ZIKA VIRUS VACCINES USING VIRUS-LIKE PARTICLES

Abstract

A flavivirus virus-like particle and methods of making and using that particle, and antibodies raised to a plurality of those particles, are provided.

Claims

1. A recombinant nucleic acid vector comprising a heterologous promoter operably linked to a nucleotide sequence encoding flavivirus prM/E, which vector lacks nucleic acid sequences encoding one or more of flavivirus NS1, NS2A, NS2B NS3, NS4A NS4B or NS5 and optionally lacks nucleic acid sequences encoding functional flavivirus capsid.

2. The recombinant vector of claim 1 wherein the heterologous promoter is a heterologous viral promoter. The recombinant vector of claim 1 which includes a portion of flavivirus capsid sequences.

4. The recombinant vector of claim 1 wherein the capsid sequence includes amino acids 98 to 112 of the capsid protein encoded by SEQ ID NO:1 or a protein having at least 80% amino acid sequence identity thereto.

5. The recombinant vector of claim 1 wherein the flavivirus is a Zika virus.

6. The recombinant vector of claim 1 wherein the prM/E sequences have at least 80% amino acid sequence identity to the prM/E sequences encoded by any one of SEQ ID Nos. 1-3 or 5.

7. The recombinant vector of claim 1 wherein the prWE sequences are operably linked to a heterologous secretion signal.

8. The recombinant vector of claim 7 wherein the heterologous secretion signal is a TPA, IL-2, IgG kappa light chain, CD33, or Oikosin secretion signal.

9. A vaccine comprising an effective amount of a flavivirus like particle comprising a lipid bilayer comprising flavivirus prM/E but which particle lacks one or more of flavivirus NS1, NS2A, NS2B, NS3, NS4A, NS4B or NS5 and optionally lacks functional flavivirus capsid.

10. The vaccine of claim 9 further comprising one or more adjuvants.

11. The vaccine of claim 10 wherein the adjuvant comprises alum, monophosphoryl lipid A (MPLA), squalene, aluminum hydroxide absorbed TLR4 agonist, dimethyldioctadecylammonium, tripalmitoyl-S-glyceryl cysteine, trehalose dibehenate, saponin, MF59, AS03, virosomes, AS04, CpG, imidazoquinoline, poly I:C, flagellin, or any combination thereof

12. The vaccine of claim 9 wherein the flavivirus is a Zika virus.

13. The vaccine of claim 9 wherein the prM/E sequences have at least 80% amino acid sequence identity to the prM/E sequences encoded by any one of SEQ ID Nos. 1-3 or 5.

14. A method to prevent, inhibit or treat flavivirus infection in a mammal, comprising: administering to the mammal a composition comprising an effective amount of a flavivirus like particle comprising a lipid bilayer comprising flavivirus prM/E but which particle lacks one or more of flavivirus NS1, NS2A, NS2B, NS3, NS4A, NS4B or NSS and optionally lacks functional flavivirus capsid, or a composition comprising an effective amount of anti-flavivirus antibodies.

13. The method of claim 14 wherein the mammal is a female mammal.

14. The method of claim 14 wherein the mammal is a human.

15. The method of claim 14 wherein the flavivirus is a Zika virus.

16. The method of claim 17 wherein the prM/E sequences have at least 80% amino acid sequence identity to the prM/E sequences encoded by any one of SEQ ID Nos. 1-3 or 5.

17. The method of claim 14 wherein the composition comprising the flavivirus like particle is administered intramuscularly, subcutaneously or intranasally.

18. The method of claim 14 wherein the composition inhibits flavivirus infection.

19. The method of claim 14 wherein the composition treats flavivirus infection.

20. The method of claim 14 wherein the composition comprising antibodies comprises antibodies pooled from multiple donors that were infected with the flavivirus.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0012] FIGS. 1A-E. In vitro characterization of Zika virus like particles. A) Schematic of pCMV-prM/E expression cassette. B) Western blot analysis of Zika virus like particles. Lanes are, 1) Bio-rad precision plus kaleidoscope protein standards. 2): pCMV-prM/E transfection pre sucrose cushion purification supe. 3) 3.510.sup.4 PFU ZIKV positive control. 4) pCMV-prM/E transfection post sucrose cushion purification pt. 5) pCMV-GFP transfection post sucrose cushion purification pt. C-E) Sucrose cushion purified Zika VLPs observed using transmission electron microscopy. C) VLPs stained with Tungsten. Diameter is indicated. Background protein staining also apparent. D) VLP stained with Tungsten. Membrane proteins visible on the surface of VLP are indicated with arrow. Background protein staining apparent. E) VLP stained with Uranyl acetate. Membrane proteins visible on the surface of VLP are indicated with an arrow.

[0013] FIGS. 2A-F. Protection of ZIKVLPS in AG129 mice. A) Neutralizing antibody titers (+/SD) of vaccinated AG129 mice pre boost and pre challenge. B) Average weight loss (+/SD) of AG129 after ID challenge with 200 PFU ZIKV over a 14 day period. C) Survival of 11 week old AG129 after ID challenge with 200 PFU ZIKV over a 14 day period. D) Viremia (+/SD) in serum samples from mice two days post challenge by qRT-PCR. Values are total RNA copies per reaction. E) Viremia (+/SD) in serum samples from mice two days post challenge by TCID.sub.50. F) PRNT.sub.50 and PRNT.sub.90 values (+/SD) of serum samples taken from ZIKVLP vaccinated AG129 mice post challenge, and pre challenge serum from PBS/alum mice.

[0014] FIGS. 3A-B. ZIKVLP serum transfer to nave AG129 mice. A) Average weight loss (+/SD) of 8 week AG129 transferred serum from mice vaccinated with ZIKVLPs after ID challenge with 20 PFU of ZIKV over a 14 day period. B) Survival of AG129 after challenge with ZIKV over a 14 day period.

[0015] FIG. 4. LD50 of ZIKV in AG129 mice. Survival of AG129 after ZIKV over a 14 day period.

[0016] FIG. 5A-B. A) Weight loss of AG129 after ID challenge with 20 PFU ZIKV over a 12 day period. B) Survival of AG129 after ID challenge with 200 PFU ZIKV over a 12 day period.

[0017] FIGS. 6A-B. Sequence of a vector with an exemplary coding sequence to express prM/E (SEQ ID NO:5).

[0018] FIG. 7. Schematic of a pCMV (A) and pTriex4-neo (B) vector for expression of prM/E.

[0019] FIG. 8A-C. Images showing GFP expression in HEK293 cells. A) pTri px4-neo GFP expression, B) pCMV GFP expression, and C) pCMV GFP expression.

[0020] FIG. 9. Western blot analysis of pTriex versus pCMV prM/E expression. Lane 1: Zika virus +; lanes 3,9: pCMV-GFP cells (pt.) and supernatant (sup.); lanes 4,10: pCMV-Columbia pt., sup.; lanes 5,11: pCMV-French-Poly pt., sup.; lanes 6, 12: pTriex-Columbia pt., sup.; and lanes 7, 13: pTriex-French-Poly pt., sup.

[0021] FIG. 10. Anti-Zika antibodies in mice before and after VLP exposure. Mice were injected IP with about 10.sup.6 TCID.sub.50 of ZIKV. 5 weeks later the mice were bled, then injected with crude VLP supernatant. Mice were bled 7 days after injection and antibodies analyzed by ZIKV ELISA.

[0022] FIG. 11. Western blot of sucrose purified VLPs. Lane 1: marker; lane 2: VLP 100,000 g precipitation; lane 3: Zika virus +; lane 4: pCMVFrench-Poly post sucrose purification; and lane 5: pCMV-GFP post sucrose purification. Cells in T-75 flasks were transfected with pCMV-prM/E, or pCMV-GFP, and supernatants were collected after 3 days, then clarified by centrifugation (15,000 g, 30 minutes), then layered onto a 20% sucrose cushion, and pelleted at 112,000 g for 3.5 hours.

[0023] FIG. 12. Sucrose fractional analysis. Lane 1: marker; lane 2: Zika virus +; lane 3: Cell debris (pt.) from clarification step; lane 4: Supernatant above sucrose cushion post centrifugation; lane 5: marker; lane 6: VLP post purification batch 1: days 0-3; and lane 7: VLP post purification batch 2: days 3-10. A second batch was harvested from transfected flasks (days 3-10). Purified as before, fractions from each sucrose purification step were analyzed to ensure there was no loss during purification.

[0024] FIG. 13. Comparison of protein expression for VLPs produced from pCMV and pTriex constructs.

[0025] FIG. 14. Mouse study. 11 AG129 mice of mixed sex and age were used. VLPs were administered IM along with 1 mg Alum. Challenge virus (100 PFU) was administered ID.

[0026] FIG. 15. Antibody levels two weeks post boost.

[0027] FIG. 16. Survival and morbidity. All controls were moribund on day 9.

[0028] FIGS. 17A-C. Dose response of ZIKVLPS in AG129 mice. A-B) PRNT.sub.50 and PRNT.sub.90 values (+/SD) of serum samples taken from AG129 mice administered a prime and boost of 0.45 g (A) or a prime only of 3.0 g (B) ZIKVLPs pre and post challenge. C) Survival of 11 week old AG129 after ID challenge with 200 PFU ZIKV over a 14 day period.

[0029] FIGS. 18A-C. Protection of ZIKVLPS in BALB/c mice. A) PRNT.sub.50 and PRNT.sub.90 values (+/SD) of serum samples taken from BALB/c mice administered a prime only of 3.0 g ZIKVLPs post challenge. B) Viremia (+/SD) in serum samples from mice two days post challenge by qRT-PCR. Values are total RNA copies per reaction. C) Average weight loss (+/SD) of BALB/c mice after ID challenge with 200 PFU ZIKV over a 14 day period.

DETAILED DESCRIPTION

Definitions

[0030] As used herein, the terms isolated refers to in vitro preparation, isolation of a nucleic acid molecule such as a vector or plasmid of the invention or a virus-like particle of the invention, so that it is not associated with in vivo substances, or is substantially purified from in vitro substances. An isolated virus-like particle preparation is generally obtained by in vitro culture and propagation and is substantially free from infectious agents. As used herein, substantially free means below the level of detection for a particular infectious agent using standard detection methods for that agent. As used herein, the term recombinant nucleic acid or recombinant DNA sequence or segment refers to a nucleic acid, e.g., to DNA, that has been derived or isolated from a source, that may be subsequently chemically altered in vitro, so that its sequence is not naturally occurring, or corresponds to naturally occurring sequences that are not positioned as they would be positioned in the native genome. An example of DNA derived from a source, would be a DNA sequence that is identified as a useful fragment, and which is then chemically synthesized in essentially pure form. An example of such DNA isolated from a source would be a useful DNA sequence that is excised or removed from said source by chemical means, e.g., by the use of restriction endonucleases, so that it can be further manipulated, e.g., amplified, for use in the invention, by the methodology of genetic engineering.

[0031] A signal peptide (sometimes referred to as signal sequence, secretory signal, e.g., an Oikosin 15 secretory signal, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) is a short (about 5 to 30 amino acids long) peptide present at the N-terminus of proteins that are destined towards the secretory pathway. These proteins include those that reside either inside certain organelles (the endoplasmic reticulum, golgi or endosomes), secreted from the cell, or inserted into most cellular membranes. Although most type I membrane-bound proteins have signal peptides, the majority of type II and multi-spanning membrane-bound proteins are targeted to the secretory pathway by their first transmembrane domain, which biochemically resembles a signal sequence except that it is not cleaved. Signal sequences generally have a tripartite structure, consisting of a hydrophobic care region (h-region) flanked by an n- and c-region. The latter contains the signal peptidase (SPase) consensus cleavage site. Usually, signal sequences are cleaved off co-translationally, the resulting cleaved signal sequences are termed signal peptides.

Exemplary Embodiments

[0032] Zika virus infection transmitted by Aedes mosquitoes is now receiving considerable attention due to its associated with microcephaly and Guillain-Barre syndrome. According to the CDC, there have been over 500 cases of travel-related Zika infections in America to date, with no locally-acquired vector-borne cases reported; in contrast, over 700 cases have been reported in US territories, of which nearly all were locally-transmitted.

[0033] Computational analysis has identified ZIKV envelope glycoproteins as a good candidate for vaccine development, as these are the most immunogenic (Shawan, 2015). Several approaches are currently being explored to develop a ZIKV vaccine, including inactivated, recombinant live-attenuated viruses, protein subunit vaccines, or DNA vaccines. A VLP vaccine approach against ZIKV may eliminate concerns of live attenuated vaccines and insufficient inactivation of killed vaccines for pregnant women and other populations at high risk of suffering the devastating effects of ZIKV infections.

[0034] VLPs are structurally mimic the conformation of native virions but do not generate progeny viruses (VLPs are non-infectious) and do not contain any viral genetic material. VLPs are known to be highly immunogenic and elicit higher titer neutralizing antibody responses than subunit vaccines based on individual proteins (Wang et al., 2013). Such VLPs present viral spikes and other surface components that display linear or conformational epitopes in a repetitive array that effectively results in recognition by B-cells (Metz and Pijlman, 2016). This recognition leads to B cell signaling and MHC class II up-regulation that facilitates the generation of high titer specific antibodies. VLPs from viruses, including hepatitis B virus, West Nile virus and Chikungunya virus, elicit high titer neutralizing antibody responses that contribute to protective immunity in preclinical animal models and in humans (Akahata et al., 2010; Spohn et al., 2010; Wang et al., 2012).

[0035] As mentioned above, a VLP vaccine approach against ZIKV eliminates concerns of live attenuated vaccines and insufficient inactivation of killed vaccines for pregnant women and other populations at high risk of suffering the devastating effects of ZIKV infections. The generation of ZIKV-VLPs containing the prM and E genes as well as the immunogenicity and efficacy testing in the AG129 mouse model is described herein. A position in the secretory signal was identified that likely allows for higher than normal levels of VLP secretion, due to the absence of an auto (NS2b-3) cleavage signal. Using bioinformatic signal sequence prediction tools, the putative signal sequences of ZIKV starting from positions aa 98-aa 112 were examined, and a site was selected that putatively resulted in the highest secretion score. The prM and E genes from ZIKV (Colombian isolate; GenBank accession no. KU646827) were combined with a secretory signal (positions aa 98-aa 112), were cloned into a mammalian expression vector (pCMV-prM/E). HEK-293 cells were transfected and supernatants were harvested from the cells at approximately 10 days post transfection. Transfected HEK-293 cells secreted VLPs with relatively high yields, likely due to the inclusion of a secretory signal that allows for higher than normal levels of VLP secretion. The cell supernatants contained a fraction of extracellular particles that were purified by ultracentrifugation though a sucrose cushion. These particles reacted with known ZIKV antibodies by Western Blot. Western blot analysis also revealed relatively high yields of VLPs after purification, indicating the potential for scalable production. To test the efficacy of this VLP vaccine, AG129 mice susceptible to ZIKV were vacinated with 2 g of total protein (about 400-500 ng of VLPs) formulated with 1 mg of adjuvant, and the mice boosted with the same vaccine two weeks later. At two weeks post boost, serum from vaccinated animals was collected and tested for anti-ZIKV neutralizing antibodies. Three weeks post boost mice were challenged with 200 PFU of ZIKV (about 400 LD.sub.50s). All control animals (n=6) died by 9 days post challenge, while vaccinated mice survived with no morbidity/illness (as of 11 days post-challenge). Passive transfer of antibodies from vaccinated mice was efficacious in protecting susceptible mice from Zika infections. Thus, the present findings show the protective efficacy of a ZIKV-VLP vaccine and highlight the important role that neutralizing antibodies play in protection against ZIKV infection. Further, passive transfer may be employed as a treatment for immune-compromised patients that cannot receive a vaccine.

[0036] In one embodiment, a recombinant nucleic acid vector is provided comprising a heterologous promoter operably linked to a sequence encoding ZIKV, prM/E. In one embodiment, the vector lacks nucleic acid sequences encoding ZIKV NS1, NS2A, NS2B, NS3, NS4A, NS4B or NS5 and optionally lacks nucleic acid sequences encoding functional ZIKV capsid, e.g., a protein that aggregates so as to form a viral capsid having a diameter of about 50 to 60 nm. In one embodiment, the heterologous promoter is expressed in mammalian cells. In one embodiment, the heterologous promoter is a heterologous viral promoter. In one embodiment, only a portion of ZIKV capsid sequences is included, e.g., a C-terminal portion of a ZIKV capsid that is linked to prM/E sequences as in the polyprotein that is expressed by wild-type flavivirus. In one embodiment, the portion of the capsid sequence includes amino acids 98 to 112 of the capsid protein encoded by SEQ ID NO:1 or a protein having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 99% or more amino acid sequence identity thereto. In one embodiment, the prM/E sequences have at least 80% %, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 99% or more amino acid sequence identity to the prM/E sequences encoded by any one of SEQ ID Nos. 1-3 or 5. In one embodiment, the portion of the capsid sequence lacks a NS2B-3 cleavage site. In one embodiment, the prM/E sequences are operably linked to a heterologous secretion signal. In one embodiment, the vector further comprises an intron and/or enhancer sequence, e.g., 5 to a prM/E coding sequence.

[0037] A recombinant host cell comprising the vector is also provided. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human or simian cell. In one embodiment, the genome of the cell is augmented, e.g., stably augmented, with nucleic acid sequences encoding ZIKV NS2B, e.g., the source of NS2B may be heterologous or homologous to the source for prM/E. In one embodiment, the genome of the cell is augmented, e.g., stably augmented, with nucleic acid sequences encoding ZIKV capsid, e.g., the capsid may be heterologous or homologous to prM/E. In one embodiment, the vector is integrated into the genome of the host cell.

[0038] Also provided is a method to prepare ZIKV VLPs. The method includes contacting a culture of isolated host cells that do not express ZIKV NS1, NS2A, NS2B, NS3, NS4A, NS4B or NS5 and optionally do not express functional ZIKV capsid, with the recombinant vector and collecting VLPs from supernatant of the culture. Thus, in one embodiment, the isolated host cells do not have ZIKV sequences prior to contact with the vector. In one embodiment, the collected particles have a diameter of about 10 to 100 nm, e.g., 20 to 60 nm, 40 to 70 nm or 40 to 60 nm. In one embodiment, the host cell expresses ZIKV NS2B. In one embodiment, the host cell expresses ZIKV capsid protein and optionally NS2B.

[0039] Further provided is a preparation comprising a ZIKV VLPs. The VLP comprises a lipid bilayer comprising ZIKV prM/E but lacks ZIKV NS1, NS2A, NS2B, NS3, NS4A, NS4B or NS5 and optionally lacks functional ZIKV capsid. Such a preparation may be used in a vaccine or immunogenic composition. The vaccine or immunogenic composition may have about 10 to 1000 g, e.g., 200 to 400 g or 400 to 800 g, or about 1 to about 500 mg, e.g., about 20 to 50 mg, about 100 to 300 or about 300 to 400 mg, of VLP. The vaccine or immunogenic composition may further comprise one or more adjuvants. In one embodiment, an adjuvant is included at about 0.01 to about 10 mg, about 1 to about 20 mg, or about 10 mg to about 100 mg.

[0040] Further provided is a method to prevent, inhibit or treat ZIKV infection in a mammal. The method includes administering an effective amount of the recombinant vector, a host cell having the vector or the vaccine or immunogenic composition having the VLPs. In one embodiment, the mammal is a female mammal. In one embodiment, the vector, host cell, vaccine or immunogenic composition is administered intradermally, intramuscularly or intravenously to the mammal.

[0041] In one embodiment, a method to passively prevent, inhibit or treat ZIKV infection in a mammal is provided. The method includes obtaining serum or plasma having anti-ZIKV antibodies from a mammal exposed to ZIKV and optionally isolating antibodies from the serum or plasma; and administering an effective amount of the serum or plasma, or isolated antibodies, to a different mammal at risk of or having a ZIKV infection. In one embodiment, the mammal is immunocompromised. In one embodiment, the anti-flavivirus antibodies are isolated from the serum before administration. In one embodiment, the mammal is a human.

Exemplary Adjuvants

[0042] Adjuvants are compounds that enhance the specific immune response against co-inoculated antigens. Adjuvants can be used for various purposes: to enhance the immunogenicity of highly purified or recombinant antigens; to reduce the amount of antigen or the number of immunizations needed for protective immunity; to prime the efficacy of vaccines in newborns, the elderly or immuno-compromised persons; or as antigen delivery systems for the uptake of antigens by the mucosa. Ideally, adjuvants should not induce immune responses against themselves and promote an appropriate immune response (i.e., cellular or antibody immunity depending on requirements for protection). Adjuvants can be classified into three groups: active immunostimulants, being substances that increase the immune response to the antigen; carriers being immunogenic proteins that provide T-cell help; and vehicle adjuvants, being oil emulsions or liposomes that serve as a matrix for antigens as well as stimulating the immune response.

[0043] Adjuvant groups include but are not limited to mineral salt adjuvants, e.g., alum-based adjuvants and salts of calcium, iron and zirconium; tensoactive adjuvants, e.g, Quil A which is a saponin derived from an aqueous extract from the bark of Quillaja saponaria: Saponins induce a strong adjuvant effect to T-dependent as well as T-independent antigens. Other adjuvant groups are bacteria-derived substances including cell wall peptidoglycan or lipopolysaccharide of Gram-negative bacteria, that enhance immune response against co-administered antigens and which is mediated through activation of Toll-like receptors; lipopolysaccharides (LPS) which are potent B-cell mitogens, but also activate T cells; and trehalose dimycolate (TCM), which simulates both humoral and cellular responses.

[0044] Other adjuvants are emulsions, e.g., oil in water or water in oil emulsions such as FIA (Freund's incomplete adjuvant), Montanide, Adjuvant 65, and Lipovant; liposomes, which may enhance both humoral and cellular immunity; polymeric adjuvants such as biocompatible and biodegradable microspheres; cytokines; carbohydrates; inulin-derived adjuvants, e.g., gamma inulin, a carbohydrate derived from plant roots of the Compositae family, is a potent humoral and cellular immune adjuvant and algammulin, which is a combination of -inulin and aluminium hydroxide. Other carbohydrate adjuvants include polysaccharides based on glucose and mannose including but not limited to glucans, dextrans, lentinans, glucomannans, galactomannans, levans and xylans.

[0045] Some well known parenteral adjuvants, like MDP, monophosphoryl lipid A (MPL) and LPS, also act as mucosal adjuvants. Other mucosal adjuvants poly(DL-lactide-coglycolide) (DL-PLG), cellulose acetate, iminocarbonates, proteinoid microspheres, polyanhydrides, dextrans, as well as particles produced from natural materials like alginates, geletine and plant seeds.

[0046] Adjuvants for DNA immunizations include different cytokines, polylactic microspheres, polycarbonates and polystyrene particles.

[0047] In one embodiment, adjuvants useful in the vaccines, compositions and methods described herein include, but are not limited to, mineral salts such as aluminum salts, calcium salts, iron salts, and circonium slats, saponin, e.g., Quid A including QS21, squalene (e.g., AS03), TLR ligands, bacterial MDP (N-acetyl muramyl-L-alanyl-D-isoglutamine), lipopolysaccharide (LPS), Lipid A, montanide, Adjuvant 65, Lipovant, Incomplete Freund's adjuvant (IFA), liposmes, microparticles formed of, for example, poly(D,L-lactide (coglycolide)), cytokines, e.g., IFN-gamma or GMCSF, or carbohydrates such as gamma inulin, glucans, dextrans, lentinans, glucomannans and/or glactomannans.

