Cross-Reference to Related Applications

This application claims benefit of U S provisional application no 61/210,305, filed March 16, 2009, the contents of which are incorporated herein by reference

Field of the Invention

This invention relates to replication-defective flavivirus vaccine vectors against respiratory syncytial virus (RSV), 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 Flavivirus pathogens include yellow fever (YF), dengue types 1-4 (DEN 1-4), 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 chimeπc vaccines), JE (INV and LAV) to prevent encephalitis in horses and stillbirth in pigs in Asia, louping ill flavivirus (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)

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 m 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 ents of viral particles, and the NS proteins are non-structural proteins, which lved in intracellular virus replication Flavivirus replication occurs in the sm Upon infection of cells and translation of genomic RNA, processing of the polyprotem starts with translocation of the prM portion of the polyprotem into the lumen of endoplasmic reticulum (ER) of infected cells, followed by translocation of E and NSl portions, as directed by the hydrophobic signals for the prM, E, and NSl proteins Ammo-termini of prM, E, and NSl 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 earned out by the viral NS2B/NS3 seπne 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 protem is produced by cleavage of prM shortly pπor to virus release by cellular furm or a similar protease E, the major protein of the envelope, is the pπncipal 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 fiaviviruses discussed above, other vaccine platforms have been developed One example is based on chimeric fiaviviruses 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 pseudomfectious IV) technology (Mason et al , Virology 351 432-443, 2006, Shustov et al , J 1 11737-11748, 2007, Widman et al , Adv Virus Res 72 77-126, 2008, 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 pseudomfectious 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 cychzation 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-PIV 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) rephcon] 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 n the PIV, resulting in the induction of neutralizing antibody response A T- ponse should also be induced via MHCI presentation of viral epitopes This h has been applied to YF 17D virus and WN viruses and WN/JE and EN2 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 m 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 m vivo due to comfection 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 ΔC 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 ftaviviruses, viral sequences can be modified in both s-PFVs and d PIVs using, e g , synonymous codon replacements, to reduce nucleotide sequence homologies, and mutating the complementary cychzation 5' and 3' elements

Respiratory syncytial virus (RSV) is a negative-sense, single-stranded RNA virus of the family Paramyxovindae Its name is based on the activity of the RSV fusion or F glycoprotein, which is on the surface of the virus and causes cell anes of mfected cells to merge, resulting m the formation of syncytia RSV the respiratory tract, and is the major cause of lower respiratory tract infections ng pneumonia) and hospital visits during infancy and childhood For p e, in the United States, 60% of infants are infected during their first RSV season, and nearly all children will have been infected by 2-3 years of age (Glezen et al , Am J Dis Child 140(6) 543-546, 1986) Of those infected, 2-3% will develop bronchiolitis, or inflammation of the small airways in the lung, and require hospitalization (Hall et al , N Engl J Med 360(6) 588-598, 2009) Further, RSV infection is increasingly being found as an infection of the elderly Current treatment is generally focused on supportive care, including administration of fluids and oxygen

Summary of the Invention

The invention provides replication-deficient pseudoinfectious flaviviruses that each include a flavivirus genome including (i) one or more deletions or mutations m 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 (u) a sequence encoding a respiratory syncytial virus (RSV) peptide or protein, or a fragment or analog thereof As descπbed elsewhere herein, the vectors of the invention are replication deficient due to the one or more deletions or mutations, and can be complemented in trans (see below for details) Any of the deletions/mutations descπbed herein, as well as other deletions/mutations resulting in replication deficiency, can be used in the vectors of the invention

In one embodiment, the respiratory syncytial virus (RSV) protein is the RSV F protein, or a fragment or analog thereof In various examples, the RSV F protein lacks a trans-membrane domain, e g , it is truncated so that it is produced in secreted form In other examples, the respiratory syncytial virus (RSV) protein is the RSV G protein, or a fragment or analog thereof

In various embodiments, the one or more deletions or mutations is within capsid (C) sequences of the flavrvirus genome, is within pre-membrane (prM) and/or envelope (E) sequences of the flavivirus genome, is within capsid (C), pre-membrane (prM), and envelope (E) sequences of the flavivirus genome, and/or is within nonal protein 1 (NSl) sequences of the flavivirus genome In other examples, the us genome includes sequences encodmg a pre-membrane (prM) and/or e (E) protein

The flavivirus genome of the replication-deficient pseudoinfectious flaviviruses can be, for example, selected from that of yellow fever virus, West Nile virus, tick-borne encephalitis virus, Langat virus, Japanese encephalitis virus, dengue virus (1-4), and St Louis encephalitis virus sequences, and chimeras thereof (also see below) In certain examples, the chimeras include pre membrane (prM) and envelope (E) sequences of a first flavivirus, and capsid (C) and non-structural sequences of a second, different flavivirus In other examples, the genome is packaged in a particle including pre-membrane (prM) and envelope (E) sequences from a flavivirus that is the same or different from that of the genome Further, sequences encoding the RSV protein can be inserted in the place of or in combination with the one or more deletions or mutations of the one or more proteins

In certain examples, sequences encoding the respiratory syncytial virus peptide or protein, or a fragment or analog thereof, are inserted in the flavivirus genome within sequences encoding the envelope (E) protein, within sequences encoding the non-structural 1 (NSl) protein, within sequences encoding the pre-membrane (prM) protein, intergemcally between sequences encoding the envelope (E) protein and nonstructural protein 1 (NSl), intergemcally between non-structural protein 2B (NS2B) and non-structural protein 3 (NS3), or as a bicistromc insertion in the 3' untranslated region of the flavivirus genome

The invention also includes pharmaceutical compositions including one or more of the replication-deficient pseudoinfectious flaviviruses described above and elsewhere herein Compositions of the invention can also a pharmaceutically acceptable earner or diluent, and, optionally, an adjuvant

Other compositions of the invention include a first replication-deficient pseudomfectious flavivirus, such as one of those described above and elsewhere herein, and a second, different replication-deficient pseudomfectious flavivirus including a genome having one or more deletions or mutations in nucleotide sequences encoding one or more proteins selected from the group consisting of capsid -membrane (prM), envelope (E), non-structural protein 1 (NSl), non-structural 3 (NS3), and non structural protem 5 (NS ₅ ), wherein the one or more proteins d by the sequences in which the one or more deletion(s) or mutation(s) occur in the second, different replication-deficient pseudomfectious flavivirus are different from the one or more protems encoded by the sequences in which the one or more deletion(s) or mutation(s) occur in the first replication-deficient pseudomfectious flavivirus

The invention also provides methods of inducing an immune response to respiratory syncytial virus (RSV) in a subject, involving administering to the subject one or more replication-deficient pseudomfectious flaviviruses or a composition as described above and elsewhere herein The subject may be at risk of but not have an infection by respiratory syncytial virus (RSV), or the subject may have an infection by respiratory syncytial virus (RSV) In certain examples, the subject is an infant, young child, or elderly person The methods of the invention can be for inducing an immune response against a protein encoded by the flavivirus genome, in addition to RSV In such methods, the subject may be at risk of but does not have an infection by the flavivirus corresponding to the genome of the pseudomfectious flavivirus, which includes sequences encoding a flavivirus pre-membrane and/or envelope protein In another example, the subject has an infection by the flavivirus corresponding to the genome of the pseudomfectious flavivirus, which includes sequences encoding a flavivirus pre-membrane and/or envelope protein

