WifQLE- ELL BASED VACCINE AGAI ST ΊΛΚΑ VIRUS

Gr oss Reference -to Related A leatinu

This application, claims priority to O,S, Provisional Application No. 62/352,796 filed on .June 21, 2016, which is incorporated herein by reference in its entirety.

Technical EieM

The present disclosure relates to -methods ^' and compositions !bftfee- prevention and/or treatment of iavivita infection. In particular, the -present disclosure relates to whole-ceil vaccine compositions that trigger protective immunity against Zika virus infection.

Summary

The presen t disclosure provides Cor an in iimoge»kt-o.ti^sition om iismg.: cel ls (i) comprising at least one flavivirus antigen (e.g.;, at least one Zika virus antigen), aud (ii)

expressin at least one cytokine.

The present disclosure ^' also provides for an immunogenic composition comprising; a first population of cells comprising at least one fJavivtrus antigen (e.g., at least one Zika virus antigen), and a second population of cells expressing at least one cytokine.

The present disclosure provides for a method of preventing or treating itavivirus infection (e.g„ ika vims infection) in a subject, the method comprising administering to the subject an imnnniogenie composition comprising; cells (i) eoniprising at least one tlaviviras antigen. (e.g. _s at least on Zika vims antigen), and (ii) expressing at least one cytokine.

The present di sc losure provides for a method of preventing or treating ¾vi virus infection (e.g. , .Zika virus, infection) in a subject, lite method comprising administering to the subject am immunogenic composition comprising ; a first population of cells comprising at least one fl.a.vi vims antigen (e,g. , at least one Zika virus antigen), and a second populatio of cells

expressing at least one cytokine.

The present disclosure provides tor a method of preventing or if ating ¾vivirus infection (e.g,, Zika vires infection) in a subject, the method comprising: (a) adniinistering to the subject ^' a first population of cells comprising at least one flavi virus antigen (e.g., at least one Zika virus antigen), and (b) administering to the subject a second population of cells expressing at least one cytokine.

In certain embodiments, the first population of cells and the second population of cells are of the same type of cells, in certain embodiments, the first population of cells and the second population of cells ate of different, types of cells.

Non-limiting examples of flaviviriises include Zika virus, the dengue vims (DEHV), the yellow fever virus (YFV), the West Nile virus (WNV), the Japanese encephalitis virus (JHV), the St. Louis encephalitis virus, and the tick-borne encephalitis virus.

(kilogray).

The subject may be a woman. In certain embodiments _* the subject is a. woman who is pregnant.

Brief inscription of the Drawings

Figures XA-ilX Detection by iraraunostaining of viral proteins in the cells for use as whole-ceil based vaccine against ZIKV. Mouse neuroblastoma cell line N2a was transfeeted. with pcD ^' A- 3.1 vector containing mouse GM«CSF gene. The transfeeted cells -were designated as N2a* GM.SCF or N2a-GM. N2a-GM cells- were then Infected with ZIKV strain M 776 (Uganda). Viral infection was monitored b staining viral E2 protein using ant-E2 antibodies 4G2. Figures I A and IB: cell having an infecfiviiy rate of about 40% - 50%. Figures IC and ID: cells having an inactivity rate of about 80% ~ 9Q%,

Figure 2 shows results from the cellular e y ne-iinked imffiimoso b nt assay (cELISA) assay, Sera from ice immunized with 2a-GM (naive) cells did not react either with N2a-GM (naive) Cells ("Group 1 on N2A~GM~n ive") o with. N¾-GM-Z1 V cells ("Group 1 on N2A-GM- Z1KA"). Sera from mice vaccinated with N2a-GM-ZIKV ceils was immunologically reactive to N2a~GM~ZlKV cells ("Group 2 on K2A-GM~Zfi A"), but not reactive to N2a.~GM ceils ("Group 2 on M2A~G -iiaive").

Figure 3 shows antibody response (vertical bars according toibe lefr y~a¾s _s cELISA assay on N2a-G - iiK-V cells) and antibody neutralization potential (solid line with closed circles, according to the .tight y-axis, PENT) for three groups of mice irmiianized with N2a-GM-ZIKV cells (group Ah ] 2a~ZlKV cells (group B), or -purified ZIKV (group C), respectively.

Figure shows antibody response (vertical bars according to the left y-a>ds _> cELISA assay on H2a~GM-ZIKV cells) for, as well as- t e number of colonies infected, with Z V in the presence of a fixed dilution (1 :3000) of the serum (solid line with closed circles, according to the right y- axls) from _* three groups of mice immunized with N2a-GM-ZIKV cells (group A), N2a~ZiKV ceils (group B), or purified ZIKV (group€}, especti el _*

Figures 5A-5KL Testing ZIK infectivity in di fferent cell types. Different types of cells, including Vero cells (Figures 5A and 5B) _; ^' M2a (naive) ceils (Figures 5A and 5B), N2a~GM€SF cells■(Figures.5E a d 5¥ Nl new cell Mae (Figures SO and 5¾), humarr fibroblasts WS-1 (Figures Si and SJ human fibroblasts PCS201.~012 (Figure 5K), were infected with ZIKV- containing cell cultute media (P.2) aad imniunostained using anii-E2 antibodies 4G2 at different time points post-kfectioa, e.g., 24 hours, or 48; tars post-infection. Figures 5A, 5C, SB, SG, and 51 show celfe 24 hours post-infection. Figures SB, 5D, 5F _f 511 5 J and 5 show cells 48 hours post-iafectioa.

DETAILED DESCRIPT!f

The present disclosure provides methods and compositions for the prevention arid/o treatment of Zika virus infection; 1» certai embodiments, die inm notherapy uses cells comprisin Zika virus antigens and expressing a cytokine, sucit as granulocyte macrophage- colony-stimulating factor (GM-CSF),. in. certain embodiments, the present disclosure relates to immunotherap using cells comprising Zika virus antigens nd cells expressing a cytokine, such as GM-CSP. The immunotherapy induces or enhaaces immune responses against Zika viruses.

In certain embodiments, di present disclosure relates to compositions a d to methods for inducing o enhancing an immune response to a fiavi virus antigen (e,g a Zika virus antigen}. An. immune response to the target antigen may provide a prophylactic or therapeutic effect.

Viral infections can be treated and or prevented by administering reagents that modulate the immune system The present compositions and methods inhibit and/or treat the ilavivirus infection (e.g. _* Zika. vims infection), and/or ameliorating on or more symptoms associated wit the ilavivirus infection (e.g., Zika virus infection). The present compositions and methods are useful in the prophylaxis and/or treatment of a disease caused by flavivifuses (e.g., Zika viruses).

An appropriate cell expressing at least ne virus antigen (e.g., cell comprising at least one inactivated or attenuated virus) can be modified to express one or more cytokines, and

administered to an uninfected subject, to serve as a vaccine and elicit an enhanced immune response to- confer the ability to resist subsequent infection b the virus. Alternatively, the antigen and the cytokines may he administered without a host cell to a subject (e.g., as cell lysate). Furthermore, the present composition can be administered to a subject infected by the viruses to treat the viral infection.

in certain embodiments, the present composition and method relate to a whole cell-based vaccine composition. In certain embodiments,, the present whole cell vaccine composition provides multiple antigens that can be targeted by both the innate and adaptive immune systems. In certain embodiments, the present whole-cell based vaccine serves as an adjuvant on its own, because of their abilit to- s imulate the immu e system in a non-specific manner . In certain embodiments, the present whole-cell vaccine comprises allogeneic cells providing MHC~ allotypes (alternative histocompatibilit complexes)* which are powerful stimulators of the i mune res onse, in certain embodiments,, the present wnole cell-based vaccine offers a minrai delivery of viral antigens to antigen presenting cells (APC as the primary recognition of infectious agents involves recognitio of infected cells, rather than individual ^' viral particles.

In certain embodiments, the present composition and method deliver both GM-CSF and inactivated Zifca virus to a subject, an therefore, esta lis both bnmoral as well as cellular immunity against Zifc vims antigens in a subject.

In -certain embodiments, the present disclosure provides for an .immunogenic composition comprising: ceils comprising at least, one ilaviviiiis antigen or a fragment thereof (e.g., at least one Zika virus antigen or a fragment thereof), wherein, the cells also express a cytokine (such a GM-CSF).

In certain embodiments, the present disclosure provides fo an immunogenic composition comprising: cells (i) comprising at least one lavivirus antigen (e,. . _> at least one Zifca virus antigen), and (ii) expressing at least one cytokine.

In certain ernbodiments, the present disclosure provides for an immunogenic composition comprising a first population of cells comprising at least one fla viviros antigen (e.g... at least one Zifca vims antigen) or a fragment thereof, and second populatio of cells expressing at least one cytokine (such as GM-CSF). In one embodiment, ceils that express a cytokine (e.g., GM-CSF) and cells that comprise at least one ilaviviras antigen (e÷g., at least one Zifca virus antigen) (such as inactivated ilaviviras, e.g., inactivated Zika vims) are of the same type of cells, in another e-rabodtaent, cells that express at least one cytokine (such as GM-CSF) and cells that comprise at least one jlavlvifns antigen e.g., at least one Zifca vims antigen) ^' {such as inactivated flavivlrns, e.g., inactivated Zika virus) are of different types of cells.

In the composition, the ratio of t he n umber of the first population cells compri sing at least one flavivirt!s antigen (e.g., at least one Zika virus antigen) to the number of the second population of eel Is expressing at least one cytokine may range from about 50: 1 to about I ; 10, 40: 1 to about ί Ά 30: 1 to about 1:6, 20:1 to about 1 :5 _> 20:1 to about 1 :3, from about 20: 1 to about 1 :2, from about 15:1 to about 1:1.5, f om about 10:1 to about 1 :1, f om about 8:1 to about 1 : 1, from about #: 1 to about 1 :2, from about 5; 1 to about 1 : 1 , rom about 4 1 to about 1 : 1, from about 3:1 to about 1: 1 , from: about 2:1 to about 1:1, fcorn about 1.5:1 to about 1:1, from about 0.8:1 to about 1 :1 , from about 0,6: 1 to about 1 ; 1, from about 0.5:1 to about 1:1, from about 0,4: 1 to about 1:1, or from about 03: 1 to about 1 L In certain embodiments, the ratio of the number of the first population ceils comprising at least one flavivtxus antigen (e.g., at least one Zika virus antigen) to the number of ike second population of cells expressing at least one cytokine may be about 1:1, about 2:1, about 3: L about 4:1, or about 5:1.

The eel! may comprise at least one virus antigen, or may comprise nucleic acids encoding at least one virus antigen. In one embodiment, th cell com rise at least one inactivated vims.

Itt certain embodiments,, the present composition and method use the irradiated whole- cell pancreatic vaccine (G VAX) which is a vaccine derived from human pancreatic cell l ines (PANG 6.03 and PANC 10.05). Pancreatic GVAX has been genetically imodifieti to secrete human granulocyte maerophage-eolony stimulating factor (GM-CSF), an immune-raoduiatory cytokine that can activate antigen presenting cells (APCs, monocytes, and DCs) locally at the vaccine site (Dranolf et ai Inimnnoj. Rev 188:147-154 (2002); Mach etal.€ \m Qpin Iinrnu l 12:571-575 (2000); Dranoff et a ^' l. Proc Natl Acad Sci USA 90:3539-3543(1993).. Furthemiore, macrophages., granulocytes and NiCT cells have also been shown to respond to G ~CSF, thereby improving tumor antigen presentation. Autologous and allogeneic cancer cells that have been genetically modified to express a cytokine„ e.g., GM-CSF, followed by administration to a patient for the treatment of cancer may be used in the present methods and compositions. U .S. Patent Nos. 5,637,483; 5,904,920; 6,277,368; 6,350,445 and 9,669,081 , each, of which is incorporated b reference herein. A form of GM-CSF-expressing genetically modified cancer cells for the treatment of pancreatic cancer is described in U.S. Patent Nos. 6,033.674 and

5,985,290, both of which are incorporated by reference herein. A universal innriuno odularory cytokine-expressing bystander cell line is also described in U.S. Pat. No. _f 464.,973, incorporated by reference herein.

The cell may express the cytokine. The cell ma express and secret the cytokine:. Non- l it iting examples of cytokines thai may be used i the presen t composition and method include GM-CSF, 1L~2 _? 1L-4 _S TNF-alpha, 1L~6 _? CD2, JCAM _* and combinations thereof In one

embodiment, the cell expresses GM-CSF,

hi certain embodiments, the present disclosure provides for an immunogenic composition comprising: cells (i) comprising at least one fiavivifns antigen (e.g., at least one Zika vims antigen), and (ii) expressing at least one immunomodulatory molecnle _* In certain e bodi ents, the present disclosure provides for an imnmnogenie com ositi n comprising a first ^' population of cells com risin at least one fiavf virus antigen (e _* g., at least one 2ika virus antigen), and second population of cells expressing at least one immunomodulatory molecule.

For example , the nrnnunomo uktory molecule can be a rec m i iaut o in cytokine, chemokrae, o iRiojanostimiil toiy' agen or nucleic acid encoding ^' .cytokines, diemokines, or immimostimulstory agents designed to enhance the immunologic response. Nonrlimitmg examples of immunornodutatory .cytokines include interferojxs (e.g., IFN-α _» iFN-fi and IFK-y), intetleufcins (e.g., ti-i, TL-2 ₅ IL-3, 1L-4, IL-5, IL-6, IL-7, IL-S, lL-9, 1L- 10, 11-12, IL-!SJL^O, and IL~2 i), tumor necrosis factors (e.g., TNF-a and TNF~f ). erytlrro oieri (EPQ), FLT-3 ligand, glp!O, ^' TCA-3, MCP- M!F, ΜΙΙ α, ίΡ-ϊρ\ Rantes, macrophage colony stinmdating factor (M-CSF). granulocyte colony stimulating factor (G-CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF), as well as functional fragments of any of the foregoing. Any iaiinunomodulatory chemokme that binds to a chemokke receptor, i.e., a CXC, CC, C. or CX3C chemokine receptor, also can be used in the present composition, and method Examples of chemokises include, but are not limited to, Mi lu, Mip-ΐβ, Mip-3fc (Larc), ίρ-3β. Rantes, Hcc-1 , pif-l, Mpif~2, Mep-l, Μφ-2, Mcp-3, Mcp~4, c -5, Eotaxin, Tare, Elc, 1309.11-8, Gcp»2 Gro-a. Gro-β, Gro-γ, Nap-2, Ena-78, Gcp-2, Ip-10, Mig, I-Tac, Sdf-l, and Bca-1 (Blc), as well as fractional fragments of any of the foregoing.

As used herein, the terms ^ mmun ge ic composition" and "vaccine composition" are lnierchan»eable.