Pharmaceutical Compositions

[0048] Pharmaceutical compositions of the present invention, suitable for inoculation or for parenteral or oral administration, comprise flavivirus VLPs, optionally further comprising sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The compositions can further comprise auxiliary agents or excipients, as known in the art. See, e.g., Berkow et al., 1987; Avery's Drug Treatment, 1987. The composition of the invention is generally presented in the form of individual doses (unit doses).

[0049] Vaccines may contain about 0.1 to 500 ng, 0.1 to 500 g, or 1 to 100 g, of VLPs. In one embodiment, the vaccine may contain about 100 g to about 500 g of VLPs. In one embodiment, the vaccine may contain about at least 100 ng of VLPs. In one embodiment, the vaccine may contain about at least 500 ng of VLPs. In one embodiment, the vaccine may contain about at least 1000 ng of VLPs. In one embodiment, the vaccine may contain about at least 50 g of VLPs. In one embodiment, the vaccine may contain less than about 750 g of VLPs. In one embodiment, the vaccine may contain less than about 250 g of VLPs. In one embodiment, the vaccine may contain less than about 100 g of VLPs. In one embodiment, the vaccine may contain less than about 40 g of VLPs. The vaccine forming the main constituent of the vaccine composition of the invention may comprise a combination of different flavirus VLPs, for example, at least two of the three types, Chinese, West African or East African.

[0050] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and/or emulsions, which may contain auxiliary agents or excipients known in the art. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water. Besides the inert diluents, such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents. See, e.g., Avery's, 1987.

[0051] When a composition of the present invention is used for administration to an individual, it can further comprise salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the composition. For vaccines, adjuvants, substances which can augment a specific immune response, can be used. Normally, the adjuvant and the composition are mixed prior to presentation to the immune system, or presented separately, but into the same site of the organism being immunized. Examples of materials suitable for use in vaccine compositions are provided.

[0052] A pharmaceutical composition according to the present invention may further or additionally comprise at least one chemotherapeutic compound, for example, immunosuppressants, anti-inflammatory agents or immune enhancers, chemotherapeutics including, but not limited to, gamma globulin, amantadine, guanidine, hydroxybenzimidazole, interferon-, interferon-, interferon-, tumor necrosis factor-alpha, thiosemicarbarzones, methisazone, rifampin, ribavirin, a pyrimidine analog, a purine analog, foscarnet, phosphonoacetic acid, acyclovir, dideoxynucleosides, a protease inhibitor, or ganciclovir.

[0053] The composition can also contain variable but small quantities of endotoxin-free formaldehyde, and preservatives, which have been found safe and not contributing to undesirable effects in the organism to which the composition is administered.

Pharmaceutical Purposes

[0054] The administration of the composition (or the antisera that it elicits) may be for either a prophylactic or therapeutic purpose. When provided prophylactically, the compositions of the invention which are vaccines, are provided before any symptom of a pathogen infection becomes manifest. The prophylactic administration of the composition serves to prevent or attenuate any subsequent infection or one or more symptoms associated with the disease.

[0055] When provided therapeutically, a VLP vaccine is provided upon the detection of a symptom of actual infection. The therapeutic administration of the vaccine serves to attenuate any actual infection. See, e.g., Avery, 1987.

[0056] Thus, a VLP vaccine composition of the present invention may thus be provided either before the onset of infection (so as to prevent or attenuate an anticipated infection) or after the initiation of an actual infection.

[0057] A composition is said to be pharmacologically acceptable if its administration can be tolerated by a recipient patient. Such an agent is said to be administered in a therapeutically effective amount if the amount administered is physiologically significant. A composition of the present invention is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient, e.g., enhances at least one primary or secondary humoral or cellular immune response against at least one strain of an infectious flavivirus.

[0058] The protection provided need not be absolute, i.e., the flavivirus infection need not be totally prevented or eradicated, if there is a statistically significant improvement compared with a control population or set of patients. Protection may be limited to mitigating the severity or rapidity of onset of symptoms of the flavivirus infection.

Pharmaceutical Administration

[0059] A composition of the present invention may confer resistance to one or more pathogens, e.g., one or more flavivirus strains, by either passive immunization or active immunization. In active immunization, an inactivated or attenuated live vaccine composition is administered prophylactically to a host (e.g., a mammal), and the host's immune response to the administration protects against infection and/or disease. For passive immunization, the elicited antisera can be recovered and administered to a recipient suspected of having an infection caused by at least one flavivirus strain.

[0060] In one embodiment, the vaccine or immune serum is provided to a mammalian female (at or prior to pregnancy or parturition), under conditions of time and amount sufficient to cause the production of an immune response which serves to protect both the female and the fetus or newborn (via passive incorporation of the antibodies across the placenta or in the mother's milk).

[0061] The present invention thus includes methods for preventing or attenuating a disorder or disease, e.g., an infection. As used herein, a vaccine is said to prevent or attenuate an infection if its administration results either in the total or partial attenuation (i.e., suppression) of a symptom or condition of the infection, or in the total or partial immunity of the individual to the disease.

[0062] At least one VLP or composition thereof, of the present invention may be administered by any means that achieve the intended purposes, using a pharmaceutical composition as previously described.

[0063] For example, administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, oral or transdermal routes. Parenteral administration can be by bolus injection or by gradual perfusion over time. One mode of using a pharmaceutical composition of the present invention is by intramuscular or subcutaneous application. See, e.g., Avery, 1987.

[0064] A typical regimen for preventing, suppressing, or treating a flavivirus related pathology, comprises administration of an effective amount of a vaccine composition as described herein, administered as a single treatment, or repeated as enhancing or booster dosages, over a period up to and including between one week and about 24 months, or any range or value therein.

[0065] According to the present invention, an effective amount of a composition is one that is sufficient to achieve a desired biological effect. It is understood that the effective dosage will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect wanted. The ranges of effective doses provided below are not intended to limit the invention and represent suggested dose ranges. However, the dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art. See, e.g., Avery's, 1987; and Ebadi, 1985.

[0066] The invention will be further described by the following non-limiting examples.

EXAMPLE 1

Experimental Procedures

Cells and Viruses

[0067] African Green Monkey kidney cells (Vero) and Human embryonic kidney 293 (HEK293) were obtained from ATCC (ATCC; Manassas, Va., USA) and grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, Utah), 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 100 U/mL of penicillin, 100 g/mL of streptomycin, and incubated at 37 C. in 5% CO.sub.2. ZIKV strain H/PF/2013 (GenBank:KJ776791), was obtained from Xavier de Lamballerie (European Virus Archive, Marseille France). Virus stocks were prepared by inoculation onto a confluent monolayer of Vero cells.

Animals

[0068] Mice of the 129/Sv background deficient in alpha/beta interferon (IFN-/) and IFN- receptors (AG129 mice) were obtained from B&K Universal Limited (Hull, England) and were bred in the pathogen-free animal facilities of the University of Wisconsin-Madison School of Veterinary Medicine. Groups of mixed sex mice were used for all experiments.

Production and purification of ZIKV VLPs

[0069] The prM and E genes of ZIKV strain H/PF/2013 with nascent signal sequence were cloned into a pCM/V expression vector under the control of a cytomegalovirus (CMV) promoter and CMV polyadenylation signal (pCMV-prM/E). Endotoxin free, transfection grade DNA was prepared using Maxiprep kit (Zymo Research, Irvine, Calif.). VLPs were expressed by transfecting 90% confluent monolayers of HEK293 cells in a T-75 flasks with 15 g of pCMV-prM/E using Fugene HD (Promega, Madison, Wis.) transfection reagent according to manufacturer protocol. The 10 ml supernatant was harvested 72 hours after transfection, and clarified by centrifugation at 15,000 RCF for 30 minutes at 4 C. Clarified supernatants were layered onto a 20% sucrose cushion and ultra-centrifuged in a SW-28 rotor at 112,000 RCF for 3.5 hours at 4 C. Pellet (PT) and supernatant (SUP) fractions at each step were saved for analysis by SDS-PAGE and Western blot. Post sucrose cushion PT were resuspended in Phosphate Buffered Saline (PBS) pH 7.2. Total protein in VLP preparations was quantified by Bradford assay. VLP specific protein was determined by comparing Zika specific bands on SDS-PAGE gels to known concentrations of BSA using ImageJ software.

Western Blot

[0070] VLP fractions were boiled in Laemmli sample buffer (BioRad, Hercules, Calif., USA) and resolved on a 4-20% SDS-PAGE gel (Biorad) by electrophoresis using a Mini-PROTEAN 3 system (BIO-RAD, CA). Gels were electroblotted onto nitrocellulose membranes using a Turboblot system. Membranes were blocked in 5% (W/V) skim milk and probed with mouse hyper immune ascites fluid primary antibody (1:5000) and goat anti-mouse HRP conjugated secondary antibody (1:5000). Membranes were developed using a solid phase 3,3,5,5-tetramethylbenzidine (TMB) substrate system.

Transmission Electron Microscopy

[0071] Samples were negatively stained for electron microscopy using the drop method. A drop of sample was placed on a Pioloform (Ted Pella, Inc.) carbon-coated 300 Mesh Cu grid, allowed to adsorb for 30 seconds, and the excess removed with filter paper. Next, a drop of methylamine tungstate or uranyl acetate (Nano-W, Nanoprobes Inc.) was placed on the still wet grid, and the excess removed. The negatively stained sample was allowed to dry, and was documented in a Philips CM120 (Eindhoven, The Netherlands) transmission electron microscope at 80 kV. Images were obtained using a SIS MegaView III digital camera (Soft Imaging Systems, Lakewood, Color.).

Vaccination and Viral Challenge

[0072] For VLP formulations, 0.45 g of sucrose cushion purified VLPs was mixed with 0.2% Imject Alum (Thermo Scientific) according to manufacturer's protocol. Groups of AG129 mice were injected intramuscularly (IM) with VLPs mixed with alum (n=5) or PBS mixed with alum (n=6) at 6 weeks of age, and again at 8 weeks of age. Sub-mandibular blood draws were performed pre boost and pre challenge to collect serum for analysis by neutralization assays and for passive transfer studies.

[0073] Vaccinated mice were challenged with 200 PFU of ZIKV strain H/PF/2013 in 25 l volumes by intradermal (ID) injection into the right hind footpad. Following infection, mice were monitored daily for the duration of the study. Mice that were moribund or that lost greater than 20% of starting weight were humanely euthanized. Sub-mandibular blood draws were performed on day two post challenge (PC) and serum collected to measure viremia.

[0074] For passive transfer studies, 5 naive mice were injected intraperitoneally (IP) with 500 l of pooled serum from VLP vaccinated, diluted serum (1:5 n=4, 1:10, n=4), or serum from PBS/alum (n=5) treated mice. At 12 hours post transfer, mice were challenged with 20 PFU in 25 l as above.

Viremia Assays

[0075] Viremia was determined by TCID50 assay. Briefly, serum was serially diluted ten-fold in microtiter plates 263 and added to duplicate wells of Vero cells in 96-well plates, incubated at 37 C. for 5 days, then fixed and 264 stained with 10% (W/V) crystal violet in 10% (V/V) formalin. Plates were observed under a light microscope to determine the 50% tissue culture infective doses (TCID50s). Serum samples were also tested for viral RNA copies by qRT-PCR. RNA was extracted from 0.02 ml of serum using the ZR Viral 267 RNA Kit (Zymo Research, Irvine, Calif.). Viral RNA was quantified by qRT-PCR using the primers and probe designed by Lanciotti et al. (Lanciotti et al., 2008). The qRT-PCR was performed using the iTaq Universal Probes One-Step Kit (BioRad, Hercules, Calif.) on an iCycler instrument (BioRad, Hercules, Calif.). Primers and probe were used at final concentrations of 500 nM and 250 nM respectively. Cycling conditions were as follows: 50 C. for 10 minutes and 95 C. for 2 minutes, followed by 40 cycles of 95 C. for 15 seconds and 60 C. for 30 seconds. Virus concentration was determined by interpolation onto an internal standard curve made up of a 5-point dilution series of in vitro transcribed RNA.

Neutralization Assay

[0076] Serum antibody titers were deteiliiined by microneutralization assay. Briefly, serum was incubated at 56 C. for 30 minutes to inactivate complement and then serially diluted two-fold in microtiter plates. 200 PFUs of virus were added to each well and incubated at 37 C. for 1 hour. The virus-serum mixture was added to duplicate wells of Vero cells in 96-well plates, incubated at 37 C. for 5 days, then fixed and stained with 10% (W/V) crystal violet in 10% (V/V) formalin, then observed under a light microscope. The titer was determined as the serum dilution resulting in the complete neutralization of the virus.

Plaque Reduction Neutralization Test

[0077] Serum samples were serially diluted, mixed with 200 PFU of the ZIKV H/PF/2013 strain and incubated for 1 hour at 37 C. This serum/virus mixture was added to confluent layers of Vero cells in 96 well plates and incubated for 1 hour at 37 C., after which the serum/virus mixture was removed and overlay solution (3% CMC, 1DMEM, 2% FBS and 1Anti/Anti) was added. After 48 hours of infection, the monolayers were fixed with 4% PFA, washed twice with PBS, and then incubated with ZIKV hyperimmune mouse ascitic fluid (1:2000, UTMB) diluted in blocking solution (1PBS, 0.01% Tween-20 and 5% Milk) and incubated overnight at 4 C. Plates were washed three times with PBS-T and then peroxidase-labeled goat anti-mouse secondary antibody (1:2000) was incubated on monolayers for 2 hours at 37 C. Following incubation, cells were washed a final three times with PBS-T and developed using 3-amino-9-ethylcarbazole (AEC)-peroxidase substrate. The amount of formed foci were counted using an 292 ELISPOT plate reader (ImmunoSPOT-Cellular Technology); quality control was performed to each scanned well to ensure accurate counting. Neutralization percentages (Nx) were calculated per sample/replicate/dilution as follows:

[00001]$\mathrm{Nx}=\{100-[100.Math.\left(\frac{A}{\mathrm{Control}}\right)$

Where A corresponds to the amount of foci counted in the sample and Control is the geometric mean of foci counted from wells treated with cells and virus only. Data of corresponding transformed dilutions (Log(1/Dilution)) against neutralization percentages per sample was plotted and fitted to a sigmoidal dose-299 response curve to interpolate PRNT.sub.50 and PRNT.sub.90 values (GraphPad Prism software).

TABLE-US-00001 SEQIDNO:9: mknpkkksggfrivnmlkrgvarvspfgglkrlpaglllghgpirmvlailaflrftaik pslglinrwgsvgkkeameiikkfkkdlaamlriinarkekkrrgadtsvgivgllltta maaevtrrgsayymyldrndageaisfpttlgmnkcyiqimdlghmcdatmsyecpmlde gvepddvdcwcnttstwvvygtchhkkgearrsrravtlpshstrklqtrsqtwlesrey tkhlirvenwifrnpgfalaaaaiawllgsstsqkviylvmilliapaysircigvsnrd fvegmsggtwvdvvlehggcvtvmaqdkptvdielvtttvsnmaevrsycyeasisdmas dsrcptqgeayldkqsdtqyvckrtlvdrgwgngcglfgkgslvtcakfacskkmtgksi qpenleyrimlsvhgsqhsgmivndtghetdenrakveitpnspraeatlggfgslgldc eprtgldfsdlyyltmnnkhwlvhkewfhdiplpwhagadtgtphwnnkealvefkdaha krqtvvvlgsqegavhtalagaleaemdgakgrlssghlkcrlkmdklrlkgvsyslcta aftftkipaetlhgtvtvevqyagtdgpckvpaqmavdmqtltpvgrlitanpviteste nskmmleldppfgdsyivigvgekkithhwhrsgstigkafeatvrgakrmavlgdtawd fgsvggalnslgkgihqifgaafkslfggmswfsqiligtllmwlglntkngsislmcla lggvliflstavsadvgcsvdfskketrcgtgvfvyndveawrdrykyhpdsprrlaaav kqawedgicgissvsrmenimwrsvegelnaileengvqltvvvgsvknpmwrgpqrlpv pvnelphgwkawgksyfvraaktnnsfvvdgdtlkecplkhrawnsflvedhgfgvfhts vwlkvredyslecdpavigtavkgkeavhsdlgywiesekndtwrlkrahliemktcewp kshtlwtdgieesdliipkslagplshhntregyrtqmkgpwhseeleirfeecpgtkvh veetcgtrgpslrsttasgrvieewccrectmpplsfrakdgcwygmeirprkepesnlv rsmvtagstdhmdhfslgvlvillmvgeglkkrmttkiiistsmavlvamilggfsmsdl aklailmgatfaemntggdvahlaliaafkvrpallvsfifranwtpresmllalascll qtaisalegdlmvlingfalawlairamvvprtdnitlailaaltplargtllvawragl atcggfmllslkgkgsvkknlpfvmalgltavrlvdpinvvglllltrsgkrswppsevl tavglicalaggfakadiemagpmaavgllivsyvvsgksvdmyieragditwekdaevt gnsprldvaldesgdfslveddgppmreiilkvvlmticgmnpiaipfaagawyvyvktg krsgalwdvpapkevkkgettdgvyrvmtrrllgstqvgvgvmqegvfhtmwhvtkgsal rsgegrldpywgdvkqdlvsycgpwkldaawdghsevqllavppgerarniqtlpgifkt kdgdigavaldypagtsgspildkcgrviglygngvvikngsyvsaitqgrreeetpvec fepsmlkkkqltvldlhpgagktrrvlpeivreaiktrlrtvilaptrvvaaemeealrg lpvrymttavnvthsgteivdlmchatftsrllqpirvpnynlyimdeahftdpssiaar gyistrvemgeaaaifmtatppgtrdafpdsnspimdtevevperawssgfdwvtdhsgk tvwfvpsvrngneiaacltkagkrviqlsrktfetefqktkhqewdfvvttdisemganf kadrvidsrrclkpvildgervilagpmpvthasaaqrrgrigrnpnkpgdeylygggca etdedhahwlearmlldniylqdgliaslyrpeadkvaaiegefklrteqrktfvelmkr gdlpvwlayqvasagitytdrrwcfdgttnntimedsvpaevwtrhgekrvlkprwmdar vcsdhaalksfkefaagkrgaafgvmealgtlpghmterfqeaidnlavlmraetasrpy kaaaaqlpetletimllgllgtvslgiffvlmrnkgigkmgfgmvtlgasawlmwlseie pariacvlivvflllvvlipepekqrspqdnqmaiiimvavgllglitanelgwlertks dlshlmgrreegatigfsmdidlrpasawaiyaalttfitpavqhavttsynnyslmama tqagvlfgmgkgmpfyawdfgvpllmigcysqltpltlivaiillvahvmylipglqaaa araaqkrtaagimknpvvdgivvtdidtmtidpqvekkmgqvlliavavssailsrtawg wgeaqalitaatstlwegspnkywnsstatslcnifrgsylagasliytvtrnaglvkrr gggtgetlgekwkarlnqmsalefysykksgitevcreearralkdgvatgghavsrgsa klrwlvergylqpygkvidlgcgrggwsyyaatirkvqevkgytkggpgheepmlvqsyg wnivrlksgvdvfhmaaepcdtllcdigesssspeveeartlrvlsmvgdwlekrpgafc ikvlcpytstmmetlerlqrrvggglvrvplsrnsthemywvsgaksntiksvsttsqll lgrmdgprrpvkyeedvnlgsgtravvscaeapnmkiignrierirsehaetwffdenhp yrtwayhgsyeaptqgsasslingvvrllskpwdvvtgvtgiamtdttpygqqrvfkekv dtrvpdpqegtrqvmsmvsswlwkelgkhkrprvctkeefinkvrsnaalgaifeeekew ktaveavndprfwalvdkerehhlrgecqscvynmmgkrekkqgefgkakgsraiwymwl garflefealgflnedhwmgrensgggveglglqrlgyvleemsripggrmyaddtagwd trisrfdlenealitnqmekghralalaiikytyqnkvvkvlrpaekgktvmdiisrqdq rgsgqvvtyalntftnlvvqlirnmeaeevlemqdlwllrrsekvtnwlqsngwdrlkrm avsgddcvvkpiddrfahalrflndmgkvrkdtqewkpstgwdnweevpfcshhfnklhl kdgrsivvpcrhqdeligrarvspgagwsiretaclaksyaqmwqllyfhrrdlrlmana icssvpvdwvptgrttwsihgkgewmttedmlvvwnrvwieendhmedktpvtkwtdipy lgkredlwcgslighrprttwaenikntvnmvrriigdeekymdylstqvrylgeegstp gvl

RESULTS

Expression and Purification of Soluble, Zika VLPs

[0078] To generate Zika VLPs (ZIKVLPs), the prM/E genes with a native signal sequence were cloned into a pCMV expression vector (pCMV-prM/E) (FIG. 1A), transfected HEK293 cells and harvested supernatants (supe) 3 days post transfection. 78 g total protein was recovered from post sucrose purification of which 21.6 g was VLP protein. Western blot analysis of this pCMV-prM/E supe. revealed expression of about 50 kDa size band (FIG. 1B, lane 2) that corresponded in size to the predicted size of the Zika viurs E gene, and additionally matched positive control Zika virus stocks (FIG. 1B, lane 3). To test the hypothesis that expression of Zika prM and E genes spontaneously form extracellular particles, supernatants from pCMV-prM/E and pCMV-GFP (negative control) transfected cells were centrifuged on a sucrose cushion (SC) sufficient for pelleting of flavi virus particles from cell culture proteins (Merino-Ramos et al., 2014). pCMV-prM/E SC purified pellet (pt) appeared to contain high levels of E protein, while pCMV-GFP pt. did not, indicating that staining was specific to expression of 100 prM and E genes.

[0079] To determine if the immune reactive extracellular particles were virus like in nature, transmission electron microscopy (TEM) was performed on pCMV-prM/E SC pt. material. TEM revealed flavi virus 103 like particles with a size that ranged from 30-60 nm (data not show), and a typical size of about 50 nm (FIG. 1C). High magnification images demonstrated surface structures characteristic of flaviral envelope proteins (FIGS. 1D, E).

Administration of ZIKVLPs is Immunogenic and Protects Highly ZIKV Susceptible // Interferon Deficient Mice

[0080] Mice that received ZIKVLPs developed low levels (GMT=1:9.2) of neutralizing antibodies (nAbs) at 109 two weeks post administration, that increased two weeks after boost (GMT=1:32). Five weeks after primary vaccination, all mice were challenged with 200 PFU of ZIKV by the ID route. Mice administered ZIKVLP maintained weight, while mice that received PBS/alum experienced significant weight loss associated morbidity throughout the challenge period.

[0081] All control mice (n=6) died 9 days after ZIKV challenge. Mice administered ZIKVLP survived with no apparent morbidity. Finally, ZIKVLP vaccinated mice had significantly lower levels of viremia on day 2 post challenge than control mice detected by qRT-PCR (p=0.0356) and 116 TCID50 assay (p=0.0493).

ZIKVLPs Elicit Plaque Reducing Neutralizing Antibody Titers in Mice That Can Be Passively Transferred to Nave Mice.