Also included in the mvention are nucleic acid molecules corresponding to the genomes of pseudomfectious flaviviruses as described herein and complements thereof The invention also provides methods of making a replication-deficient pseudoinfectious flavivirus as descπbed herein These methods involve introducing a nucleic acid molecule as described above into a cell that expresses the protein corresponding to any sequences deleted from the flavivirus genome of the replication- deficient pseudoinfectious flavivirus The protein can be expressed in the cell from, for example, the genome of a second, different, replication-deficient pseudoinfectious flavivirus In various examples, the protein is expressed from a replicon (e g , an rus replicon, such as a Venezuelan Equme Encephalitis virus replicon)

By "replication-deficient pseudoinfectious flavivirus" or "PIV" is meant a us 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, NSl, 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 for use of the PIV as a vector to deliver a heterologous lmmunogen, 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-PIV, 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-PIV 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)

The invention provides several advantages For example, the PIV vectors and PrVs of the invention are highly attenuated and highly efficacious after one-to-two doses, providing durable immunity. Further, unlike inactivated vaccines, PIVs mimic irus infection, which can result m increased efficacy due to the induction of B- and T-cell responses, higher durability of immunity, and decreased dose ments In addition, similar to whole viruses, PIV vaccines target antigen- p ng cells, such as dendritic cells, stimulate toll-like receptors (TLRs), and mduce balanced Thl/Th2 immunity PIV constructs have also been shown to grow to high titers m 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 PFV vectors of the invention provide an option for developing vaccines against non-flavivirus pathogens, such as RSV, for which no vaccines are currently available

Other features and advantages of the mvention will be apparent from the following detailed description, the drawings, and the claims.

Brief Descnption of the Drawings

Fig 1 is a schematic illustration of single component PIV (s-PIV) technology Fig 2 is a schematic illustration of two-component PIV (d-PFV) technology Fig 3 is a schematic illustration of a general experimental design for testing immunogemcity 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 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), YFl 7D, PIV-WN (RV-WN), and YF/WN LAV

(CVWN) with hamster-adapted Asibi (PIV-YF and YF17D 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 chimeπc flaviviruses

Fig 7 is a table showing WN/TBE PIV titers and examples of immunofluorescence of cells containing the indicated PIVs

Fig 8 is a set of graphs showing the replication kinetics of YF/TBE LAV and PIV- WN/TBE in Vera and BHK cell lines (CV-Hypr = YF/Hypr LAV, CV-LGT = YF/LGT LAV, RV-WN/TBEV = PIV-WN/TBEV)

Fig 9 is a series of graphs showing survival of mice inoculated IC with PFV- d YF/TBE LAV constructs in a neurovirulence test (3 5 week old ICR mice, N/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 IP with PIV- WN/TBE(Hypr) (RV-WN/Hypr), YF/TBE(Hypr) LAV (CV-Hypr), and YF/LGT LAV (CV-LGT) constructs and YF17D m 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)

Fig 12 is a schematic representation of PIV constructs expressing rabies virus G protein, as well as illustration of packaging of the constructs to make pseudomfectious 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 (ΔC Rabies G, ΔPrM-E Rabies G, and ΔC-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 (ΔC-Rabies G, ΔPrM-E-Rabies G, and ΔC-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 set of immunofluorescence images showing expression of full- RSV F protein (strain A2) by the ΔprM-E component of d-PIV-WN in helper er transfection.

Fig. 20 is a schematic representation of wild-type RSV F and RSV trF.

Fig. 21 is a schematic representation of three PIV(WN)-RSVtrF (Al strain) constructs: ΔC-RSVtrF sPIV, ΔprME-RSVtrF dPIV helper, and ΔCprME-RSVtrF. Immunofluorescence of helper cells after transfection (Day 4) is also shown.

Fig. 22 is a series of images showing titration of WNΔC-RSV trF PIV in Vero cells visualized by immunostaining.

Fig. 23 is an image showing a Western blot analysis of two ΔprME-RSVtrF stocks, 2 days post infection.

Fig. 24 is an image showing a Western blot analysis of Vero cells infected with the indicated amounts of VP2400, vFP2403, and PIV-F.

Fig. 2 ₅ is a set of graphs showing endpoint titers obtained using the indicated constructs and routes of administration in two sets of experiments (RSVi27 and RSVi32) indicating the anti-RSV-F IgG antibody titres obtained by ELISA. "F" represents vector with the F insert (truncated), while "e" represents the empty vector alone. "FI_RSV" is a formalin inactivated RSV virus, while "RSV" is a live RSV virus preparation.

Fig. 26 is a set of graphs showing serum neutralization titers obtained using the indicated constructs and routes of administration in two sets of experiments (RSVi27 and RSVi32). "F" represents vector with the F insert (truncated), while "e" represents the empty vector alone. "FI_RSV" is a formalin inactivated RSV virus, while "RSV" is a live RSV virus preparation (see Fig. 25). 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 immunogens inserted into the vectors as well as, optionally, the vectors themselves The invention also mcludes compositions including combinations of PIVs and/or PIV vectors, as descπbed herein, and methods of using such compositions to induce immune responses against inserted immunogen es and/or sequences of the PIVs themselves The focus of the invention is tors containing respiratory syncytial virus (RSV) immunogens, such as F or G immunogens, in one embodiment (see, e g , truncated F protein, below) These vectors can be used in methods to prevent or treat RSV infection, and also m combination methods involving use of, for example, any of the other vectors described herein (such as vectors including immunogens of other pathogens and/or cancer, and/or allergy-related immunogens) The vectors, compositions, and methods of the invention are descπbed further below

PIV Vectors

The PrV vectors of the invention can be based on the smgle- or two- component PrVs descπbed 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 l and Fig 12) 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 cychzation (i e , the sequence encoding ammo 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-IO), 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 m such cells by use of a viral vector-derived rephcon, such as an alphavirus replicon (e g , a rephcon based on Venezuelan Equine Encephalitis virus (VEEV), Smdbis virus, Semliki Forest virus (SFV), Eastern Equine Encephalitis virus (EEEV), Western Equine Encephalitis virus (WEEV), or Ross River virus) To decrease the ity of productive recombination between the PIV vectors/PIVs and menting sequences, the sequences in the rephcons (encoding C, prM, and/or E) ude nucleotide mutations For example, sequences encoding a complementing C protein can include an unnatural cychzation 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 protem 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 descπbed 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 12) In another example, the first component includes a deletion of C, prM, and E, and the second component includes a deletion of NSl (Fig 12) Both components can include cis- acting promoter elements required for RNA replication and a complete set of nonstructural 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 amino terminal fragment of the capsid protein This sequence can be followed by a protease cleavage sequence such as, for example, a ubiqmtine or foot-and-mouth disease virus (FAMDV)-specific 2A 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 rephcons, as discussed above in reference ngle component PIV approach Exemplary cell lines that can be used in the mponent genome approach include Vero (e g , ATCC CCL-81), BHK-21 (e g , CCL-IO), C7/10, and other cells of vertebrate or mosquito origin