The cells may be proliieration-incompeient. In certain embodiments, methods may be used to inactivate cells to make them incapable of ceil division but retaining the abilit to express cytokines. The cells ma be unable to u er o mitosis, hut still retain the capability to express proteins such as cytokines. Cells may be inactivated by tendering them proliferation incompetent by irradiation. I certain embodiments, a dose ranging from about 3500 rads to about 30,000 tads is used to irradiate cells, in certain embodiments, methods such as treatments with mitomycin C , cy cloheximi.de, and analogous agents, and/or incorporat i o of a suicide gene by the cell etc. are used to render the cells prolifeatiou-iucompetent.

In one em od m n of t e invention, ce ls are ren ere prolifer tion incompetent by irradiation prior to adrainistraiion to the subject.

The present compos tion may ^' elicit T-cel!, and/or B-ceiL, responses against a flavivirus. The present vaccine composition can be used for prevention and/or treatment of flavivit-us infections (e.g., Zika. virus infections). Additionally, vaeetne(s) of the present disclosure can be used to prevent flavivirtts infection (e.g., Zika virus infection) of a fetus during pregnancy, For example, administration of vaccine to a woma before and/or during pregnancy can pre vent flavi virus infection (e.g. , Zika vims infection) of a fetus.

In one embodiment she present disclosure panicles a metho of preventing and/or treating Bavivirus infection (e,g÷ _> Zika virus infection) in a subject, the method comprising tire step of administering t the subject a vaccine composition comprising: cells (i) comprising at least one flaviviras antigen (e.g.,,, at least one Zika virus antigen), and (ii) expressing at least one cytokine,

In one embodiment, the present disclosure provides a method of preventing and/or treating .flavivinis infection (e.g., Zika vims infection) in a subject, the method comprising the step of ^' administering to the subject vaccine composition compri sing; a first population of cells .comprising at least one flavi virus antigen (e.g., at least one Zika virus antigen} and a second population of ceils expressing at least one cytokine.

I I in one embodiment _* me present disclosure provides a method of eliciting an imiaftae response to a flaviyirus (e,g. _; Zika virus) ^' in. a subject, the method comprising the ste of administering to the subject a vaccine composition comprising; cells (i) comprising at least one flavivirus antigen (e.g., at least one Zika virus antigen), and (ii) expressing at least one cytokine. i n one embodiment,, the present disclosure pro des a method of elic iting an immune response to a flavivirus (e.g., Zika virus) in a subject, the method comprising the step of administering to the subject a vaccine composition, comprising: a first population of cells comprising at least one flavivirus antige (e.g., at least one Zika vims antigen), and a second population of cells expressing at least one cytokine.

In one embodi ment, the present disclosure provides a method of preventi ng and/or treating flavivirus infection (e.g., Zika virus infection) in a subject, the method comprising the step f: (a) administering to the subject a first population of celts comprising at least -one flavivirus antigen (e.g., at least one Zika virus antigen), and (b) administering to the subject a second population Of cells expressing at least one cytokine.

In one embodiment, the present disclosure provides a method of eliciting an immune response to a flavivirus (e.g., Zika virus) in a subject, the method, comprising the step of: (a) administering to the subject a first population of cells comprising at least one -flavivirus antigen (e,g., at least one Zika virus antigen), and (b) administering to the subject a second population of cells expressing at least one cytokine.

The first population of ceils comprising at least one flavivirus antigen (e÷g.,. at least one Zika virus antigen), and the second population of ceils expressing at least one cytokine may be co-administered to a subject. The first population of cells comprising at least one flavivirus antigen (e.g., at least one Zika virus antig n) may be administered to a subject prior to, concurrently with, or subsequent to, the second population of cells expressing at least on cytokine.

As used herein, the term "co-adrainistering" refers to a process where the first populatio of cells comprising at least one flavivirus antigen (e.g., at least one Zika virus antigen) and the second population of cells expressing at least one cytokine are encountered by the subject's immnn system at essentially the same time. The two populations of cells may or may not be administered by the same vehic le, If they are administered in two separate vehicles., they can be administered sufficiently closely, b h 'in time and by route of administration, tha they ate ■encountered, essentially siumltaneous!y by the s«bject% immune system to achieve the de ired specificity.

The present disclosure relates to compositions and methods of regulating the immune response of a subj ect to a virus antigen by administering a mixture' of the v ms antigen, and one or more cytokines, and/or adhesion or accessory molecules, in such a manner that the immune System of the subject is stimulated, in certain embodiments, the antigen, the cytokine, and/or the adhesion or accessory molecule, are coadministered ia a therapeutically effective amount, which results in the systemic immune response _*

The present composition and method provide for transfer or release of the cytokine., and/or the adhesion or accessory molecule, in direct proximit or in combination with the virus antigen. The cytokine and the vims antigen may be administered in the same vehicle, or may be administered in two separate vehicles. If they are administered In two separate vehicles, they are administered sufficiently closely , both in time and by route of administration, thai they are encountered essentially simultaneously by the individual's immune system. That is, the cytokine, andbr the adhesion or accessory molecules, can be co-administered with the vims antigen in any manner which provides transfer or delivery of the cytokine in the context of the v irus antigen in relatio to which the Immune response is to be regulated. For example _* this can be accomplished by rising slow or sustained release delivery systems, or direct injection. In certain, embodiment, as a result of the co- administration, the nonspecific cytokine has the specific effect of amplifying- or altering the specific iminnne response to the virus antigen. The emphasis is on local interaction of the cytokine and the virus antigen to mimic the physiological occurrence of simultaneous presentation of cytokine and antigen, to maximize efficacy and minimize toxicity.

The present composition f vaccine composition or immunogenic composition) fract ons by preparing the immune system to mount a response to virus _* In certain embodiments, a vaccine c an comprise an antigen, which is a virus or a component of die vims, or a fragment thereof, that is introduced into subject to be vaccinated in a non-toxic, non-infectious and/or non-pathogenic form, in certain embodiments, virus antigens include whole Mv« attenuate viruses (modified to reduce their virulence) or inactivated viruses, individual viral components (e.g, , protein or polysaccharides) and the genetic material of the virus (e.g, _:1 RNA or DNA), The

disorder or condition (e.g., afflicted by viral infection).

As -used herein, "susceptible to" or "prone to" or "predisposed to" a specific disease or condition (e.g., viral infection) and the l ke refers to a subject who based on genetic,

environmental, health,, and/or other risk factors is more likely to be infected by the virus than the general population. An increase in likelihood of bein infected by the virus may be an increase of about 10%., 20% , 50%, 100%, 150% , 200%, or more.

As used herein, the terms "treat," treating," "treatment/ ^* and the like refer to reducing or ameliorating a disorder (e.g., viral infection) and/or symptoms associated therewith. The term "effective amount'* refers to am amount of the compositio or cells that is sui cient to stimulate* enhance, or elicit^ the mmune response of a subject against a flawwus (e.g,, Ztka. vk s).

An "immonological response" or "immune response" to an antigen, or vaccine or co sition comprising the antigen, is the development its a .rna malian subject of a umoral ^• and/or a cellular immune response to the antigen. An 'immunological response" or "immune response" as used herein encompasses at least one or more of the following effects; tire

production of antibodies by B-cells; and/or the activation of suppressor -cells and/or ^' Γ-eells directed specifically to an antigen or antigens present in the vectors, composition or vaccine of interest. In some embodiments, the "mmumological response*' or "immune response"

encompasses the inactivatioii of suppressor T~eells. A "cel lular immune response" is one mediated by T-lymphoeytes and/or other white blood cells, including without limitation H cells and macrophages. Functionally cellular immunity includes antigen specific cytotoxic T cells (CTL). Antigen specific T cells, CTL, or cytotoxic T cells as used herein refers to cell which have .specificity for peptide antigens presented in association with proteins encoded by the major histocompatibility comple (MHC) or human leukocyte antige s (HLA) as the proteins are referred to in humans. CTLs of the present invention include activated C TL which have become triggered by specific antigen in the context of MHC; and memory CTL or recall CTL to refer to T cells that have become reactivated as a result of re-exposure to antigen as well as cross- reactive CTL. CTLs of the present disclosure include CP4+ and D8+ T cells. Activated antigen specific CTLs of the present disclosure promote the destruction, and or lysis of ceils of the subject infected with the pathogen or cancer cell to which the CTL are specific via amongst other things, secretion of chemokines and cytokines Including without limitation macrophage inflammatory protein I a ( IP-la), MIF- 1B _S and RANTES; and secretion of soluble factors that su press the disease state, Cellular immunity of the present disclosure also refers to antige specific response produced by the T helper subset of T cells. Helper T cells act to help stimulate the function, arid .focus the activity of nonspecific effector cells against cells displaying peptide in association with MHC molecules on their surface;, A cellular immune response also refers to the production of cytokines, chemokines and other such molecules produced by acti vated cells and/or other whit blood cells including those derived from CD4 and CDS T cells and NK cells, A composition or vaccine that elicits a cellular Immane response ma serve to sensitive a mammalian subject ^' by the presentation of antigen in association with MEC molecules at the cell surface. The cell-mediated immune response is directed at, or near, cells presenting antigen at their surface. in addition, antigen-specific T-lytaphoeytes can be generated to allow for the future protection of an immunized host The ability of a particular antigen to stimulate a cell- mediated immunological response may be determined by a number of assays known i the art. Such as by lyiBphoprolifefation (lymphocyte ^' ctivation) assays, CT ^' L cytotoxic cell assays, or b assaying for T ymphoeytes specific for the antigen i a sensitised subject Such assays are -well known in the art. See, e,.g... Brlekson et a!., J. Immunol, (1993) 151:4189-4199; Doe etai. Ear _* I. Immunol. (1994) 24:2369-2376. Methods of measuring cell-mediated immune response include measurement of inftaeeliuiar cytokines Or cytokine secretion b T~cell populations, or by measurement of epitope specific T-ee ls e,g. _t by the tetramer technique) (reviewed by

cMichael et at, I Exp, Med. 187(9)1367-137., 1998; Mcheyzer-WilUams et ai, Immunol. Rev, 150:5-21, 1996; La!vaiu et al 1 Exp. Med, 186:859-865, 1 97), An immunological.

response, or immune response, as used herein encompasses one which stimulates the production of CTJLs, and/or the production o activation of helper T-cells and/or an antibody-mediated immune response.

The present method and composition may regulate the knraune response to a itaviviros (e.g. , Zika virus) in a subject. The term "regulating the immune response" refers to any alteration in any cell type involved i the immune response. The definition is meant to include m increase or decrease in- the numbe of cells, an increase or decrease in the activity of the ceils, or any oilie changes which can occur within me immune system. The cells may be, but are not limited to , T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages,, eosinophils, mast cells, dendritic cells, or neutrophils. The definition encompasses both a stimulation or enhancement of the immune system to develop a sufficiently potent response to a deleterious target, as well as a suppression of the immune system to avoid a destructive response to a desirable target, in the case of stimulation of the immune system, the definition includes future protection against subsequent viral challenge.

Zika ^' Virus and J¾viylr uses The present compositions and methods can be nsed for the treatment and/or prevention, of infection caused by a ffevivirus _* The Flav virus family (Flavivjridae) are single-stranded {+} RNA v ras. In certain embodiments, flavivirases - include arthropod-transmitted human pathogens. Nan-limiting examples of ftaviviruses include Zika vims, the dengue virus (DENY), the yellow fever virus (YFV), the West Nile virus (WNV), the Japanese encephalitis virus (JEV), the St. Louis encephalitis virus, and the tick-borne encephalitis virus.

The present compositions and methods can be used for the treatment and/or prevent on of infection caused by Zika. vims, inc luding any strain of Zika vims. Within the mosq«ito~bome ciade of flaviviruses, ZlK ^r is a ..member of the Spondweni. group; both genetically and serologically. ZIK! V Is related closely io the four serotypes of DEN V with approximately 43% amino acid identity and extensive antibody cross-reactivity (Alfean et ah i, Virol 89, 11773- 1 1785 (2015), Current data suggests that ZIKV is likely io have originated in East Africa and subsequently spread to West Afric and then to Asia, resulting in distinct lineages (Nigerian strain, MR766 strain, and the Asian strain). All strains currently associated with the outbreak in the Americas belong to the Asian genotype and are most closely related to strains from Yap, Cambodia, Thailand, ami French Polynesia, (Petersen et at , N Engl J Med 201$; .374:1552- 1563).

In certain embodiments, in order to generate a vaccine composition with broad specificity and cross-reactivity against more than one Zifca viras strain, cells used for the generation of the vacc ne composi tion are infec ted with two or more strains of Zika viruses, in certain

embodiments, cells infected with distinct strains of Zifca viruses are combined and ^' formulated into one vaccine composition.

A virus antigen (e.g., a Zika virus antigen) may refer to any protein., carbohydrate, nucleic acid (RNA or DMA), or other component capable of eliciti ng an immune response. A virus antigen (e.g., a Zika virus antigen) includes* but is not limited to, the whole virus with all of its associated antigens as an antigen, as wel as any component separated from the virus, proteins purified troni the virus, or. uni ue carbohydrate- moiet es ^' associated with the virus. In certain embodiments, the virus antigen comprises the Savivinxs (or Zika virus) E-glyeeprotein or fia.vivin.5S envelope glycoprotein, eapsid protein, nucleocapsid, and/or a viral glycoprotein. In e ta n embodiments, the virus antigen (e _f g, _s a Zika virus antigen) comprises at least one nonstmclural protein m w-- at least one structural protein of the vims (e.g„ Zika vims). I» certain mbodiment, the Zika virus antigen comprises at least one nonstractural protein and/or at least one structural protein of the virus (e.g.; Zika virus).

The present cell may contain at least one virus antigen (e.g., at least one flavi virus

-ant en, or at least one Zika virus antigen). For example, the present eel! may contain I, 2, 3, ., 5, ,6, 7, 8, 9. 10 or more virus antigens (e.g., fiavivirus antigens, or Zika vims antigens).

Non-limiting examples of the virus antigens include the envelope (E) protein, the membrane (M) protein, viral RMA, a eaps d (C) protein, a structural, protein., a membrane precursor (Pr ) protein, a part or complete envelope, a nonstructural protein (e g., MSI* NS2A, S2B, NS3, N.S4A, NS4B, and Ή$5) of the vims (e.g., tlavivirus, or Zika vims). Bo!lati et ai, Structure and functionality in t aviviros S-proteins: Perspectives for drug design, Antiviral Research, 2010, 87(2);.! 25-148. Kostyuehenko et al., -Structure of the thermally stable Zika virus, Harare (2016} doi i 0.1038/nature 17994.