[0082] The plaque reduction neutralization test (PRNT) assay is widely considered to be the gold standard for characterizing and quantifying circulating levels of anti-dengue and other flaviviral neutralizing antibodies (nAb) (Thomas et al., 2009). A PRNT assay was developed for rapidly measuring ZIKV specific neutralizing antibodies. Pooled serum samples collected from mice pre-challenge, as well as individual serum samples collected from mice post-challenge were tested by this PRNT assay. Pre-challenge, pooled serum from mice administered ZIKVLP had a calculated 90% plaque reduction (PRNT.sub.90) titer of 1:34. The PRNT.sub.90 titer increased 2 weeks post challenge (GMT=126 662).

[0083] To test the role of anti-ZIKV antibodies in protection against challenge, groups of mice received ZIKVLP 128 antiserum, undiluted (n=5), diluted 1:5 (n=4), or 1:10 (n=4). As a negative control mice (n=5) were transferred serum from mice previously vaccinated with PBS alum.

[0084] Negative control mice rapidly lost weight starting after day 7 and all died day 9 post challenge. Mice that received undiluted serum maintained weight throughout the 12 day period post challenge, and showed no signs of infection. Mice that received diluted anti-ZIKV antibodies were not protected from challenge, although survival and weigh loss were slightly extended relative to negative control mice 134.

DISCUSSION

[0085] Most experts and public health workers agree that a Zika vaccine is urgently needed. In February 2016, the World Health Organization declared that the recent clusters of microcephaly and other neurological disorders in Brazil constitute a public health emergency of international concern. Their recommendations included enhanced surveillance and research, as well as aggressive measures to reduce infection with Zika virus, particularly amongst pregnant women and women of childbearing age. ZIKV is now receiving considerable attention due to its rapid spread in the Americas, and its association with microcephaly (Mlakar et al., 2016) and Guillain-Barre syndrome (Pinto Junior et al., 2015). In our studies, we designed a ZIKV-virus-like particle (VLP) vaccine, demonstrated expression in vitro by western blot and transmission electron microscopy, and tested the protective efficacy and role of antibodies in protection in the AG129 mouse model.

[0086] Although the transfection and purification procedures for this ZIKV-VLP have yet to be optimized, we had an overall calculated yield of 2.2 mg/ml. Similar expression levels have been reported for other flavivirus VLP expression strategies (Pijlman, 2015). Future work will optimize VLP production and purification parameters, which should significantly increase both yield and purity. Stably transfected HEK cells that continuously express VLPs allow for scalable production to meet global demand for a ZIKV vaccine.

[0087] ZIKV-VLPs, formulated with alum, induced detectable neutralizing antibodies and protected animals against lethal challenge (>400 LD50s) with no morbidity or weight loss. Pre-challenge GMT neutralizing titers were 1:32, and pooled pre-challenge serum PRNT.sub.90 and PRNT.sub.50 titers were 1:34 and 1:157 respectively. At a relatively low dose of 450 ng, the present results indicate that the ZIKV VLPs are highly immunogenic. Additionally, the antibody titers we obtained are consistent with those reported for other highly immunogenic flavivirus VLP vaccines (Ohtaki et al., 2010; Pijlman, 2015).

[0088] Vaccinated mice challenged with >400 LD50s had low levels of viremia (mean=127, geometric mean=25.4 TCID50/ml) detected after challenge. Copies of RNA ZIKV genomes in serum of mice were significantly higher than levels of viremia. However, the disparity between viral genome copies and viremia has been observed for other flaviviruses including dengue (Bae et al., 2003). Since AG129 mice are highly susceptible to viral challenge, it is possible that the challenge dose given for the active vaccination study was artificially high. Additionally, methods for challenging mice from infected mosquito bite should be developed to most accurately mimic natural infection. Animal studies can determine if the ZIK VLP vaccine can protect female mice from contracting ZIKV during pregnancy using established models for such studies (Miner et al., 2016). ZIK-VLP vaccines may be tested in a non-human primate translational model which most accurately mimics human infection.

[0089] A VLP vaccine approach against ZIKV has significant advantages over other technologies as it will eliminate concerns of live attenuated vaccines and insufficient inactivation of killed vaccines for pregnant women and other populations at high risk of suffering the devastating effects of ZIKV infections. In recent years, recombinant virus-like particle (VLP)-based vaccine strategies have been frequently used for novel vaccine design. VLPs are known to be highly immunogenic and elicit higher titer neutralizing antibody responses than subunit vaccines based on individual proteins (Ariano et al., 2010).

[0090] The role of neutralizing antibodies in protecting against ZIKV was demonstrated by antibody passive transfer studies as naive AG129 mice receiving pooled serum from VLP vaccinated animals were fully protected. These results are consistent with previous findings that indicate the important role of antibodies in protecting against many mosquito-borne viruses, such as Japanese encephalitis, yellow fever and chikungunya. In this study, full protection was observed when animals received undiluted serum, with no weight loss or other clinical signs observed. While these studies highlight the importance of serum antibodies in ZIKV protection, upcoming studies will determine the minimum antibody titer needed for protection, whether the ZIKV-VLP can elicit CD8+ responses, and the overall role of cellular immunity in protection. It is also important to determine whether anti-ZIKV antibodies elicited by the VLPs play any role in dengue protection or disease enhancement.

[0091] In this study, the AG129 IFN receptor-deficient mouse model was used for evaluation of the ZIKV-VLP. Recently, the suitability of mice deficient in IFN-/ and - receptors as an animal model for ZIKV was demonstrated, as they are highly susceptible to ZIKV infection and disease, developing rapid viremic dissemination in visceral organs and brain and dying 7-8 days post-infection (Aliota et al., 2016). The AG129 mouse model exhibits an intact adaptive immune system, despite the lack of an IFN response, and it has been used extensively to evaluate vaccines and antivirals for DENV (Brewoo et al., 2012; Fuchs et al., 2014; Johnson and Roehrig, 1999; Sarathy et al., 2015).

[0092] In the present study, aluminum hydroxide (commonly known as alum) was used as the adjuvant for the ZIKV-VLP preparations. Since its first use in 1932, vaccines containing aluminum-based adjuvants have been successfully administered in humans demonstrating excellent safety. A variety of adjuvant formulations may, however, be employed with ZIKV VLPs to enhance immunogenic potential including adjuvants that facilitate antigen dose sparing, enhanced immunogenicity, and/or broadened pathogen protection.

[0093] Thus, a VLP based Zika vaccine is described herein that elicits protective antibodies in mice, and is safe, suitable for scalable production, and highly immunogenic. Fast-tracking development of this ZIKV vaccine is a public health priority and is crucial for restoring confidence and security to people who wish to have children or reside in, or visit areas in which ZIKV is endemic.

EXAMPLE 2

Exemplary Zika Virus Polyprotein Sequences:

[0094] Accession No. KU646827 (Which is Incorporated by Reference Herein)