Additional examples of d-PFV approaches that can be used m the invention are based on use of complementing genomes including deletions in NS3 or NS ₅ sequences A deletion in, e g , NS 1 , NS3 , or NS5 proteins can be used as long as several hundred ammo acids in the ORF, removing the entire chosen protein sequence, or as short as 1 ammo acid inactivating protein enzymatic activity (e g , NS5 RNA polymerase activity, NS3 helicase activity, etc ) Alternatively, point ammo 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 ΔNS deletions can be combined in one helper molecule The same heterologous gene (such as an RSV F or G protein (e g , truncated RSV F protein) 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 ιmmunogen(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 of the invention can be comprised of sequences from a single flavivirus type (e g , West Nile, tick-borne encephalitis (TBE, e g , strain Hypr), Langat (LGT), yellow fever (e g , YF 17D), 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, Loupmg ill, Powassan, Negishi, Absettarov, Hansalova, and Apoi viruses), or can comprise sequences from two or more different flaviviruses. Sequences of some strains of these viruses are readily available from generally accessible sequence databases, sequences of other strains can y determined by methods well known m the art In the case of PIV vectors s including sequences of more than one flavivirus, the sequences can be those meric flavivirus, as descπbed 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 YFl 7D Other examples include the YF/WN, YF/TBE, YF/LGT, WN/TBE, and WN/LGT chimeras descπbed 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 chimeπc 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 chimeπc flaviviruses 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 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 ΔC 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 m 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-flavivims immunogens in PIVs and chimeπc flaviviruses of the invention can result in dual vaccines that elicit protective immunity against both a flavivirus vector virus pathogen rget heterologous immunogen (e g , RSV immunogens, such as those ed herem)

As discussed above, the PIV vectors and PIVs of the invention can comprise seq es of chimeπc 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 non-structural 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 (as well as non-chimeπc flaviviruses, e g , West Nile virus) used in the invention, used as a source for constructing PFVs, 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 3'UTR), such as any of those descπbed in WO 2006/116182 For example, the C protein or 3'UTR deletions can be directly applied to YF/WN, 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 9 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 PIV vectors and PIVs, and as live attenuated chimeric flavivirus vectors 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 nonstructural proteins of the chimeras can include those from yellow fever virus (e.g., YF 17D) 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 s, it is advantageous to use a signal sequence comprising yellow fever and quences (see below). In one example, the signal sequence includes yellow quences in the amino terminal region (e.g., SHDVLTVQFLIL) and TBE sequences in the carboxy terminal region (e.g., GMLGMTIA), resulting in the sequence SHDVLTVQFLILGMLGMTIA. 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). 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. Additional information concerning these 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 NSl 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 (e.g., certain RSV F or G protein sequences, as described herein), as well as complete proteins, can be expressed lntergemcally, at the N- and C-termini of the polyprotem, or bicistromcally (e g , withm the ORF under an IRES or in the 3'UTR under an IRES, see, e g , WO 02/102828, WO 2008/03614 ₆ , 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, ΔC, ΔprM-E, ΔC- ΔNS1, ΔNS3, and ΔNS5 Thus, m one example, in the case of s-PIVs and the mponent of d-PIVs, immunogen encoding sequences can be inserted in place of capsid sequences Immunogen-encoding sequences can also, optionally, be inserted in place of deleted prM E sequences in the ΔprM-E component of d-PIVs In another example, the sequences are inserted in place of or combined with deleted sequences in ΔC-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- termim 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 ΔC 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 πbosome 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 polyprotem 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/m association with the ER membrane

In more detail, the above-described approaches to making insertions can employ the use of, for instance, appropriate vector-deπved, insert-derived, or unrelated signal and anchor sequencess included at the N and C termini of glycoprotem inserts Standard autoproteases, such as FMDV 2A autoprotease (~20 acids) or ubiquitm (gene ~ 500 nt), or flanking viral NS2B/NS3 protease e sites can be used to direct cleavage of an expressed product from a growing tide chain, to release a foreign protein from a vector polyprotem, and to ensure viability of the construct Optionally, growth of the polyprotem chain can be terminated by using a termination codon, e g , following a foreign gene insert, and synthesis of the remaining proteins m the constructs can be re-imtiated 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 (Orlmger 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 m 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 usmg 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, 8, 340-341, and 436-437), or in place of short deletions (e g , deletions of 1, 2, 6, 7, or 8 amino acids) beginning at the indicated ammo acids (or within 1-5 s thereof, m either direction)

In 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 NSl (or in regions surrounding the indicated sequences, as described above) In other examples, insertions can be made mtergemcally For example, an insertion can be made between E and NSl proteins and/or between NS2B and NS3 proteins (see, e g , WO 2008/100464) In one example of an mtergemc 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 NSl sequences In another example of an intergemc 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 πbosome entry site (IRES, e g , an IRES derived from encephalomyocarditis virus, EMCV), 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 respiratory syncytial virus (RSV) F protein (including truncated F) 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. ogens

PIVs (s-PIVs and d-PIVs) based on flavivirus sequences and live, attenuated chimeric flaviviruses (e.g., YF/WN, 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 immunogens. The focus of the invention is the delivery of RSV immunogens, such as RSV fusion or F protein (or RSV G) immunogens (e.g., truncated F proteins; see below, for example the truncated F protein sequence in Example 3). PIVs and chimeric flavivirus vectors delivering a particular RSV immunogen can, optionally, be delivered with vectors delivering one or more other RSV immunogens, or one or more immunogens from another pathogen (e.g., viral, bacterial, fungal, and parasitic pathogens), one or more immunogens from cancer, and/or allergy-related immunogens. Specific, non-limiting examples of immunogens that can be delivered according to the invention are provided as follows.

As noted above, a central focus of the invention is delivery of the RSV proteins such as, in one embodiment, the RSV fusion or F glycoprotein and, in particular, truncated forms of this protein. The RSV F glycoprotein is one of the major immunogenic proteins of the virus. It is an envelope glycoprotein that mediates both fusion of the virus to the host cell membrane, and cell-to-cell spread of the virus. The amino acid sequence of the F protein is highly conserved among RSV subgroups A and B and is a cross-protective antigen.