In one embodiment, the vims antigen is a viral peptide, polypeptide or protein _* In one embodiment, the antigens of a f!avivirus (e.g., Zika vi s) are at least one inactivated or attenuated whole virus ,

An antigen refers to a molecule containing one Or more epitopes (either linear,

conf rm tional or both) or immunogenic determinants that will-stimulate a host's immune- system,, such as a mammal's immune system, to make a humoral and/or cellular antigen-specific response. The terra is used, interchangeably with the term 'iranOTnogen." An antigen, may be a virus (e.g:, an inactivated vims, _: or an attenuated virus), a whole protein, a truncated protein, a fragment of a protein or a peptide. Antigens ma he naturally occurring, genetically engineered variants of the protein, or may be eoclon optimized for expression in a particular mammalian subject or host. Generally, -a B-cell epitope will include at least about 5 amino acids but can e as small as 3-4 amino acids, A T-cell epitope, such .as a G L epitope, will include at least about 7-9 amino acids, and a helper 1 -ceil epitope at least about 12-20 amino acids..Normally, an epitope will include between about 7 and 15 amino acids, such as, 9, 10, 12 or 15 amino acids. The term ^' "antigen* denotes both snhutiit antigens, (i.e., antigens which are separate and discrete from whole organism: with which the antigen is associated in. nature). Antibodies such -as. anti-idiotype antibodies, or fragments thereof, and synthetic peptide mlmotopes., that is synthetic peptides which can mimic an antigen or antigenic ^' determinant, are also captured under the definition of antigen as used herein _* Furthermore, an "antigen" refers to a protein, which includes

modifications, such as deletions, additions and substitutions, generally .conservative in nature, to the naturally occurring sequence, so long as the protein maintains the ability to elicit an

Immunological response, as defined herein. These modifications ma be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the antigens. Antigens of ^" the present disclosure may also be codon o imized by methods known in the art to improve their expression or irmhunogenicity in the host. As used herein, a "cross reaction" immunogenic determinant refers to a determinant, epitope, or antigen which is capable of eliciting an immune response to related but not identical antigenic determinants.

Viruses (e.g.,, Zika virus) may be inactivated by irradiation, a physical method, a chemical method, or a combination thereof

Viruses (e.g., Zika vims) can be inactivated by gamma irradiation, ultraviolet (UV) irradiation, heat, or other methods know in the art.

Viruses (e.g., Zika vims) may be inactivated using physical and/or chemical method. A range of inactivafion agents or methods have been described to inactivate viruses for vaccine purposes. Examples of viral inactivation methods include, gamma irradiation (Martin et al.

Vaccine 2$(ί 8):3143-3151 (2010b)}; Afeharirl and. Mal.lfcaeh.er Immunol Cell ffio 8.8(2)· 103- 104 (20tQ}) _s UV treatment (Budowsky et al. Arch Virol 68 (3- :239-24? ( f 981)}, heat (Minis and Plavsic, N Engl J Med 352(14): 1 1 1-1412 (2012)}, ascorbic acid (Madhttsndana e al. Int J Infect Pis 8(11: 1-25 (2004)), ethy lenimine derivatives (Largfat and Nebel, J Clin Microbiol 11(2): 120-122 (1980)), psorlens ( aves et ah Vaccine 29(15): 2691-2696 (2011)), hydrogen peroxide (Arnanna et al, Nat _. Med 18(6):974~979 (2012)), and other methods (Stauffer et at Recent Pat Antsinfect Drug Pfscov 1(3);2 1-296 (2006)). Sanders et al. provide a detailed discussion, of inactivated viral vaccines in Vaccine .Analysis: Strategies, Principles, and Control, Chapter 2. Spri ger, 2014,

1» certain embodiments, a virus (e,g., ZiiSV) ca be inactivated by gamma irradiation. A perso skilled in the art would recognize that the suitable irradiation dose to inactivate a virus in cells may vary upon the virus, speci fic iral strain, nuhiher of cells carrying a virus, etc. ki certain embodiments, the gamma irradiation dose for inaciivaiiori of the virus (e.g., ZiKV} ranges from about 20 kGy to about 40 k<3y, from about 25 kGy to about 40 kGy, from aboat 25 kGy to about 35 kGy, from about 20 kGy to about 35 kGy, or from about 25 kGy to about 30 kGy.

In certain embodiments, a virus (e.g., ZIKV) cm be inactivated using UV irradiation. In certain embodtments, UV with a wavelength ranging fforn about 230 mm to about 280 nro (e.g., at energies 900-1000 joule/m") is used for the inactivation of a virus (e.g., ZIKV). In certain embodiments, UV with a wavelength of about 254 nra is used for the inaeiivatio of a vims (e,g>, ZIKV).

In certain embodiments,, a virus (e.g., ZIKV) is Inactivated by beat treatment at a temperature ranging from about 45"C to about 80"€1 froni about 45"C to about 7$°C, f orn about 50*C to about 75 ^f, C, from about 5 fC to about 70%, or from about 5 ⁽ f to about 60¾,

I» certain embodiments, virus (e.g. , ZIK i inactivated by irradiation or heat treatment for about 10 minutes to about 5 hours, about 20 minutes to about 3 hours, about 20 minutes to about 2 hours, about 30 minutes to about 1 hour, or longer.

In one embodiment, the regimen used for inaeiivation of Zlka virus in infected cells also renders die ceils proliferation incompeten

In certain embodiments, the virus (e.g., Zlka virus) is inactivated by a chemical agent, such as fottnahn (formaldehyde), beta-propiolae ne (i¾Fl ), hydrogen: peroxide, lutataldehydej -aeety!etfryieneimine, binary ethyieneinnns, tertiary eihyleneiniine, ascorbic acid, capryiie acid, psolarens, detergents including non-ionic- detergents etc. U.S. Patent Publication No.

201700.1.4502.

In order to test the presence of ZIK V in a subject methods relying on the detection of viral nucleic acid b R -PCR ^' and/or the detection of igM antibodies by IgM-captare enzyme- linked immunosorbent assay (MAC-ELIS A ) may be used . Wh ile the detection of vi ral nucl eic acid i serum provides a definitive diagnosis, in most eases vitemia is transient, and diagnosis b RT-PCR has been most successful ^'■ within I week after the onset of -clinical - illness. However, viral RNA can be detected n serum approximatel 10 weeks after infection in a pregnant woman whose fetus has implications of congenita); infection. There k data that suggest that ZIXV RNA can be detected longer In wine than In. serum _* If these findings are further verified, the period durin which a definitive diagnosis of Zika virus Infection can he .established by RT-PCR would be extended (Paterso et at N Engl J Med 2016; 374:1552-1563). Testing /regimens for the diagnosis of prenatal and antenatal Zika virus infection may rely on. the RT-PCR of amniotic fluid. In addition to RT-PCR, tismnmohistochernkal testing have been used to detect Zika virus infection in tissues of fetal losses and full-term infants who died shortly after birth.

be autologous or allogeneic

In certain embodiments, the present, cells comprise human cancer cells that are genetically modified to secrete GM-CSE. In certain embodiments, the present cells are pancreatic cancer cells, colon cancer ceils, lung cancer cells, breast cancer cells, ovarian cancer cells, prostate cancer ceils, melanoma ceils, or combinations thereof. In one embodiment, the present vaccine composition comprises allogeneic pancreatic ductal adenocarcinoma (PDA) tomor cells engineered to secrete GM-CSF. Far example, the vaccine comprises: GM-CSF-seereting PDA vaccine (G¥ AX),

2.1 in certain embodiments, the ceils are permissive to infection by ZiKV. In one embodiment, vaccine compositions and methods ^' of the present disclosiire can employ an cell type whic is pemiissiv for ZIKV infection, where the ceils are maintained in a surviving mode immediately upon infection with Zika viras (i.e., cells are not killed by the virus immediately follo wing the infection).

In certain embodiments, the ability of the cells to secrete GM-CSF- f cilitates the

.recruitment and activation of PCs and macrophages, which increases the effecti veness of antigen presentation to -cells.

in certain embodiments, the present ceils comprise skin immune cells, including dermal fibroblasts, epidermal kera&nocytes, and immature dendritic ceils (X Virol. 2015 Sep 1 ; 89(17): 8880-8896). in certain em odiment the present cells comprise epithelial cells. In certain embodiments, the present ceils comprise- nenroprogeniiot cells, MlaJkar et al. ₅ N. Emi). J Med. 374951-958. (2016); Sarao ei al. PloS Hegl. Trop. Pis. 10 (2016); Tang et al. Cell Stem Cell. 18(5):-587-90 (2016),

in certai embodiments, the present cells are Vero cells, N2a cells, human fibroblast WS- 1 cells, human fibroblast PCS201--G12 cells, RC-5 -cells. WI-38 ee¾ BH -21 cells, CBO cells, C6 C3, or combinations thereof.

At least one vims antigen (e.g., Zika viras antigen) may be introduced into, or loaded onto, the present cells. In certain embodiments, nucleic acids encoding at least one viras antigen (e,g _t , Zika viras antigen) ma be introduced Into the eelis.

The cells may be antigen -presenting cells (APCs). Non-limiting examples of antigen- presenting cells including dendritic cells, macrophages., B cells, cells of myeloid lineage, Langerhans ceils, epithelial cells, or any nucleated cells.

The APC may be autologous or allogeneic; The AFC may be isolated from a subject The APC may also be derived from cells isolated from a -subject.

Dendritic Cells (DCs) can be generated w /v or ex vivo from innnaiirre precarsors (e.g., ^• monocytes), CD34+ cells (i.e., cells expressing€034), etc, IIS. Patent Me. 8,728,806.

In certain embodiments, the cell ma -co-express human leucocyte antige (HLA). The: HI, A expression by the cell, may be the result of a . atural process or due to recombinant expression ofHLA, The HLA. expression may be as a result of HLA expression, by the cell by endogenous processes, Alternatively, the HLA expression ma occur as a result of recombinant engineering and protein production.

Cells may be contacted with nucleic acids encoding on or more vims ntigeos (e.g. _i Zlka virus antigens), for example, cells can be transfected with expression vectors or infected with viral vectors for i troducing .nucleic, acids encoding one or .more virus niigeiis (e.,g. , Zika virus antigens) inio the cells.

Other techniques for introducing nucleic acids into ceils include* but are not limited to, eiec ^' ttoporatkm, jmkroinjection, ^' hypotonic shock, scrape loading, catioriic liposomes, and calciu phosphate eopreeipitation _*

The time a id amount of antigens, or nucleic acids encoding the antigens,, necessar for the antigen presenting cells to process and present the antigens, can be determined, for example, by assaying T cell cytotoxic activity in vitro or using antigerj-presentirsg eels as targets of CTLs, Other methods that can detect the presence of antigen on the surface of antigen-presenting cells are also contemplated by the presented invention. The antigen-presenting ceds loaded with the antigen can be used to stimulate CTL proliferation In vivo or ex νϊνα, The ability of the loaded dendritic cells to stimulate a CTL response can be measured by assaying the ability of the effector cells to lyse target ceils. For example, the non-radioactive LDH cytotoxicity assay or the europium release assa can be used, Volgmarin et at* J. Immunol. Methods 119:45-51 , 1.989.

Expression can be optional ly eifeeted by targeting -the. expression construct to specific cells, such as with viral vector or a receptor ligand, or by using a tissue-specific promoter, or combinations thereof,

In certain embodiments, the present cells are loaded with one or more virus antigens (e.g., Zika virus antigens). As used herein, a cell "loaded" (or "pulsed") with peptide shall mean that the cell has been incubated with the peptide under conditions pennitting entry into, and/or attachment onto, the cell of the peptide. For example, APCs (e.g., dendritic cells) can be incubated with one or more virus antigens (e.g., Ska virus antigens) under conditions that are needed to load the MH€ of the APC (e.g., the dendritic ceil). Suitable conditions for antigen loading are provided that, permit an APC to contact, process and/or present one or more antigens

limited to, non-covalent complex formation (Chariot), osmotic lysis of pinocytic vesicles (Influx pinocytic cell-loading .reagent), electric power (eieetropo.ration), lipid-based delivery system (Bioporter), mtcroirrfection, small protein transduction domains (PTDs) from viral proteins, bacterial secretion system type III (T3SS), oell-penetratiug peptide (CPP) conj gation, arid protein transection. in certain embodiments of the adoptive immunotherapy methods described above, the cells of interest (i e,, mature DCs) can be purified prior to administration to the subject.

.Purification of the cells can. be done usin a variety of methods n wn in the art, including methods in which antibodies to specific ceil surface molecules are employed, Ibese methods include both positi ve an negative selection methods. For example, cells generated in vitro cm be isolated by staining the cells with fhioreseently labeled antibodies to cell surface markers followed by sorting of the cells that express both of these markets on their cell surface using fluorescence .activated cell, sorting (FACS). These and other purification/isolation methods are well known to those of skill in the art. he present cells may be inactivated or rendered proliferation incompetent by any suitable technique kn wn- in the ar such, as gamma irradiation of UV irradiation. Proliferation incompetent cells are ceils that have been treated in such a way that renders them unable to underg mitosis, .bat still maintain the capacity to express proteins, such as a cytokine (e.g., GM~ CSF), ^' In certain embodiments, f r cells infected with ZI V, cells can be treated to .inactivate the vires and rendered proliferation incompetent.

In certain embodiments, the cells are rendered proliferation incompetent using gamma irradiation. A person skilled in the art would recognize that the suitable irradiation dose to inactivate/growth arrest a cell or population of cells ma vary upon the cell type and/or number of cells. In one embodiment, the gamma irradiation dose for rendering cells proliferation incompetent ranges fro about 100 Gy (Gray) to about 300 Gy. In another embodiment, th gamma irradiation dose for rendering cells proliferation incompetent ranges front about 150 to about 30 Gy.

I l certain embodiments, cells are rendered proiifeiation. incompetent using OV irradiation.

In certain embodiments, to prepare cells comprising inactivated iilavivituses (e,g. , Zika virus), cells are first infected with flavivirases (e.g., Zika viruses). Following infection, tlayivlruses (e _* g<, Zika viruses) can be inactivated in such, cells to prevent propagation of th viruses upon administration to a subject.

In. certain embodiments, the gamma irradiation levels for rendering the cells proliferation incompetent are approximately two orders of magnitude lower than those required for virus inacti vation. Thns, irradiating cells at the levels required for the virus inactivation may

completely obliterate them, eliminating the capability of these cells to express and secrete GM- CSF. To ckeujweHi thi s limitation, ells expressiug GM~CSF may be used as bystanders ½ combination with virus-loaded cells> This rmmyBtsai on scenario can be accom lished by initially dividing ceils into two fractions: (i) cells infected with flaviviruses (e.g. _. , Zika viruses); and (ii) cells not infected with favivifuses (e.g., Ska viruses). Then only fraction (i) of the cells (but not fraction <¾)) will, be treated to inactivate the viruses. Under these circumstances, fraction (ii) will not need to be exposed to high radiation levels, as it lacks viruses. Sock approach ensures that cells that flaviviruses (e.g _*> Zika viruses) are irradiated at the levels required, for tendering the cells proliferation Incompetent, which allows them to maintain the ability to express GM-CSF. The two fractions of cells (cells that lack taviviruses (e.g., 23ka viruses) and are rendered proliferation incompetent, and cells that comprise inactivated fiavi viruses (e.g. , Zifca viruses)) are then combined and administered to a subject.