TABLE-US-00002 (SEQIDNO:6) IRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVIVIAQDKPTVDIE LVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWG NGCGLFGKGSLVTCAKFACSKKIVITGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGH ETDENRAKIVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLMINNKHWLVHK EWTHDIPLPWELNGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGA LEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVIVEV QYAGTDGPCKVPAQIVIAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSY IVIGVGEKKITHHAVHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGK GIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTA VSADVGCSVDFSKKETRCGTGVFVYNDVEAIATRDRYKYHPDSPRRLAAAVKQAWEDG ICGISSVSRMENIMWRSVEGELNAILEENGVQLTVVVGSVKNPMWRGPQRLPVPVNEL PHGWKAWGKSYFVRAAKTNNSFVVDGDTLKECPLKHRAWNSFLVEDHGFGVFHTSVWL KVREDYSLECDPANTIGTAVKGKEAVHSDLGYWIESEKNDTWRLKRAHLIEMKTCEWP KSHTLWTDGIEESDLIIPKSLAGPLSHHNTREGYRTQMKGPWHSEELEIRFEECPGTK ATHVEETCGTRGPSLRSTTASGRVIEEWCCRECTMPPLSFWAKDGCWYGMEIRPRKEP ESNLVRSMVTAGSTDHMDHFSL (SEQIDNO:1) atcaggtgcataggagtcagcaatagggactttgtggaaggtatgtcaggtgggacttgg gttaatgtcgtcttggaacatggagattgtgtcaccgtaatggcacaagacaaaccgact gtcgacatagagctggttacaacaacagtcagcaacatggcggaggtaagatcctactgc tatgaggcatcaatatcagacatggcttcggacagccgctgcccaacacaaggtgaagcc taccttgacaagcaatcagacactcaatatgtctgcaaaagaacgttagtggacagaggc tggggaaatggatgtggactttttggcaaagggagcctggtgacatgcgctaagtttgca tgctccaagaaaatgaccgggaagagcatccagccagagaatctggagtaccggataatg ttgtcagttcatggctcccagcacagtgggatgatcgttaatgacacaggacatgaaact gatgagaatagagcgaaggttgagataacgcccaattcaccaagagccgaagccaccctg gggagttttgaaagcctaagacttgattgtgaaccgaggacaggccttaacttttcagat ttgtattacttgactatgaataacaagcactggttggttcacaaggagtggttccacgac attccattaccttggcacgctggggcagacaccggaactccacactggaacaacaaagaa gcactggtagagttcaaggacgcacatgccaaaaggcaaactgtcgtggttctagggagt caggaagaagcagttcacacgacccttgctggagctctggaggctgagatgaatggtgca aagggaaggctgtcctctggccacttgaaatgtcgcctgaaaatggacaaacttagattg aagggcgtgtcatactccttgtgtaccgcagcgttcacattcaccaagatcccggctgaa acactgcacgggacagtcacagtggaggtacagtacgcagggacagatggaccttgcaag gttccagctcagatggcgatggacatgcaaactctgaccccagttgggaggttgataacc gctaaccccgtaatcactgaaagcactgagaactctaagatgatgctggaacttgatcca ccatttggggactcttacattgtcataggagtcggggagaagaagatcacccaccactgg cacaggagtggcagcaccattggaaaagcatttgaagccactgtgagaggtgccaagaga atggcagtcttgggagacacaacctgggactttggatcagttggaggcgctctcaactca ttgggcaagggcatccatcaaatttttggagcagctttcaaatcattgtttggaggaatg tcctggttctcacaaattctcattggaacgttgctgatgtgattggatctgaacacaaag aatggatctatttcccttatgtgcttggccttagggggagtgttgatcttcttatccaca gccatctctgctgatgtgaggtgctcggtggacttctcaaagaaggagacgagatatggt acaggggtgttcgtctataacgacgttgaagcctggagggacaggtacaagtaccatcct gactccccccgtagattggcagcagcagtcaagcaagcctgggaagatggtatctgcggg atctcctctgtttcaagaatggaaaacatcatgtggagatcagtagaaggggagctcaac gcaatcctggaagagaatggagttcaactgacggtcgttgtgggatctgtaaaaaacccc atgtggagaggtccacagagattgcccgtgcctgtgaacgagctgccccacggctggaag gcttaggggaaatcgtacttcgtcaaagcagcaaagacaaataacagctttgtcgtggat ggtgacacactgaaggaatgcccactcaaacatagagcatggaacagctttcttgtggag gatcatgggttcgaggtatttcacactagtgtctggctcaaggttagagaagattattca ttagagtatgatccagccgttattggaacagctgttaagggaaaagaggctatacacagt gatctagactactgaattgagagtgagaagaatgacacatggagactgaagagggcccat ctgatcgagatgaaaacatgtgaatggccaaagtcccacacattgtggacagatggaata gaagagagtgatctgatcatacccaagtctttagctgggccactcagccatcacaatacc agagagggctacaggacccaaatgaaagggccatggcacagtgaagagcttgaaattcgg tttaaggaatacccaggcactaaggtccacgtgaaggaaacatgtggaacaagagaacca tctctgagatcaaccactgcaagcggaagggtgatcgaggaatggtgctgcagggagtgc acaatgcccccactgtcgttctgggctaaagatggctgttggtatggaatggagataagg cccaggaaagaaccagaaagcaacttagtaaggtcaatggtgactgcaggatcaactgat cacatagatcacttctccctt KU955593(full-length) (SEQIDNO:7) MKKPKKKSGGFRIVNMLKRGVARVSPFGGLKRLPAGLLLGHGPI RMVLAILAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKEKK RRGTDTSVGIVGLLLTTAMAVEVTRRGNAYYMYLDRSDAGEAISFPTTMGMNKCYIQI MDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVT LFSKSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKV IYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIE LVTTTVSNMAEVRSYCYEASISDMASDSRCFTQGEAYLDKQSDTQYVCKRTLVDRGWG NGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHET DENRAKVEITPNSPRAEATLGGFGSLGLTCEPRTGLDFSDLYYLTMNNKHWLVHKEWF HDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAE MDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAG TDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVG EKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFG AAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLMCLALGGVLIFLSTAVSADVGC SVDFSKKETRCGTGVFVYNDVEAWRDRYKYHPDSPRRLAAAVKQAWEDGICGISSVSR MENIMWRSVEGELNAILEENGVQLTVVVGSVKNPMWRGPQRLPVPVNELPHGWKAWGK SYFVRAAKTNNSFVVDGDTLKECPLKHRAWHSFLVEDHGFGVFHTSVWLKVREDYSLE CDPAVIGTAAKGKEAVHSDLGYWIESEKNDTWRLKRAHLIEMKTCEWPKSHTLWTDGI EESDLIIPKSLAGPLSHHNTREGYRTQMKGPWHSEELEIRFEECPGTKVHVEETCGTR GPSLRSTTASGRVIEEWCCRECTMPPLSFRAKDGCWYGMEIRPRKEPESNLVRSMVTA GSTDHMDKFSLGVLVILLMVQEGLKKRMTTKIIISTSMAVLVAMILGGFSMSDLAKLA ILMGATFAEMNTGGDVAHLALIAAFKVRPALLVSFIFRANWTPRESMLLALASCLLQT AISALEGDLMVPINGFAIAWLAIRAMVVPRTDNITLAILAALTPLARGTLLVAWRAGL ATCGGFMLLSLKGKGSVKKNLPFVMALGLTAVRLVDPINVVGLLLLTRSGKRSWPPSE VLTAVGLICALAGGFAKADIEMAGPMAAVGLLIVSYVVSGKSVDMYIERAGDITWEKD AEVTGNSPRLDVALDESGDFSLVEDDGPPMREIILKVVLMAICGMNPIAIFFAAGAWY VYVKTGKRSGALWDVPAPKEVKKGETTDGVYRVMTRRLLGSTQVGVGVMQEGVFHTMW HVTKGSALRSGEGRLDPYWGDVKQDLVSYCGPWKLDAAWDGHSEVQLLAVPPGERARN IQTLPGIFKTKDGDIGAVALDYPAGTSGSFILDKCGRVIGLYGNGVVIKNGSYVSAIT QGRREEETPVECFEPSMLKKKQLTVLDLHPGAGKTRRVLPEIVREATKTRLRTVTLAP TRVVAAEMEEALRGLPVRYMTTAVNVTKSGTEIVDLMCHATFTSRLLQPIRVPNYNLY IMDEAHFTDPSSIAARGYISTRVEMGEAAAIFMTATPPGTRDAFPDSNSPIMDTEVEV PERAWSSGFDWVTDHSGKTVWFVPSVRNGNEIAACLTKAGKRVIQLSRKTFETEFQKT KHQEWDFVVTTDISEMGANFKADRVIDSRRCLKPVILDGERVILAGPMPVTHASAAQR RGRIGRNPNKPGDEYLYGGGCAETDEDHAHWLEARMLLDNIYLQDGLIASLYRPEADK VAAIEGEFKLRTEQRKTFVELMKRGDLPVWLAYQVASAGITYTDRRWCFDGTTNNTIM EDSVPAEVWTRYGEKRVLKPRWMDARVCSDHAALKSFKEFAAGKRGAAFGVMEALGTL PGHMTERFQEAIDNLAVLMRAETGSRPYKAAAAQLPETLETIMLLGLLGTVSLGIFFV LMRNKGIGKMGFGMVTLGASAWLMWLSEIEPARIACVLIVVFLLLVVLIPEPEKQRSP QDNQMAIIIMVAVGLLGLITANELGWLERTKSDLSHLMGRREEGATIGFSMDIDLRPA SAWAIYAALTTFITPAVQHAVTTSYNNYSLMAMATQAGVLFGMGKGMPFYAWDFGVPL LMIGCYSQLTPLTLIVAIILLVAHYMYLIPGLQAAAARAAQKRTAAGIMKNPVVDGIV VTDIDTMTIDPQVEKKMGQVLLIAVAVSSAILSRTAWGWGEAGALITAATSTLWEGSP NKYWNSSTATSLCNIFRGSYLAGASLIYTVTRNAGLVKRRGGGTGETLGSKWKARLNQ MSALEFYSYKKSGITEVCREEARRALKDGVATGGHAVSRGSAKLRWLVERGYLQPYGK VIDLGCGRGGWSYYAATIRKVQEVKGYTKGGPGHEEPMLVQSYGWNIVRLKSGVDVFH MAAEPCDTLLCDIGESSSSPEVEEARTLRVLSMVGDWLEKRPGAFCIKVLCPYTSTMM ETLERLQRRYGGGLVRVPLSRNSTHEMYWVSGAKSNTIKSVSTTSQLLLGRMDGPRRP VKYEEDVNLGSGTRAVVSCAEAPNMKIIGNRIERIRSEHAETWFFDENKPYRTWAYKG SYEAPTQGSASSLINGVVRLLSKPWDVVTGVTGIAMTDTTPYGQQRVFKEKVDTRVPD PQEGTRQVMSMVSSWLWKELGKHKRFRVCTKEEFINKVRSNAALGAIFEEEKEWKTAV EAVNDPREWALVDKEREHHLRGECQSCVINMMGKREKKQGEFGKAKGSRAIWYMWLGA RFLEFEALGFLNEDHWMGRENSGGGVEGLGLQRLGYVLEEMSRIPGGRMYADDTAGWD TRISRFDLENEALTINQMEKGHRALALAIIKYTYQNKVVKVLRPAEKGKTVMDITSRQ DQRGSGQVVTYALNTFTNLVVQLTRNMEAEEVLEMQDLWLLRRSEKVTNWLQSNGWDR LKRMAVSGDDCVVKPIDDRFAHALRFLNDMGKVRKDTQEWKPSIGWDNWEEVPFCSHH FNKLHLKDGRSIVVPCRHQDELIGRARVSPGAGWSIRETACLAKSYAQMWQLLYPHRR DLRLMANAICSSVPVDWVPTGRITWSIHGKGEWMTTEDMLVVWNRVWIEENDHMEDKT PVTKWTDIPYLGKREDLWCGSLIGHRPRTTWAENIKNTVNMMRRIIGDEEKYVDYLST QVRYLGEEGSTPGVL (SEQIDNO:2) agttgttgatctgtgtgaatcagactgcgacagttcgagtttgaagcgaaagctagcaac agtatcaacaggttttattttggatttggaaacgagagtttctggtcatgaaaaacccaa agaagaaatccggaggattccggattgtcaatatgctaaaacgcggagtagcccgtgtga gcccctttgggggcttgaagaggctgccagccggacttctgctgggtcatgggcccatca ggatggtcttggcgattctagcctttttgagattcacggcaatcaagccatcactgggtc tcatcaatagatgaggttcagtggggaaaaaagaggctatggaaataataaagaagttta agaaagatctggctaccatgctgagaataatcaatgctaggaagaagaagaagagacgaa gcacagatactagtgtcggaattgttggcctcctgctgaccacagccatggcagtggagg tcactagacgtgggaatgcatactatatgtacttggacagaagcgatgctggggaggcca tatcttttccaaccacaatggggatgaataagtgttatatacagatcatggatcttggac acatgtgtgatgccaccatgagctatgaatgccctatgctagatgaggaggtagaaccag atgacgtcgattgttggtgcaacacgacgtcaacttgggttgtgtacggaacctgccacc acaaaaaaggtgaagcacggagatctagaagagctgtgacgctcccctcccattccacta ggaagctgcaaacgcggtcgcagacctggttggaatcaagagaatacacaaagcacctga ttagagtcgaaaattggatattcaggaaccctggcttcgcgttaacagcagctgccatca cttggcttttgggaagctcaacgagccaaaaagtcatatacttggtcatgatactgctga ttgccccggcatacagcatcaggtgcataggagtcagcaatagggactttgtggaaggta tgtcaggtgggacttgggttgatgttgtcttggaacatggaggttgtgttaccgtaatgg cacaggacaaaccgactgtcgacatagagctggttacaacaacagtcaacaacatagcgg aggtaagatcctactgctatgaggcatcaatatcggacatggcttcggacagccgctgcc caacacaaggtgaagcctaccttgacaagcaatcagacactcaatatgtctgcaaaagaa cgttagtggacagaggctggggaaatggatgtggactttttggcaaagggagcctggtga catgcgctaagtttgcttgctctaagaaaatgaccgggaagagcatccagccagagaatc tggaataccggataatgctatcagttcatggctcccagcacagtgggataatcgttaatg atacaggacataaaactgatgagaatagagcgaaagttgagataacgcccaattcaccaa gagccgaagccaccctggggggttttggaagcctaggacttgattgtgaaccgaggacag gccttgacttttcagatttgtattacttgactatgaataacaagcactggttggttcaca aagagtggttccacgacattccattaccttgacatgctgaagcagacaccggaactccac actggaacaacaaagaagcactggtagagttcaaggacgcacatgccaaaaggcagactg tcgtggttctagggagtcaagaaggagcagttcacacggcccttgctggagctctggagg ctgagatagatgatacaaaggaaaggctatcctctagccacttgaaatgtcacctgaaaa tggataaacttaaattgaaggacgtgtcatactccttatgtaccacagcgttcacattca ctaaaatcccgactgaaacactgcacaggacagtcacagtgaaggtacaatacgcaagga cagatggaccttgcaaggttccagctcagatggcggtggacatgcaaactctgaccccag ttgagaggttgataaccgctaaccctgtaatcactgaaagcactgagaactccaagatga tactggaactagatccaccatttagagactcttacattgtcataggagtcgggaaaaaga agatcacccaccactagcacagaagtggcaacaccattagaaaagcatttgaagccactg tgagaggtgccaagagaatggcagtcttgggagacacagcctaggactttggatcagttg ggggtgctctcaactcactgggcaagggcatccatcaaatttttggagcagctttcaaat cattgtttggagaaatgtcctagttctcacaaattctcattgaaacgttgctggtgtagt tgggtctgaatacaaagaatggatctatttcccttatgtgcttggccttaggaggagtgt tgatcttcttatccacagccgtctctgctgatgtggggtgctcggtggacttctcaaaga aggaaacgagatgcggtacaggggtgttcgtctataacgacgttgaagcttggagggaca gatacaagtaccatcctgactcccctcgtagattggcagcagcagtcaagcaaacctggg aagatgggatctgtgagatctcctctatttcaagaatgaaaaacatcatgtgaagatcag tagaaggggagctcaacgcaatcctggaagagaatggagttcaactgacggtcgttgtgg gatctgtaaaaaaccccatgtggagaggtccacagagattgcccgtgcctgtgaacgagc tgccccatggctagaaggcttaggggaaatcgtacttcgtcaagacagcaaagacaaata acagctttgtcatggatggtgacacactgaaggaatgcccactcaaacatagagcatgga acagctttcttgtggaggatcatgagttcggggtatttcacactagtgtctggctcaagg ttagagaagattattcattagagtgtgatccagccgtcattggaacagccgctaagggaa aagaggctgtacacagtgatctaagctactgaattgagaatgagaaaaacgacacatgga agctgaagagggcccacctgatcgagatgaaaacatgtaaatggccaaagtcccacacat tgtggacagatggaatagaagaaagtgatctgatcatacccaagtctttagctgggccac tcagccatcacaacaccagagagggctacaggacccaaatgaaagggccatggcatagtg aagagcttgaaattcggtttgaggaatacccaggcactaaggtccacgtggaggaaacat gtggaacaagaagaccatctctgagatcaaccactgcaagcagaagggtaatcgagaaat ggtgctgcagggagtgcacaatgcccccactatcgttccgggctaaagatggttgttggt atggaatggagataaggcccaggaaagaaccagaaagtaacttagtaaggtcaatggtga ctgcaggatcaactgatcacatgaatcacttctcccttgaagtgcttgtgattctactca tggtacagaaagggctaaagaaaagaatgaccacaaagatcatcataagcacatcaatgg cagtgctggtagctatgatcctgggaggattttcaatgagtgacctggctaagcttgcaa ttttgatgggtgccaccttcgcggaaatgaacactggaggagatgttgctcatctggcgc tgatagcggcattcaaagtcagacctgcgttgctggtatctttcattttcagagctaatt ggacaccccgtaagagcatactgctgaccttggcctcgtgtcttctgcaaactgcgatct ccgccttggaaagcgacctaatgattcccatcaatggttttactttggcctggttgacaa tacgagcgatggttgttccacgcactgacaacatcaccttggcaatcctggctgctctga caccactggcccggggcacactgcttgtggcgtggagagcaggccttgctacttgcgggg ggttcatgctcctttctctgaagggaaaaggcaatgtgaaaaagaacttaccatttgtca tggccctgggactaaccgctgtgaggctggtcgaccccatcaacgtggtgggactgctgt tgctcacaaggagtaqgaagcggagctggccccctagtgaagtactcacagctgttggcc tgatatgcgcattgactggagagttcgccaaggcggatatagagatggctgagcccatga ccgcggtcggtctgctaattgtcagttacgtggtctcaggaaagagtgtggacatgtaca ttgaaagagcaagtgacatcacatggaaaaaagatgcggaaatcactggaaacagtcccc ggctcgatgtggcactagatgagagtggtgatttctccctagtggaggatgatggtcccc ccatgagagagatcatactcaaagtggtcctgatggccatctgtggcatgaacccaatag ccataccctttgcagctgaagcgtgatacgtgtatgtgaaaactggaaaaaggagtggtg ctctatggaatgtgcctgctcccaaggaagtaaaaaagagggagaccacagatggagtgt acagagtaatgactcgtagactgctaggttcaacacaagttggagtgggagtcatgcaag agggggtcttccacactatgtggcacgtcacaaaaggatccgcgctgagaagcggtgaag ggagacttgatccatactgggaagatgtcaagcaggatctggtgtcatactatggtccat ggaaactagataccgcctgagacgggcacagcgaagtgcagctcttggccgtgccccccg gagagagagcgaggaacatccagactctgcccggaatatttaagacaaaggatggggaca ttggagcagttgcgctggactacccagcaggaacttcaggatctccaatcctagataagt gtgagagagtaataggactctatggtaatggggtcgtgatcaaaaatgagagttacgtta atgccatcacccaagagaggagagaggaagagactcctattgagtacttcgaaccttcga tgctgaagaagaagcagctaactgtcttagacttgcatcctggagctgggaaaaccagga gagttcttcctgaaatagtccgtgaagccataaaaacaagactccgcactgtgatcttag ctccaaccagggttatcgctgctgaaatggaggaagcccttagaaggcttccagtgcgtt atataacaacaacagtcaatgtcacccattctggaacagaaatcgttgacttaatgtgcc atgccaccttcacttcacgtctactacagccaatcagagtccccaactataatctgtata ttatggatgaggcccacttcacagatccctcaagtatagcagcaagaggatacatttcaa caaaggttgaaatgggcgaggcggctgccatcttcatgactgccacgccaccaggaaccc atgacgcattcccggactccaactcaccaattatggacaccgaagtggaagtcccagaga gagcctggagctcaggctttgattgggtgacggatcattctggaaaaacagtttggtttg ttccaagcgtgaggaatggcaatgagatcgcagcttgtctgacaaaggctggaaaacggg tcatacaactcagcagaaagacttttgagacagagttccagaaaacaaaacatcaagagt gggacttcgtcatgacaactgacatttcagagataggcgccaactttaaagctgaccgtg tcatagattccaggagatgcctaaagccggtcatacttgatggcgagagagtcattctgg ctggacccatgcctgtcacacatgccagcgctgcccagaggagggggcgcataggcagga accccaacaaacctggagatgagtatctgtatgaaggtggatgcgcagagactgatgaag accatgcacactggcttgaagcaagaatgcttcttgacaacatttacctccaagatggcc tcatagcctcgctctatcgacctgaggccgacaaagtagcagctattgagggagagttca agcttaggacggagcaaaggaagacctttgtggaactcatgaaaagaggagatcttcctg tttggctggcctatcaggttgcatctgccggaataacctacacagatagaagatggtgct ttgatggcacgaccaacaacaccataatggaagacagtgtgccggcagaggtgtggacca gatacggagagaaaagagtgctcaaaccgaggtggatggacgccagagtttgttcagatc atgcggccctgaagtcattcaaagagtttgccgctgggaaaagaggagcggcctttggag tgatggaagccctgggaacactgccaggacatatgacagagagattccaggaggccattg acaacctcgctgtgctcatgcgggcagagactgaaagcagaccctacaaagccgcagcgg cccaattaccggagaccctagagactatcatgcttttggggttgctgggaacagtctcgc tgggaatctttttcgtcttgatgcggaacaagggcatagggaagatgggctttggaatgg tgactcttggggccagcgcatagcttatgtggctctcagaaattaagccagccagaatta catgtgtcctcattattgtgttcctattgctggtggtactcatacctgagccagaaaagc aaagatctccccaggacaaccaaatggcaatcatcatcatggtagcagtgggtcttctgg gcttgattaccgccaatgaactcggatggttggagagaacaaagagtgacctaagccatc taatgggaaggagagaggagggggcaactataggattctcaatggacattgacctgcggc cagcctcagcttgggctatctatgctgctctgacaactttcattaccccagccgtccaac atgcagtgaccacttcatacaacaactactccttaatggcgatggccacgcaagctggag tgttgttcggtatgggtaaagggatgccattctatgcatgggactttggagtcccgctgc taatgataggttgctactcacaattaacacccctgaccctaatagtggccatcattttgc tcgtggcacactacatgtacttgatcccagggctgcaagcagcaactgcgcatgctgccc agaagagaacggcagctggcatcatgaagaaccctgttgtggatggaatagtggtgactg acattgacacaatgacaattgacccccaagtggagaaaaagatgggacaggtgctactca tagcagtagctgtctccagcgccatactgtcgcggaccgcctgggggtggggtgaggctg gggccctgatcacagctgcaacttccactttgtaggagggctctccgaacaagtactgga actcctccacagccacctcactgtgtaacatttttaggggaagctacttggctggagctt ctctaatctacacagtaacaagaaacgctggcttgatcaagagacgtgggggtggaacgg gagagaccctgggagagaaatggaaggcccgcctgaaccagatgtcggccctggagttct actcctacaaaaagtcaggcatcaccgaggtgtgcagagaagagacccgccacgccctca aggacggtgtggcaacgggaggccacgctgtgtcccgaggaagtgcaaagctgagatggt tggtggagaggggatacctgcagccctatggaaaggtcattgatcttggatgtggcagag ggggctggagttactatgccgccaccatccgcaaagttcaagaagtgaaaggatacacaa aagaaggccctggtcatgaagaacccatgttggtgcaaagctatgggtagaacatagtcc gtcttaagagtgaggtggacgtctttcatatggcggctgagccgtgtgacacgttgctgt gtgatataggtgagtcatcatctagtcctgaagtggaagaagcacggacgctcagagtcc tctccatggtgggggattggcttgaaaaaagaccaggagccttttgtataaaagtgttgt gcccatacaccagcactatgatggaaaccctggagcgactgcagcgtaggtatgggggaa gactggtcagagtgccactctcccgcaactctacacatgagatgtactgggtctctggag cgaaaagcaacaccataaaaagtgtatccaccacgagccagctccttttggggcgcatgg acgggcccaggaggccagtgaaatatgaagaggatgtgaatctcggctctggcacgcggg ctgtggtaagctgcgctgaagctcccaacatgaagatcattggtaaccacattgaaagga tccgcagtgagcacgcggaaacgtggttctttgacgagaaccacccatataggacatggg cttaccatggaagctacgaggcccccacacaagggtcagcgtcctctctaataaacgggg ttgtcaggctcctgtcaaaaccctgggatgtggtgactggagtcacaggaatagccatga ccgacaccacaccgtatggtcagcaaagagttttcaaggaaaaagtggacactagggtgc cagacccccaaaaaggcactcgtcagattatgagcatggtctcttcctgattgtggaaag agttaggcaaacacaaacgaccacgaatctgtaccaaagaagagttcatcaacaagattc gtagcaacgcagcattaggggcaatatttgaagaggaaaaagagtggaagactgcagtgg aagctgtgaacgatccaaggttctgggctctagtggacaaggaaagagagcaccacctga gagaagagtgccagagctatgtgtacaacatgatgggaaaaagagaaaagaaacaagggg aatttggaaaggccaagggcagccgcgccatctggtacatgtggctaggggctagatttc tagagttcgaagcccttggattcttgaacgaggatcactggatgaggagagagaattcag gaggtggtgttgaaaggctagaattacaaagactcggatatgtcttagaagagatgagtc gcataccaggaggaaggatgtatgcagatgatactgctggctggaacacccacatcagca ggtttgatctgaagaatgaagctctaatcaccaaccaaatgaagaaaggacacaggacct tggcattggccataatcaagtacacataccaaaacaaagtggtaaaggtccttagaccag ctgaaaaagggaagacagttatggacattatttcaagacaagaccaaagggggagcggac aagttgtcacttacgctcttaatacatttaccaacctagtagtgcagctcattcgaaata tggaggctaaggaagttctagagatgcaagacttgtggctgctgcagaggtcagagaaag tgaccaactggttgcagagcaatggataggataggctcaaacgaatggcagtcagtggag atgattgcgttgtgaaaccaattgatgataggtttgcacatgctctcaggttcttgaatg atatgggaaaagttaggaaggacacacaagagtggaaaccctcaactggataggacaact gggaagaagttccgttttgctcccaccacttcaacaagctccatctcaaagacgggaggt ccattgtggttccctgccgccaccaagatgaactgattggccgagctcgcgtctcaccgg gggcgggatggagcatccgggagactgcttgcctagcaaaatcatatgcgcaaatgtggc agctcctttatttccacaaaagggacctccgactgatggccaatgccatttgttcatctg tgccagttaactgggttccaactgggagaactacctggtcaatccatggaaaaggagaat ggatgaccactgaagacatgcttgtggtgtggaacagagtgtggattgaggagaacgacc acatggaagacaagaccccagttacgaaatggacagacattccctatttgggaaaaaggg aagacttatggtgtaggtctctcatagggcacagaccacgcaccacctgggctgagaaca ttaaaaacacaatcaacataatgcgtaggatcataggtgataaagaaaaatacgtgaact acctatccacccaagttcgctacttgggcgaagaagggtccacacctggagtgctataag caccaatcttagtgttgtcaggcctgctagtcagccacagcttggggaaagctgtgcagc ctgtgacccccccaggagaagctggaaaaccaaacccataatcaggccaagaacgccatg acacggaaaaagccatgctgcctgtgagcccctcagagaacactgagtcaaaaaacccca cgcgcttggaggcgcaggatgagaaaagaaggtggcgaccttccccaccctttaatctgg ggcctgaactggagatcagctgtggatctccagaagagggactagtggttagaggagacc ccccggaaaacgcaaaacagcatattgacgctgggaaagaccagagactccatgagtttc caccacgctggccgccaggcacagatcgccgaatagcggcgaccggtgtagggaaatcca tgagtct KU866423 (SEQIDNO:8) MKNPKKKSGGFRIVNMLKRGVARVSPFGGLKRLPAGLLLGHGPI RMVLAILAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKEKK RRGADTNVGIVGLLLTTAMAAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQI MDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVT LPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKV IYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIE LVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWG NGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHET DENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWF HDIPLPWRAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAE MDGAKGRLSSGKLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAG TDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPFFGDSYIVIGVG EKKITHHWHRSGSTIGKAFEATVRGARRMAVLGDTAWDFGSVGGALNSLGKGIHQIFG AAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSADVGC SVDFSKKETRCGTGVFVYNDVEAWRDRYKYHPDSPRRLAAAVKQAWEDGICGISSVSR MENIMWRSVEGELNAILEENGVQLTVVVGSVKNPMWRGPQRLPVPVNELPHGWKAWGK SYFVRAAKTNNSFVVDGDTLKECPLKHRAWNSFLVEDHGFGVFHTSVWLKVREDYSLE CDPAVIGTAVKGKEAVHSDLGYWIESEKNDTWRLKRAHLIEMKTCEWPKSHTLWTDGI EESDLIIPKSLAGPLSHHNTREGYRTQMKGPWHSEELEIRFEECPGTKVHVEETCGTR GPSLRSTTASGRVIEEWCCRECTMPPLSFQAKDGCWYGMEIRPRKEFESNLVRSMVTA GSTDHMDHFSLGVLVTLLMVQEGLKKRMTTKIIISTSMAVLVAMILGGFSMSDLAKLA ILMGATFAEMNTGGDVAHLALIAAFKVRPALLVSFIFRANWTPRESMLLALASCLLQT AISALEGDLMVLINGFALAWLAIRAMVVPRTDNITLAILAALTPLARGTLLVAWRAGL ATCGGFMLLSLKGKGSVKKNLPFVMALGLTAVRLVDPINVVGLLLLTRSGKRSWPPSE VLTAVGLICALAGGFAKADIEMAGPMAAVGLLIVSYVVSGKSVDMYIERAGDITWEKD AEVTGNSPRLDVALDESGDFSLVEDDGPPMREIILKVVLMTICGMNPIAIPFAAGAWY VYVKTGKRSGALWDVPAPKEVKKGETTDGVYRVMTRRLLGSTQVGVGVMQEGVFHTMW HVTKGSALRSGEGRLDPYWGDVKQDLVSYCGPWKLDAAWDGHSEVQLLAVPPGERARN IQTLPGIFKTKDGDIGAVALDYPAGTSGSPILDKCGRVIGLYGNGVVIKNGSYVSAIT QGRREEETPVECFEPSMLKKKQLTVLDLHPGAGKTRRVLPEIVREAIKTRLRTVILAP TRVVAAEM5EALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFTSRLLQPIRVPNYNLY IMDEAHFTDPSSIAARGYISTRVEMGEAAAIFMTATPFGTRDAFPDSKSPIMDTEVEV PERAWSSGFDWVTDHSGKTVWFVPSVRNGNEIAACLTKAGKRVIQLSRKTFETEFQKT KHQEWDFVVTTDISEMGANFKADRVIDSRRCLKPVILDGERVILAGPMPVTHASAAQR RGRTGRNPNKPGDEYLYGGGCAETDEDHAHWLEARMLLDNIYLQDGLIASLYRPEADK VAAIEGEFKLRTEQRKTFVELMKRGDLPVWLAYQVASAGITYTDRRWCFDGTTNNTIM EDSVPAEVWTRHGEKRVLKPRWMDARVCSDHAALKSFKEFAAGKRGAAFGVMEALGTL PGHMTERFQEAIDNLAVLMRAETGSRPYKAAAAQLPETLETIMLLGLLGTVSLGIFFV LMRNKGIGKMGFGMVTLGASAWLMWLSEIEPARIACVLIVVFLLLVVLIPEPEKQRSP QDNQMAIIIMVAVGLLGLITANELGWLERTKSDLSHLMGRRSEGATIGFSMDIDLRPA SAWAIYAALTTFITPAVQHAVTTSYNNYSLMAMATQAGVLFGMGKGMPFYAWDFGVPL LMIGCYSQLTPLTLIVAIILLVAHYMYLIPGLQAAAARAAQKRTAAGIMKNPVVDGIV VTDIDTMTIDPQVEKKMGQVLLIAVAVSSAILSRTAWGWGEAGALITAATSTLWEGSP NKYWNSSTATSLCNIFRGSYLAGASLIYTVTRNAGLVKRRGGGTGETLGEKWKARLNQ MSALEFYSYKKSGITEVCREEARRALKDGVATGGHAVSRGSAKLRWLVERGYLQPYGK VIDLGCGRGGWSYYAATIRKVQEVKGYTKGGPGHEEPMLVQSYGWNIVRLKSGVDVFH MAAEPCDTLLCDIGESSSSPEVEEARTLRVLSMVGDWLEKRPGAFCIKVLCPYTSTMM ETLERLQRRYGGGLVRVPLSRNSTHEMYWVSGAKSNTIKSVSTTSQLLLGRMDGFRRP VKYEEDVNLGSGTRAVVSCAEAPNMKIIGNRIERIRSEHAETWFFDENHPYRTWAYKG SYEAPTQGSASSLINGVVRLLSKPWDVVTGVTGIAMTDTTPYGQQRVFKEKVDTRVPD PQEGTRQVMSMVSSWLWKELGKHKRPRVCTKEEFINKVRSNAALGAIFESEKEWKTAV EAVNDPRFWALVDKEREHHLRGECQSCVYNMMGKREKKQGEFGKAKGSRAIWYMWLGA RFLEFEALGFLNEDHWMSRENSGGGVEGLGLQRLGYVLEEMSRIPGGRMYADDTAGWD TRISRFDLENEALITNQMEKGHRALALAIIKYTYQNKVVKVLRPAEKGKTVMDIISRQ DQRGSGQVVTYALNTFTNLVVQLIRSMEAEEVLEMQDLWLLRRSEKVTNWLQSNGWDR LKRMAVSGDDCVVRPIDDRFAHALRFLNDMGKVRKDTQEWKPSTGWDNWEEVPFCSKH FNKLHLKDGRSIVVPCRHQDELIGRARVSPGAGWSIRETACLAKSYAQMWQLLYFHRR DLRLMANAICSSVPVDWVPTGRTTWSIHGKGEWMTTEDMLVVWNRVWIEENDKMEDKT PVTKWTDIPYLGKREDLWCGSLIGHRPRTTWAENIKNTVNMVRRIIGDEEKYMDYLST QVRYLGEEGSTPGVL (SEQIDNO:3) atgaaaaacccaaaaaagaaatccgaaggattccggattgtcaatatgctaaaacacgga gtagcccgtgtgagcccctttgggggcttgaagaggctgccagccggacttctgctgggt catgggcccatcaggatggtcttggcgattctagccttcttgagattcacggcaatcaag ccatcactgggtctcatcaatagatggggttcagtggggaaaaaagaggctatggaaata ataaagaagttcaagaaagatctggctgccatgctgagaataatcaatgctaggaaggag aagaagagacgaggcgcagatactaatgtcggaattgttggcctcctgctgaccacagct atggcagcggaagtcactaaacgtggaagtgcatactatatatacttggacagaaacgat gctggggaggccatatcttttccaaccacattggggatgaataagtgttatatacagatc atggatcttggacacatgtgtgatgccaccatgagctatgaatgccctatgctggatgag gagatggaaccagatgacatcgattattggtacaacacgacgtcaacttgggttgtgtac ggaacctgccatcacaaaaaaggtgaagcacggagatctagaagagctgtgacgctcccc tcccattccactaggaagctgcaaacgcggtcgcaaacttggttggaatcaagagaatac acaaaacacttgattagagtcaaaaattagatattcaagaaccctggcttcacgttaaca gcagctgccatcacttggcttttgggaaactcaacaaaccaaaaagtcatatacttgatc atgatactgctaattgccccggcatacagcatcaagtgcataggagtcaacaatagagac tttgtggaaggtatgtcaggtgggacttgggttgatgttgtcttggaacatggaggttgt gtcaccgtaatggcacaggacaaaccgactgtcgacatagagctggttacaacaacagtc aacaacatgacggaggtaagatcctactgctataaggcatcaatatcgaacatagcttcg aacagccgctgcccaacacaagatgaagcctaccttgacaagcaatcagacactcaatat gtctgcaaaagaacgttagtggacagaggctggggaaatggatgtggactttttggcaaa gggagcctggtgacatgcgctaagtttgcatgctccaagaaaatgaccgggaagagcatc cagccagagaatctagagtaccggataatgctgtcagttcatagctcccagcacagtaga atgatcgttaatgacacagaacatgaaactgatgagaatagagcgaaggttgagataacg cccaattcaccaagagccgaagccaccctggggggttttggaagcctaggacttgattgt gaaccgaggacaggccttgacttttcagatttgtattacttgactatgaataacaagcac tagttggttcacaaggagtggttccacgacattccattaccttggcacactggagcagac accggaactccacactggaacaacaaagaaacactggtagagttcaaggacgcacatgcc aaaaggcaaactgtcgtggttctagggagtcaagaaggagcagttcacacggcccttgct ggagctctggaggctgagatggatggtgcaaagggaaggctgtcctctggccacttgaaa tgtcgcctgaaaatagataaacttagattgaagggcgtgtcatactccttgtgtaccaca gcgttcacattcaccaagatcccggctgaaacactgcacggaacagtcacagtggaagta cagtacgcagggacagatggaccttgcaaggttccagctcagatggcggtggacatgcaa actctgaccccagttgggaggctgataaccgctaaccccgtaatcactgaaagcactgag aactccaagatgatgctgaaacttgatccaccatttgggaactcttacattgtcatagga atcgaggaaaagaagatcacccaccactggcacaggagtggcagcaccattgaaaaagca tttgaagccactgtgagaggtgccaggagaatggcagtcttgggagacacagcctgggac tttggatcagttggaggcgctctcaactcattgggcaagggcatccatcaaatttttgga gcagctttcaaatcattgtttagaggaatgtcctgattctcacaaattctcattggaaca ttgctgatgtgattgagtctgaacacaaagaatgaatctatttcccttatgtgcttagcc ttagggggagtgttgatcttcttatccacagccgtctctgctgatgtggggtgctcggtg gacttctcaaagaaggagacgagatgcggtacaggggtgttcgtctataacgacgttgaa gcctggaggaacaggtacaagtaccatcctgactccccccatagattgacagcagcagtc aagcaagcctgggaaaatggtatctgtgggatctcctctgtttcaagaatggaaaacatc atgtggagatcagtagaaggggagctcaacgcaatcctggaagagaatggagttcaactg acggtcgttgtgggatctgtaaaaaaccccatgtggagaggtccacagagattgcccgtg cctgtgaacgagctgccccacggctggaaggcttgggggaaatcgtacttcgtcagagca gcaaagacaaataacagctttgtcgtagatggtgacacactaaaggaatacccactcaaa cataaagcatgaaacagctttcttgtagaggatcatgggttcggggtatttcacactagt gtctggctcaaggttagagaagattattcattagagtgtgatccagccgttattggaaca gctgttaagggaaaggaggctgtacacagtgatctaggctactggattgagagtgagaag aataacacatagaggctgaagagagcccatctgatcgagatgaaaacatgtgaatggcca aagtcccacacattgtggacagatggaatagaagagagtgatctgatcatacccaagtct ttagctgggccactcagccatcacaataccagagagggctacaggacccaaatgaaaggg ccatgacacagtaaagagcttaaaattcagtttgaagaatgcccaggcaccaaggtccac gtggaagaaacatgtggaacaagaggaccatctctaaaatcaaccacagcaagcggaaga gtgatcgaggaatggtgctacagggaatgcacaatgcccccactgtcgttccaggctaaa gatggctgttggtatggaatggagataaggcccaggaaagaaccagaaagtaacttagta aggtcaatggtgactgcaggatcaactgatcacatggatcacttctcccttggagtgctt gtgattctgctcatggtgcaggaagagctgaagaagagaatgaccacaaagatcatcata agcacatcaatggcaatgctggtagctatgatcctgggaggattttcaatgaatgacctg gctaagcttgcaattttgatgagtgccaccttcgcggaaatgaacactggaggagatgta gctcatctggcgctgatagcggcattcaaagtcagaccagcgttgctggtatctttcatc ttcagagctaattgaacaccccgtgaaaacatgctactggccttagcctcgtgtctttta caaactgcgatctccgccttggaaggcgacctgatggttctcatcaatgattttgctttg gcctggttggcaatacgagcgatgattgttccacgcactgataacatcaccttggcaatc ctggctgctctgacaccactggcccggggcacactgcttgtggcgtggagagcaggcctt gctacttgcaaggggtttatgctcctctctctgaagggaaaaggcaatatgaaaaagaac ttaccatttgtcatgaccctggaactaaccactgtgagactgatcaaccccatcaacgtg gtgggactgctgttgctcacaaggagtaggaagcggagctggccccctagcgaagtactc acagctgttggcctgatatgcgcattggctggagggttcgccaaggcagatatagagatg gctggacccatgaccgcggtcagtctgctaattgtcaattacatagtctcaagaaagagt gtggacatgtacattgaaaaagcaggtgacatcacatgggaaaaagatgcggaagtcact ggaaacagtccccggcttgatgtggcgctagatgagagtggtgatttctccctggtggag gatgacggtccccccatgagagagatcatactcaaggtggtcctgatgaccatctgtggc atgaacccaatagccataccctttgcagctgaaacgtggtacgtatacatgaaaactgga aaaaggagtggagctctatgggatgtgcctactcccaaagaagtaaaaaaggaggagacc acagatggagtgtacagagtgatgactcgtagactgctaggttcaacacaagttggagtg ggagttatgcaagagggggtctttcacaccatgtggcacgtcacaaaaggatccgcgctg agaagcgatgaaagaagacttaatccatactggggagatgtcaaacaggatctggtgtca tactatggtccatggaagctagatgccgcctgggacgggcacagcgaggtgcagctcttg gccgtgccccccggagagagagcgaggaacatccagactctgcccggaatatttaagaca aaggatggggacattggagcggttgcgctggattacccagcaggaacttcaggatctcca atcctagacaagtgtgagagagtaataggactttatggcaatggggtcatgatcaaaaat aggagttatgttagtaccatcacccaagggaggagggaagaagagactcctgttgagtgc ttcgagccttcgatgctgaagaagaagcagctaactgtcttagacttgcatcctggagct gggaaaaccaggagagttcttcctgaaatagtccgtgaagccataaaaacaagactccgt actgtgatcttagctccaaccagggttgtcgctgccgaaatggaggaagcccttagaggg cttccagtgcgttatatgacaacaggagtcaatgtcacccactctggaacagaaatcgtc gacttaatgtgccatgccaccttcacttcacgtctactacaaccaatcaaagtccccaac tataatctgtatattatggatgaggcccacttcacagatccctcaagtataggagcaaga ggatacatttcaacaagggttgagatgggcgaggcggctgccatcttcatgaccgccacg ccaccaggaacccgtgacacatttccggactccaactcaccaattatgaacaccgaagtg gaagtcccagagagagcctggagctcaggctttgattgggtgacggatcattctggaaaa acagtctggtttgttccaagcgtgaggaacggcaatgagatcgcagcttgtctgacaaag gctggaaaacggatcatacagctcagcaaaaagacttttgagacagagttccagaaaaca aaacatcaagagtgagactttatcgtgacaactgacatttcaaaaatgggcaccaacttt aaagctgaccgtgtcatagattccagaagatgcctaaagccagtcatacttgatggcgag agagtcattctggctggacccatgcctgtcacacatgccagcgctgcccagaggaggggg cgcataggcaggaatcccaacaaacctggagatgagtatctgtatggaggtgggtgcgca gagactgacaaagaccatacacactagcttgaaacaagaatgctccttaacaatatttac ctccaagatggcctcatagcctcgctctatcgacctgaagccgacaaagtagcagccatt gagggagagttcaagcttaggagggagcaaaggaagacctttgtggaactcatgaaaaga ggagatcttcctgtttggctggcctatcaggttgcatctgccggaataacctacacagat agaagatagtgctttgatggcacgaccaacaacaccataatgaaagacagtatgccgaca gaggtgtggaccagacacgaagagaaaagagtgctcaaaccaaggtggatggacgccaga gtttgttcagatcacgcggccctgaagtcattcaaggagtttgccgctgggaaaagagga gcggcttttggagtgatggaagccttgggaacactgccaggacacatgacagagagattc cagaaagccattgacaacctcgctgtgctcatgcgggcaaagactgaaagcagaccttac aaagccgcagcggcccaattgccggagaccctagagaccattatgcttttggagttgctg ggaacagtctcgctgggaatctttttcgtcttgatgaggaacaagggcatagggaagatg ggctttggaatggtgactcttggggccagcgcatggctcatgtggctctcggaaattgag ccagccaaaattacatgtgtcctcattattgtgttcctattgctagtggtgctcatacct gagccagaaaaacaaagatctccccaagacaaccaaatggcaatcatcatcatggtagca gtaggtcttctgggcttgattaccgccaatgaactcggatggttggagagaacaaagagt gacctaagccatctaatgggaaggagagaggagggggcaaccataggattctcaatggac attaacctgcagccagcctcagcttaggccatctacgctaccttgacaactttcattacc ccagccgtccaacatacagtgaccacttcatacaacaactactccttaatggcgatggcc acgcaagctggagtgttgtttggtatgggcaaagggatgccattctacgcatgggacttt ggagtcccgctgctaatgataggttgctactcacaattaacacccctgaccctaatagta gccatcattttgctcgtggcgcactacatgtacttaatcccaagactgcagacagcaact gcgcatgctgcccagaagaaaacggcagctggcatcatgaagaaccctgttgtggatgga atagtggtgactgacattgacacaatgacaattgacccccaagtggagaaaaagatggga caggtgctactcatagcagtagccgtctccagcgccatactgtcgcggaccgcctggggg tagagggagactggggccctgatcacagctgcaacttccactttgtagaaaggctctccg aacaagtactggaactcctctacagccacttcactgtgtaacatttttagggaaagttac ttggctggagcttctctaatctacacagtaacaagaaacgctggcttggtcaagagacgt gggggtggaacaggagagaccctgggagagaaatggaaggcccgcttgaaccagatgtcg gccctggagttctactcctacaaaaagtcaggcatcaccgaggtgtgcagagaagaggcc cgccacgccctcaaggacgatgtggcaacgggaagccatgctgtgtcccaaggaagtgca aagctgagatgattggtggagcggggatacctgcagccctatggaaaggtcattgatctt ggatgtggcagagggggctggagttactacgccgccaccatccgcaaagttcaagaagtg aaaggatacacaaaaggaggccctgatcatgaagaacccatgttggtgcaaagctatggg tagaacataatccgtcttaagagtgaggtggacatctttcatatggcgactgaaccgtgt gacacgttgctgtgtgacataggtgagtcatcatctagtcctgaagtggaagaagcacgg acgctcagagtcctttccatggtgggggattggcttgaaaaaagaccaggagccttttgt ataaaagtgttgtgtccatacaccagcactatgatagaaaccctagagcgactgcagcgt aggtatgagggaagactggtcagagtgccactctcccacaactctacacataagatgtac tgggtctctggagcgaaaaacaacaccataaaaaatgtgtccaccacgaaccagctcctc ttggggcgcatggacgggcccaggaggccagtgaaatatgaggaggatgtgaatctcggc tctggcacgcgggctgtggtaagctgcgctgaagctcccaacatgaagatcattggtaac cacattgaaaagatccacagtgaacacgcggaaacgtggttctttgacaagaaccaccca tataggacatgggcttaccatgaaagctataaggcccccacacaaaggtcagcgtcctct ctaataaacggggttgtcaggctcctgtcaaaaccctgggatgtggtgactggagtcaca ggaatagccatgaccgacaccacaccgtatggtcagcaaagagttttcaaggaaaaagtg gacactaaggtgccagatccccaagaaaacactcgtcaggttataagcatgatctcttcc tggttgtggaaagagctagacaaacacaaacggccacgagtctgtaccaaagaagaattc atcaacaaggttcgtagcaatgcagcattaggggcaatatttgaagaggaaaaagagtgg aagactgcagtggaagctgtgaacgatccaaggttctgggctctagtggacaaggaaaga gagcaccacctgagagaaaagtgccagagttatatgtacaacatgatgagaaaaaaagaa aagaaacaaggggaatttggaaaggccaagagcagccgcgccatctggtatatgtggcta ggggctagatttctagagttcgaagcccttggattcttgaacgaggatcactggatgggg agagagaactcaggaggtggtgttgaagggctgggattacaaagactcggatatgtccta gaagaaatgagtcgcataccaagaggaaagatgtatgcagataacactgctagctggaac acccacatcagcaggtttgatctggaaaatgaagctctaatcaccaaccaaatggaaaaa gggcacagggccttggcattggccataatcaagtacacataccaaaacaaagtggtaaag gtccttagaccagctgaaaaagggaagacagttatggacattatttcgagacaagaccaa aagaggagcaaacaagttatcacttacgctcttaacacatttaccaacctagtagtgcaa ctcattcgaagtatgaaggctgaggaagttctagagatacaagacttgtggctgctgcgg aggtcagagaaagtgaccaactggctgcagagcaacggatgggataggctcaaacgaatg gcagtcagtggagatgattgcgttgtgaggccaattgatgataggtttgcacatgccctc aggttcttgaataatatggggaaagttaagaaggacacacaaaaatggaaaccctcaact ggataggacaactgggaggaagttccattttgctcccaccacttcaacaagctccatctc aaggacgggaggtccattgtggttccctgccgccaccaagatgaactgattggccgggcc cgcgtctctccaggggcgggatggagcatccgggagactgcttgcctagcaaaatcatat gcgcaaatgtagcagctcctttatttccacaaaagggacctccgactgatggccaatgcc atttgttcatctgtgccagttgactgggttccaactggaagaactacctggtcaatccat ggaaagggagaatggatgaccactgaagacatgcttgtggtgtggaacagagtgtggatt gaggagaacgaccacatggaagacaagaccccagttacgaaatggacagacattccctat ttgggaaaaagggaagacttgtggtgtggatctctcatagggcacagaccgcgcaccacc tgggctgagaacattaaaaacacagtcaacatggtgcgcaggatcataggtgatgaagaa aagtacatggactacctatccacccaagttcgctacttgggtgaagaagggtctacacct ggagtgctgtaa
prM/E proteins include those having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 99% or more amino acid sequence identity to the prM/E proteins encoded by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0095] Capsid proteins include those having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 99% or more amino acid sequence identity to the proteins encoded by one or more of SEQ ID NO:1 SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0096] An exemplary intron/enhancer sequences useful in a vector include: atcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccgcggccgggaa cggtgcattggaacgcggattccccgtgccaagagtgactcaccgtccggatctcagcaagcaggtatgtactctccag ggtgggcctggcttccccagtcaagactccagggatttgagggacgctgtgggctcttctatacatgtaccttttgcttgc ctcaaccctgactatcttccaggtcaggatcccagagtcaggggtctgtattttcctgctggtggctccagttcaggaaca gtaaaccctgctccgaatattgcctctcacatctcgtcaatctccgcgaggactggggaccctgtgacgaac (SEQ ID NO:4), or a nucleotide sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 99% or more nucleotide sequence identity to SEQ ID NO:4.