RSV F protein comprises an extracellular region, a trans-membrane region, and a cytoplasmic tail region. A truncated protein delivered according to the invention can be, for example, one in which the trans-membrane and cytoplasmic tail regions of the F protein are absent (see, e g , Example 3, below) Lack of expression of the trans-membrane region results in a secreted form of the RSV protein

RSV F protem, as used herein, includes both full-length and truncated RSV fusion proteins, which may have the sequences described herein, or have variations in their amino acid sequences including naturally occurring in various strains of RSV and those introduced by PCR amplification of the encoding gene while retaining the immunogenic properties, a secreted form of the RSV F protein lacking a transane anchor and cytoplasmic tail, as well as fragments capable of generating ies which specifically react with RSV F protein and functional analogs A first is a functional analog of a second protein if the first protein is immunologically related to and/or has the same function as the second protein It may be for example, a fragment of the protein, or a substitution, addition, or deletion mutant thereof The RSV F glycoprotein can be from, e g , subgroup A or B (Wertz et al , Biotechnology 20 151-176, 1992)

In a further embodiment of the present invention, RSV G glycoprotein can be delivered The G protem is a approximately 33 kDa protein and is heavily O- glycosylated, giving rise to a glycoprotein having a molecular weight of about 90 kDa (Levine, S , Kleiber-France, R , and Paradiso, P R (1987) J Gen Virol 69, 2521- 2524) The 298 ammo acid residue RSV G protein belongs to the type II glycoproteins with the transmembrane domain (TM) located near the N-termmus (putative location residues 38 to 66 underlined in Sequence Appendix 7 The RSV F and G proteins, or fragments or analogs thereof, can be from, for example, group A (e g , Al or A2) or B RSV

Other examples of immunogens that can be delivered according to the invention are protective immunogens of the causative agent of Lyme disease (tick- borne spirochete Borreha burgdorferi) In one example, PIVs including TBE/LGT sequences, as well as chimeπc 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 these immunogens 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- chimeπc 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 lmmunogemcity, OspA can be mutated to reduce chances of autoimmune responses and/or to eliminate sites for unwanted post- translational modification m vertebrate animal cells, such as N-linked glycosylation, which may affect immunogemcity of the expression product Mutations that decrease munity can include, e g , those described by Willett et al , Proc Natl Acad A 101 1303-1308, 2004 In one example, FTK-OspA, a putative cross- T cell epitope, Bb OspAi _{65 173} (YVLEGTLTA) is altered to resemble the corresponding peptide sequence oϊBorreha afzelh (FTLEGKVAN) In FTK-OspA, the corresponding sequence is FTLEGKLTA

The sequence of OspA is as follows

1 mkkyllgigl llaliackqn vs sldeknsv svdlpgemkv lvs keknkdg kydliatvdk

61 lelkgtsdkn ngsgvlegvk adkskvklti sddlgqttle vfkedgktlv skkvtskdks

121 steekfnekg evsekntra dgtrleytgi ksdgsgkake vlkgyvlegt ltaekttlvv

181 kegtvtlskn lsksgevsve lndtdssaat kktaawnsgt stltitvnsk kt kdlvftke

241 ntitvqqyds ngtklegsav eitkldeikn alk

The full-length sequence and/or immunogenic fragments of the full-length sequence can be used Exemplary fragments can include one or more of domains 1 (ammo acids 34-41), 2 (amino acids 6 ₅ -75), 3 (ammo 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 compπsing 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 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/KK/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. MoI. 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 PFV 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, munogenicity/protection examined in available animal models (e.g., as ed in Gipson et al., Vaccine 21:3875-3884, 2003; Labuda et al., Pathog. 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:

Additional details concerning the TBE-related PIVs and LAVs are provided in Example 2, below.

Other PIV and LA V- 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)) can also be used. 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-hmitmg examples, we descπbe 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 RSV F protein in place of or in combination with various deletions m one- and two- nent PIV vectors (see Example 3, below)

As is demonstrated in the Examples, below, s-PIV constructs may be geously 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 ΔC 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 ΔprM-E, ΔC-prM-E, and/or ΔNS1 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 ΔC-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 or G 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 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 HAO (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 mclude PAKLLKERGFFGAIAGFLE (HAO), PAKLLKERGFFGAIAGFLEGSGC (HAO), NNATFNYTNVNPISHIRGS (NBe), MSLLTEVETPIRNEWGCRCNDSSD (M2e), MSLLTEVETPTRNEWECRCSDSSD (M2e), EVETLTRNGWGCRCSDSSD (M2e), EVETPTRN (M2e), VETPIRNEWGCRCNDSSD (M2e), and SLLTEVETPIRNEWGCR (M2e) nal M2e sequences that can be used in the invention include sequences from the extracellular domain of BM2 protein of influenza B (consensus MLEPFQ, e g , LEPFQILSISGC), and the M2e peptide from the H5N1 avian flu (MSLLTEVETLTRNGWGCRCSDSSD)

Other examples of pathogen immunogens that can be delivered in the vectors of the invention include codon-optimized SIV or HIV gag ( ₅ 5 kDa), gpl20, gplδO, SIV mac239 rev/tat/nef genes or analogs from HIV, and other HfV immunogens, immunogens from HPV viruses, such as HPV16, HPV18, etc , e g , the capsid protein Ll which self-assembles into HPV-hke particles, the capsid protein L2 or its immunodominant portions (e g , ammo 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 resultmg fusion protein mutant incapable of inducing transformation (Boursnell et al , Vaccine 14 1485-1494, 1996) Other immunogens include protective immunogens from HCV, CMV, HSV2, 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, such as RSV immunogens as described herein, 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 Further, the genes can be truncated at N- and/or C-termim, or by internal deletion(s), or modified by specific amino acid changes to increase visibility to the immune system and immunogenicity Immunogemcity can be increased by chimeπzation 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," Schπns and O'Hagan Eds , 2006, Elsevier mic Press Amsterdam, Boston)

With respect to construction of dual vaccines against rabies and other us 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 flagelhn, multimeπzation 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 immunogemcity 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 \ ectors 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 m Li et al , Vaccine 25(14) 2567-2574, 2007), CHIKV envelope proteins El and E2 (or the C-E2-E1, E2- ettes), HCV structural proteins C-El -E2 forming VLPs (Ezelle et al , J Virol 12325-12334, 2002) or other proteins to induce T-cell responses, Ebola virus otem GP (Yang et al , Virology 377(2) 255-264, 2008)

