious flavivirus comprising a genome comprising Japanese encephalitis virus sequences, and a replication-deficient
pseudoinfectious flavivirus comprising a genome comprising dengue virus sequences;
(b) a replication-deficient pseudoinfectious flavivirus comprising a genome comprising yellow fever virus sequences, and a replication-deficient pseudoinfectious flavivirus comprising a genome comprising dengue virus sequences; and
(c) a replication-deficient pseudoinfectious flavivirus comprising a genome comprising tick-borne encephalitis or Langat virus sequences and an inserted sequence encoding a Borrelia burgdorferi immunogen, and a replication-deficient pseudoinfectious flavivirus comprising a genome comprising tick-borne encephalitis or Langat virus sequences and an inserted sequence encoding a tick saliva protein immunogen, or a replication-deficient pseudoinfectious flavivirus comprising a genome comprising tick- borne encephalitis or Langat virus sequences and inserted sequences encoding a Borrelia burgdorferi immunogen and a tick saliva protein immunogen.
43. A pharmaceutical composition comprising the live, attenuated chimeric flavivirus of any of claims 30-32.
44. A method of inducing an immune response to tick-borne encephalitis virus or Langat virus in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 43.
45. The method of claim 44, wherein the subject does not have but is at risk of developing infection by tick-borne encephalitis virus or Langat virus.
46. The method of claim 44, wherein the subject is infected with tick-borne encephalitis virus or Langat virus.
47. A replication-deficient pseudoinfectious flavivirus comprising a flavivirus genome comprising one or more deletions or mutations in nucleotide sequences encoding one or more proteins selected from the group consisting of capsid (C), pre-membrane (prM), envelope (E), non-structural protein 1 (NS1), non-structural protein 3 (NS3), and non-structural protein 5 (NS5), wherein the flavivirus genome comprises yellow fever virus sequences in which sequences encoding pre-membrane and envelope proteins are replaced with sequences encoding pre-membrane and envelope proteins of a tick-borne encephalitis virus or a Langat virus, and sequences encoding the signal sequence between the capsid and pre-membrane proteins of the flavivirus genome comprise a hybrid of sequences encoding yellow fever virus and tick-borne encephalitis or Langat virus capsid/pre-membrane signal sequences, or a variant thereof.
48. The replication-deficient pseudoinfectious flavivirus of claim 47, wherein the sequences encoding the capsid/pre-membrane signal sequence of the flavivirus genome comprise yellow fever virus sequences in the 5' region and tick-borne encephalitis or Langat virus sequences in the 3 ' region.
49. A replication-deficient pseudoinfectious flavivirus comprising a flavivirus genome comprising one or more deletions or mutations in nucleotide sequences encoding one or more proteins selected from the group consisting of capsid (C), pre-membrane (prM), envelope (E), non-structural protein 1 (NS1), non-structural protein 3 (NS3), and non-structural protein 5 (NS5), wherein the flavivirus genome comprises West Nile virus sequences in which sequences encoding pre-membrane and envelope proteins are replaced with sequences encoding pre-membrane and envelope proteins of a tick-borne encephalitis or a Langat virus, and the sequences encoding the signal sequence between the capsid and pre-membrane proteins of the flavivirus genome comprise sequences encoding a tick-borne encephalitis or a Langat virus capsid/pre-membrane signal sequence, or a variant thereof.
50. A replication-deficient pseudoinfectious flavivirus comprising a flavivirus genome comprising one or more deletions or mutations in nucleotide sequences encoding one or more proteins selected from the group consisting of capsid (C), pre-membrane (prM), envelope (E), non-structural protein 1 (NS1), non-structural protein 3 (NS3), and non-structural protein 5 (NS5), wherein any capsid (C) and non-structural (NS) proteins in said flavivirus genome are from Langat virus and any pre-membrane (prM) and envelope (E) proteins are from a tick-borne encephalitis virus.
51. The replication-defective pseudoinfectious flavivirus of claim 6, comprising multiple heterologous immunogens.
52. The replication-defective pseudoinfectious flavivirus of claim 51, wherein said multiple heterologous immunogens are from a human immunodeficiency virus or a simian immunodeficiency virus.
53. The replication-defective pseudoinfectious flavivirus of claim 51 or 52, wherein said multiple immunogens comprise heterologous transmembrane and/or signal sequences.
54. The replication-defective pseudoinfectious flavivirus of claim 53, wherein said heterologous sequences are from rabies virus G protein.
PCT/US2013/024495 2012-02-01 2013-02-01 Replication-defective flavivirus vaccines and vaccine vectors WO2013116770A1 (en)

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