[0097] An exemplary vector sequence useful to produce VLPs is shown in FIG. 6 (SEQ ID NO:5).

[0098] An exemplary African lineage Zika isolate has the following nucleotide sequence (SEQ ID NO:11 which encodes the protein provided at Accession No. HQ234500 which is incorporated by reference herein):

TABLE-US-00003 atgaaaaacccaaagaagaaatccggaggattccggattgtcaatatgctaaaacgcgga gtagcccatgtaaaccccttgaggggtttgaagaggctgccggccggactcctgctgggc catggacccatcagaatggttttggcgatactagccttcttgagattcacagcaatcaag ccatcactgggcctcatcaatagatagggttccgtggggaagaaggaggctatggaaata ataaaaaagttcaagaaagatcttgctgccatgttgagaataatcaatgctaggaaggag aggaagagacatggagctaatgccaacatcggaatcgtcaacctcctgctgactacagtc atggcagcagagatcactagacgcgggagtgcatactacatgtacttggacaggagcgat gctggtaaggccatttctttcgttaccacactggggatgaacaaatgccatgtgcagatc atggacctcgggcatatgtgtgacgccaccatgagttatgagtgccccatgctggacgag ggagtggagccagatgacgtcgattgctggtgcaacacgacatcaacttgggttgtgtac ggaacctgtcatcataaaaaaggtgaagcacgacaatccagaagagccgtgacgcttcct tctcactctacaaggaagttgcaaacacgatcgcagacttgactagaatcaagagaatac acaaagcacctgatcaaggttgagaattggatattcaggaaccccggatttgcgctagtg gctgtagctattgcctggctcctgggaagctcgacgagccaaaaagtcatatacttggtc atgatattgttgattgccccggcatacagtatcaggtgcataggagttagcaataaagac ttcgtggagggcatgtcaggtgggacctgggttgatgttgtcttggaacatggaggttgt gtcaccgtgatggcacaggacaagccaacagttgacatagagttggtcacgacaacggtt agcaacatggccgaagtgagatcctactgctacgaggcatcaatatcggacatggcttca gacagtcactgcccaacacaaagtgaagcctaccttgacaagcaatcagacactcaatat gtctataaaagaacattggtggacagaggttgggaaaatggatgtggactttttggcaag gggagcttggtgacgtgtgccaagtttacatgctccaagaaaatgacagggaagagcatc cagccggagaacttggagtaccggataatgctatcagtgcatggatcccagcacagtggg atgattgtgaatgacgaaaacagagcaaaagtcaaggttacacccaattcaccaaaagca gaagcaaccttgggaagttttgaaagcctgagacttgattgtgaaccaaggacaggcctt gacttttcagatctgtattacctgaccatgaacaataacgattggttggtgcacaaagag tggtttcatgacatcccattaccttggcattctggtgcagacactgaaactccacactgg aacaacaaagaggcactggtgaagttcaaggacgcccacgccaaaaggcaaactgttgta gttctggggagccaagaagaagccgttcacacggctctcgctggagctctggaggctgag atggatggtgcgaagggaaggctatcctcaggccatttgaaatgccgcctaaaaatggac aagcttaggttgaagggtgtgtcatattccctgtgtaccgcagcgttcacattcaccaag gttccagctgaaacattgcatggaacagtcacaatggaggtgcagtatacagggaaggat agaccctgcaaggtcccagcccagatggcgatggacatacagaccctgaccccagttgga aggctgataacggctaaccctgtgatcactgaaagcactgagaattcaaagatgatgttg gagctcgacccaccatttggggattcttacattgtcataggagtcggggacaagaaaatc acccatcactggcatcggagtagtagcatcatcggaaaggcatttgaagccactgtgaga ggcgccaagagaatggcagtcttgggagacacagcctgggactttggatcagttggaggt gtgtttaactcattgggcaagggtattcaccagatctttggagcagctttcaaatcactg ttcggaggaatgtcctgattctcacagatcctcataggcacactgttggtgtggttgggt ctgaacacaaagaatggatctatctccctcacatgcttggccttgggaagagtgatgatc ttcctttccacggctatttctgctgatgtgaggtgttcagtggacttctcaaaaaaggaa acgagatgtggcacgggggtgttcatctacaatgacgttgaagcctggagggatcgatac agataccatcctgactccccccgcagattggcagcagctgttaagcaggcttgggaagag gggatttatgggatctcctccatttcgagaatggaaaacatcatatggaaatcagtggaa ggggagcttaatgcgatcctagaggaaaatggagtccaactaacagttgtagtgggatct gtaaaaaaccccatgtggagaggtccacgaagattgccagtgcccgtaaatgagctgccc catggctggaaagcctgggggaaatcgtactttgttagggcggcaaagaccaacaacagt tttattgtcgacggtgacacactgaaggaatgtccgctcaaacatagaacatggaatagc ttccttgtagaggatcacgggtttggggtcttccacaccagtgtttggctgaaggtcaga gaggactattcattagagtgtgacccagccgtcataggaacagctgtcaagggaaaggag gctgcacacagtgatctaggctattggattgagagtgaaaagaatgacacatggaggctg aagagggctcatctgattgagatgaagacatgtgagtggccaaagtctcacacactgtgg acagatggagtagaagaaaatgatctaatcatacccaagtccttagctgatccactcagc caccacaacaccagagaggattatagaactcaagtgaaaggaccatggcatagtgaagag ctcgaaatccggtttgaggaatgcccaggcaccaaggttcatgtggaggagacatgcgga actagaggaccatctttaagatcaaccactgcaagtggaagggtcatagaggaatggtgc tatagggaatacacaatgcctccactatcgttccgggcaaaagacgactgctgatatgga atggagataaggcccagaaaggaaccagagagcaacttagtgaggtctatggtgacagca ggatcaaccgatcacatggatcacttctctcttggagtgcttgtgattctactcatggtg caggaagatttgaaaaagagaatgaccacaaagatcataatgagcacatcaatggcaata ctggtagccatgatcttgggaagattctcaatgagtgacctgactaagcttatgatccta atggatgccactttcgcagaaatgaacactggagaagatgtagctcacttggcattagta gcggcatttaaagtcagaccagccttgttggtttccttcatcttcagagccaactggaca ccccgtgagagcatgctgctagccctggcttcgtgtctcctgcagactgcgatttccgct cttaaaggcaagctgatgatcctcgttaatgaatttgctttggcctagttggcaatacga acaatggccgtgccacgcactgataacatcactctagcaattctgaccgctctaacacca ttagccagaggcacactgcttgtggcatggagagcgggcctcgccacttgtagagggttc atgctcatttccctgaaagggaaaggtagtgtgaagaagaacctgccatttgtcatggcc ttgggattgaccactgtgaggatagtgaaccccattaatgtgataggactactgttacta acaaagagtggaaaacggaactggccccctagtgaagtgcttacagctgtcggcctaata tgtgcactggccggagggtttgccaaggcagacatagagatggctgggcccatggctgca gtaggcctgctaattgtcagttatgtggtcacgggaaagagtgtggacatgtacattgaa aaaacaggtaatattacatgggaaaaagacgcgaaagtcactggaaacagtcctcagctt aacgtggcactagataagagtgatgatttctctttggtagaggagaatggcccacccatg agagagatcatactcaaggtggtcctgatggccatctgtggcatgaacccaatagccata cccttcgctgcaggagcgtggtatgtgtatgtaaagactgggaaaaggagcggtgccctc tgggacgtgcctactcccaaaaaagtaaaaaagggagagactacagatggaatgtacaga gttatgactcgcagactgctgggttcaacacaggttggagtaggagtcatgcaagaagga gtcttccataccatgtggcacgtcacaaaaggagccgcattgaggagcggtgaaggaaga cttgatccatactggggggacgtcaagcaggacctggtgtcatattgtgggccgtggaag ttgaatgcaacctgggatagactaaatgaggtgcagcttttggccgtaccccccgaagag agggctaaaaacattcagactctgcctggaatatttaaaacaaagaatggggacatcgga gcagttgctctagactaccctgcaggaacctcaggatctccgatcctagacaaatgcgga agagtgataggactttatggcaatggggttgtgatcaagaatggaagctatgttagtgcc ataacccagggaaaaagggagaaggagactccggttgagtgctttgaaccctcgatgcta aggaagaagcaactaacagtcttggatctgcatccaggagccgggaaaaccaggagagtt cttcctgaaatagtccgtgaagccataaagaagagacttcgcacagtgatcttagcacca accagggttgttgctgctgagatggaggaagccctaagaggacttccggtgcgttacatg acaacagcaatcaacgtcacccattctgggacaaaaatcattgatttgatgtgccatgcc accttcacttcacgcctactacaaccaatcagagtccccaactacaacctttatatcatg gatgaggctcatttcacagatccttcaagcatagctgcaagaggatacatatcaacaagg gttgaaatgggcgaggcggctgctatcttcatgactgctacaccaccaggaacccgcgat gcgtttccagattccaactcaccaatcatggacacagaagtggaagtcccagagagagcc tggaactcaggctttgactaggtgacagaccattctggaaaaacaatttagtttgttcca agtgtgagaaacggaaatgaaatcgcagcctgtctgacaaaagctggaaagcgggttata cagctcagcaggaagacttttgagacagagtttcagaagacaaaaaatcaagagtgggac tttgtcataacaactgacatttcagagatgggtgccaacttcaaggctgaccggatcata gattccaggaaatgcctaaagccagtcatacttaatggtgagagagtcatcctggctggg cctatgcccgtcacgcacgccagtgctgctcagaggagaggacgtataggcaggaacccc aacaaacctggagatgagtatatgtatggaggtgggtgtgcagagactgatgaagaccat gcacactagcttgaagcaagaatgcttctcgacaacatttacctccaggatagcctcata gcctcgctctatcgacctgagactgacaaggttgccgccattgaaggagagttcaagcta aggacagagcaaaggaagacctttgtagaactcatgaagagaggagaccttcccgtttgg ctggcctatcaagtagcatctgccggaataacttacacagacagaagatggtgctttgat ggcactaccaacaacaccataatggaagacagtgtaccagcagaggtgtggaccaagtat ggaaagaagaaagtgctcaaaccgaagtggatgaatgccaaggtctgttcagatcatgcg actttgaaatcgttcaaagaatttgccgctaggaagagaggagcgactttggaagtaatg gatgccctaggaacattgccaggacacatgacagagaggtttcaggaagccattgacaat ctcgctgtgctcatgcgagcagagactggaagtaggccctacaaagcagcggcagctcaa ctgccggagaccctagagaccattatgctcttgggtttattgggaacagtttcgctagga atcttctttgtcttgatgcagaacaaaggcatcaggaagataggcttcgaaatggtaacc cttggggccagcgcatggctcatgtggctttcggaaattgaaccagccagaatcgcatgt gtcctcattgtcgtgtttctgttactggtggtgctcatacctgagccagagaagcaaaga tctccccaggacaatcaaatggcaatcatcatcatggtggcagtgggccttctggatttg ataactgcaaacgaactcggatagctggaaagaacaaaaagtgatatagctcatctaatg ggaaggaaagaagaggggacaaccgtaggattctcaatggatattgatctgcggccagcc tccgcctgggctatttatgccgcattgacaactctcatcaccccagccgtccaacatgcg gtgaccacctcatacaacaactactccctgatggcgatggccacacaagctagagtgcta tttgacatgggcaaagggatgccattttatgcataggactttggagtcccgctgctaatg atgggttgttactcacaattaacacccctgaccctgatagtggccatcattctgcttgtg gcacactacatgtatttgatcccaggtttgcaggcagcagcagcacgtgccgcccagaag aggacagcagctggcatcatgaagaatcccgttattgatgaaatagtgatgactgacatt aacacaataacaattaacccccaagtggagaagaagataggacaaatgttactcatagca gtagctgcctccagtgccgtgctgctgcggaccgcttggggatggggggaggctggggct ctgatcacagcagcaacctccaccttatgggaaggctctccaaacaaatactggaactcc tctacagccacttcactgtgcaatatcttcagaggaaattatttagcagggacttccctt atttacacagtaacaagaaatgccggtctggttaagagacgtggaggtgaaacgggagag actctgggagagaagtggaaagcccgcctgaaccagatgtcggctttggagttctattct tacaaaaagtcaggcatcaccgaagtgtgtagggaggaggcacgccgcgccctcaaggat ggaatggccacaggaggacatgctgtatcccggagaagcgcaaagcttagatggttggta aagagaggatacctgcagccccatggaaagattgttgacctcggatgtggcaaagggggc tggagttattacgctgccaccatccgtaaagtgcaggaggtcagaggatacacaaaggga ggtcctgatcatgaagaacccatgctggtgcaaagctatgggtggaacatagttcgcctc aagagtggagtggacgtctttcacatggcggctgagccgtgtgacactttgctgtgtgac attgacgagtcatcgtccaatcctgaagtggaagagacgcgaacactcaaagtgctctcc atggtgggagactggctcgagaaaagaccaggggccttctgcataaaggtgctgtgccca tacaccagtactatgatggagaccatggagcgactgcaacgtaggtatgggggaggattg gtcagagtgccattgtcccgcaactccacacatgagatgtattgggtctctggagccaaa aataacatcataaagaatatgtccaccacaaatcagctcctcttggaacgcatagatggg cctaggaggccagtgaaatatgaagaggatgtgaacctcggctcaggcacacgagctgtg gcaagctgtgctgaggctcccaacatgaagatcattggtaggcgcattgagagaatccgc aatgaacatgcaaaaacatggttctttaatgaaaaccacccatacaggacatgggcctac catggaaactacaaagcccccacgcagaagtcagcatcatccctcgtgaacagggttatt agactcctgtcaaagccctaggatgtagtgactgaagtcacaggaatagctatgactgac accacgccatacggccaacaaagagtcttcaaagaaaaggtggacactagggtgccagac ccccaagaaggcacccgccgagtaatgaacatgatctcgtcttggctatggaaggagctg gaaaaacgcaagcggccacgtgtctacaccaaaaaagagttcatcaataaggtacacagc aatgcagcactaggaacaatatttgaagagaaaaaagaatggaagacagctgtagaagct gtgaatgatccgagattttgggctctagtggacaaggaaagagaacaccacctgagagga gagtgtcacagctgtgtgtacaacatgatgggaaaaagagaaaagaagcaaggagaattc gggaaagcaaaaagcagccgcacaatctagtacatatagttgagagccagatttctgaaa tttgaggctcttggattcttgaatgaagaccattagatgggaagagaaaactcaggaggt ggcgttgaagggctaggactgcaaaggcttggatacattctagaagaaatgaaccgggcg ccaggaggaaagatgtatgcagatgacaccgctggctgggatacccgtattagcaggttt gatctggagaatgaagccctgatcactaaccagatggaaaaagggcacagagctctggcg ttggccgtaattaaatacacataccaaaacaaagtggtaaaggttctcagaccagctgaa ggagggaaaacagtcatggacatcatctcaagacaagaccagagagggagcggacaagtt gttacttatgctctcaacacattcaccaacctggtggtgcagcttatccggaacatggag gctgaagaggtgctagagatgcatgatctatggctattgaggaaaccagagaaagtgacc agatagttgcagagcaatgaataggacagactcaaacgaatagcagtcaatggagatgac tgcgttgtaaagccaattgatgataggtttgcacatgccctcaggttcttgaatgacatg ggaaaagttaggaaagacacacaggaatggaaaccctcgactggatggagcaattgggaa gaaatcccgttctgttcccaccacttcaacaagctgcacctcaaggataggagatccatt atggtcccctgccgccaccaagatgaactgattggccgagcccgtatctcaccaggggca ggatggagcatccgagagactgcctgtcttgcaaaatcatatgcccagatgtggcagctt ctttatttccacagaagagacctccgactgatggccaatgccatctgttcggccgtgcca gccgactaggtcccaactgggagaaccacctggtcaatccatagaaagggaaaatggata actaatgaggacatgctcatggtgtgaaatagagtgtggattgaggagaacgaccacatg ggggacaagacccctgtaacaaaatggacagacattccctatttgggaaaaagggaggac ttatggtgtggatcccttatagggcacagacctcgcaccacttgggctgagaacatcaaa gacacagtcaacatggtgcgtagaatcatagataatgaaaaaaggtacatggactaccta tccacccaagtacgctacttggatgaggagaggtccacacctggaatgctg