C ytoklnes

The present composition may express or comprise at least one cytokine:. Cytokines include the general class of hormones of the cells of the imnume system, including !ymphokmes, monokines, and others. The present composition may express o comprise an imm.unomodulating cytokine, which Is any cytokine that is involved in immune system regulation or has an effect ■upon modulating the immune response.

Cytokines suitable for use in the present vaccine compositions include, but are not limited to, GM-CSF, m interleukin, an interferon, and tumor necrosis factor. Specific examples of such cytokines include interferon alpha (lFN-α), inter ei&ift^ · (iL~2J _* inter leak g (IL~4), interleukin- 2 (IL* 12), TNF -alpha, and granulocyte niacro hage-colony stimulating factor (GM- CSF), r combinations thereof, Non-limiting examples of cytok nes Include, GM-CSF, IL-l (IL- ialpha or IL-ibeta), lL-2, 1L-3, IL-4, IL-5, IL-6. 1L-7, 1L~8, lL-% IL-ΪΟ, 1L~1 L 1L-I2, IL-J .5, M- CSF, G-CSF, UF, LT, TGF-beta ₅ gamraa-iFN (or alpha-f FN or beia-IFN), T F-alplia, BCGF, CD2, or !CAM. A. 5. Hamblin, 1993, D, Male, ed., Oxford University Press, Hew York, .Y. Guidebook to Cytokines and Their Receptors, 1995, N. A, Nicola, ed. (Oxford University Press, New York, NX).

I » certain embodiments, the cytoki ne is substantially similar to the human form of th protein, oris derived from the protein of the human sequence (i.e.:, of huma origin), In certain. em odiments, cytokines of oilier m¾mmak with substantia! homology to the human forms of.IL-· 2, GM-CSF,, TNF~aIp a, and others, may e used in. me present composition or method when demonstrated to exhibit similar activity on the immune system. In. certain embodiments, the present composition or method uses proteins that are substantially analogous t any particular cytokine, but have relatively minor changes -of protein sequence,

I» addition to the cytokines, adhesion or accessory molecules or combinations thereof ($ h as a NKG2 D Sigand, CD40L, C D80, CD86, and the like), may be employed alone or in combination with the cytokines.. Dranoff (2002) Immunol Rev.. 188: 1 7- .154; Jain, et al (2003) Ann. Surg. Oncol ! 0:810-820; Borrello and Pardoll. (2002) Cytokine Growth Factor Rev.

13; 185-1 3; Chen, et at. (2005) Cancer Immunol. Imnmnother. 27: 1.-11 ; jaergaard, et at (2005) J. Neurosurg, 103:156-164; Tai, et al. (2004) J. Biomed. Sci. 1 1:228-238; Schwaab, etal (2004) J. Urol. 171:1036-1042; Fiiese, et at. (2003) Cancer Res. 63:8996-9006; Brlones, et a). (2002) Cancer Res. 62:3195 _^ 3199; Vieweg and Dannull (2003) Urol Clin. North Am. 30:633-643; Mineheff et al (2001) Crit Rev. Oncol. liematok 39:125-132.

In certai embodiments, immunomodulatory cytokines such as GM-CSF can he used as- adjuvants to whole-ceil based vaccine to enhance the vaccine efficacy. The GM-CSF may he Mi-length human GM-CSF, or may have an amino acid sequence 80%, 85%, 90%, or 95% identical to a &II lengt human GM-CSF, which has one ot more biological activities of lull length GM-CSF. Examples of biological activities of GM-CSF include its capacity to stimulate macrophage differentiation and proliferation, or ac tivation of antigen presenting dendritic cells . Additionally, the GM-CSF sequenc may include one or more mutations which can be amino acid substitutions, deletions, o additions. Methods for the evaluation and the measurement of the biological activity of GM-CSF proteins are known n the aft. U.S. Patent No, 7,371,370, In certain embodiments, cytokines, antigens, or hormones of other mammals with substantial homology to the human forms of the cytokines, antigens, and hormo eSj will be useful in the invention when demonstrated to exhibit -similar activity on the immune system.

In addition to cytokines, ^' vaccin compositions of the present disclosure can be

engineered t secrete cytokines and factors that promote homing of DCs to germinal centers as wel l as licensing of dendritic cell s. Such factors kcl¾de Kpo olysaec atide (LPS),(¾gand for Toll-like receptor 4), Foiy(I C} (recognized y 1 oil-like receptor 3), and CpG (ligaiid for Toll- Tike receptor -9),

Vectors

The virus antigeu($) ma be i trodu ed into t e cell, or loaded onto the celt using any vector. The cell may be genetically engineered using an vector to express cytokine.

As used herein,, the term " vector" refers to a polynucleotide capable of transporting another nucleic acid to which it has been linked. The present vectors can ^' be, for example, a plasrrud vector* a single- or dotjfete-strande phage vector, or a single- or double-stranded RNA or DNA viral vector. Such vectors inchide, but are not Mtriited to, chromosomal, episomal and vims-derived vectors, e.g., vectors derived from bacterial plasrnids, bacteriophages, yeast episonies, yeast chrornosornal elements, and viruses S ch as baouiovituses, papova viruses, SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived fto combinations thereof, such as those derived .from, plasrnid and bacteriophage genetic elements, cosmids and phagemids.

Expression vectors can be Used to replicate and/of express the nucleotide sequence encoding, e.g., a vims antigen, and/or a cytokine in a cell (e.g., a mammalian ceil such as ^' hitman cell). A variety of expression vectors useful for introducing into cells the polynucleotides of the in ventions are well known in the art

Recombinant vectors are prepared usin standard techniques known in the art, and contain suitable control elements operably linked to the nucleotide -sequence encoding the target antigen. See, for example, Plotkin, et al (eds,) (2003) Vaccines, 4 ed,, W.B. Saunders, Co., Pfrifa,, Pa.; Sikora, et al. (eds.) (1996) Tumor Jrnmunology Cambridge University Press, Cambridge. UK Hackett and Ham (eds.) Vaccine Adjuvants, Humana Press, Totowa, N.J.; Isaacson (eels.) (1 92) Rec mbinant DNA Vaccines, Marcel Dekker,.NY. NW; Morse., et al. (eds.) (2004) Handbook of Cancer Vaccines, Humana Press, Totowa, N.J.), Liao, et al. (2005) Cancer Res. 65:9089-9098; Dean (2005) Expert Opin, Drug Deliv. 2 :227-236; Aden, et al. (2003) Expert Rev. Vaccines 2:483-493; Dela Cru¾, et al. (2003) Vaccine 21 : 3317-1326;

Manses, et al (2000) ^' Eur, 1. Pharni, Biopharm.: 5:0:413-417; Exder (1 9$) Vaccine 16; 1439- 1443; Dlsis, el al. (1:996) J. immunoi, 156:3151-3158). Peptide vaccines are described (see, e.g _t , MeCabe, et al, (1995) Cancer Res. S5rl 741-174?; Minev, et al (1994) Cancer Res. 54:4155- 4161· Snyder, et al. (2004) J. Virology 78:7052-7060,

^' Non-limiting viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, siadbis viral vectors, lierpesviral vectors, SV-40 vectors, and pox viral vectors, siicb as vaccinia viral vectors, bacu!oviral vectors, alphaviraS vectors. Fowl po viral vectors, AV-pox viral vectors, modified vaccinia Ankara (MVA) viral vectors, and other .r com inant viruses,

in one embodiment, the vector is an adeno-assoeiated vims (AAV, or adenovirus- assoeiat d virus) vector. See also, e.g.,, Walsh et a!,, !993 _> Proc. Soc÷ Exp, Biol, Med. 204:289- 300; U.S. Pat. No. 5,436,146). Any of the AAV. serotypes may be used, including, but: not limited to,. AA VI, AAV2, AAV3, AAV4, AAV5, AAV6, AAV?, AAVS, AAV9, AA IO,

AAV 11 , etc.

Adenoviruses are described in, e,g.., Kosenfeld et al, 1991 _? Science 252:431-434;

Roseirfeld et al... 1992, Cell 6S: 143- 155; Masiraugeli et al,, .1993, J, Clin, Inves 91:225-234 Kozarsky and Wilson, 1993, Curr. Opm. Genetics Develop, 3:499-503; Bout et al, 1994, Huma Gene Therapy 5:3-10; PCT Publication No. WO 94/12649; and Wang et al., 1995, Gene Therapy 2:775-783). US. Patent No, 7,244,617.

Greater detail about retroviral vectors is available in Boese et al, 1994, Biotherapy 6:2 1-302, in one embodiment, the present expression vector is a lentivirtts (inekid ng ht aan immtmpdeficieacy virus (HIV)), which is a sub-type of retrovirus.

Plasniids thai may fee used as the present expression vector include, but are not limited to, pcDHA3.1, peONAS. i-hygro, pGL3, pCDMS (Seed, 1987, "An LFA-3 cDNA encodes a phospholipid-liuked membrane protein homologous to its receptor CD2", Nature _* 840-842) and pMT2PC {Kaufman et al, 1987, ^' "Translations! efficiency of polycistronic mRNAs and their utilization to express heterologous genes in mammalian cells", FMBO J. 6; 187-193). Any suitable plasmid ma he used in the present in vention.

Bacterial vectors include, for example, .Salmonella _* Shigella, Yersinia, Lactobacillus,. Streptococcus, Baeil!s Calraeae-Guena,. Bacillus ^' anthraeis, and Escherichia coii. The bacterium can be engineered to contain a -nuclejc-acid-eiicodirig- reco binant -antigen.,: a heterologous antigen, or an antigen derived from a tumor, cancer cell, or infecti ve agent Moreover, the bacterium ca be modified to he afte t^e&ih anotae* aspect, -fne- n0n4|sterial bacterial vaccine can be absent of any nucleic acid encoding a recombinant antigen (see, e.g÷, Xu, et al (2003) Vaccine 21:644-648; Pasetri, et ai. (2003) J. Virol. 77:5209-5219; Loessner and Weiss (2004) Expert Opin. Biol Ther, 4:157-168; Graagetie, et al. (2002) Vaccine 20:3304-3309; Byrd, et al. (2002) Vaccine 20:2197-2205; Edelrnan _s et l. (1999) Vaccine 17:904-914; Domeneeh. et ai, (2005) Microbes and Infection 7: 860-866).

Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., episomal mmmaialian vectors). Other vectors (e.g., non-episotiaal mammalian vectors) ate integrated into the genome of a .host cel l upon introduction into the host cell, and thereby are repli cated along with the host genome.

In certain eiiibodiments, a vector comprising nucleic acid sec ence encoding a cytokine (and/or a nucleic acid sequence encoding a virus antigen) may be ti&nsferred to a ceil in vitro, using any of a number of methods known in the art, which include electtoporation, membrane fusion with liposomes,. Lipofectamine treatment, incubation with calcium phosphaie-DNA precipitate, DEAE-dextran mediated transfection, infection with modified viral nucleic acids, direct microinjection into single ceils, etc. Procedure's for the cloning and expression of ^* modified forms of a nati ve protein using recombinant DN A technology are generally known in the art, as described in Ausubel, et al, 1992 and Sarahrdok, et al., 1989. imm» nofcherapy

In one e bod ment vaccine coniposiiions aud rneihods of the present disclosure can be used to prevent and/or treat symptoms and adverse outcomes associated with flavivirus infection (e,g., ZJKV infection). In another embodiment, vaccine compositions and methods of the present disclosure can be used for the prevention of microcephal and other brai anomalies associated with Zika .infection in newborns. In further embodiment, vaccine compositions and methods of the present disclosure can be used to prevent negative outcomes associated with Zika infection in adults, such as increased risk of Guillain-Barre syndrome.

The present composi tion ma ^' be adnrinisiered to a sub ject t prevent or trea t viral infection, and/or th symptoms: associated therewith. In. certain embodiments _* the present composition may be administered to a subject prior to infection, by the virus (e.g., Zika virus). In.

virus).

in certain embodiments, cells expressing GM-CSF and cells comprising inactivated inactivated t½ytv.iruses (e _^ g., Zika virus) are rendered proliferation incompetent and formulated int two separate vaccine eon*positions. Under sncb eircnnistarices, where mere are two or more separate vaccine compositions, vaccine compositions can be adiBtofstered simisltaneonsiy, separately or sequentially.

In certain embodiments, the present composition comprises GVAX comprising at least one vims antigen (e.g., Zika vires antigen). 0 VAX may refer to an. inactivated tumor cell.

containing a nucleic acid that encodes granulocyte maerephage-eolony stimulation factor (GM- CSF), where the tumor cell line was T-26 cells, a cell line that expresses the gp70. AH I is an epitope of gp70, an tnunmiGdomtnartt antigen of C 26 cells. Yoshimura, et al (2006) Cancer Res. 66:1096-1104; Jain, et al. (2003) Annals Surgical Oncol 10:810-820; Zhoa, et at (2005) Cancer l es. 6S:I0?9-IQ8S; Chang, et al. (2000) int. J. Cancer 86:725*730; Borrello and Pardoll (2002) Cytokine Growth Factor Rev. 13 :185-193; Thomas, et al (1 98) Human Gene Thef. 9:835-843), ^' U.S. Patent No, 9,198560:,

According to the methods of the present disclo u e _s vaccine compositions described herein may be used for the prevention and/or treatment of fiavivims infection (e.g., Zika virus infection) in a subject, either ^' alone or in combination with other methods suitable tor the preventio and or treatment of ftavivirus infection (e.g., Zika virus infection).

Methods -are provided for administration, of the present immunogenic composition to a subject in. need of imniunostimulatlon,

Dosages may be titrated to optimize safety and efficacy. Typically* dosage-effect relationships from, in vitro studies can. provide useful guidance on the proper doses for patient administration. Studies in animal models can also be used for guidance regarding effective dosages for treatment of flavivirus infection (e.g., Zika virus infection) in accordance with the present disclosure.

In certain embodiments, one or more than one administration of (he present composition s can be delivered to the subject in a course of treatment Dependent upon the particular course of treatment, multiple administrations -may be given at a single time point with the treatment repeated at various time intervals. For example, an in itial or pr iming (or prime) treatment may be followed by one or more booster (or boost) treatments. In certain embodiments, the primin (or prime) and booster (or boost) treatments are delivered by the same route of administration and/or Any appropriate route of administration may be employed, for example, administration may be topical, arenteral intravenous, rotraarterial _s subcutaneous, tratumoral iniramaseui&r, intracranial, ntraorbital, oplit hnie, intraventrkanar.. intrahepatic, intracapsular, intrathecal, intracistetnai, intraperitoneal, intranasal, aerosol, suppository, or oral administration. For example, therapeutic formulations, may be in the form of liquid solutions, or sus nsions; for oral administration,, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders., nasal drops, or aerosols.