In addition to the immunogens descπbed above, the vectors described herem may include one or more ιmmunogen(s) deπved 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, vancella-zoster virus, poxvirus), ssDNA virus (e g , parvovirus, papillomavirus (e g , El, E2, E3, E4, E5, E6, E7, E8, BPVl, 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 , picornavirus, coxsackie virus, hepatitis A virus, pohovirus, 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 deπved 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-I 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-I lmmunogens, 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-I subtype B accounts for only a small minority of mfections, and subtype HIV-I C appears to be the most common infecting subtype Thus, in certain embodiments, it may be desirable to select lmmunogens from HIV-I subtypes B and/or C It may be e to include lmmunogens from multiple HIV subtypes (e g , HIV-I subtypes B HIV-2 subtypes A and B, or a combination of HIV-I and HIV-2 subtypes) in a mmunological composition Suitable HIV lmmunogens include ENV, GAG, POL, NEF, as well as variants, derivatives, and fusion proteins thereof, for example lmmunogens 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), Borreha spp (e g , Borrelia burgdorferi), Brucella spp (e g , Brucella abortus, Brucella cams, Brucella melitensis, Brucella suis), Campylobacter spp (e g , Campylobacter jejuni), Chlamydia spp (e g , Chlamydia pneumoniae, Chlamydia psittaei, Chlamydia trachomatis), Clostridium spp (e g , Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetam), Corynebacteπum spp (e g , Corynebacterium diptheriae), Enterococcus spp (e g , Enterococcus faecalis, enterococcus faecum), Escherichia spp (e g , Escherichia coll), Francisella spp (e g , Francisella tularensis), Haemophilus spp (e g , Haemophilus influenza), Helicobacter spp (e g , Helicobacter pylon), Legionella spp (e g , Legionella pneumophila), Leptospira spp (e g , Leptospira interrogans), Listeπa 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 rickettsn), Salmonella spp (e g , Salmonella typhi, Salmonella typhinuπum), 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, dium coli, Cestoda spp., Cimicidae spp., Clonorchis sinensis, Dicrocoelium cum, Dicrocoelium hospes, Diphyllobothrium latum, Dracuncuhαs spp., coccus 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-I), melanocyte differentiation antigens (e g , Melan A/MART-1, tyrosinase, gplOO), mutational antigens (e g , MUM-I, p53, CDK-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- 1/Melan A (Kawakami et al , J Exp Med , 180 347-352, 1994), gp75 (TRP-I) (Wang et al , J Exp Med , 186 1131-1140, 1996), tyrosinase (Wolfel et al , Eur J Immunol , 59 764, 1994), NY-ESO-I (WO 98/14464, WO 99/18206), melanoma lycan (Hellstrom et al , J Immunol , 130 1467-1472, 1983), MAGE family s (e gl, MAGE-I, 2, 3, 4, 6, and 12, Van der Bruggen et al , Science 254 1643- , 991, 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-I, Gaugler et al , Immunogenetics 44 323-330, 1996, U S PatentNo 5,939,526), N-acetylglucosaminyltransferase-V (Gmlloux et al , J Exp Med 183 1173-1183, 1996), pl5 (Robbins et al , J Immunol 154 5944-5950, 1995), U- catenm (Robbins et al , J Exp Med , 183 1185-1192, 1996), MUM-I (Couhe et al , Proc Natl Acad Sci U S A 92 7976-7980, 1995), cyclm 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-aW (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) (Hams et al , Breast Cancer Res Treat, 29 1-2, 1994), carcinoembryomc 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-I gene products, Jerome et al , J Immunol , 151 1654-1662, 1993), EBNA gene products of EBV (e g , EBNA-I, 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), KSA (U S Patent No 5,348,887), kinesm 2 (Dietz, et al , Biochem Biophys Res Commun 275(3) 731-738, 2000), HIP-55, TGFβ-1 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), HlFT, NY-BR-I (WO 01/47959), NY-BR-62, NY-BR-75, NY-BR-85, NY-BR-87, and NY-BR-96 (Scanlan, M Serologic and Biomformatic Approaches to the Identification of Human Tumor s, in Cancer Vaccines 2000, Cancer Research Institute, New York, NY), pancreatic cancer antigens (e g , SEQ ID NOs 1-288 of U S Patent No 31) Immunogens may also be derived from or direct the immune response agamst 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 can include one or more substitutions or deletions, e g , substitutions of conservative amino acids as described herein 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 lmmunodominant/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 interfeπng sequences, e g , withm 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 ammo acids The ranges also include, e g , 20-400, 30-100, and 50-100 ammo acids

The invention also includes compositions including mixtures of two or more PIVs and/or PIV 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 PIV cocktails included m 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 descπbed above In addition, m the case of the PIV-DEN, the PFV can include sequences of just one dengue serotype selected from the group consisting of dengue es 1-4, or the cocktail can include PIVs expressing sequences from two, three, ur of the serotypes Further, the TBE/Borreha burgdorfen/tick saliva protein 4TRP, Isac, Salp20) vaccines descπbed herein can be based on including the different immunogens withm 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 mvention includes the PIV and LAV vectors, as well as corresponding nucleic acid molecules, pharmaceutical or vaccine compositions, and methods of their use and preparation The PIV and LAV vectors of the invention can be used, for example, in vaccination methods to induce an immune response to RSV and/or the flavivirus vector, and/or another expressed immunogen, as descπbed herein These methods can be prophylactic, in which case they are earned out on subjects (e g , human subjects or other mammalian subjects) not having, but at risk of developing infection or disease caused by RSV or flavivirus and/or a pathogen from which another expressed immunogen is deπved Such methods include instances in which a subject becomes infected by RSV, but is able to ward off the infection and significant symptomatic disease, because of the treatment according to the invention The methods can also be therapeutic, in which they are earned out on subjects already having an infection by one or more of the relevant pathogens, such as RSV Such methods include the amelioration of one or more symptoms of the infection, whether partial or complete Further, the viruses and vectors can be used individually or in combination with one another or other vaccmes 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) Of particular interest with respect to vaccination against RSV are infants and young children, including pre-mature infants, as well as middle aged and elderly people Thus, for example, human patients age 1 day to five years (e g , 2 months to 3 years, or 4 months to two years), or age 50 to 65 and above

Formulation of the PIV and LAV vectors of the invention can be earned out ethods that are standard in the art Numerous pharmaceutically acceptable s for use in vaccine preparation are well known and can readily be adapted for he 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 and LAV vectors can simply be diluted m a physiologically acceptable solution, such as sterile saline or sterile buffered saline

The PIV and LAV vectors of the invention can be admimstered using methods that are well known in the art, and appropπate amounts of the viruses and vectors to be admimstered 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 admimstered For example, in the case of live, attenuated viruses of the invention, the viruses can be formulated as sterile aqueous solutions containing between 10 ² and 10 ^s , 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 PFVs can be administered at similar doses and in similar volumes, PIV titers however are usually measured in, e g , focus-forming units determined by immunostaimng of foci, as these defective constructs tend not to form virus-like plaques Doses can range between 10 ² and 10 ⁸ FFU and admimstered 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, intranasal (e g , by inhalation or nose drops), intravenous, or oral routes In specific examples, dendπtic 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 vaccmes 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, 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 ministration 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, polyphosphazme, 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 coh (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 vaccme 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", Schyns and O'Hagan Eds , 2006, Elsevier Academic Press Amsterdam, Boston, etc ) Optionally, a patch containing a layer of an appropriate toxin-deπved adjuvant, can be applied over the injection site Toxin promotes local inflammation attracting lymphocytes, which leads to a more robust immune response Additional details concerning the invention are provided in the Examples, below hi 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 trated for the first time that d-PIV constructs were avirulent in this model and t two-component PIVs do not undergo uncontrolled (unlimited) spread in vivo not cause clinical signs Secondly, we performed comparisons of the immunogemcity 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 PIV-YF Thus, production of VLPs can vary between different, similarly designed PIV constructs Specifically, we propose that PIV-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 polyprotem 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 (m 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) mto PIV genomes, recovery of PIV progeny and measuring VLP secretion Non-flavivirus 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-termmus 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 (in constructs where C protein sequence is unnecessary, e g , ΔC PIV) and laced in front of foreign gene Optimization of vector signal can be performed om 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 m 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 Vaccm 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 descnbed 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 (ΔprM-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 m a universal helper packaging cells line, and sequential use of different recombinant PIV-vectored vaccines in the dividual, as discussed above In addition, we confirmed previous observations V constructs can be stably propagated to high yields in vitro, and that no nation 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 descπbed m the Examples, below

Example 1. Pseudoinfectious virus platform development studies

Attenuation in suckling mouse neurovirulence (NV) model

Materials used in the studies descπbed 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 vims 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 chimeπc 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 m 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 257:303-372, 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 logio PFU and 3 logio PFU, respectively), with longer average survival time (AST) of animals that died, as expected. Thus, PIV cts are completely avirulent in this sensitive model (at least 20,000 - 200,000 ss neurovirulent than the licensed YF 17D vaccine).