[0099] An exemplary Asian lineage Zika isolate has the following sequence (SEQ ID NO:12 which encodes the protein provided at Accession No. HQ234499 which is incorporated by reference herein):

TABLE-US-00004 ATGAAAAACCCAAAAAAGAAATCCGGAGGATTCCGGATTG TCAATATGCTAAAACGCGGAGTAGCCCGTGTGAGCCCCTT TGGGGGCTTGAAGAGGCTACCAGCTGGACTTCTGCTGGGT CATGGACCCATCAGGATGGTCTTGGCGATACTAGCCTTCT TGAGATTCACGGCAATCAAGCCATCACTGGGTCTCATCAA TAGATGGGGTTCCGTGGGGAAAAAAGAGGCTATGGAAATA ATAAAGAAGTTCAAGAAAGATCTGGCTGCCATGCTGAGAA TAATCAATGCTAGGAAGGAGAAGAAGAGACGTGGCGCAGA CACCAGTGTCGGAATTGTTGGCCTCCTGCTGACCACAGCC ATGGCAGTGGAGGTCACCAGACGTGGGAGTGCATACTATA TGTACTTAGACAGAAGCGATGCTGGGGAGGCCATATCTTT TCCAACCACACTGGGGGTGAATAAGTGTTACATACAGATC ATGGATCTTGGACACATGTGTGATGCCACAATGAGCTATG AATGCCCTATGTTGGATGAGGGGGTAGAACCAGATGACGT CGATTGCTGGTGCAACACGACATCGACTTGGGTTGTGTAC GGAACCTGCCATCACAAAAAAGGTGAGGCACGGAGATCTA GAAGAGCTGTGACGCTCCCCTCTCATTCCACTAGGAAGCT GCAAACGCGGTCGCAGACCTGGTTGGAATCAAGAGAATAC ACAAAGCACTTGATCAGAGTCGAAAATTGGATATTCAGGA ACCCTGGCTTTGCGTTGGCAGCAGCTGCCATTGCTTGGCT TTTGGGAAGCTCAACGAGCCAAAAAGTCATATACTTGGTC ATGATACTGTTGATTGCCCCGGCATACAGTATCAGGTGCA TAGGAGTCAGCAATAGGGATTTTGTGGAAGGTATGTCAGG TGGGACCTGGGTTGATGTTGTCTTGGAACATGGAGGTTGT GTTACCGTAATGGCACAGGACAAGCCAACTGTTGATATAG AGTTGGTCACAACAACGGTTAGCAACATGGCGGAGGTAAG ATCCTACTGCTACGAGGCATCAATATCGGACATGGCTTCG GACAGCCGCTGCCCAACACAAGGTGAAGCCTACCTTGACA AGCAGTCAGACACTCAATATGTTTGCAAAAGAACGTTAGT GGACAGAGGTTGGGGAAATGGATGTGGACTCTTTGGCAAA GGGAGCCTGGTGACATGCGCCAAGTTTGCATGCTCCAAGA AAATGACTGGGAAGAGCATCCAGCCAGAGAACCTGGAGTA CCGGATAATGCTGTCAGTTCATGGCTCCCAGCACAGTGGG ATGATTGTTAATGACANAGGACATGAAACTGATGAGAATA GAGCGAAGGTTGAGATAACGCCCAATTCACCAAGAGCCGA AGCCACCCTGGGAGGTTTTGGAAGCCTAGGACTTGATTGT GAACCGAGGACAGGCCTTGACTTTTCAGATTTGTATTACT TGACTATGAATAACAAGCATTGGTTGGTGCACAAGGAGTG GTTCCATGACATTCCACTACCTTGGCATGCTGGGGCAGAC ACCGGAACTCCACATTGGAACAACAAAGAAGCATTGGTAG AGTTCAAGGACGCACATGCCAAAAGGCAAACTGTCGTGGT TCTAGGGAGTCAAGAAGGAGCCGTTCACACGGCTCTTGCT GGAGCCCTGGAGGCTGAGATGGATGGTGCAAAGGGAAGGC TGTCCTCTGGCCACTTGAAATGTCGCTTGAAAATGGACAA ACTTAGATTGAAGGGCGTGTCATACTCCTTATGTACCGCG GCGTTCACATTCACCAAGATCCCGGCTGAAACGCTGCATG GGACAGTCACAGTGGAGGTACAGTATGCAGGGACAGATGG ACCCTGCAAGGTTCCAGCTCAGATGGCGGTGGATATGCAA ACTCTGACCCCAGTTGGGAGGTTGATAACCGCTAACCCTG TGATCACTGAAAGCACTGAGAATTCAAAGATGATGTTGGA ACTTGACCCACCATTTGGGGATTCTTACATTGTCATAGGA GTTGGGGATAAGAAGATCACCCACCACTGGNACAGGAGTG GCAGCACCATCGGAAAAGCATTTGAAGCCACTGTGAGAGG CGCCAAGAGAATGGCAGTCTTGGGAGACACAGCCTGGGAC TTTGGATCAGTCGGAGGTGCTCTCAACTCATTGGGCAAGG GCATCCATCAAATTTTTGGAGCAGCTTTCAAATCATTGTT TGGAGGAATGTCCTGGTTCTCACAAATCCTCATAGGAACG TTGCTGGTGTGGTTGGGTCTGAACACAAAGAATGGATCTA TTTCCCTTACGTGCTTGGCCTTAGGGGGAGTGTTGATCTT CCTATCTACAGCCGTCTCTGCTGATGTGGGGTGTTCGGTG GACTTCTCAAAGAAGGAAACGAGATGCGGTACGGGGGTGT TCGTCTATAACGACGTTGAAGCCTGGAGGGACAGGTACAA GTACCATCCTGACTCCCCTCGTAGATTGGCAGCAGCAGTC AAGCAGGCCTGGGAAGATGGGATCTGTGGGATCTCCTCTG TTTCAAGAATGGAAAACATTATGTGGAGATCAGTAGAAGG GGAGCTCAACGCAATTCTGGAAGAGAATGGAGTTCAACTG ACGGTCGTTGTGGGATCTGTAAAAAACCCCATGTGGAGAG GTCCGCAGAGGTTGCCTGTGCCTGTGAATGAGCTGCCCCA CGGTTGGAAGGCCTGGGGGAAATCGTACTTTGTCAGGGCA GCAAAGACCAACAACAGCTTTGTTGTGGATGGTGACACAC TGAAGGAATGCCCGCTCAAACACAGAGCATGGAACAGCTT TCTTGTGGAGGATCACGGGTTCGGGGTATTTCACACTAGT GTCTGGCTTAAAGTCAGAGAGGATTACTCATTAGAGTGTG ATCCAGCCGTCATAGGAACAGCTGCTAAGGGAAAGGAGGC CGTGCACAGTGATCTAGGCTACTGGATTGAGAGTGAAAAG AACGACACATGGAGGCTGAAGAGGGCTCACCTGATCGAGA TGAAAACATGTGAATGGCCAAAGTCCCACACACTGTGGAC AGATGGAATAGAAGAAAGTGATCTGATCATACCTAAGTCT TTAGCTGGGCCACTCAGCCACCACAACACCAGAGAGGGCT ACAGGACTCAAGTGAAAGGGCCGTGGCATAGTGAAGAGCT TGAAATCCGGTTTGAGGAATGTCCAGGCACCAAGGTCCAC GTGGAGGAAACATGTGGAACGAGAGGACCGTCCCTGAGAT CAACCACTGCAAGCGGAAGGGTGATCGAGGAATGGTGCTG CAGGGAATGCACAATGCCCCCATTGTCGTTCCGGGCAAAA GATGGCTGTTGGTATGGAATGGAGATAAGGCCCAGGAAGG AACCAGAGAGTAACCTAGTAAGGTCAATGGTGACTGCAGG ATCAACTGATCACATGGATCACTTCTCCCTTGGAGTGCTT GTGATTCTGCTCATGGTGCAGGAAGGGCTGAAGAAGAGAA TGACCACAAAGATCATCATAAGCACATCAATGGCAGTGTT GGTAGCTATGATCCTGGGAGGATTTTCAATGAGTGACTTG GCTAAGCTTGCAATTCTGATGGGTGCCACCTTCGCGGAAA TGAACACTGGAGGAGATGTAGCTCATCTGGCGCTGATAGC GGCATTCAAAGTCAGACCCGCGTTGCTGGTCTCTTTCATC TTCAGAGCCAATTGGACACCCCGTGAGAGCATGCTGCTGG CCTTGGCCTCGTGCCTTCTGCAAACTGNGATCTCCGCCCT GGAAGGCGACCTGATGGTTCTCATCAATGGTTTTGCTTTG GCCTGGTTGGCAATACGAGCGATGGCTGTTCCACGCACTG ACAACATCACCTTGGCAATCCTGGCTGCTCTGACACCACT GGCCCGAGGCACACTGCTTGTAGCGTGGAGAGCAGGCCTT GCTACTTGTGGGGGGTTCATGCTCCTCTCTCTGAAGGGGA AAGGTAGTGTGAAGAAGAACCTACCATTTGTCATGGCCTT GGGACTAACCGCTGTGAGGCTGGTTGACCCCATCAACGTG GTGGGACTGCTGTTGCTCACAAGGAGTGGGAAGCGGAGCT GGCCCCCTAGTGAAGTACTCACAGCTGTTGGCCTGATATG TGCACTGGCCGGAGGGTTCGCCAAAGCAGATATAGAGATG GCTGGGCCCATGGCTGCAGTTGGCCTGCTAATTGTTAGTT ACGTGGTCTCAGGAAAGAGTGTGGACATGTACATTGAAAG AGCAGGTGACATCACATGGGAAAAAGATGCGGAAGTTACT GGAAACAGCCCCCGGCTCGATGTGGCACTAGATGAGAGTG GTGATTTCTCCCTGGTGGAGGATGATGGTCCCCCCATGAG AGAGATCATACTCAAGGTGGTCCTGATGACCATCTGTGGC ATGAACCCAATAGCCATACCCTTTGCAGCTGGAGCGTGGT ATGTGTATGTGAAGACTGGAAAGAGGAGTGGTGCTCTATG GGATGTGCCTGCTCCCAAGGAAGTAAAAAAGGGGGAGACC ACAGATGGAGTGTATAGAGTGATGACTCGCAGACTGCTAG GTTCAACACAAGTTGGAGTGGGAGTCATGCAAGAGGGGGT CTTCCACACTATGTGGCACGTCACAAAAGGATCCGCGCTG AGGAGCGGTGAAGGGAGACTTGATCCATACTGGGGAGATG TTAAGCAGGATCTGGTGTCATACTGTGGCCCGTGGAAGCT AGATGCCGCTTGGGACGGACACAGCGAGGTGCAGCTTTTG GCCGTGCCCCCCGGAGAGAGAGCGAGGAACATCCAGACTC TGCCCGGAATATTCAAGACAAAGGATGGGGACATCGGAGC AGTTGCTCTGGACTACCCAGCAGGAACTTCAGGATCTCCG ATCCTAGACAAGTGTGGGAGAGTGATAGGACTCTATGGCA ATGGGGTCGTGATCAAAAATGGAAGTTATGTTAGTGCCAT CACCCAAGGGAGGAGGGAGGAAGAGACTCCTGTTGAATGC TTCGAACCTTCGATGCTGAAGAAGAAGCAGCTAACTGTCT TGGATCTGCATCCTGGAGCTGGGAAAACCAGGAGAGTTCT TCCTGAAATAGTCCGTGAAGCCATAAAAACAAGACTCCGC ACGGTGATCCTGGCTCCAACCAGGGTTGTCGCTGCTGAAA TGGAGGAAGCCCTTAGAGGGCTTCCAGTGCGTTACATGAC AACAGCAGTTAATGTCACCCACTCTGGGACAGAAATCGTT GATTTAATGTGCCATGCCACCTTCACTTCACGCCTACTAC AACCCATTAGAGTCCCCAACTACAATCTTTACATTATGGA TGAGGCCCACTTCACAGATCCCTCAAGTATAGCAGCAAGA GGATACATATCAACAAGGGTTGAGATGGGCGAGGCGGCTG CCATCTTCATGACCGCCACACCACCAGGAACCCGCGACGC ATTTCCGGACTCTAACTCACCAATCATGGACACAGAAGTG GAAGTCCCAGAGAGAGCCTGGAGCTCAGGCTTTGATTGGG TGACGGATCATTCTGGAAAAACAGTTTGGTTTGTTCCAAG CGTGAGGAACGGCAACGAGATCGCGGCTTGTCTGACAAAA GCTGGAAAACGGGTCATACAGCTCAGCAGAAAGACTTTTG AGACAGAGTTCCAGAAAACAAAAAATCAAGAGTGGGACTT CGTCGTAACAACTGACATCTCAGAGATGGGCGCCAACTTC AAAGCTGACCGGGTCATAGATTCCAGGAGATGCCTGAAGC CGGTCATACTTGATGGCGAGAGAGTCATTCTGGCTGGACC CATGCCTGTCACACATGCCAGCGCTGCCCAGAGGAGGGGG CGCATAGGCAGGAATCCCAACAAACCTGGAGATGAGTATA TGTATGGAGGTGGGTGCGCAGAGACTGATGAAGACCATGC ACACTGGCTTGAAGCAAGAATGCTTCTTGATAACATTTAC CTCCAAGATGGCCTCATAGCCTCGCTCTATCGACCTGAGG CCGATAAGGTAGCAGCCATTGAGGGAGAGTTCAAGCTTAG GACGGAGCAAAGGAAGACCTTTGTGGAACTCATGAAAAGA GGAGATCTTCCTGTTTGGCTGGCCTATCAGGTTGCATCTG CCGGAATAACCTACACAGATAGAAGATGGTGTTTTGATGG CACGACCAACAACACCATAATGGAAGACAGTGTGCCGGCA GAGGTGTGGACCAGATACGGAGAGAAAAGAGTGCTCAAAC CGAGGTGGATGGACGCCAGAGTTTGTTCAGATCATGCGGC CCTGAAGTCATTCAAAGAATTTGCCGCTGGGAAAAGAGGA GCGGCCTTTGGAGTGATGGAAGCCCTGGGAACACTGCCAG GACACATGACAGAGAGGTTTCAGGAAGCCATTGACAACCT CGCTGTGCTCATGCGGGCAGAGACTGGAAGCAGGCCCTAC AAAGCCGCGGCGGCCCAATTACCGGAGACCTTAGAGACCA TCATGCTTTTGGGTTTGCTGGGAACAGTCTCGCTGGGAAT CTTCTTTGTCTTGATGCGGAACAAGGGCATAGGGAAGATG GGCTTTGGAATGGTGACCCTTGGGGCCAGTGCATGGCTTA TGTGGCTCTCGGAAATTGAGCCAGCCAGAATTGCATGTGT CCTCATTGTCGTGTTTCTATTGCTGGTGGTGCTCATACCT GAGCCAGAAAAGCAGAGATCTCCCCAGGACAACCAAATGG CAATTATCATCATGGTAGCAGTGGGTCTTCTGGGCTTGAT AACCGCCAATGAACTCGGATGGTTGGAGAGAACAAAAAGT GACCTAGGCCATCTAATGGGAAGGAGAGAGGAGGGGGCAA CCATGGGATTCTCAATGGACATTGACTTGCGGCCAGCCTC AGCTTGGGCTATCTATGCCGCTCTGACAACTCTCATCACC CCAGCCGTCCAACATGCGGTAACCACTTCATACAACAACT ACTCCTTAATGGCGATGGCCACGCAAGCCGGAGTGTTGTT TGGCATGGGCAAAGGGATGCCATTCTATGCGTGGGACTTC GGAGTCCCGCTGCTAATGATGGGTTGCTACTCACAATTAA CACCCTTGACCTTAATAGTGGCCATCATTCTGCTCGTGGC GCACTACATGTACTTGATCCCAGGTCTACAGGCAGCAGCG GCGCGCGCTGCCCAGAAGAGAACGGCAGCTGGCATCATGA AGAACCCTGTTGTGGATGGAATAGTGGTGACTGACATTGA CACAATGACAATTGACCCCCAAGTGGAGAAAAAGATGGGA CAAGTGCTACTCATAGCAGTAGCCATCTCCAGTGCCGTTC TGCTGCGCACCGCCTGGGGGTGGGGGGAGGCTGGGGCCCT GATCACAGCCGCAACTTCCACTTTGTGGGAAGGCTCTCCG AATAAATACTGGAACTCCTCCACAGCCACTTCACTGTGTA ACATTTTTAGGGGAAGTTACTTGGCTGGAGCTTCTCTTAT TTACACAGTAACAAGAAACGCTGGCCTGGTCAAGAGACGT GGAGGTGGAACGGGAGAGACCCTGGGGGAGAAATGGAAGG CCCGCCTGAACCAGATGTCGGCCCTGGAGTTTTACTCCTA CAAAAAGTCAGGCATCACCGAAGTGTGCAGAGAAGAAGCC CGCCGCGCCCTCAAGGACGGAGTGGCAACAGGAGGCCATG CTGTGTCCCGAGGAAGCGCAAAGCTTAGATGGTTGGTGGA GAGAGGATACCTGCAGCCCTATGGAAAGGTCATTGATCTT GGATGTGGCAGAGGGGGCTGGAGTTACTACGCCGCCACCA TCCGCAAAGTTCAAGAGGTGAAAGGATACACAAAGGGAGG CCCTGGTCATGAAGAACCCACGTTGGTGCAAAGCTATGGA TGGAACATAGTCCGTCTTAAGAGTGGGGTGGACGTCTTTC ACATGGCGGCGGAGTCGTGTGACACTTTGCTGTGTGACAT AGGTGAGTCATCATCTAGTCCTGAAGTGGAAGAAGCACGG ACGCTCAGAGTACTCTCCATGGTGGGGGATTGGCTTGAAA AAAGACCAGGGGCCTTTTGTATAAAGGTGTTGTGCCCATA CACCAGCACCATGATGGAAACCCTAGAGCGACTGCAGCGT AGGTATGGGGGAGGACTGGTCAGAGTGCCACTCTCCCGCA ACTCTACACATGAGATGTACTGGGTCTCTGGAGCGAAAAG CAACATCATAAAAAGTGTGTCCACCACGAGCCAGCTCCTC TTGGGACGCATGGACGGGCCCAGGAGGCCAGTGAAATATG AGGAGGATGTGAATCTCGGCTCCGGCACGCGAGCTGTGGC AAGCTGCGCCGAAGCTCCCAACCTGAAGATCATTGGTAAC CGCGTTGAGAGGATCCGCAGTGAGCATGCGGAAACGTGGT TCTTTGATGAGAACCACCCATACAGGACATGGGCTTACCA TGGGAGCTACGAGGCCCCTACACAAGGGTCAGCGTCTTCT CTCATAAACGGGGTTGTCAGGCTCCTGTCAAAGCCCTGGG ATGTGGTGACTGGAGTCACAGGAATAGCCATGACCGACAC CACACCGTATGGCCAGCAAAGAGTTTTCAAGGAAAAAGTG GACACTAGGGTGCCAGACCCCCAGGAAGGCACTCGTCAGG TGATGAACATGGTCTCTTCCTGGCTATGGAAGGAGCTAGG TAAACACAAACGGCCACGAGTTTGCACCAAAGAAGAGTTC ATCAATAAGGTTCGCAGCAATGCAGCACTGGGGGCAATAT TTGAAGAGGAGAAAGAATGGAAGACTGCAGTGGAAGCTGT GAACGATCCAAGGTTCTGGGCCCTAGTGGACAAGGAAAGA GAGCACCACTTGAGAGGAGAGTGTCAGAGCTGTGTGTACA ACATGATGGGAAAAAGAGAAAAGAAGCAAGGGGAATTTGG AAAGGCCAAGGGCAGCCGCGCCATTTGGTACATGTGGCTA GGGGCTAGATTTCTAGAGTTTGAAGCCCTTGGATTCTTGA ACGAGGATCACTGGATGGGGAGAGAGAATTCAGGAGGTGG TGTTGAAGGGCTGGGATTACAAAGACTTGGATATGTTCTA GAAGAAATGAGCCGCACACCAGGAGGAAAGATGTATGCAG ATGATACCGCTGGCTGGGACACCCGCATCAGTAGGTTTGA TCTGGAGAATGAAGCTCTGATCACCAACCAAATGGAGAAA GGGCACAGGGCCTTGGCGTTGGCCATAATCAAGTACACAT ACCAAAACAAAGTGGTAAAGGTCCTTAGACCAGCTGAAAG AGGGAAGACAGTTATGGACATCATCTCAAGACAAGACCAA AGAGGGAGCGGACAAGTTGTTACTTACGCTCTTAATACAT TCACCAACCTGGTGGTGCAGCTCATTCGGAACATGGAGGC TGAGGAAGTTCTAGAGATGCAAGACTTGTGGCTGTTGAGG AGGCCAGAGAAGGTGACCAGCTGGTTGCAGAGCAACGGAT GGGATAGGCTCAAACGAATGGCAGTCAGTGGAGATGATTG TGTTGTGAAACCAATTGATGATAGGTTTGCACATGCCCTC AGGTTTTTGAATGACATGGGGAAAGTTAGGAAGGACACAC AGGAGTGGAAACCCTCAACTGGATGGAGCAACTGGGAAGA AGTTCCGTTTTGCTCCCATCACTTCAACAAGCTTTACCTC AAGGACGGGAGGTCCATTGTGGTCCCCTGTCGCCACCAAG ATGAACTGATTGGCCGAGCCCGCGTCTCACCAGGGGCGGG ATGGAGCATCCGGGAGACTGCTTGCCTAGCAAAATCATAT GCACAAATGTGGCAGCTTCTTTATTTCCACAGAAGGGACC TCCGACTGATGGCCAACGCCATTTGTTCATCTGTGCCAGT TGACTGGGTTCCAACTGGGAGAACCACCTGGTCAATCCAT GGAAAGGGAGAATGGATGACCACTGAGGACATGCTTGTGG TGTGGAACAGAGTGTGGATTGAGGAGAACGACCACATGGA GGACAAGACCCCAGTCACGAAATGGACAGACATTCCCTAT TTGGGAAAAAGGGAAGACTTATGGTGTGGATCTCTTATAG GGCACAGACCACGCACTACTTGGGCTGAGAACATTAAAGA CACAGTCAACATGGTGCGCAGGATCATAGGTGATGAAGAA AAGTACATGGACTACCTATCCACTCAAGTTCGCTACTTGG GTGAAGAAGGGTCCACACCTGGAGTGTTA