The administration of the present cells o composition (vaccination) may he given once, twice, three times, foilr times, five to s, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen rimes, fifteen times, or more, within a treatment regime to a subject/patient The admmtsiration of the present ceils or composition (vaccination) may be given every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every S: days, every 9 days, every _0 days, every 1 1 days, every 12 days, every 13 days, every 1 days, every 16 days, ever 18 days, every 20 days, every i month, every 2 mouths, every 3 months, every 6 months, or at different frequencies.

The present cells may be administered to a subject (e,g. _s a huma -subject) at a dose ranging from about 1 X Iff* cells to about 1 X lQ ⁿ cells, from about I X ⁴ cells to about 1 X 10 ^K * cells, from about I X 10 ^" cells to about I X 1.0 ^s cells, from about I X 1.0* cells to about 1 X 0 ^s cells, from about: 1 X 10 ⁶ cells to about 1 X 10 ^' cells., from about 1 X 10 ⁷ cells to about 1 X J ø* cells, about 1 X 10 ⁵ cells, about 1 X 1 * cells, about 1 X i0 ⁷ cells _* about f 10 ⁸ cells, or about I X 10 ^?> ceils.

In certain embodiments, the present cells are administered at a concentration of about 1.0 ^* cells/ml to about I0 ^' cells/ml, about JO ³ cells/ml, about Iff cells/ml, about Ϊ0 ⁵ ceils ml, about lO ⁶ cells/ml, about 10 ⁷ cells/ml, about 10 cells/ml, about l f cells/ml, about 10 ⁱ⁰ cells/ml, or about l¥ ¹ eelis l. In certain -embodiments, the total volume of the present composition adm nistered is about 0.001 mL-about 10 raL, e.g., 0.01 niL, 0.1 niL, or 1 mh. In one

embodiment, the present composition is administered at a concentration of about 2 X 10' cells/ml in total vol ame of about 0.1 mh. In one embodiment, the present composition is administered subentaseousiy. in certain embodiments, toe vaccine Is ateiaisteted t ke or more, e„g., 3 times, 4 times, 5 times, 6 times, 7 times, 8 tiroes, 9 times, 10 times, 15 times., 20 times, 25 times* 30 times, 35 times, 40 titties, 50 times, 60 titties, 70 times, 80 times, 90 times or more, for example, the vaccine is administered at least once per week, e.g., at least twice per week, at least three times per week, at least fou times per week, at least five times per week, at least six times per week, at least seven times per week. 1» certain embodiments, the vaccine is administered at least once per day, e.g., at least twice per day, at least every eight hours* at least every four hours, at least every two hours, or- at leas every hoar, in certain embodiments, the present compositions are administered for a duration of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks _* 3 weeks, 4 weeks, five weeks, si weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 moeths, 11 months, 12 months, 2 years, 3 years, 4 years, 5 years or more. In certain embodiments, the present composition is administered one dose every two weeks for 4 to weeks or until the viral infection is treated.

The present invention provides a method for eliciting in a subject an immune response to at least one vires antigen. The method comprises administering to the subject cells comprising at least one virus antigen (or loaded with -at least one virus antigen),, or cells comprising nucleic acids encoding at least one vims antigen. The cells may also express at least one cytokine. The present ceils or composition, when administered to the subject, may elicit a im uoe response to the vims (e.g., Zifca virus), and/or ma elicit an immune response to at least one virus antigen.

In certain embodiments, the present invention provides a method of preventing: and/or treating iral .m!eetiot!, the met od comprising administering to a SBbject a therapeutically or prophySacticall effective amount of a pharmaceutical composition (e.g., an immunogenic composition, or a vaccine composition), wherein the composition comprises: ceils (i) comprising at least one flavivirus antigen (e.g., at least one Zika virus antigen), and (ii) expressing at least one cytokine.

In certain embodiments, the present in vention provides a method of preventing and/or treating viral infection, the method comprising administering to a subject a therapenticaily o prophylaeticslly effective amount of a pharmaceutical composition (e.g., an immunogenic composition, or a vaccine composition), wherein the composition comprises; a first population of cells comprisin at least one fiavivirus antige (e.g, , at least one Zifca virus antigen), and second population of cells expressing at least one ey¾Mne, sncb as G -CSF.

In certain embo iments, die present cells antige»-present¾g cells) comprising one or more virus antigens (or a fragment thereof) may be used to contact lymphocytes under conditions sufficient to produce virus aaiigen-specific lymphocyte capable of eliciting an:

Immune response against the virus. Thus;, the antigen-presenting cells also can be osed to provide lymphocytes, including T lymphocytes and B lymphocytes, for eliciting a immune response against a cell that comprises a vires antigen. In one embodiment, a preparation of T lymphocytes is contacted with the antigen-presenting cells described above for a period of time, tor priming the T lymphocytes to the at least one vims antige presented by the antigen-presenting cells. Accordingly, T lymphocytes and B lymphocytes thai are primed to respond to cell s that comprise a virus antigen can be prepared,

T lymphocytes can be obtained from any suitable source such as peripheral blood, spleen, and lymph nodes. The T lymphocytes can be used as crude preparations or as partially purified or substantially purified preparations, which can be obtained, by standard techniques including, bnt not limited to, methods involving immimomagnetic or Sow cytometry techniques using antibodies.

In another embodiment, T cells can be removed from a subject and treated in vitro with the present cells (e.g. _* , antigen-presenting cells), wherein the resulting CXL are reiofused autologous ly or aflogeneically to the subject,

Acc ordi ngly , the antigen-prirned antigen-presenting cells of the preserti disclosure and tire antigen-specific T lymphocytes generated with these antigen-preseming: ceils c n: fee used as Imniunomodulating composition for prophylactic or therapeutic applications for viral infection. In some embodiments, the virus antigen-primed atrtigen-presenting cells of the present disclosure can be used for generating CD8+ CTL,€D4+ C¾ and/or B lymphocytes for adoptive transfer to the subject, Thus, for example, virus antigen-specific CTLs can be adoptively transferred for therapeutic purposes in subjects afflicted with viral infection.

The present compositions- r meth ds may function t provide or enhance an Immune response. Generally _* the immune response can include humoral immune response, -cell-mediated i mun response, or both. For example, antigen presentation through an. »¾m«aologica-l pathway involving MH.C class O molecules or direct B~eeil sti ulatio can produce a humoral response; and, antigens presented through a pathway Involving M C 1 molecules can elicit cell-mediated immune response, A humoral response can be d ^' eteranned b a standard immunoassay fo antibody levels in a semm sample from the subject receiving the pharmaceutical composition, A ^■ cellular immune response is. a response thai Involves T cells and -can b determined in vitro or in vi vo, For example, a general cellular immune response can be determined as the T cell proliferative activity ^' in cells (e.g., peripheral blood leukocytes (PB:Ls)) sampled from the subject at a suitable time following the administering of a harma eutical composition. Following incubation of e.g., FB Cs with a stimulator for an appropriate period, t¾]ihymidine

incorporation can be determined. The subset of T cells that is proliferati can be determined using flow cytometry. T cell cytotoxicity can also be determined.

The immune response that is elicited or enhanced ma be sufficient for prophylacti or therapeutic treatment of viral infection, or a syrnpiom associated therewith, Accordingly, a beneficial effect of the present compositions- and/or methods will, generally at least in part be immune-mediated, although, an immune response need not be positively demonstrated in order for the composi tions and methods described herein to fall within th e scope of the present disclosure.

While one round of vaccination may be sufficient to generate sustained and protective immu e response* periodic booster (or boost) treatments ma be administered. I one

embodiment booster (or boost) treatments are administered 6-12. months following the initial vaccine administration, or following previous booster (or boost) administration.

An effective ainounf of a prime or boost vaccine may be .given in one dose, but is not restricted to one dose. Thus, the administration can be two, three, four,, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more, administrations of the vaccine. Where there is more than one administration of a vaccine the administrations can be spaced by time intervals- of one minute* two minutes, three, four, fi e, six, seven, eight, nine, ten, or more minutes, by intervals of about one hour, two hours, three, tour, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, IS, 19, 20, 21, 22, 23, 2 hours, and so on. The adn'sinistratioBS can also be spaced by time intervals f one day , two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, 11 days, 12 days, 13 days, 14 days, 15 days, .16 days, .1.7 days, 18 days, 19 days, 20 days, 21 days, and combinations■ thereof. The invention is .net limi ted to dosing intervals that are spaced qually in time, but encompass doses at non-equal intervals* such as a priming schedule consisting of administration at 1 day, 4 days, 7 days, and 25 days, just to provide a non-limiting example.

The following may be take into consideration in determioiug the relative timing of the prime vaccine and boost vaccine. It has been found that dminis ration of an antigen, or nucleic acid encoding an antigen, can stimulate expansion of antigen-specific immune cells, resulting in a peak, followed b contraction of the number of antigen specific immune cells (see, e.g., BadOvinac , et al. (2002) Nature ^' Immunol, 3 :61.9-626). Initiation of the boost vaccination Can be administered before the peak is reached, coincident wit the peak, or after the peak.

Admi stration of the boost vaccinati n can be initiated when a population of antigen- specific immune ceils has expanded (increased in ^■ number) to at least 20% the maxima! number of antigen-specific immune ceils that is eventually attained; to at least 30%; to at least 40%; to a t least 50%; to at least 60%; to at least.70%; to at least 80%; to at least 90%; to at least 95%; to at least 99% the maximal number of antigen-specific immune ceils that is eventually attained. Additional schedules of prime-boost vaccines are available, for example, the boost vaccination can fee initiated w hen the population of antigen-specific cel ls has contracte to under 90% t he maximal number of antigen-specific cells; under 80%; under 70%; under 60%; under 50%; under 40%; under 30%; under 20%; under 10%; under 5%; under 1.0%; under 0.5%; under 0.1%; under 0,05%; or under 0.01% the maximal number of antigen-specific immune ceils. The antigen-specific cells can be identified as specific for a vector-specific antigen (specific for empty vector), otsp cifie lor a heterologous antigen expressed by a nucleic acid contained in. the vector.

hi other aspects, administration of the boost vaccination can be initiated a t -about day s after the prime vaccination is initiated; about 10 days after the prime vaccinatio is initiated; about 15 days; about 20 days; about 25 days; about 36 days; about 35 days; about 40 days; about 45 days; about 50 days; about 55 days; about 60 days; about 65 days; about 70 days; about 75 days; about 80 days, about 6 months, and ab ut 1 year after administration of the prime vaccination is initiated.

The boost vaccination can be administered 5- 10 days after the prime vaccination; 10-15 days after the prime vaccination; 15-20 days after the prime vaccination; 20-25 days after the prime vaccmation; 25-30 days after me prime vaccination; 30-40 days after the prime- vaccination; 40-50 ^' days alter the prime vaccination; 50-66 days after the prime vaccination; 60- 70 days after the prime vaccination; and so on.

In some embodiments of the invention the boost dose will enhance the prime dose imrnnne response by at least two-fold, at times between about three- aad five-fold or five-fold to ten- fold, or from ten-fold to I DO-feld or greater, in some eiiibodiroen ts of the inventi on the prime dos and boost dose wilt ave a synergistic effect on tire immune response, in some

embodiments of the invention the enhanced im une response will include a T-cel!: response., and in some embodiments the T-cell response will be a CDS† T-cell response, I some embodiments of the invention the prime dose and boost dose will break the mammal's tolerogenic state towards the target antigen _*

Treating a subject using the present compositions and methods may refer to reducing the symptoms of the disease, reducing the occurrence of the disease, reducing _. the severity of the disease, and/or preventing a disease from occurring. As such, to treat a subject means both preventing disease occurrence (prophylactic treatment) and treating a subject thai has a disease (therapeutic treatment). In particular, treating a subject is accomplished providing or enhancing an iiniBune response in the subject.

The present, cell may comprise one or more virus antigens, including 2, 3, 4, 5 _* 6, 7, 8, % 10 or more antigens. In certain embodiments, the present cell comprises one or more inactivated (or attenuated) viruses (e,.g., one or more inactivated (or attenuated) Zika viruses). Additionally, multiple, independently generated cells can be administered to a. subject.

The present cells may be autologous, allogenic (e.g. , from a different donor subject thai is MHC matched or mismatched with the recipient subject) or heterologous to the recipient subject. The cells can be introduced into a snbject. by any mode that elicits the desired trnmone response to the virus (e.g.;. Zifes virus).

Vaccine compositions of the present disclosure can be administered, for example, intramuscularly, intradera ally, subdem aHy, subcuianeously, orally, intraperitonea!iy,

intratheeally, or intravenously. Modes of administration also include systemic rectal, topical, intraocular, buccal, intravagiuai, intraeisiernal, ifttracerebroven ricidar, intratracheal, nasal, transdermal, witMn/on. implants, or parenteral. reuies _* The term "parenteral" includes subcutaneous, intrathecal, mnavenous, intramuscular, intraperitoneal, o in&sion. Compositions comprising a composition of the invention cat} be added to a physiological fluid, such as blood. Inhaled therapy is also provided,

the. quantity of the vaccine compositions described in the present disclosure for effective therap will depend upon, a variety of factors^ including the type of strain of ZiKV infection _* means of administration, physiological state of the patient, other medications administered, and other factors. The vaccination or administrat o parameters, the dosing schedule, etc., can. -be determined by routine experimentation. A mammalian subject can initially be given a low dose of the vaccine composition. The dose and/or -the relative amounts of the vaccine composition can be varied while monitoring die immune response.

Suitable pharmaeeuricali acceptable carriers for the vaccine .compositions of the present disclosure refers to fluid vehicles that can be injected into a host without significant adverse effects. Suitable pharmaceutically acceptable carriers known in the art include, sterile water, saline, glucose solution, and physiologically acceptable aqueous buffers or solutions, including phosphate-buffered saline. Camera may Include auxiliary agents including,, diluents, stabilizers (i.e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like. Appropriate amounts of the ceils are mixed fth the selected carrier t form the final vaccine composition. n one embodiment, all the components of the vaccine can be provided together in one carrier, but if desired, one or more components can be provided in a separate carrier and administered in combination with the other components.

The surro ate . oint of vaccination can include the specific anii-Zl V antibod levels (titers), T-ce!l activation (by the JFN-gamma levels) and viremta levels upon virus .challenge. The inveiitors will perform these experiments In rhesus monkeys since they are susceptible to ZIKV infection and display almost all manifestations characteristic of human infection. Additionally, a set of experiments using non-human primates will he performed in order to establish whether the vaccination approach, of the present disclosure protects fetuses form infection and the development of neurologic conditi ons. The present disclosure provides a phannaceiittcal composition, (e.g., an immunogenic composition or a vaccine composition) comprising: cells comprising at leas one vims antigen or a fragment thereof (e.g., at least one Zika virus antigen or a fragment thereof), wherein the cells also express a cytokine, such as (jM~CSF,

The present disclosure also provides a pharmaceutic al composition (e.g., an

immunogenic composition or a vaccine composition) comprising: cells (1) comprising at. least one fJavivirus antigen (e.g., at least one Zika vims antigen), and (ri) expressing at least one cytokine.

in some embodiments, the composition further comprises an adjuvant as described above. When administered to a subject, the pharmaceutic l composition, elicits or enhance an immune response to a virus (eg., Zika virus).