The YF d-PrV 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 logio FFU of YF d-PIV and WN d-PIV, respectively, were homogenized and used for titration). Thus, the d-PIVs 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 apeak titer of- 6 logio 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-PFV 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/effϊcacy 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 logio FFU/dose) or chimeric LAV and YF 17D LAV controls (4 logio PFU) Select PIV- WN, -JE and -YF groups were boosted on day 21 with 5 logio FFU of corresponding constructs (Table 3) 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 ntrol group Accordingly, animals immunized with both PIV-WN and LAV were protected from lethal challenge on day 35 with wt WN virus (IP, 5 ₀ ), but not mock-immumzed animals (Table 3) When WN neutralizing ant bodies 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 LD ₅₀ ) (Table 3), which is in agreement with the knowledge that neutralizing antibody titers as low 1 10 are protective against flavivirus mfections

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 mfect 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- WN) 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 appropπate 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 (Malm et al , Miciobes and Infection, 2 1677-1685, 2000), etc ] in place of the YF signal for prM A similar experiment was performed m ~ 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 PRNT ₅₀ titers on day 38 (pre-challenge) were 1 320, 1 640, and 1 1280 m groups that received 5, 6, and 6 (pπme)+5 (boost) logio FFU doses, respectively This was somewhat lower compared to YF/WN LAV 4 logio PFU control (> 1 2560) PIV-JE and -YF mduced detectable specific zing antibody responses, albeit with lower titers compared to YF/JE LAV and controls All animals immunized with PFV-WN and YF/WN were solidly d 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 vireima 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) virerma 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 m 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 lmmunogemcity/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 m 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 For instance, for PIV-WN after 1 ^st dose, SC immunization resulted in WN-speciflc PRNT50 titer of 1 40, while IP inoculation resulted in much higher titer 1 320 (and after the 2 ^nd dose, titers were similar) A more pronounced effect was observed for other constructs after both the 1 ^st and 2° ^d doses Interestingly, PIV- YF/WN was very highly immunogenic by IP route (titer 1 320 after I ^s ' IP dose vs 1 20 by SC, and 1 1,280 after 2 ^nd 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-presentmg cells are more abundant in the abdomen as opposed to tissues under the skin), is an nt consideration for use of PIV vaccines In humans, efficient targeting of c cells of the skm, 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-descπbed 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 PRNT ₅₀ in animals immunized with 5 logio FFU of the second component of WN d-PFV, containing the ΔC-prM-E deletion and thus not encoding VLPs, but packaged into the WN envelope m BHK- CprME(WN) helper cells, and no YF-specifϊc neutralizing activity was found in sera from animals immunized with 4 logio 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 PIV-JE packaged into WN envelope The absence of neutralizing response against packaging envelopes permits manufacturing different PIV vaccines m 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 m 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) ly, WN-specific titers against PIV-JE + PIV-WN mixture and PIV-WN alone milar (1 320 vs 1 640 and 1 5,120 vs 1 5,120 for one and 2 doses, vely) No or little cross-specific response was induced by either PIV-JE or - WN The result was also confirmed by measuring PRNT _5O 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 Vera cells, for d-PIVs Samples harvested after each passage were titrated in Vero cells by immunostaimng Constructs grew to high titers, and no recombination restoπng whole virus was observed For instance, PIV-WN consistently grew to titers 7-8 log ₁₀ FFU/ml in BHK-CprME(WN) helper cells (containing a VEE replicon expressing the WN virus C-prM-E proteins), and WN d- PIV grew to titers exceeding 8 logio 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-δ951, 2008), or plasmids for chimeric LAVs (e g , pBSA-ARl, a single-plasmid version of us clone of YF/JE LAV, WO 2008/036146), respectively, using standard s in the art of reverse genetics The prM-E sequences of TBE virus strain Hypr nk 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 (Vero for chimeπc viruses, and helper BHK cells for PIV) were transfected with RNA transcπpts 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 (plasimd 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 coveπng the 5'UTR-C- prM-E-begmmng of NSl region are shown in Sequence Appendix 2) The p42 deπved YF17D/Hypr chimera contained a hybnd YF17D/Hypr signal peptide for the prM protein, while the p45 deπved YF17D/Hypr chimera contained a hybnd YF17D/WN signal peptide for prM (Sequence Appendix 1) The former chimeric virus produced very high titers at both PO (immediately after transfection) and Pl (the next passage in Vero cells), up to 7 9 logio PFU/ml, which were 0 5 logio 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-deπved YFl 7D/LGT chimera had the same prM signal as the p42- denved virus, it also produced very high titers at PO and Pl passages (up to 8 1 logio PFU/ml) and formed large plaques A deπvative of the p42-deπved 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 hx I, WO 2006/116182) As expected, the p ₅ 9 virus grew to lower titers (5 6 logio PFU/ml at PO and Pl , respectively), and formed small plaques ined 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 deπved from plasmids p39 and p40 (Fig 7, Sequence Appendix 1 for C/prM junction sequences, and Sequence Appendix 3 for complete 5'UTR-ΔC-prM-E-beginnmg of NS 1 sequences) These contained complete Hypr or WN prM signals, respectively Both PIVs were successfully recovered and propagated in BHK-CprME(WN) or BHK-C(WN) helper cells (Mason et al , Virology 351 432-443, 2006, Widman et al , Vaccine 26 2762-2771, 2008) The PO and Pl sample titers of the p39 variant were 0 2 1 0 logio 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 7) The higher rate of replication of the p39 candidate than p40 candidate was confirmed in a growth curve experiment (Fig 8) In the latter expeπment, 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 fiavivirus 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 P21 wild type strain or attenuated E5 strain), and chimeric YF 17D ne/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 LA V 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 logio FFU of PIV -Hypr (p39 and p40) variants by both routes survived and showed no signs of sickness, similar to mock-inoculated animals (Table 7), and thus PIV-TBE vaccines are completely avirulent. Mice inoculated IC with YF 17D control (1 - 3 logio PFU) showed dose-dependent mortality, while all animals inoculated IP (5 logio 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., enced by complete mortality and shorter AST at the 2 logio PFU dose, the dose tested for YF/TBE LAV candidates. The non-neurovirulent phenotype of E, 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). In addition, p43 and p59 candidates were non-neuroinvasive, while p42 and p45 caused partial mortality after IP inoculation (5 logio PFU/dose) (Table 7; Fig. 10). 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 (LD ₅₀ ~ 0.1 PFU) and IP (LD ₅₀ < 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 expeπment in Table 7), 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 PRNT ₅₀ against wt TBE Hypr virus (Table 8, 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 YF 17D and Mock negative controls] Titers in individual test samples as well as GMTs for each group are provided in Table 8 Titers in test samples were similar within each e g , in groups immunized with PIVs, indicating high uniformity of immune e in animals As expected, no TBE-specific neutralizing antibodies were d 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- lmmumzation with a high IP dose (500 PFU) of wt Hypr TBE virus Mortalities from partial observation (on day 9 post-challenge, observation being continued) are provided in Table 8, 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 PTV-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) also had high antibody titers (GMT 1 6,085) and were protected (Table 8, Fig 11) Interestingly, PTV -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 πght prM signal, e g , the TBE-specific signal, to achieve high-level replication/VLP secretion, which in this experiment m 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 ized. The genes were cloned into PIV-WN vector plasmids using standard s of molecular biology well known in the art, and packaged PIVs were ed following in vitro transcription and transfection of appropriate helper (for s- PrVs) or regular (for d-PIVs) cells.