[0100] An exemplary Spodweni virus lineage has the following nucleotide sequence (SEQ ID NO:13 which encodes the protein provided at Accession No. DQ859064, which is incorporated by reference herein:

TABLE-US-00005 atgaaaaacccaaaaagagccggtaggagccggcttgtcaatatgctaaaacgcggtgca gcccatgtcatccctccagaaggaggactcaagaagctgcctgtaggattgctattaggt cggggtccgatcaaaatgatcctggccatactggcattcctacgatttacaacaataaaa ccgtccactggcctcatcaacagatggggaaaagtgggcaaaaaagaggccatcaaaatc ctcacaaaattcaaggctgacgtgggcaccatgctgcgtatcatcaacaatcggaagaca aaaaagagaggagtcaaaactgaaattgtgttcctggcattgctgatgtctattgttgct atggaagtcacaaaaaagggggacacctattacatgtttgcggacaagaaggacgccgga aagatggtgacctttgagactgaatctggacccaaccgttactccatccaagcaatggac attggacatatgtgtccagctacaatgagctatgaatgtcccgtgctggaaccacagtat gagccagaggatgtcgactgttggtgcaactcgacaggagcatggattgtgtatggcaca tgcacccacaaaacaacggaagagacaagacgttccagacgttcaatcaccctgccatct catgcctcacaaaaattggagaccagatcatcgacatagcttaaatcgcgcaaatactcc aaatatctaataaaagtggaaaactggatcctccgcaatccaagatatgcgttggtgact gcagtgattggatggactctgggcaggagtcgcagccagaagatcatctttgtcactctg ctcatgttggtagcccccgcatacagcatcagatgcattggaattggaaacagagacttc attgagggaatgtccagtggcacctgggtgaacattgtcctggaacatggtgattgtgtg acaataatgtcaaacgacaaacccacattggactttgaactggtgacaacgaccgcaagt aacatggctaaggtcaagtcctactactatgaaactaacatatccgagatggcatcggac aggaggtgccccacacagggggaagcttatcttgacaaaatggccgactcccagtttgtg tgcaagcgtgggtacgttgacaggggctggggaaacggatgtggactctttggaaaagga agcattgtcacttgcgctaagttcacatgtgtgaaaaagctcacagggaaaagcattcaa ccggaaaatctcaaataccggatcgttatttcggtacacgcttcccaacatagaggaata attaacaatgacaccaatcaccaagacaacaaggaaaacagaacacgcattaatatcaca gctagcgctccccgtgttgaggtggaacttggctcctttggatccttctcgatggagtgt gaaccccggtcaggattgaactttggtgacctgtattacctcaccatgaacaacaagcat tggctggttaatagaaattggtttcacgatctttccttaccatggcatacagaagccaca tcaaacaatcatcactagaacaacaaggaggcgctggtaaaattcaaaaaagcccacgca aagaagcagacggctgtgatcctagaaagtcagaaaggaactgttcacacagcactggcc ggcgcactggaggctgagtctgatggacacaaagcgactatctactctggacacttgaag tatcgcttgaagctagacaaactgcgcctgaagggaatgtcatatgcactctgcacagga gcattcaccttcgctcgcaccccctctgaaacaattcacggcaccgccacagtggaactg caatatgcaggtaaagatgggccgtgcaaagttcccatagtaattaccagtaacaccaat aggatagcctcgacaggcaggctgatcacagcgaatccggtgatcacggaaagtggaaca aactcaaagatgatggtcgagattgaccctccgtttggtgattcttacattattgtgggc actggcacaacaaaaattacccaccattggcacagagccggtagttcaattggacgtgca tttgaggctaccatgagaggagcaaaacggatggcggtcctcggcaacaccgcttgagac tttagctctattggggacatgttcaactccgttagaaagtttgtccaccaggtatttgga tcaacatttaaggcattgtttggagacatgtcctggttcacacagctectgatagaattt ctgctcatatggatgggtttgaacgcacgcggtggaaccgtggccatgagcttcatgggc attggggctatgctgattttcctagccacctcggtgtcaggagacacaggatgctcggtt gacatatccagaagggaaatgcggtgcgggagcgacatattcgtgtacaatgacgttgac gcatgacaaagccgctacaaataccatcctgaaacccccagaactttggccactgccata aaaacagcttggaaagaagggacctgtaacattacctcagtgagcagaatgaaaaaccta atgtggagctctgtggctggagagttgaatgcaatccttgaggacaattcagtgccattg acagtcgtcgttggcgagccaaaatatccactgtacaatgctccaaagaggctgaaacca ccagcatcagagttaccgcaggggtggaagtcctgaggaaagtcatactttgtctcagcc gcaaaaaacaacaactcctttgtagtagatgataacaccatgaaggaatgcccaaaacag aagcgagcatagaacaacttgagaatagaggatcatgggttcggagtcttccacactagc atctggctgaaattccatgaggacaactccaccgaatgtgacacagctatcataggaacg gcggttcgcgggaaggaagccgttcatagtgacttgggctactggatagagagtgagcgc aatgacacatggaggctctctcgagcgcacctgatcgaagcaaagacatgtgaatggcca cggtcgcacacactgtggacggacggagtgaaagagagcgagctgatcattccacgtggc ttaaccggtcctttcaaccatcataacacgcatactggctacaagactcagaataaaggt ccctggcatttaggtgatgttgaaattcagttcgccacgtgccccggaacaaccgtggtc caggaccaagagtgcagggacaggggcgcttctctacgcacgaccacagctagtggaagg gtaatcaatgaatggtgctacaggtcatgcaccatgcctccactcagtttcaagacaaaa gatgaatgttgatatgcaatggagatacgtcctgtgaaagaacaagagtcaaacctcgtg cgatcacacgtcactgccggaagcacaaaccacatagaccatttctctctcagattaata gtggtcatgttgatggtgcaagaaggtatgaagaagagaatgacatcaaaagcaataatc acctcagcggcctttctcctggcggttatgatagtgggaggtttcacgtaccaggatttt aggaggctagtggtattggtggatgctgcatttgctgaaatgaacactggagatgacgtt acgcacctagcgctgatggcagcgtttaaaatgaggccagcgatgctggtctcattcatg ttcagagccttgtggacccccagagagtcactgcttttaactctggctacctgcctcctg caggtgtcagtgacaccactggatcattccatcatgatcgtggttgatgggattgcgctg tcctggttgtgtctgaaagccatcttggtgccgcgtaccccaaacatagcccttcctctt ctcgctatgctgtcacccatgctccaaggtaccaccattgtggcatggcgagctatgatg gcggccctggctgtcataaccttggcttccatgaagcatggaaggggtgtaaaaaaaacg tttccctacaccatcggatgcatccttaacagcatagacttaattgaaaacttggggtta gttggcctcctcttgttgacagcctcaaaaaagaggagttggcctccgagtgaggtgatg acggctgtcggactgatctgtgcaattgtgggcggactaaccaagaccgacattgacatg acgggacccatggcaaccatagaactgctgatggtgagctatgtgatttctgacaagagt atggacatatacattaaaaaggtgtgtgacatatcatgagacaagaacgctgaaataaca gacacaagtccgcggctgaatgtagctctcgacaacagtaaagatttctcacttatccag gatgacgggccccccactcgagagattgtgttgaaggtgtttctgatgtgtgtttgcggt gtcagccccatagccatcccctttgcagccgctgcttggttcgtgtacattaaatcaggg aaaaaaagcggcgccatgtaggacattccatccccaagagaagtgaaaaaaggggaaaca acggctggagtatacagaatcatgacacgtaaattgctgggcagcacacaggtgggagcc ggagtaatgcataaaggtgtttttcacacaatgtgacacgtcacaaaaggttcggccctt cggagtggtgagggacgcctagatccatactggggaaacgtgaagcaggatttgatctct tactgcggaccatggaaactggatgggaaatgggacggcgtgtcggaagtccaactgata acggtcgccccaggtaagcgcgccagaaatatgcagacaaaaccaagagtgttcaagacc actgatggagaaatcagggccttggcccttaacttcccaggcggaagttcagactccccg ataattgacaaaaatgaacatgtaattggcctgtatggaaatggtgtcatggtcaagagt ggaagctacgtgagtgccatcatgcagacagagaagatggaggaacccgcagttgactgc tttgaggaggacatgctgagaaaaaagaagctgacggtgctcgacctccatccaggagct ggaaaaactcgaagagtgctccctcaaatcgtcaaggctgcaattaagaaacgcctacgc acggtaatcctagcacccacccgagtagtggcagctgagatagctgaggcactaaaagac cttccaataaggtacatgactccggcaatttcagccacccataatggcaataagattatt gaccttatgtgccacgccacttttacatcaaggctaatgcaaccaattagggtgcctaat tacaatctatatataatggatgaggcccacttcacagatcctgcaagcatcgctgcaaga aggtacatagcaacaagagtggacatgggaaacgccgcagccatcttcatgacggccacc cctcctggcagcactaaagctttcccggattcaaacgcccccatcacagatgttgaaaca gagattcctaacaaggcgtggaattctggatttaaatggatcactgattacccagagaaa accgtttggtttgtccctagtgtcagaatgggcaatgagatctcggcctgcctcacaaaa gccggcaaatcggttatccaactcagccggaaaacctttgaaacagagtaccagaagaca aagaatggtgaatgggactttgtcgtaaccactgacatctcagaaatggaagccaacttc aaggccgacagagtcatagactcacgaaaatgcttgaagccagtgattctggatgacatg gaagaaaaagttattcttgccaggccgatggcagtaacaccatccagcgcaactcaacgc agaggaagaattggaagaaaccccaacaaaactggagatgagttctattacggggggggc tgtgccgcaacggatgatgaccatgctcattgggtagaggctaggatgctgcttgacaac atctacctccaggacaacctcgttgcatctctgtacaaaccagaacaaggaaaggtctcg acaatagaaggggagttcaaactgagaggaaaacagagaaaaaccttcgtggagctgatg aagagagggaacttgccaatgtgattgtcatatcaagtgacggcctccagactcaactat actgaccggcgctggtgctttgatggaaaaaacaacaacaccatcctggaggactgcgtc cccgtcgaggtgtggacaaaatttggagagaaaaagattctgaagcccagatggatggac gctcagatctgctctgatcatgcctctttgaagtctttcaaagagtttgctgcaggaaag agaacaatagccactggcttaattgaagcttttgagatgcttcccgggcacatgactgag agattccaggaggccgtcgacaatttggccgtgttgatgagggccgaggcaggctctagg acacacagaatggctacagcacagctccctaagacaatagaaaccatcctgctcctcagc ctgctggcattcgtgtcacttggtgtattttttatactgatgagggcaaaaggattagga aaaatggggtccggcatgatcgtgctggcaggaagtggctggctcatgtggatgtctgag gtggaaccagcccgcatagcttgtgtggtgatcatagtgtttctgctaatggtcgttctg attccggaaccagagaagcagcgctctccccaggacaatcaactggctctaattatcttg atcgcgacgggcctcatcacgctcatcgcggccaatgagctaggttggttagaaagaaca aagagtgacctcaccaggctgttttggaaagaacacgctgagccaacaggaaggagaaga ttttccttctcgctggacattgacctgcggccggcatcggcctgggcaatatatgccgct atgacaaccctgatcacaccgacagtccaacacgctgtgaccacatcgtacaacaactac tctctcatagctatgaccactcaggccggaattctttttggcatgagacgggaggtgcct ttttacaaatgggactttggcgtgccactccttatgctaggctgctactcacaacttacc ccactcaccctgatcgtgactctcgtgatgctaaccgctcactatctctatctcatcccc gggctccaggcaacggccgccagggccgcccaacgaaggacggctgctggaataatgaaa aacccagtggtggatggaattgtggtaactgacatagacccaatccaaatcgatccaaat gtcgaaaagaagatgggccaggtcatgctcatctttgtggctttggcgagcgcgattctc atgaaaacggcatggggttagggagaagctggtgcccttgcatcggcagcagctgccacc ctatgagaagggactcccaacaagtactagaattcatcaacgactacatccttgtgcaac atatttcggggaagttatctggcaggtccctccctcatctacaccgtcacacgcaatgca ggtatcatgaagaaaaggggcggtggaaatggagaaacggtgggcgagaaatggaaggag cgcttgaatcggatgaccgcgcttgaattctacgcctacaagcggtcaggaataactgaa atgtgcagagaacccaccagaaaagccttgaaggatggagtcgtcacaggagaacacgct gtctcccgcaaaagcgcaaagctacaatggatgatggaacatggccacatcaatctagtg ggacgcgttgtcgacctcggatgtggaaggggtggctggagttactacgccgcatctcaa aagcaagtcctcgaggtgagaggctacacaaaagggggagcgggccacgaggagcccatg aatgtccaaagttatggttagaacatagtgcgactcaagagtggagtggacgttttttat ctaccatcagaaccatgtgacacgctactctgtgacattggagagtcatcctcgaaccca gcagtagaagaaacccggactctgagaatgctcggaatggttaaaacctggctggaacga ggcgtaaagaacttctgcatcaaagtgctctgcccgtacaccagtgccatgattgagcgg ctggaagccctccagcgtcgctacggaggaggcctggtgagggttccactctccagaaat tccacccacgaaatgtactgggtctctggaacaaaatcaaacatcatcaggaatgtgaat accaccagccagctgctcatgcacagaatgaacatccccacgcggaaaacaaagtttgaa gaaaacgtcaatctggagaccggaaccagggcaattgaaaacagagctaaccctcccgac atgaaaaaactaggcagccggattgagcggttgagaaaggaatatggatccacttggcac tacgatgaaaaccacccctacaggacatggcattaccacggcagttatgaggctgacacg caagactccgcctcctcaatggtcaacggcgtggtgcgtctcctctcaaaaccatgagat gcattgagctcagtcaccaacattgctatgacggacacaactccgtttgaacagcaacgg gtgttcaaggagaaagtggacacccggactccagaccccaagcaaggcacgcaaagaatc atggccataacatcacaatggctgtgggaccgcctagcaagaaacaagacccctcggatg tgcacgcgacaggaattcataaacaaggtcaacagtcacgcggcgttgggacccgttttt agagaacaacagggatggggttcagcggccaaageggtagtagatcctaggttttgggag ctcgttgacaatgaaagagaagcccatttgagaggggaatgcttgacctgtgtctacaac atgatggggaaaagagaaaagaaactcggtgaattcgggaaggcaaaaagcagcaaagcc atttggtacatgtggctgggagcccgcttcctcgagttcgaggccctgggcttcctcaat gaagaccactggttaagcagagagaactctggagggggagttgagggcttgggcctccaa aaacttggatacatccttgaagagatcagcaggaagccaggaggcaaaatgtatgccgat gacacggctggctgggacacccgcatcacgaaatacgacctagaaaatgaggcgcgcatt ttggaaaaaatgaacgggatccacaaaaaactcgcacaggccatcatcgagttgacatac aagcataaggttgtgagagtcttgagaccagcaccacaagggaaggtcgttatggacatc atctccaggccagaccaaagggggagtgggcaggtggttacttatgccctcaacacctat acaaacttagtggtgcagctgatccgtaacatggaagcagaggctatcatcaatgaaaga aacatggaagagctccaaaacccatggaaaatcatcaattggctaaaaggaaatggatgg gacagactccactcgatgacagtaaatggagataactgtatcgtgaaaccaatagatgat aggttcgcctatgcactgaatttcctcaatgacatgggcaaggtcagaaaagatgtccag gaatggaagccctcgccggggtggacaaactgggaagaagtgcccttttgctcccaccac ttcaacaagctcccgatgaaggatggaagaacaataatagttccctgccagcaccaagat gagttgataggcagggctaaagtttctccaggaaaaggctgatcactcaatgaaacagca tgcttgggcaagtcttatgcccagatgtggctactgttgtactttcacaggagagatctc cgactcatggcaaacgcaatctgctctgctgtaccggtgagttgggtgcccacggggaga acaacctggtccatccataggcgtgaagagtggatgacaacagaggacatgctagaggta tggaacagagtgtggatcatagagaatgagtacatggaggacaagacccctgtcacagag tggaccgatgttccatacttgggaaagagagaagacttgtggtgcggctcccttattgga cacaggccaagaagcacatgggcagagaacatctgggctgccatttatcaagtgcgccga gcaatcggcgaaactgaagaatatagagactacatgagcacacaggtccgctatggctcg gaggaagagccaagcgctggtatgttgtaa

EXAMPLE 3

[0101] Exemplary vectors expressing GFP were transfected into HEK293 cells and expression was assessed (FIGS. 7-8). prM/E sequences were also expressed from the two vectors in HEK cells and supernatants and cells analyzed 48 hours later (FIG. 9). Supernatants were concentrated by centrifugation at 100,000 g for 60 minutes. Western blots were analyzed using University of Texas Medical Branch (UTMB) mouse ascites. More VLPs were secreted from pCMV-FP transfected cells (lane 11 in FIG. 9) than pTriex transfected cells (lane 13). Sucrose purified fractions were subjected to Western blot (FIGS. 10-11). pCMV-prM/E SC purified pellet (pt) appeared to contain high levels of E protein while pCMV-GFP pt did not, indicating that staining was specific to expression of prM and E genes. In summary, a pCMVvector expressed more protein than a pTriex vector. VLPs collected at days 3-10 provided for about 60 g total protein from about 100 mL. On day 3 the productivity of the cells was about 50 g per 15 mL (3.3 g per mL, or 3.3 mg/L). For stably transfected cells, a marker, e.g., a Zeocin resistance gene, may be introduced into the vector that expresses prM/E.

[0102] ZIKV VLPS (ZIKVLPs) formulated with alum were injected into 6-8-week-old interferon deficient A129 and AG129 mice. Control mice received PBS/alum. Animals were challenged with 200 PFU (>400 LD.sub.50s) of ZIKV strain H/PF/2013. All vaccinated mice survived with no morbidity or weight loss while control animals either died at 9 days post challenge (AG129) or had increased viremia (A129). Neutralizing antibodies were observed in all ZIKVLP vaccinated mice.

EXAMPLE 4

Materials and Methods

Cells and Viruses

[0103] African Green Monkey kidney cells (Vero) and Human embryonic kidney 293 (HEK293) were obtained from ATCC (ATCC; Manassas, Va., USA) and grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, Utah), 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 100 U/ml of penicillin, 100 g/ml of streptomycin, and incubated at 37 C. in 5% CO2. ZIKV strain H/PF/2013 (GenBank:KJ776791), was obtained from Xavier de Lamballerie (European Virus Archive, Marseille France). Virus stocks were prepared by inoculation onto a confluent monolayer of Vero cells.

Animals

[0104] Mice of the 129/Sv background deficient in alpha/beta interferon alpha/beta/gamma (IFN-//IFN-) receptors (AG129 mice) were obtained from B&K Universal Limited (Hull, England) and were bred in the pathogen-free animal facilities of the University of Wisconsin-Madison School of Veterinary Medicine. 5-week-old BALB/c mice (The Jackson Laboratory, Maine, USA) were used for wild-type vaccination studies. Groups of mixed sex mice were used for all experiments.