In one embodiment, the present pharmaceutical composition comprises antigen- presenting cells contacted in viim ox ex vivo with at least One virus antigen. I another embodiment, the present invention provides a composition comprising antigen-presenting cells contacted in vitro with nucleic acids encoding at least one virus antigen.

The present pharmaceutical composition can be ^' useful as vaccine compositions for proptiylactic or therapeutic rrcatoent of a viral infection, such as for preventing or treating Zika virus infection in the subject.

In certain embodiments, the present composi tio is administered to a subject, either alone, or hi combination with, one or more other modalities of therapy.

The pharmaceutical composition may further comprise a pharmaceutically acceptabl carrier, diluent, or excipient. Techniques for formulating and administering also can be found in Remington's Pharmaceutical Sciences, Mack Piiblishing Co., Easton, Fa., latest edition.

Pharmaceutically acceptable carriers known i the art. include, but are not limited to, sterile water, saline, glucose, dextrose, or buffered solutions. Agents such as diluents, stabilizers (e.g., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, additives that enhance viscosity, and the like. Preferably, the medium or carrie will produce minimal or no adverse ef ecis.

The phamiaceulical composition ma further comprise a adjuvant Preferably, the

4.1 adjuvant employe provides for increased ini aaogenieity. The adjuvant can be one that provides fa slow release of antigen (e.g., altposome * or it can be an adjuvant that is

immunogenic. For example, the adjuvant can be a known adjuvant or other substance that promotes nucleic acid uptake, recruits immune system cells to the site of administration , or facilitates the immune activation of responding l mphoid cells. Adjuvants include, but are not limited to, oil and water emulsions, aluminum hydroxide, grucau, dextran sulfate, iron oxide, sodium alginate, Baeto-Adjuvant, synthetic polymers such as poly amino acids and co-polymers of amino acids, saponin, paraffin oil, arid mummy I d peptide. U.S. Patent No. 9,011,835,

in some embodiments, the adju vant is comprised of incomplete Preand'S adjuvant (Monianide IS A 51) or Corynebacterram granufosum F40.

The vaccine compositions of the invention may further comprise various excipients _s adjuvants, carriers, auxiliar substances, modulating agents, and the like, A carrier, which is optionally present, is a molecule tliat does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized niacromolecules such as proteins, polysaccharides, pofylaetic acids, poiygivcollic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Examples of particulate carriers include those derived from polymethyl methaerylate polymers, as well as raicropafticl.es derived, from poly lactides) and poly(lactide-co-glycolides), known as PLG. See., e.g., Jeffery etai, Pharrn. Res. (1993) 10:362-368; cGee J , e af , J Micfoencapsii!. 14(2): 197-210, 1.997 0'Oagin D T, et ai, Vaccirie 1 1:(2): 149-54,. 1.993. Such carriers are well known to those of ordinary skill the art Additionally, these carriers may function as ininiunostiniulating agents ("adjuvants

Furthermore, the antigen may be conjugated to a bacterial toxoid, such as. toxoid from diphtheria, tetanus, cholera, etc., as well as toxins derived from E coll. Such adjuvants include, but are not limited to: (1) aluminum salts { to );, such a - aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oiJ h ater emulsion formulations (with or without other specific imniimostimnlatin agents such as mnramyi peptides (see below) or bacterial cell wall components), such as for example (a) MFS9 (international Publication Ma WO 90/MS37), ^• contaktng 5% Squaleae, 0,5% Tween 80, and 0.5% Span 85 (optionally containing variou amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfiulili¾er such -as Mode! ί 10 ¥ microfluidlzer (Microti ui dies, Newton, Mass.), (b SAF, containing 10% Scpaiane, 0.4% . Tween SO, 5% pluronic- iocked polymer L121 , and MDP either microfliudized into a sulmilcron emulsion or vortexed to generate a larger particle size ^• emulsio.fi, and (e) Ribi.TM. adjuvant system (RA5), (Ribi Imraunochem, Hamilton, Mont) containing .2% Scfuafene, 0,2% Tween 80, and one or mote bacteria? cell wall components from the group consisting of mouophospaoryhptd A (M L), trehalose dimyeolate (TDM), and ceil wall skeleton (C S), preferably MFL^CWS (Detoxn); (3) saponin adjuvants, sack as

Sti nlo.n.TM, (Cambridge Bioscience, Worcester, Mass.) may be used or particle generated therefrom such as ISCOMs (ininiunostlniu!ating - complexes); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (If A) (5) cytokines, such as mier!eukras (IL- Ϊ , lL-2, etc), . macrophage colony stimulating factor (M-CSF) _S tumor necrosis factor (TNF), beta cherrtokines (MiP, I -alpha, 1-beta Eantes, etc); (6) detoxified mutants of a bacter ial ADP- ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT% articular f LT-K63 (where lysine is substituted . for the wild-type amino acid at position 63) LT-R72 (where arginhie is -substituted for the wild-type amino acid at position 72), CT-S10 (where serine is substituted for the- wild-type amino acid at position 109), and PT- .9/G 129 (where lysine is substituted for the wild-type amino acid at position 9 and glycine -substituted at position 1 9) (see, e.g., Inteiiiatioriat Publication. Nos. WO93/132 2 arid W092/I9265); and (7) other substances that act as iinCTHtaostiniulatliig agents to enhance the effecti veness of the eonmosltion.

The dosage and regimen will be d ter in d, at least in part, be determined by tire potenc of the modality, the vaccine delivery employed, the need of the subject.

The pharmaceutical compo ition can be dministered m a therapeutically or a

prophylactleally effective amount. Administering the pharmaceutically acceptable composition of the present Invention to the subject can be carried out using known procedures, and at dosages and for periods of time sufficient to achieve a desired effect. For example, a therapeuticall or prophyiactically effective amount of the pharmaceutical composition can vary according to factors such as the age, sex, and weight of the subject. Dosage regime can be adjusted by one of ordinary skill in the art to elicit the desired innnune response including immune responses that provide therapeutic or prophylactic effects, The pharmaceutically acceptable .composition can be administered to the su jec at art suitable s¾e> The route of administering can be parenteral, intrai iscular, subcutaneous, intradermal, iutraper itoneal, intranasal, intravenous (inditding via an indwelling catheter), via an afferent lymph vessel, or by any other route and die subject's condition. Preferably, the dose will be administered i«. an amount and for a period of time effective in brid ing about a desired ^• response, be it eliciting the immune response or the prophylactic or therapeutic treatment of the viral infection and/or symptoms associated therewith,

Adi¾inistering can be properly timed, and can depend on the clinical condition of the subject, the objectives of administering _* an r other therapies also being contemplated, or administered, in some embodiments, an initial dose can be administered, and the subject monitored for ati immunological and/or clinical response. Suitable means of immunological monitoring Include using patient's pe ipheral blood lymphocyte (PBL ) as tespo.nde.fs,. An immunological reaction also can be determined by a delayed inflammatory response at the site of administering:

One or more doses subsequent to the initial dose can be given as appropriate, typically on a monthly, semimonthly, or a weekly basis, until the desired effect is achieved. Thereafter, additional booster or maintenance doses can be gi ve as required, particularly when the immunological or clinical benefit appears to subside.

Single or multiple administrations of the present composition can be carried out with cell numbers and treatment being selected by a care provider (p.§>, a hysician), in certain embodiments, the present cells are a minis ere in a phar aeeutiealiy acceptable carrier,.

Suitable carriers can be the growth medium in which the cells wer grown, or any suitable buffering med u such, as phosphate buffered saline, The cells can be administered alone or as an adjunct therapy in conjunction with other therapeutics.

In one embodiment, the present composition is admmisiered sysiemieally, e,g. _? by injection. Alternately, one can administer locally rather than sysiemieally, for example, via injection directly into tissue. The pharmaceutical composition ma be in a depot or sustained release formulation. Furthermore, one can administer in a targeted drug- delivery system, fo example, in a liposome that is coated with iissiie-speeitlc antibody . The liposomes can be targeted to and taken up selecti vely b the tissue. harm ceutical■ ebmpos oiis may be administered directly, eudoscopiealiy, intratracheally, tntrattwntoraUy, intravenously, intraiesionally, inu'aranscularly, intraperitonealiy, regionally, percutaneously, topically, intrarieriaily. jntravesically, or subetrtaieousjy.

Compositions i»ay be a mmisiered. 1, 2,. 3, 4„ 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 7, IS, 19, 20 or more tim.es, and they may be administered every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, IB,. 19, 20, 21, 22, 23, 24 hours, or I, 2, 3, 4, 5, 6, 7 days, or L 2, 3, 4, 5 weeks, or L 2, 3, 4 , 5, 6, 7, 8, 9, 10, I L 12 months.

These compositions can be stored in unit or muiti~do.se containers, for example, sealed ampoules or vials, as art aqueous solution or as a h ophilized t¾nt};oJatioji for reconstiiutJon. In one embodiment, a therapeutic composition containing an agent of this invention is administered in. a unit dose, for exam le _* The term "luiit dose" when used in reference to a therapeutic composition, of the present invention refers to physically discrete units Suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; Le., carrier, or vehicle.

The presen t pharmaceutical composi tion can he gi ven subsequent to, preceding, or eontemporarieousry with, other therapies including therapies that also elicit an. immoae response in the subject, and/or other antiviral therapies. For example, the subject may previously or concurrently be treated by anti- viral agents, surgery, other forms of immunotherapy, mm- angiogenic agents, and hormonal agents. Such other therapies preferably are provided in. such a way so as not to interfere with the immunogenicity of the compositions of th present invention.

Methods of Measuring Imiaufte Response

A variety of in vitro and i vivo assays are known in the art for measuring an hnmone response, including measuring humoral and cellular immune responses, which include but are not 1 united to, standard . immunoassays, such as MA, ELISA assays; intracellular staining; T celt assays including for example, !ymplioproliferaiion {l pfeocyte activation) assays, C L

cytotoxic cell assays, or by assayin fo T-lymp oeytes specific for the antigen in a sensitized Subject. Such assays are well known in the art . See, e,g,, Ericfcson et ah, I. Immunol. (.1993) 151:4189-4199; Doe et a ... Eur. 1, Immunol. (1994) 24:2369-2376. Recent methods of measuring cell-niediaied immune response include measurement of intracellular cytokines or cytokine secretion by T-eell populations, or by measurement of -epitope specific T-eells (e.g., by the tetramer technique) (reviewed by cMichael, A. JL and O'Callaghan, C. A., I.Exp. Med.

187(9)1367- 1371, 1998; cbeyzer-Willla m, M. CL, et a!.. _; Immunol Rev. 150:5-21, 1996;

Laivaoi, A _r et al, J. Exp. Med. 186:859-865, 1997). Assays for measuring. an immune response also include the cellular enzyme-l inked immunosorbent assa (cELiSA) assay, plaque reduction neutralization test (FR T), etc.

The release of cytokines (e.g., ΙΕΝ-γ, TN - , and/or XL- 17) ma be assayed by, e.g., ELISpet assay, to determine ^' immune responses.

The cytokine ELISPOT (Enzyme-Linked ImmunoSPOT) assa is designed to enumerate- cyto¾«e~seerering cells. The assay has the advantage of detecting only actiy3ted m:ernory T eells and has the ability to detect cytokine release in response to antigen b a single cell thereby permitting direct calculation of responder T cell frequencies. Tire high sensitivity and easy performance, allowing the determination of peptide-reactive T cells without prior in vitro expaiision _s makes the ELISPOT assay well suited to monitor T cell responses. Tanguay et al, 1994. Lymphokine Cytokine Res. 13: 259. Carter et al, 1997. Curt. Opin. Immunol. 9: 177. In. illustrati ve embodiments disclosed herein, the enzyme- linked inmiunospot (ELISPOT) assay (BD Biosciences) is used to detect and analyze ^' individual cells that secrete interferon-gamma (IFN'gamma). The ELISPOT assay is capable of detecting cytokine producing cells from both activated naive arid memory T-cell populations and derives lis specificity and sensitivity b employing high affinity capture and detection antibodies and enzyme-amplification. Additional i form tion regarding the use of ELISPOT assay is provided in I. Immunol. Methods. 20GL 2S4(l-2):59. Briefly, cells are incubated in the wells of the ELISPOT plate pre-coated with a high-affinity monoclonal antibody to which the cytokine, produced during incubation, will bind. Subsequently, cells are washed away. Areas in which the cytokines have been bound are detected with a combination of biotmyiated anti-cyiokme detection antibodies and <[>-labeied goat an ti- biotin antibodies. The last step in the assay is the addition of a reagent allowing the precipitation of silver on (^ revealing the site of cytokine secretion, (i.e., spot formation), Animal models, e,g, min-hutnan primates, are known in the art. For example, the mous is an accepted model for human immune response. Mouse NIC cell response to tumors is an. accepted model for human ^' N ^' cell response to tumors. Additionally., mouse T cells are a model for human T cells, mouse dendritic cells (DCs) area model for human DCs. mouse NKT cells are a model for human NKT cells _* - mouse innate respoase is an accepied model for human innate response, and so on. Model studies are disclosed, for example, for CD8 - T cells, central memory T cells, and effector memory T cells (see, e.g., Walzer, ef al (2002} J. Immunol. 168:2704- 2711); the two subsets of NK cells (see, e.g.. Chakir, et l. (2000). J. Immunol. 165 4985-4993; Smith, eiai. (2000) J. Exp. Med. 191 : J 341-1354; Ehriich, et al. (2005) J, Immunol. 174:1922- 1931 ; Peri it, et at (1998) J, Immunol .61 :582! -5824); NKT cells (see, e.g., Couedel et at

(1998) Eur. I im uaol 28:4391-4397; Sakamoto, et al (1 99) J. Allergy Clin. Immunol 103: S445-S451 Saikh, et al. (2003) 1. Infect. Dis. 188:1562-1570; Bmoto ₄ et a! (1 97) Infection Imniimity 65:5003-5009; Tanigachi, et al. (2003) Arum. Rev. Immunol. 2 :483-513; Sidobre, et al. (2004) Proe. Natl. Acad. Sci 101 :12254-12259); monocytes macrophages (Sunderkotier, et al, (2004) J. Immunol 172:4410-4417); the two lineages of DCs (Boonstra, et al (2003) J. Exp. Med 197:101-109; Donnenberg _? .et al. (2001) Transplantation 72:1946-1951 : Becker (2003) Virus Genes 26: 1 19-130: Ca ine, et al. (2003) J. Immunol. 173 ;6466-6477; Penna, et al (2002) J.. Immunol. 69:6673-6676; Alferink, et al.. (2003) J. Exp. Med. 197: 585-599). Mouse innate response, including the Toll-Like Receptors (TLRs), is a model for human innate immune response, as disclosed (see, e,g., Janssens arid Beyaert (2003) Clinical M-icrob, Revs, 16:637- 6 6), M use neutrophils are an accepted model Cor fcumaft nean¾phik (see, e,^ Kohayashl etal. (2003) Proc. Natl Acad. Sci. USA 100: 10948- 10953; Torres, etal. (2004) 72:2131-2139; Sibelius, et al, (1999) Infection Immunity 67; 1.125-1 BO; Tvinnerei , et al. (2004) J. Immunol. 173:1 94-2002). Murine immune response to Listeria is an accepted mode! for human response to Listeria (see, e.g., Kolb-Maiiter, ei al. (2000) Infection Immunity 68:3680-3688; Brzoza, et al (2004) J. Immunol 173:2641-2651 ).