Expression of rabies virus G protein in WN s-PIV and d-PIV

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, Epidemic Infect. 116:235-240, 1996; Li et al., Virology 356:147-154, 2006).

Full length rabies virus G protein (original Pasteur virus isolate, GenBank accession number NC OO 1542) was codon-optimized, chemically synthesized, and inserted adjacent to the ΔC, ΔprM-E and ΔC-prM-E deletions in PIV-WN vectors (Fig. 12). The sequences of constructs are provided in Sequence Appendix 4. General designs of the constructs are illustrated in Fig. 13. The entire G protein containing its own signal peptide was inserted in-frame downstream from the WN C protein either with the ΔC deletion (ΔC and ΔC-prM-E constricts) or without (ΔprM-E) and a few residues from the prM signal. Foot and mouth disease virus (FMDV) 2A 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-NSl-5 genes in the ΔC construct, or signal for NSl and NS 1-5 genes in ΔprM-E and ΔC-prM-E constructs

Packaged WN(ΔC)-rabiesG, WN(ΔprME)-rabiesG, and WN(ΔCprME)-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 tion/infection of BHK-C(WN) and/or BHK-C-prM-E(WN) helper cells, as well as BHK cells, by immunostaining and immunofluorescence assay (IFA) usmg anti- 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 m 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-mfected Vero cells, which were fixed but not permeabihzed, 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 mam prerequisite for high immunogemcity of expressed G Individual packaged PIVs can spread following infection of helper BHK cells, but cannot spread in regular cells as illustrated for WN(ΔC)-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 contaimng the G protein, if produced by PIV infected cells An identical result was obtained with the G protein of another rhabdovirus, Vesicular stomatitis virus (VSV), contrary to previous observations of non-specific packaging of Semhki 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 nonspecific 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 lmmunostaining 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(ΔC)-rabiesG PIV 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 ΔC deletion (~ 200 nt), significantly increasing the overall size of mbinant RNA genome In contrast, in WN(ΔprME)-rabiesG, and WN(ΔCprME)- PIVs, the insert is combined with a much larger deletion (~ 2000 nt) Therefore, nstructs 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 logio 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(ΔC)-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(ΔprME)-rabiesG and WN(ΔCprME)- rabiesG PIVs will induce humoral immune response only against rabies because they do not encode the WN prM-E genes WN(ΔC)-rabiesG s-PIV construct can be also co- inoculated with WN(ΔprME)-rabiesG construct in a d-PIV formulation (see m Fig 12), 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(ΔprME)-rabiesG, and a d-PIV sample, WN(ΔprME)-rabiesG + WN(ΔC) PrV (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 stamable 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 comfection with the two PIV types

The WN(ΔCprME)-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 Fig 12) For example it can be a WN virus genome containing a deletion of one of the NS proteins, e g , NSl, 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 3253-3203, 2000) We have constructed a WN-ΔNS1 genome (sequence provided in Sequence Appendix 4) and obtained evidence of co-mfection with WN(ΔprME)-rabiesG or WN(ΔCprME)-rabiesG constructs, and spread in vitro, by immunostaimng In the case of such d-PIVs, rabies G protein can be also inserted and expressed in helper genome, e g , WN-ΔNS1 genome, to increase the amount of expressed rabies G protein resulting in an increased anti-rabies immune response As with any dPIV versions, one immunogen can be from one pathogen (e g , rabies G) and the other from a second pathogen, resulting in three antigenic ities of vaccine As discussed above, ΔNS1 deletions can be replaced with or used ination with ΔNS3 and/or ΔNS5 deletions/mutations, in other examples ion of RSV F protein in WN s-PIV and d-PIV

Respiratory syncytial virus (RSV), member of Paramyxovindae 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 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 PTV, using the insertion schemes shown in Fig 12 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(ΔC) RSV F, WN(ΔprME)- RSV F, and WN(ΔCprME)- RSV F PIV constructs were used to transfect helper BHK-CprME(WN) cells Efficient replication and expression of RSV F protein was first demonstrated by immunostaimng of transfected cells with an anti-RSV F Mab, as illustrated for the WN(ΔprME)- RSV F construct in Fig 19 The presence of packaged PIVs in the supernatants from transfected cells (titer as high as 7 loglO FFU/ml) was determined by titration in Vero cells with immunostaimng Additionally, similar constructs can be used that contain a modified F protein gene Specifically, the N-terminal native signal peptide eplaced in modified F protein with the one from rabies virus G protein The ation is intended to elucidate whether the use of a heterologous signal can increase of F protein synthesis and/or replication of PIVs

It has been demonstrated that a C-terminally truncated, secreted form of RSV F could be more immunogenic than full-length protein (Li et al , J Exp Med 188 681-688, 1998) Therefore, we also cloned the available truncated RSV F gene (see Fig 20) into the WN PIV vectors Insertion designs were as in Fig 13, with the only exception that the gene did not contain the sequence encoding C-terminal F protein anchor, to produce soluble form of truncated F (trF) in the lumen of the ER, which should be efficiently secreted from cells Resulting WN(ΔC)-RSV trF, WN(ΔprME)- RSV trF, and WN(ΔCprME)- RSV trF PIVs (see Sequence Appendix 6 for the sequences of these constructs) were recovered in helper BHK-CprME(WN) cells Results of IFA for transfected cells, performed with anti-RSV F Mab, are shown in Fig 21 Efficient expression of trF product was observed, also demonstrating that all defective recombinant viruses were viable Titers of PIVs as high as 2x10 ⁶ FFU/ml were observed in cell supernatants immediately after transfection, these are expected to further increase with passages Importantly, similar numbers of foci were detected by both anti-RSVF and anti- WN antibodies in titration experiments in Vero cells (Fig 22), and intensities of staining with both antibodies were comparable indicative of high-level expression of trF product Western analysis of two ΔprME-RSVtrF stocks, two days post-mfection of Vero and BHK (WNV/C prM-E) helper cells, is shown in Fig 23 These PIVs can be evaluated further for lmmunogemcity/effϊcacy in available animal models for RSV disease, in both S-PIV and d-PIV formulations (see below for further evaluation of ΔprME RSVtrF)

Set forth below is the RSV ammo acid sequence of the truncated construct The chimeric West Nile/RSV-F signal peptide (sεkteiavi/melDiikanaMiliavtfcfass) is designed to be cleaved by signal protease after " fass", releasing N-terminus of F2 "qmtee " At the C-terminus is the sequence of autoprotease FMDV 2A fused to RSVF (nfdllklagdyesnpg). This sequence and/or only the RS V F protein portion thereof can be used in any of the vectors described herein Further, this sequence (and/or only the RSV F protein portion thereof) can be the basis for derivation of analogs and fragments for use in the invention Thus, sequences having percentage identities to this sequence, as described above, or fragments, as described above, can be used m the invention