Production and Purification of ZIKV VLPs

[0105] The prM and E genes of ZIKV strain H/PF/2013 with nascent signal sequence were cloned into a pCMV expression vector under the control of a cytomegalovirus (CMV) promoter and CMV polyadenylation signal (pCMV-prM/E, FIG. 1). Endotoxin free, transfection grade DNA was prepared using Maxiprep kit (Zymo Research, Irvine, Calif.). VLPs were expressed by transfecting 90% confluent monolayers of HEK293 cells in a T-75 flasks with 15 g of pCMV-prM/E using Fugene HD (Promega, Madison, Wis.) transfection reagent according to manufacturer protocol. The 10 ml supernatant was harvested 72 hr after transfection, and clarified by centrifugation at 15,000 RCF for 30 min at 4 C. Clarified supernatants were layered onto a 20% sucrose cushion and ultra-centrifuged in a SW-28 rotor at 112,000 RCF for 3.5 hours at 4 C. Pellet (PT) and supernatant (SUP.) fractions at each step were saved for analysis by SDS-PAGE and Western blot. Post sucrose cushion PT were resuspended in Phosphate Buffered Saline (PBS) pH 7.2. Total protein in VLP preparations was quantified by Bradford assay. VLP specific protein was determined by comparing Zika specific bands on SDS-PAGE gels to known concentrations of BSA using ImageJ software.

Western Blot

[0106] VLP fractions were boiled in Laemmli sample buffer (BioRad, Hercules, Calif., USA) and resolved on a 4-20% SDS-PAGE gel (Biorad) by electrophoresis using a Mini-PROTEAN 3 system (BIO-RAD, CA). Gels were electroblotted onto nitrocellulose membranes using a Turboblot system. Membranes were blocked in 5% (W/V) skim milk and probed with mouse hyper immune ascites fluid primary antibody (1:5000) and goat anti-mouse HRP conjugated secondary antibody (1:5000). Membranes were developed using a solid phase 3,3,5,5-tetramethylbenzidine (TMB) substrate system.

Transmission Electron Microscopy

[0107] Samples were negatively stained for electron microscopy using the drop method. A drop of sample was placed on a Pioloform (Ted Pella, Inc.) carbon-coated 300 Mesh Cu grid, allowed to adsorb for 30 seconds, and the excess removed with filter paper. Next, a drop of methylamine tungstate or uranyl acetate (Nano-W, Nanoprobes Inc.) was placed on the still wet grid, and the excess removed. The negatively stained sample was allowed to dry, and was documented in a Philips CM120 (Eindhoven, The Netherlands) transmission electron microscope at 80 kV. Images were obtained using a SIS MegaView III digital camera (Soft Imaging Systems, Lakewood. Colo.).

Vaccination and Viral Challenge

[0108] Each of the following animal studies was performed as one biological replicate. For VLP formulations, the indicated dose of sucrose cushion purified VLPs was mixed with 0.2% Imject Alum (Thermo Scientific) according to manufacturer's protocol. Groups of AG129 mice were injected intramuscularly (TM) with VLPs mixed with alum (n=5) or PBS mixed with alum (n=6) at 6 weeks of age, and again at 8 weeks of age. Sub-mandibular blood draws were performed pre boost and pre challenge to collect serum for analysis by neutralization assays and for passive transfer studies.

AG129 mice were challenged with 200 PFU of ZIKV strain H/PF/2013 in 25 L volumes by intraderml (ID) injection into the right hind footpad at 11 weeks of age. Balb/c mice were vaccinated once at 5 weeks of age as above, and challenged at 13 weeks of age with 200 PFU of H/PF/2013 in 50 l by retro orbital injection (IV route).

[0109] Following infection, mice were monitored daily for the duration of the study. Mice that were moribund or that lost greater than 20% of starting weight were humanely euthanized. Sub-mandibular blood draws were performed on day two post challenge (PC) and serum collected to measure viremia.

[0110] Eight week old AG129 mice were used for passive transfer studies Five naive mice were injected intraperitoneally (IP) with 500 L of pooled serum from VLP vaccinated, diluted serum (1:5 n=4, 1:10, n=4), or serum from PBS/alum (n=5) treated mice. At 12 h post transfer, mice were challenged with 20 PFU in 25 l as above.

Viremia Assays

[0111] Viremia was determined by TCID.sub.50 assay. Briefly, serum was serially diluted ten-fold in microtiter plates and added to duplicate wells of Vero cells in 96-well plates, incubated at 37 C. for 5 days, then fixed and stained with 10% (W/V) crystal violet in 10% (V/V) formalin. Plates were observed under a light microscope to determine the 50% tissue culture infective doses (TCID.sub.50s). Serum samples were also tested for viral RNA copies by qRT-PCR. RNA was extracted from 0.02ml of serum using the ZR Viral RNA Kit (Zymo Research, Irvine, Calif.). Viral RNA was quantified by qRT-PCR using the primers and probe designed by Lanciotti et al (Lanciotti et al., 2008). The qRT-PCR was performed using the iTaq Universal Probes One-Step Kit (BioRad, Hercules, Calif.) on an iCycler instrument (BioRad, Hercules, Calif.). Primers and probe were used at final concentrations of 500 nM and 250 nM respectively. Cycling conditions were as follows: 50 C. for 10 min and 95 C. for 2 min, followed by 40 cycles of 95 C. for 15 sec and 60 C. for 30 sec. Virus concentration was deteif lined by interpolation onto an internal standard curve made up of a 5-point dilution series of in vitro transcribed RNA, with the lowest copies per reaction being 100.

Neutralization Assay

[0112] Serum antibody titers were determined by microneutralization assay. Briefly, serum was incubated at 56 C. for 30 min to inactivate complement and then serially diluted two-fold in microtiter plates. 200 PFUs of vines were added to each well and incubated at 37 C. for 1 h. The virus-serum mixture was added to duplicate wells of Vero cells in 96-well plates, incubated at 37 C. for 5 days, then fixed and stained with 10% (W/V) crystal violet in 10% (V/V) formalin, then observed under a light microscope. The titer was determined as the serum dilution resulting in the complete neutralization of the virus.

Plaque Reduction Neutralization Test

[0113] Serum samples were serially diluted, mixed with 200 PFU of the ZIKV H/PF/2013 strain and incubated for 1 hr at 37 C. This serum/virus mixture was added to confluent layers of Vero cells in 96 well plates and incubated for 1 hr at 37 C., after which the serum/virus mixture was removed and overlay solution (3% CMC, 1DMEM, 2% FBS and 1Anti/Anti) was added. After 48 hrs of infection, the monolayers were fixed with 4% PFA, washed twice with PBS, and then incubated with ZIKV hyperimmune mouse ascitic fluid (1:2000, UTMB) diluted in blocking solution (1PBS, 0.01% Tween-20 and 5% Milk) and incubated overnight at 4 C. Plates were washed three times with PBS-T and then peroxidase-labeled goat anti-mouse secondary antibody (1:2000) was incubated on monolayers for 2 hours at 37 C. Following incubation, cells were washed a final three times with PBS-T and developed using 3-amino-9-ethylcarbazole (AEC)-peroxidase substrate. The amount of formed foci were counted using an ELISPOT plate reader (ImmunoSPOT-Cellular Technology); quality control was performed to each scanned well to ensure accurate counting. Neutralization percentages (Nx) were calculated per sample/replicate/dilution as follows:

[00002]$\mathrm{Nx}=\{100-[100.Math.\left(\frac{A}{\mathrm{Control}}\right)$

Where A corresponds to the amount of foci counted in the sample and Control is the geometric mean of foci counted from wells treated with cells and virus only. Data of corresponding transformed dilutions (Log(1/Dilution)) against neutralization percentages per sample was plotted and fitted to a sigmoidal dose-response curve to interpolate PRNT.sub.50 and PRNT.sub.90 values (GraphPad Prism software).

RESULTS

[0114] Expression and Purification of Soluble, Zika VLPs To generate Zika VLPs (ZIKVLPs), we cloned the prM/E genes with native signal sequence into a pCMV expression vector (pCMV-prM/E) (FIG. 1A), transfected HEK293 cells and harvested supernatants (supe.) 3 days post transfection. 78 g total protein was recovered from post sucrose purification of which 21.6 g was ZIKVLP protein. Western blot analysis of this pCMV-prM/E supe. revealed expression of an about 50 kDa size band (FIG. 1B, lane 2) that corresponded in size to the predicted size of the Zika virus E gene, and additionally matched positive control Zika virus stocks (FIG. 1B, lane 3). To test the hypothesis that expression of Zika prM and E genes spontaneously form extracellular particles, supernatants from pCMV-prM/E and pCMV-GFP (negative control) transfected cells were centrifuged on a sucrose cushion (SC) sufficient for pelleting of flavi virus particles from cell culture proteins (Merino-Ramos et al., 2014). pCMV-prM/E SC purified pellet (pt.) appeared to contain high levels of E protein, indicating that staining was specific to expression of prM and E genes. To determine if the immune reactive extracellular particles were virus like in nature, we performed transmission electron microscopy (TEM) on pCMV-prM/E SC pt. material. TEM revealed virus like particles with a size that ranged from 30-60 nm, and a typical size of about 50 nm (FIGS. 1C-E).

Administration of ZIKVLPs is Immunogenic and Protects Highly ZIKV Susceptible // Interferon Deficient (AG129) Mice

[0115] First, the LD50 of the H/PF/2013 strain in 12 week-old mixed sex AG129 mice was determined. Groups of mice (n=5) were infected with 5-fold serial dilutions from 2 PFU to 0.02PFU of ZIKV and monitored for 4 weeks following the last mortality. All mice infected with 2 or 0.4 PFU died within the first week of challenge (FIG. 4), while lower doses killed only 1 to 2 mice within the first two weeks. Interestingly, 2 mice infected with 0.2 PFU ZIKV became ill and were euthanized due to weight loss and paralysis 4.5 weeks following challenge. The resultant LD.sub.50 value in PFUs was calculated to be 0.19 PFU by the Reed-Muench (REED and MUENCH, 1938) method.

[0116] To determine if ZIKVLPs are immunogenic and protective in highly susceptible AG129 mice, groups of mice received a prime and boost of 450ng ZIKVLPs. AG129 mice that received ZIKVLPs developed low levels (GMT=1:9.2) of neutralizing antibodies (nAbs) at two weeks post administration (FIG. 2A), that increased two weeks after boost (GMT=1:32). Five weeks after primary vaccination, all mice were challenged with 200 PFU (>1000 LD.sub.50s) of ZIKV by the ID route. Mice administered ZIKVLPs maintained weight, while mice that received PBS/alum experienced significant morbidity throughout the challenge period (FIG. 20B). All control mice (survival 0/6) died 9 days after ZIKV challenge and had significantly lower survival (p=0.0016) than mice administered ZIKVLPs (survival 5/5, FIGS. 2B and C). Finally. ZIKVLPs vaccinated mice had significantly lower levels of viremia on day 2 post challenge than control mice detected by qRT-PCR (ZIKVLP=1.310.sup.4 RNA copies, PBS/alum 9.610.sup.7 RNA copies, p=0.0356, FIG. 2D) and TCID50 assay (ZIKVLP=1.310.sup.2 TCID50s, PBS/alum 2.810.sup.5 TCID.sub.50s p=0.0493, FIG. 2E).

ZIKVLPs Elicit Plaque Reducing Neutralizing Antibody Titers in Mice That Can Be Passively Transferred to Nave Mice.

[0117] The plaque reduction neutralization test (PRNT) assay is widely considered to be the gold standard for characterizing and quantifying circulating levels of anti-dengue and other flaviviral neutralizing antibodies (nAb) (Thomas et al., 2009). A PRNT assay was developed for rapidly measuring ZIKV specific neutralizing antibodies. Pooled serum samples collected from mice pre-challenge, as well as individual serum samples collected from mice post-challenge were tested by this PRNT assay. Pre challenge, pooled serum from mice administered ZIKVLPs had a calculated 50% plaque reduction (PRNT.sub.50) titer of 1:157. The PRNT.sub.50 titer increased 2 weeks post challenge (GMT=5122) (FIG. 2F).

[0118] To test the role of anti-ZIKV antibodies in protection against challenge, groups of mice received ZIKVLP antiserum (pooled pre challenge serum, titer in FIG. 2F), undiluted (n=5), diluted 1:5 (n=4), or 1:10 (n=4). As a negative control, mice (n=5) were transferred serum from mice previously vaccinated with PBS alum. Negative control mice rapidly lost weight starting after day 7 and all died day 9 post challenge (FIGS. 3A-B). Mice that received undiluted serum maintained weight throughout the 14 day period post challenge, and showed no signs of infection. Mice that received diluted anti-ZIKV antibodies were not protected from challenge, although survival and weight loss were slightly extended relative to negative control mice (FIGS. 3A-B).

A Single Dose of ZIKVLPs Can Protect Highly Susceptible AG129 Mice

[0119] To determine if a single dose could protect AG129 mice, groups of 6-week old AG129 mice were vaccinated with 3 g ZIKVLPs adjuvanted with alum. An additional group of mice (n=5) was vaccinated with a prime and boost of 0.45 g adjuvanted with alum for comparison. Negative control mice (n=5) received a prime and boost of PBS/alum. Vaccinated mice developed neutralizing antibodies measured by PRNT assay prior to challenge (FIG. 17A). Eight weeks following primary vaccination mice were challenged with 200 PFU (>1000LD.sub.50s) of ZIKV by the ID route. All mice administered a prime of 3 g or a prime and boost of 0.45 g ZIKVLPs survived throughout the 6 week challenge period (FIG. 17C) and maintained weight throughout the challenge period. Pre challenge neutralizing antibody titers in both single (GMT PRNT50=288, PRNT90=81) and double dose (GMT PRNT50=235, PRNT90=50) groups increased significantly (p<0.005) in all animals measured at 3 weeks post challenge (FIGS. 17A-B).

ZIKVLPS Protect Wildtype BALB/c Mice

[0120] To determine if ZIKVLPs can protect wildtype BALB/c mice against non-lethal ZIKV challenge, a group (n=6) was vaccinated with a single dose of 3 ZIKVLPS adjuvanted with alum. Negative control mice (n=5) were administered PBS/alum. Eight weeks after vaccination mice were challenged with 200 PFU ZIKV by the IV route. A single dose of ZIKVLPs elicited high titers of neutralizing antibodies (PRNT50=381, PRNT90=75) detected immediately prior to challenge (FIG. 22A). Mice vaccinated with ZIKVLPS were completely protected from viremia on day 2 post challenge (FIG. 18B), and maintained weight throughout the challenge period (FIG. 18C). Negative control animals lost minor amounts of weight beginning at day 2 post challenge, had high levels of viremia and recovered by 2 weeks post challenge. Neutralizing antibodies were undetectable in negative control mice prior to challenge, but increased significantly after challenge (FIG. 18A). Antibody titers in vaccinated mice decreased, but were not significantly different than before ZIKV challenge (FIG. 18A).

DISCUSSION

[0121] Most experts and public health workers agree that a Zika vaccine is urgently needed. In February 2016, the World Health Organization declared that the recent clusters of microcephaly and other neurological disorders in Brazil constitute a public health emergency of international concern. Their recommendations included enhanced surveillance and research, as well as aggressive measures to reduce infection with Zika virus, particularly amongst pregnant women and women of childbearing age. ZIKV is now receiving considerable attention due to its rapid spread in the Americas, and its association with microcephaly (Mlakar et al., 2016) and Guillain-Barre syndrome (Pinto Junior et al., 2015). In these studies, a ZIKV-virus-like particle (VLP) vaccine was designed and it was expressed in vitro as shown by western blot and transmission electron microscopy, and its protective efficacy and role of antibodies in protection in the AG129 mouse model tested. An overall yield of 2.2 mg/L was calculated for the VLP tested. Similar expression levels have been reported for other flavivirus VLP expression strategies (Pijlman, 2015). Future work will optimize VLP production and purification parameters, which should significantly increase both yield and purity. Stably transfected HEK cells that continuously express VLPs allow for scalable production to help meet global demand for a ZIKV vaccine, which is estimated to be 100 million doses a year.

[0122] ZIKV-VLPs, formulated with alum, induced detectable neutralizing antibodies and protected animals against lethal challenge (>400 LD.sub.50s) with no morbidity or mortality. Pre-challenge GMT neutralizing titers were 1:32, and pooled pre-challenge serum PRNT.sub.90 and PRNT.sub.50 titers were 1:34 and 1:157 respectively. At a relatively low dose of 450 ng, our results indicate that our ZIKVLPs are highly immunogenic. The antibody titers obtained are consistent with those reported for other highly immunogenic flavivirus VLP vaccines (Ohtaki et al., 2010; Pijlman, 2015). Previous work has shown a direct correlation between dose of VLPs and neutralizing antibody titers. For ZIKV, questions remain about the quantitative relationship between dose of VLPs and their effect on neutralizing antibody titers and protection from ZIKV challenge in vivo.

[0123] In the above-described studies, mice were vaccinated with ZIKVLPS and challenged with a homologous strain of ZIKV (H/PF/2013), which raises the question of ZIKVLP specific antibody cross reactivity to heterologous viruses currently circulating in the Americas. Although the H/PF/2013 virus was isolated well before the current outbreak from a patient infected in French Polynesia, there is a high degree of amino acid similarity (about 99%) to endemic South American strains of ZIKV (Faria et al., 2016; Zanluca et al., 2015). Some experts agree that the high serological cross-reactivity among ZIKV strains would allow for a monovalent vaccine (Lazear and Diamond, 2016). Nevertheless, care must be taken to empirically determine if antibody responses elicited by ZIKV LPs cross-react and protect against South American strains. Finally, any future ZIKV vaccination programs should incorporate careful surveillance of circulating strains to help suppress immunological escape, and ensure efficacy of vaccines in human populations.

[0124] Vaccinated AG129 mice challenged with >1000 LD.sub.50s had low levels of viremia (1.310.sup.2 TCID.sub.50s, FIG. 2E) detected after challenge. Copies of RNA ZIKV genomes in serum of mice were significantly higher than levels of viremia. However, the disparity between viral genome copies and viremia has been observed for other flaviviruses including dengue (Bae et al., 2003). Since AG129 mice are highly susceptible to viral challenge, it is possible that the challenge dose given for the active vaccination study was artificially high. Methods for challenging mice from infected mosquito bite should be developed to most accurately mimic natural infection. The most important criteria for any ZIKV vaccine is its ability to prevent placental and fetal pathology in ZIKV infected pregnant women. Recently developed IFN deficient pregnant mouse models can provide an opportunity to assess if vaccination of pregnant animals can protect the fetus from ZIKV-induced pathology. (Miner et al., 2016). Although models for ZIKV infection in pregnant non-human primates (NHP) are still being developed, ZIKV vaccines should be tested in NHP translational models which most accurately mimics human immune responses to vaccination.

[0125] A VLP vaccine approach against ZIKV has significant advantages over other technologies as it will eliminate concerns of live attenuated vaccines and insufficient inactivation of killed vaccines for pregnant women and other populations at high risk of suffering the devastating effects of ZIKV infections. Production of inactivated vaccines requires high titer growth of infectious virus which may pose a safety concern for workers. Additionally, the production of both attenuated and inactivated ZIKV vaccines is limited to batch production, whereas flavirus VLPs can continuously expressed from stable cell lines. In recent years, recombinant virus-like particle (VLP)-based vaccine strategies have been frequently used for vaccine design. VLPs are known to be highly immunogenic and elicit higher titer neutralizing antibody responses than subunit vaccines based on individual proteins (Ariano et al., 2010).

[0126] The role of neutralizing antibodies in protecting against ZIKV was demonstrated by antibody passive transfer studies as naive AG129 mice receiving pooled serum from VLP vaccinated animals were fully protected. These results are consistent with previous findings that indicate the important role of antibodies in protecting against many insect-borne flaviviruses, such as Japanese encephalitis, west Nile virus, and tick borne encephalitis (Chiba et al., 1999; Kimura-Kuroda and Yasui, 1988; Tesh et al., 2002), even at low levels of circulating antibodies. In this study, full protection was observed when animals received undiluted serum (PRNT.sub.50 1:157), with no weight loss or other clinical signs observed. While these studies highlight the importance of serum antibodies in ZIKV protection, there are still many important questions related to ZIKV immunology. What is the minimum antibody titer needed for protection, do ZIKVLPs elicit CD8+ responses and are these responses involved in protection, and what is the overall role of cellular immunity in protection? It is also important to determine if anti-ZIKV antibodies, particularly those elicited by ZIKVLPs, play any role in dengue protection or disease enhancement.

[0127] In this study AG129 IFN receptor-deficient mice were used. This mouse models are commonly used for the evaluation of arboviral vaccines, including dengue, chikungunya and yellow fever virus (Meier et al., 2009; Partidos et al., 2011; Prestwood et al., 2012). We recently documented the suitability of mice deficient in IFN-/ and - receptors as an animal model for ZIKV, as they are highly susceptible to ZIKV infection and disease, developing rapid viremic dissemination in visceral organs and brain and dying 7-8 days post-infection (Aliota et al., 2016), and evaluated doses as low as 1 PFU. In our current studies we observed consistent lethality at doses below 1 PFU, indicating that there are viral subpopulations refractory for the formation of CPE in cell culture, but still capable of establishing a lethal infection in highly susceptible mice. It is of great interest is that at a very low dose (0.2PFU) two of five mice became ill more than 1 month after infection, as infection with ZIKV typically produces rapid lethality in AG129 mice.

[0128] The current studies challenged mice with 200 PFU at 11 weeks of age. All control mice lost 20% weight, were moribund, and succumbed to by challenge by day 9. ZIKV challenge therefore appears to be completely lethal in both juvenile and adult AG129 mice. The AG129 mouse model exhibits an intact adaptive immune system, despite the lack of an IFN response, and it has been used extensively to evaluate vaccines and antivirals for DENV (Brewoo et al., 2012; Fuchs et al., 2014; Johnson and Roehrig, 1999; Sarathy et al., 2015). In our studies WT BALB/c mice did not succumb to infection with ZIKV consistent with previous studies where BALB/c mice were experimentally inoculated with 200 PFU of ZIKV (Larocca et al., 2016). Mice also developed high levels of viremia following IV inoculation. A single dose of VLPs prevented detection of viral RNA copies in serum of vaccinated mice at 2 days post infectionwhen viremia levels typically peak in the BALB/c model. It is possible that viral replication was completely inhibited, as there was no boost response in neutralizing antibodies observed following challenge. Finally, in repeat AG129, and Balb/c mice mouse studies, animals were protected from ZIKV challenge 8 weeks after vaccination. ZIKVLP therefore appear to elicit a potent memory response.

[0129] In the present study, aluminum hydroxide (commonly known as alum) was used as the adjuvant for ZIKV-VLP preparations. Since its first use in 1932, vaccines containing aluminum-based adjuvants have been successfully administered in humans demonstrating excellent safety. Adjuvant formulations of ZIKV-VLP may facilitate antigen dose sparing, enhanced immunogenicity, and broadened pathogen protection.

[0130] In summary, a vaccine against ZIKV is currently unavailable, nor is there any specific prophylactic treatment. A VLP based Zika vaccine that elicits protective antibodies in mice, and is safe, suitable for scalable production, and highly immunogenic, is disclosed herein. Fast-tracking development of this ZIKV vaccine is a public health priority and is crucial for restoring confidence and security to people who wish to have children or reside in, or visit areas in which ZIKV is endemic.

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