The immunological efficacy of the present me-thods and compositions may be determined based on the Distribution Free Resampling (DFR) method.

Immune responses to vaccines were assessed by harvesting splenoeytes, a source that prov ides cells of the immune sy stem, including T cells and dendritic cells (DCs). Antigen-

4/ specific inmiwne responses were measured by Incubating spfenoeytes till one or more peptides and meas ring immune ceil activity, where activity was determined by iptrace!i«lar statiing (ICS) and Enzyme-Linked munoSpot (EOSPOT) assays. In some assays, only a single peptide was added, where the peptide contained only one epitope of a viral antigen. In other assays, an. entire library of peptides was added,, encompassing ^' the entire length of the antigen.

ICS assays involve permea ilmng the splenoeytes, and treating with an antibody that binds cytokines that have accumulated inside the immune cell, where the antibody allows fluorescent tagging. Brefeidin blocks protein transport, and provokes the aceanndation of cytokines within the immune cell.

Kits

The present disclosure further pertains to a kit containing the present phaHnaeeuticat composition. The kit or container holds an effective amount of a pharmaceutical composition for carrying out the methods or producing the compositions described herein and/or instructions for producing or using the compositions for prophylactic use in, or therapy of, a patient or subject having or suspected of having or at risk of viral infection.

The kit may comprise instructions for administering the present composition to a subject having ot at risk of viral infection (e,g. Zika virus infection). The instructions will generally Include information abont the use of the composition for the treatment or prevention of viral infection (e ₊ g, , Zika virus _. , infection}, in other embodiments, the insttue tions include at least one of the fol lo wing; description of the therapeutic agent _s dosage schedule and ad inistration for treatment or prevention of viral infection or symptoms thereof; precautions; warnings;

Indications; eoimier-hidications; overdosage information; adverse reactions; animal

pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container, U .S. Patent Publication No. 203.7/0106067. A kit may also contain instructions for combining the components so as to formulate an

Immunogenic compositio suitable for adnitnlstmtion to a mammal. The compositions of the present invention can be supplied in unit dosage or kit oot*. Kits can comprise various components of the pharmaceutical composition or vaccines thereof provided in separate containers as well as various other active ingredients or agents.

Administering to both human and aon-human vertebrates is contemplated within the scope of the present invention. Veterinary applications also are contempl ated. Generally , ike subject is any living organism in which an immune response can be elicited. Examples of Subjects Include, without limitation;, humans, livestock, dogs, cats, ice, rats, and transgenic species thereof.

in so embodiments, the kit comprises- a sterile container that contains a therapeutic or prophylactic composition; such containers can ^' be ^' boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art, Such containers can be made of plastic,, glass _* ^' laminated paper, metal foil, or oilier materials suitabl e for holding medicaments,

¾e!m|t¾aas

As used herein, "attenuation" and "attenuated" encompasses a virus, or gene in the vims, and the like, that is modified to reduce toxicity to a host. The host can be a human or animal host, of an organ, tissue, of cell. The virus can be attenuated, to reduce binding to a host cell, to reduce spread from one host cell to another host cell, of to reduce intracellular growth in a host ceil, Attenuation- can be assessed by easurin , e, _: g:., an indicator of toxicity, the L¾>, the fate of clearance fr m an gan, or the competitive index (see, e,g, _y Auefbueh, et at- (2001} infect. Immunit 69:5953-5957). Generally, an attenuation results art increase in the LDje by at least 25%; more generally by at least 50%; most generally by at least 100% (2-fold); ^' normally by at least 5-fold; more normally by at least 10-fold; most normally by at least 50-fold; often b at least lOO-ibld: more often by ai least 500-fbld; and most ften: by at least 1000- fold; usually at least 5000-fold; more usually by at least 10,000-fold; and most usually by at least 50,000-fold; and most ofte by at least 100,000-ibid, "Attenuated gene ^4* encompasses a gene that mediates toxicity, pathology, or virulence, to a host, growth within the host, or survival within the host, where the gene is mutated i a way that mitigates, reduces, or eliminates the toxicity, pathology, or virulence. "Mutated gene" encompasses deletions, point mutations, insertion mutations, and frameshift .mutations in regulatory regions of the gem, coding regions of the gene, non-coding re ians of the gene, or any cornbuiation thereof

"Effective amount* as used in treatment encompasses:, without limitation, an amount that can ameliorate, reverse, mitigate, or prevent a symptom or sign of a medicai condition or disorder. Unless dictated otherwise, explicitl or otherwise, ai "effecti e amount" is not limited to a minimal amount sufficient to ameliorate a condition, or to an amount that results In an optimal or a maximal amelioration of the condition. "Effective amount" within the context of administration of a vaccine composition is that which causes an immune response in the mammal, in certain e odimeats* an "effective amount" of a vaccine composition or as immunogenic composition refers to the amount of target antigen which elicits measurable immune response in a mammalian subject as compared to the mmu e response in the manunaiian subject in the absence of adminisu ation of the antigen.

In one embodiment, a subject is a mammal such as a primate, and, more preferably, a human . Examples of non-human pr imates include marmosets, monkeys, chimpanzees, gorillas, orangutans, and gibbons. The term "subject" also includes domesticated animals, such as cats, dogs, etc , livestock (for e ample _s cattle (cows), horses, pigs, sheep, goats, etc.), laboratory animals (for example, ferret, -chinchilla, mouse, rabbit, rat, gerhi!, guinea pig, etc.) and avian species (for example, chickens, turkeys, duc s, pheasants, pigeons, doves, parrots, cockatoos, geese, etc.). Subjects can also include fish (for example, zebratish, goldfish, tilapia, salmon, and trout), amphibians and reptiles.. As used herein, a. "subject" is the same as a "patient," and the ter s can be used interchangeably .

By "control" or "reference" is meant a standard of comparison. As used herein, "changed as compared to ¾ control" sample or subject Is understood as having a level that is statistically different than a sample from a normal, untreated, or control sample:. Control samples include, fo example, cells in -culture,, one or more laboratory test anim ls, or one or more -human, subjects. Methods to select and test control samples are within the ability of those in the art.

As used herein, "operably linked" is understood as joined, preferably by a coval ent linkage, e.g„ joining an arnino-terminus of one peptide, e,g _÷ , expressing an enzyme, to a carboxy teeuinus of another peptide, e.g. , expressing a signal sequence to target the protein to a specific cellular eompartrnent; joining a promoter sequence with a protein coding sequence, in manner that the two ormore eomponeins that are operably linked either retain their original activity, or gain at) activity ijponjoirung such that the activity of the operably linked portions can be assay ed and have detectable activity, e.g.. enzymatic activity, protein expression activity.

The following ar examples of the present myeutfca* and ar not to be construed as Knr ng.

Exauiple I Overview of IKV vaedrt -Studies

Whole-cell vaccination (WCV) against ZIKV is produced using irradiated human cancer cells., whic can be infected with the- virus and which can also be genetically modified to express and secrete cytokines that promote antigen, presenting eelfe to engulf vims~lniee¾d eells, process the viral antigens, and present them to T-cells.

Previously described- 0¥ AX pancreas yaecine is com rise of biun n pancfeat e cattcer cell lines transfected with GM-CSF. For pancreatic cancer treatment, these cells are grown in vitro, irradiated by gamma-rays and administered intradennally as a therapeutic vaccine.

Previous studies have shown GVA expressing GM-CSF is capable of inducing a robust humoral and T-cell response against pancreatic tumors in viva.

In order to generate ZIKV using GV AX expressing GM-CSF, the inventors will first test permissivity (permissiveness) of GVAX pancreas cells (PANC 6.03-PANC-10.05-G -CSF) to support Z KV infection, in parallel, the inventor will evaluate p rmissivSty of GV AX pancreas ceils to ZIK infection to the cell ^' lines known to be pemiissive to ZIK ^T . As described in the present disclosure, various, cell t pes ha e been shown to be susceptible to ZIK infection., where nonliroiting examples include Vero, LLC- K2, HEK293T, etc. following the transaction of cells with ZIKV, pemiissiviiy of GVA cells can be evaluated using a number of techniques, including RT-PCR, ZIKV plaque assay, MOX and/or flow cytometry using ant - ZiK antibody 4G2.

A mouse epithelial cell line (that will be used for the generation of the ccin and the tested it in the preclinical animal model) will also be selected based on its capabilit to support the virus. Once identified, such mouse epithelial cell line will be transfected with murine G -

ability to secrete G -CSF and maintain the viable yints.

I» c se ^: fh& gai¾fls-8HSRrad atio« giram required for fell Zika vims inaetivation restdts in failure of cells to secrete GM-CSF, the inventors will next explore the by-stander approach, wherein the Zika vin¾s~permissive GM-CSF-transfected cell line will be split in two fractions: one fraction will be infected with Zika virus and irradiated under the regimen required for a complete viral inac-tivatiott. Another fraction (comprising non-infected cells) will be- irradiated under the regime required for rendering the cells proliferation incompetent. Both fractions will be combined and used in a vaccine composition. In conclusion, non-infected cells will serve as a source of GM-CSF, while Zika- infected cells will serve as- a source of vital antigens.

In addition to using cells infected with Zika virus for the purposes of vaccine preparation, the inventors will also test ceils traasfected with Zika replieon. Under such circumstances, there would be «ø need for the inaciivation of Zika virus, and cells would be rendered proliferatio incompetent using standard methods in the art.

Following the generation o vaccine composition comprising proliferation incompetent cells that (i) express GM-CSP. and (ii) c m ri e: mactivated Zik virus, the inventors will evaluate the vaccine composition using i viv animal, models, includin mous models as well as non-homan primates Macaque rhesus. Since rodents (mice) are susceptible to Z KV infection, felly im.mum competent mice will be vaccinated with vaccine compositions described here. In order to assess the eflicacy arid immunogenic potential -of Zika vaccine, the inventors will measure various parameters, including, but not limited to, ami-ZIKV antibody titers, T-eeJl activation (ELISPOT) and . inhibition of infection .in vitro using immune serum.

Following animal vaccination, the levels of anti-ZIKV neutralizing antibodies as well as the levels of T~celf response will be assessed in vitro using ELISA, plague neutralization assay, ELISPOT and/or other hnmunological techniques. In one set of experiments, the inventors will use a mouse model wherein animals will be vaccinated with a vaccine derived, from ZIKA. virus permissive mouse cell line and transfected with mGM~CSF:, In experiments using a non-human priraate model, the inventors will use GM-CSF Vero rtansieeted cells, which will be either in fected with i ka virus or transfected with Zika replieon.

Following the vaccination, animals will be challenged with Zika vims. The vireoria will be monitored in vaccinated animals and compared to non-vaccinated group. In order to model the repeating mosquito bites, the effect of repeating infection will also be evaluated.

In addition to testing the efficacy of the ZIK V vaccine in adult animals, the inventors will also asses the effectiveness of vaccine composition described here in a developing embryo and/or fetus. For example, adult animals will be vaccinated and allowed, to breed. During the gestation period, animals will be challenged with Zika vims and newborns will be examined, fo the persistence of the virus in their body and for the neurologic manifestation of the viral Infection. The purpose of this experimental desig is to test whether the initial viremia, which is created by a niosquito bite, can be quenched at the onset of infectio by the existing an i-viral inmumity , The follo ing- general steps will be carried out in moose studies ^* ( 1 ) permissive .mouse epithelial cancer cell line will be permanently transfected with mouse GM-C F; (2) a fraction of ^• transfected cells will be infected with Zika virus fo 24-48 ho«rs _:S followed by irradiation where the infected cells will be irradiated at, e.g., 25-40 kGy (or other doses as discussed herein) and non-infected, cells at 150-250 Gy< Subsequent to irradiation, cells will be combined and injected intradermal^' in mouse flanks; (3) after three weeks of incubation period, the procedure described in step (2) will be repeated and 8- 10 clays later blood will be drawn and an antibody titers against ZJ V measured using ELISA. and plaque inhibition assay (P1A); (4) upon reaching a sufficient atiti-vkns antibody levels, both vaccinated and non-vaccinated mice will be challenged in parallel, and the levels of viremia in blood will be .monitored for- period of time spanning from several days to several weeks,.

Low levels or the complete absence of viremia in vaccinated mice will serve as a proof of vaccine effectiveness.

E ampl 2 ^' Testi»g _. of ZIKV vaccine I». Mouse Models of Zika Virus Pathogenesis

Studies i animal models will be carried, out to evaluate he efficacy of the vaccines of the present dssciosure. Until .recently, a limited ntimbeirofsttidi.es have been performed in mice (Bell et al. Arch. Gesamte V irosf tseh. 35, 183-193 (J 97 ); Dick et ai, Tra s . R, Soc Trop, Med. Mm- 46, $09-520 (1952)· Way et at, I. Gen. Virol. 30, 123-130 (1976)). Although these early studies suggested that ZIKV can replicate and induce injury in cells of th central nervous system (CMS), it remained unclear whether this pathogenesis is related, to ZlKV-mduced nearodevelopmenial defects or Gnillam-Baire ^' syndrome (GBS) in ksmans. Whil thes studies indicated that mice could be infected with ZIKV via intracerebral inoculation, determining mechanisms of pathogenesis and evaluating candidate vaccines requires -more clinically relevant inoculation routes.

Recently, Lazear et al, evaluated ZIKV infection and disease ½ ildtype (WT) C57BL/6 mice, as well as a large panel of imraune-deficient transgenic mice, using several strains of ZIKV including a contemporary clinical isolate (Cell Host Microbe 12016) S 1.9 1- 3 J 28(16)30102-0). Specifically, the authors tested S~ to 6~week-oid WT CS7BL/6 mice as well as congenic transgenic mice lacking, key cOTponents of innate antiviral mmun ty (f narl. ^■ ·' ^■■ :, Mavs~ ^''~ ₅ IrfT ^" , Irf3 ^~ ' ^~ Irf5 ^{~ "} Irf7 ^{" ~} TKO) for susceptibility to disease induced by

contemporary human isolate of ZIKV' ^' (H/PF/2013) from French Polynesia, as well as the original ZIKV strain, MR 766. Four to six-week-old Ifnat l mice (which cannot respond to IFN-a/b) and M3~ ^! ~ fcfip ^'" 7 ^~ ' ^~ TKO mice (which produce almost no IFN-a/b) developed neurological disease and were susceptible to ZIKV infection, _. while single IrO IrfS and Mavs ^{" "} knockout mice exhibited no overt illness, Ifnarl ^" ' ^"' -mice sustained high viral loads in die brain and spinal cord, which is consistent with evidence thai ZIKV causes neiirodevelopmental defects in human fetuses. Moreover, the testes of Ifhaf I. rake had the highest viral loads, wbie can be relevant to sexual transmission © ZI V.