Recombinant poxviruses expressing RSV F

Based on the premise that protection can be obtained using a very limited, but focused antibody response, we have shown that a live vector expressing a codon optimized anchorless RSV F confers protection against RSV infection in an appropriate animal model and thus can be suitable for an infant vaccine

NYVAC is a highly attenuated vaccinia strain with a series of deletion of virulence-associated or host-range genes of the Copenhagen strain (Tartaglia et al , Dev Biol Stand 84 159-163, 1995) It has been used in a variety ofpre-clmical and clinical studies and shown to be promising Therefore, NYVAC has been included as a delivery vehicle for a comparative vaccine evaluation

To generate the recombinant NYVAC expressing codon-optimized anchorless RSV F, IVR (m vitro recombination) was performed with CEF cells infected by parental NYVAC at M O I of 10 and transfected with donor plasmid pLNZ16 Subsequently, IVR reaction products were serially diluted ten-fold from 1 10 ³ to 1 10 ⁶ , and plated on CEF cells in 100-mm plates overlaid with medium-agarose without Blue-Gal Three days after the first overlay, a second overlay containing Blue-Gal and Neutral Red was added Blue plaques were picked and plaque purification continued until a white plaque was available for amplification The isolated plaque went through three amplification steps, i e , Pl , P2, and P3 The recombinant was fully characterized at P2 to confirm identity and purity The NYVAC recombinant was designated vP2400

Fowlpox is a member of the avipoxvirus genus and can cause disease m chickens and turkeys Transmission of fowlpox virus is limited to avian species, with replication in mammalian cells resulting in abortive replication The inability of fowlpox to produce infectious virus in mammalian cells renders fowlpox a very attractive vector for human vaccine development The safety and efficacy of fowlpox- accines have been investigated in a number of clinical trials for diseases such er, HIV, and malaria Preliminary results indicate that fowlpox vaccines are d well tolerated, and have demonstrated both immune and clinical efficacy This vector was also used to compare delivery systems that express the RSV F gene product, and to allow a thorough evaluation of both immune efficacy and safety in relevant animal model systems

To generate recombinant fowlpox expressmg codon-optimized anchorless RSV F, rVR was performed with CEF cells infected by a parental fowlpox at M O I of 10 and transfected with the donor plasmid pLNZ15 (Paoletti, Proc Natl Acad Sci U S A 93 11349-11353, 1996) The rest of the steps are the same as above The fowlpox recombinant was designated vFP2403

Western blot analysis of PIV mediated expression of RSVF (See Fig 24)

Vera cells (~1 5 x 10 ⁶ ) were infected at an MOI of I ₀ with Lanes 2 and 3, vP2400 (NYVAC RSV F), Lanes 4 and 5, vFP2403 (fowlpox-RSV F), Lanes 6 and 1, PIV-F (ΔprME-RSVtrF), and Lanes 8 and 9, mock infected cells All recombinant viruses express a codon optimized anchorless RSV F Cell supernatants were harvested at 24 (Lanes 2, 4, 6, and 8) and 48 (Lanes 3, 5, 7, and 9) hours after infection Equal amounts of the supernatant samples were analyzed by SDS-PAGE and the amount of RSV F present in each sample was determined using primary antibody, i e , a mouse anti-RSV F (5353C75), followed by a goat anti-mouse IgG horseradish peroxidase (HRP) conjugate as secondary antibody The level of RSV F present in each sample was measured by comparison to a purified preparation of protein F from RSV-mfected cells (2 5 ng, Lane 10) measured using a Kodak Imager Station 4000MM Pro The results demonstrate that the amount of RSV F expressed in PIV-F infected Vero cells was significantly greater than that expressed by NYVAC and similar to that expressed by fowlpox

Immunization studies using PIV-F

For intramuscular immunization, Balb/c (6-8 weeks old) were injected bilaterally with 2 x 50 μl of PBS solution containing viral vectors expressing RSV F protein at two doses - either 10 ⁶ or 10 ⁷ PFU Animals were boosted 4 weeks later e same dose of the vaccine Mice in control groups were immunized ally with 10 ⁶ PFU RSV-Long strain or intramuscularly with an FI-RSV (100 μl) prepared according to the procedures used for the 1960's trials Four weeks after boost, mice were challenged mtranasally with either 2 2 x 10 ⁶ PFU RSV- A2 (for RSVi27) or 10 ⁷ PFU RSV-A2 (for RSVi32)

ELISA analysis of sera derived from vaccinated mice (See Fig 25)

Immune sera were analyzed for anti-RSV-F IgG antibody titers using ELISA, which was performed with an lmmunoaffinity-punfied full-length RSV protein (50 ng/ml) by two-fold dilutions of immune sera Goat anti -mouse F(ab)2 IgG (H+L) conjugated to horseradish peroxidase was used as secondary antibody The titer is a reciprocal of the last dilution at which the OD450 was greater than 0 1 and at least twice that of a control, to which no sample was added It can be seen from Fig 25 that both i m and i p immunization with PIV-F generated the highest titers of IgG of the vectors tested

Neutralization assay (See Fig 26)

Vero cells were seeded onto 24-well plates (1 5 x 10 ⁵ per well), incubated at 37°C for two days The neutralization reaction mixtures (serial diluted sera + virus + complement) were prepared m DMEM and incubated for 1 hour in a 37°C shaker The neutralization mixtures were added to the Vero cells After a 2 hour incubation in a 37°C shaker, the mixtures were removed and overlay media (methyl cellulose/DMEM) was added to each well The infected Vero cells were incubated for 4 days at 37°C, then fixed with 80% methanol and stained with a primary antibody, i e , a mouse anti-RSV F antibody (5353C75), followed by a goat anti-mouse IgG- horseradish peroxidase (HRP) conjugate as secondary antibody. The plaques were counted by eye and neutralizing titers were expressed as the dilution that caused 60% plaque reduction.

It can be seen from Fig. 26 that both i.m. and i.p. immunization with PIV-F showed the highest neutralization titers of all vectors tested.

Table 4. PIV are immunogenic in hamsters and protect against challenge ¹

Table 5. Immunization of hamsters with PIV: comparison of SC and routes

Table 6. Immune responses to PIV cocktails (mice) ¹

Table 8. Neutralizing antibody titers (PRNT ₅₀ ) in mice immunized IP (determined against wt TBE virus Hypr), and protection from challenge (postchallenge observation, day 9)

Table 9. Examples of published attenuating E protein mutations that can be used for attenuation of chimeric TBE LAV candidates 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 1) 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(1) 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, ell et al , Virology 269(1) 225-237, 2000, Gπtsun et al , J Gen Virol 82(Pt -1675, 2001

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

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

What is claimed is

Sequence Appendix 1

Sequence Appendix 2

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75

Sequence Appendix 4. WN PIV constructs expressing rabies virus G protein.

WN (ΔCprME)-Rabies PIV sequence (partial)

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SEQUENCE APPENDIX 6

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