Briefly, irsar mice will be injected with in c ivated VAX ceils xpressing GM-GSF, as well as inactivated GVAX cells infected with various strains of ZII V, Vaccination will be carried out at least 2 weeks before challenging the animals with the virus. Next, Ifaarl ^" * ^''" animals that have received the vaec e(s) will he challenged with ZIKV of a specific strain by

sabcs:taneo«s (f otp8tl)j intravenous (retro-orbital), or i p. routes with lO; ² , IQ or ^' 10 FFU of ZlKV. Survival, weight toss, and disease symptoms will be .monitored for .14-30 days, depending on the experiment. Mice will be euthanized at various days following the viral challenge, and liver, spleen, kidney, testes, brain, and spinal cord will be harvested, weighed, and ^• homogenized. Viral burden will be evaluated by q T- CR or plaque assay (Lazear et a!. Cell Host Microbe (2016) SI 931-3128(16)30102-0),.

Furthermore, Miner et ai have recently described a mouse model of placental and fetal disease associated with mater© tr nsmission of ZIK.V (Cell 2016 May 19 165(5); 1081 - 1 ) . Female mice lacking type .1 interferon signaling (Ifnari ^'"!" ) crossed to wild-type (WT) males predeced heierozygovis fetuse exhibiting similar immune status as mat observed in h man fetuses, Moreover, maternal inoculation at embryonic day (E6.5) or E7.5 resulted in fetal demise that was associated with Z1KV infection of the placenta and fetal brain. Thus, this model can be used, to test ability of vaceioe(s) of the present disc losure to prevent congeni tal malformations during pregnancy. xamnlc; 3 Generation of ceils transfocted wit ON A e»e» ng GM-CSF and infected with Ziks viruses

Mouse neuroblastoma cell line N2a was used to ^■ produce. whale cell-based vaccine against the Zika virus, N2a cells originate from. Baib/C mice and have similar genetic background.

Althoug considered allogeneic (due to a number of somatic mutations occurred it* vitxo), N2a cells cause no or very little immune response i Balb C mice.

The N2a cells were transfected with ONA encoding mouse M-CSF using pcDNA-3.1 vector containing the neo-resistance gene. The transfected ceils were designated as N2a-G SCF of 2a-GM, N2a-GM cells were infecte with 2IKV: strain M&776 (Uganda). ital Infection was monitored by staining viral B2 protein using arh-B antibodies 4G2 according to the following protocol:

1. Grow N2A-GM ceils on coversUps to 70-80% confliiency.

2. Transfer the CQverslips Into 24 well-plates with PBS.

3. Fix the cells with 10% formalin for J 5 min; wash twice (PBS).

4. Permeabilhte with 0.1% Triton X-100 for 1 S min; wash twice (PBS),

5. Block using 2% BSA P S for 1 hr wash once,

6. Add 4G2 antibodies diluted ! ;5~!:6 with PBS to cells; incubate 2 hrs at room- temperature or overnight at ¾,

7. Wash 2 times.

8. Add secondary antibody donkey-antl-rnous conjugated with. Alex Flwor4SS (1:500) .in 0.1 % Triton, and incubate for 1 lir.

9. Wash twice, add DAPi Embed and observe under the microscope.

In different experiments, cells had an infectivity rate of about 40-50% (Figures 1 A and B), or an mfectivtty rate of about 80-90% (Figures 1C and I D).

Exam l 4 Vaecinaflo of mice using whole cell-based _, vaccine (cells expressing GM-CSF and infected with Eifet iruses

Two groups of .mice wer immunized with either 2a~GM ceils or N2&~GM-oaive cells (Group I , n = 4) or N2a-GM-Zf K V cell (Grou 2, n ~ 4), For each group, mice were vaccinated twice within a time frame of two weeks: a first vaccination or prime vaccination, and a second vaccination or boost vaccination. For the first (prime) vaccination, about 1 f cells at th infectivity rate of about 40-50% were injected suhentaraeousiy into the right Sanies of the mice. For the second (boost) vaccination, about 20x10* cells at the infectivity rate of about 80-90% were injected subeutaneously in the right flanks of the mice.

Blood from limn united animals was collected 9 days after th second vaccination, and sera was .prepared. The astibody titers of the sera were measured using serial -dilutions of th e sera in standard cellular en¾yme-linked immun sorbent assay (oELISA) assay. Two types of cells were used, in. cELlSA; N2a-GM cells (naive) arid N2a-GM-ZiK¥ ceils at tfee inieerivity rate of about $0-90%, Specifically, cells were -grown at regular conditions, collected at 80-90%

eanilueney with.0.25 rypsia/EDTA. Cell suspension was placed immediately into culture Media ( 10% PBS) to inactivate Trypsiri/EDTA. Cell suspension was then spun at 25 «3O0g _: , and the media was aspirated, Cells were resuspended in ice-cold MeiOH (methanol), and placed at - 20°C for 20.rahi, PBS was added into the tubes, spun at 450g for 10 min nd replaced with, fresh PBS, and stored at 4"€ for 2-4 days.

The eELISA. test was performed as follows.

1. For cBUSA, fixed cells are washed, with PBS twice, resnspended in blocking buffer- (0,3% dry milk in PBS). The multiscreen plate ^' (Miilipore, MAHV 4510) was filled with 30 μί blocking huiier. Incubate ceils and. plate for 1 r at RT, Em ty plate using the Sect on. device, spread cells over the plate, 100 μί/well, 30-50x10 ^" cells per well.

2. Remove blocking solution, add diluted sera 50 μΐ/ ell (e.g. _f dilution for unknown media is 1 :2 with blocking solution), negati ve controls (blocking solution and fresh culture media) and positive control (if any), incubate for 2 hrs at RT.

3. Wash plate 5-6 times with blocking solution.

4. Prepare horseradish peroxidase (.HRP}-eonjuga.ted. antibodies (e.g..., _. goat anti-rnouse- IgG(H+L)-BRP, or other appropriate conjugates) diluted 1 :3000 in. the blocking solution. Add conjugate solution to die wells of die multiscree plate, 50 uj/wel!, incubate tor I hr at RT.

5. Wash the plate 6-8 times with, the blockin solution.

6. Add Ultra-TMB substrate solution (ThermoFisher, catalog number 34028), 50 μί well, incubate for 1 hr at RT

7. Watch for the blue color development. In certain embodiment, it takes 5-10 iBmuies. In certain embodiment;, it takes no more than 30 η¾ϊη. Watch, closely for the negative control well which shoul d remain uncolored. If color appears in the negative control (this is usually due to problems with washing procedure), stop reaction immediatel with 10% HC1. (2-5 glAvell). If there is no color in negati ve wells, stop the reaction, no l ater than.30 min after the start of the reaction. When reaction is stopped, blue color turns int yellow.

8. Read ateorlmrs.ee at 450nm , As shown in Figure 2, sera from mice immunized with N2&-G (naive) cells did not react either wit N2a-GM. (naive) cells ('"Group I on N2A-G -riatve" in Figure 2) or with M2a- GM-ZIKV cells ("Grou 1 on N2A-GM-ZIKA" in Figure 2). This indicates that neither N2a cells' antigens nor recombinant mouse GM-CSF secreted by trarisfected cells had

i mtmoge lcity sufficient for an antibody response. Ser from mice vaccinated with 2a-GM- ZfflLV cells was immunologically reactive to N2a-G ~ZIKV ceils ("Group 2 on N2A-GM- ZtKA" in Figure 2). but not to N2a-GM cells ("Group 2 on N2A-GM-riaive ^5r> in Figure 2), This indicates that only immune response directed against ZIKV was detected. This suggests thai syngeneic cells 2a did not trigger immune responses, and that only immune response against vims antigens was detected (Figure 2).

Figure 3 shows antibody response (vertical bars according to the left y-axis, cEOSA assay on N2a-GM-E1KV cells) and antibody neutmliza ion potential (solid line according to the right y-axis, PENT) for three groups of mice immunized with N2a-GM-2iKV cells (group A), ^' N2a-ZI Y cells (group B), or purified ZIKV (grou C), respectively , hi the plaque reduction neutralization test (PENT), neutralization of ZIKV virus-fonmng plaques was studied in the presence of sera fjrora mice vaccinated with N2a-GM-ZiKV cells (A) ₄ N2a-ZIKV ceils (B), or purified ZIKV (C). As shown in Figure 3, anti-Z KV antibody titers are higher in mice immunized w ith 2 -GM-Z3 V than anii-ZK¥ antibody titers in mice immunized with N2a- XtKV or purified I , Thi Indicates that the local secretion of GM-CSF by N2a-G cells facilitates the activation of immune cells and results in the increase of antibody production. The dilution of sera required for 50% inhibition, of virus induced plaques is the highest in group A which is in good correlation with the antibod titers.

Figure 4 shows antibody response (vertical bars according to the left y-axis, cELISA assay on N2a-G -Z1KV cells) for, as well as the number of colonies infected with ZIKV in the presence of a fixed dilution (1 :30£ ⁾ 0) of the serum from, three groups of mice immunized wi h N2a-GM-Z1KV cells (group A N¾a-Zi V cells (group B) ₅ or purified ZIKV (group C} _} respectively. Figure 4 is similar to Figure 3 except that in, this experiment the number of virus induced plaques was counted at the fixed sera dilution usin sera ires* mice Horn: all three .groups.

The data presented I» Figures 3 and 4 were die averages of 7 animals in each group.

PRN was used to u ntit the titer of aeiitraiizing aatihod for the ika vims. Sertim r m the vaccinated mice was pretneuhated with th ZIK V to allow the antibodies to react with the viruses. The serurn that had been preincubaied with the ZiKV was then added to a. racmakyer of host cells (e.g., N2A cells, or N2A-GM cells). The concentration of serum to reduce the number of plaques by 50% .compared to the seruin-r ee virus gives the measure of how much antibody is present or how effective it is. This -measurement Is denoted as the PRNT50 value. Alternatively or additionally , the dilution of serum to reduce the number of plaques by 50% compared to the serum-free vims gives the measure of how much antibody is present or how effective it is _* This measurement is denoted as the 50% neutralization value (Fi ure 3).

PRHT was done as follows.

To test sera for ofiottalization 2A-C)M cells suspension in 100 μΐ media are plated onto a 96-weH plate, at 2x10 ⁴ cells/well and used after about 24 hrs (60-70% confluence), o at 1x10 ⁴ ceiis/weli which can be used after about 48 hrs-

This step was done in separate transfer plates. The serum, was. diluted in series at step 4 starting with 1/25 growth media), and titrate down with step 4 (last dil. ί ;160Q), 60 μί of all dilutions was prepared in triplicates; Several wells with 60 μΐ οί diluent (media) tor ZiKV only which is added later as a control Single dilution of 1/4 of pre-titrated ZIKV containing media is also prepared.. 60 ui of ZIKV (1:40) are added int all wells with, ser ;, normal mouse serum (NMS) and culture media. The final dilution of sera in preincubation plate increases 2 times and becomes the range fro 1/50 to 1/3200. The plate is incubated on a plate shaker at 2500 rprn for 2 hrs at room temperature.

After 2 hrs, 100 μΐ of mixtures are added carefully to the Ή2Α cells plate (plated 1 day or

2 days before in 100 μΐ of media)- Also, fresh ore-titrated ZiKV-eontainin media is added to several plates t reach the same dilution as in preincubation plate. This is the control for infection which is done without hrs of preincubation. Final dilutions of sera, in cell plate are 1: 100*I :#400. and l V - 1 : 160. The plates incubated for 48-72 hrs, Afterwards,, the plates are washed with fresh PBS and Methylene blue is added to stain die remaining cell. The virus Induced plaques are seen at the bottbna of the plates as white s ots hich can be coiinted using plaque counting.

Es¾mp¾e 5 Testing ZIKV inactivit in different cefl Un

Different types of cells,. inclnding Vero cells (Figures 5 A md 5B% 2a (naive) cells

(Figures 5 A md SB), N2a-GMCSF cells (Figures S and SF), N2a new cell line (Figures 5G arid 5H), human fibroblasts WS-1 (Figures 51 and Sj), human fibroblasts PC S2 ⁽ ) 1-012 (Figure SK), were infected with IK V-contaioittg cell culture media (P2) aad itnmunostained using a«ti.-E2 antibodies 4G2 at different time pcnnts post~infectk>m e<.g., 24 hours, or 48 hou s postinfection _* The cells were immunostaked for the virus E2 antigen using anii-E2 antibodies 4G2 (see

Example 3 for the staining protocol). Figure 5 show different levels of peoiiissivit of cells for ZIKV infection. A multiplicity of infection (ΜΟΪ) 1, the highly .remissive cell line is Vero (90- 100%),. while human fibroblasts and N2 cells have tow petratssivity (3-5%). In order to reach high level of infection in N2a cells which were used in die- experiments of Examples 3 and 4, MOl 20 was used.

The practice of the present inventi on may employ, unless otherwise indicated, conventional techniques including those of molecular biology (including recombinant techniques), immunology, cell biology, biochemistry, and phamtaceutical practice. Such teciuncpes are explained fully In the literature, for example, Molecular Cloning: A Laborator Manual, second edition, (Sambrook et at};. Methods in Enzymobgy (Academic Press, Inc.): Current Protocols in Molecular Biolog (F. M Ausitbel ei l, eds); Current Protocols in

Immunology (John Wiley & Sons, inc. , A' J, Handbook of Pharmaceutical Exelpients ( Rowe et al., Eds); Vaccines (Plotkin and Orenstein, 2003); and Vaccine Protocols (Methods in Molecular Medicine) (Robinsln, Cranage and H dso ;, 2003),

The scope of the present invention is not limited by what has been specific ally shown and described hereinabove. Those skilled m the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions and dimensions. Numerous references, including patents and various -publications, are cited and discussed i the description. of thi invention. The citation, and discussion of such references is provided mer ely to clarify the description of the present nvention a»d is not as adnussion that any reference is prior art to the mvention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety. Variations, modifications and othe implementations of what is described herein will occnr to those of ordinary skill in the ar without departing from the spirit and scope of the invention. While certain emb d ments of the present invention have been shown and described, it will be obvious to those skilled in the ail that changes and modifications may be made without departing trout the spiri t and scope of the invention. The matter set forth in the foregoing description is offered b way of i llustration only and not as a limitation.