WO2018148499A1 - Flavivirus peptide sequences, epitopes, and methods and uses thereof - Google Patents

Abstract

The present application relates to composition of matter, processes and use of composition of matter relating to flavivirus peptides and epitopes, for example, for therapeutic or preventative vaccination against a flavivirus, and/or for inducing, enhancing, or sustaining an immune response against a flavivirus, and/or for detecting an infection with or an exposure to a flavivirus in a subject. The flavivirus may be for example the Zika and/or Dengue virus.

Description

Our Ref.2016-103-03 F^{LAVIVIRUS PEPTIDE SEQUENC} _T ^E _H ^S _E ^{, E} _R ^P _E ^I _O ^TO _F ^{PES, AND METHODS AND USES} CROSS-REFERENCE TO RELATED APPLICATION

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e_{rbenerce 6d2} ^{The present application claims the benefit of U.S. provisional patent application serial} d_{/o4c5u7m,75en3t f airleed in oconrp Foerbartuedar hyer 1e0i,n 2 b0y1 r7efe bryen Scueja inn t Shheirres etnat.ir Tethye. contents of the above-} TECHNICAL FIELD

[ _c ⁰ _o ⁰ _m ^2] _position ^T _o ^h _f ^e _m ^p _at ^r _t ^e _e ^s _r ^en _re ^t _lat ^a _i ^p _n ^p _g ^li _t ^c _o ^at _f ⁱ _l ^o _av ⁿ _iv ^r _ir ^e _u ^la _s ^te _p ^s _ep ^t _t ^o _ide ^c _s ^o _a ^m _nd ^po _e ^s _p ^it _it ⁱ _o ^o _p ⁿ _es ^o _. ^{f matter, processes and use of} SEQUENCE LISTING

[ ^{S0e0q3u]ence Li Isntin agcc (sourbdmanictteed w eitlhec 3tr7on CiFcaRll §1.52(e)(5), the present specification makes reference to a} _{The .txt file was generated on February} ^y ₁ ^a _st ^s _, ^a ₂₀ ^.t ₁ ^x ₈ ^{t f} _a ⁱ _n ^le _d ⁿ _i ^a _s ^m ₂ ^e ₁ ^d _k ^“ _b ^Se _i ^{qListing.txt” on February 9, 2018).} _{Sequence Listing are herein incorporated by reference. n size. The entire contents of the} BACKGROUND

[ ^{N00il4e] virus, d Felnavgiuveir vuisru iss a (D gEenNuVs) o,f tic vkir-ubsoersn ien e the family Flaviviridae. This genus includes the West} _{(ZIKV) and several other viruses which may ca} ⁿ _u ^c _s ^e _e ^ph _en ^al _c ⁱ _e ^ti _p ^s _h ⁽ _a ^T _li ^B _ti ^E _s, ^{) virus, yellow fever virus, Zika virus} _{(ISFs) such as cell fusing agent virus (CFAV), Palm Creek virus as (P wCeVll) a,s an indse Pcat-rsrapecific flaviviruses (PaRV). matta River virus} ^{[ (0e0n5v]elo Flaviviruses are share several common aspects: common size (40–65 nm), symmetry 10,000–p1e1d,0,0 i0co bsaasheesd),ra aln nucleocapsid), nucleic acid (positive-sense, single-stranded RNA around} _{and cause significant hu} ^d _m ^ap _an ^pea _d ^r _i ^a _se ⁿ _a ^c _s ^e _e ⁱⁿ _in ^th _t ^e _h ^e _e ^{lectron microscope. Flaviviruses are globally emerging} _{flaviviruses are maintained in animal reservoirs fo inrm nat oufre en ancedp ahraelit tirsan or hemorrhagic fever. Most through the bite of an infected mosquito or tick. Other virus transsmmiisttseiodn to ro huutmesan csan pr iinmclaurdilye}

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c_leb ^e _l ^{s of ZIKA infection have no symptoms, but when present they are usually} _{conju oer d joeningtu peai fne,v nera,u asenad, v moamyit cinaugs,e or fe lovsesr, o rfa asphp,e hteitaed.ache, pain behind the eyes,} ^{[ m00ic7r]ocepha Flyu hrtahser b,ee an c caounsafilrm reeldati ionn tshheip 20 b1e5tw}

b ^{Bereanzil ZiaInK oVutb anredak a, a cnodng seignnitsal o syndrome including a inneden ca sseeen re ipnor ZtsIK ofV s-einxfueaclt terdan msmicies.si ZoInK aVre h mas also been linked to Guillain-Barfre m Sicyrnodcreopmhaely (G hBavSe) wo tmheen W wesatsern co Hnfeimrmisepdhe troe, ounting. Recently there was a major outbreak of ZIKV} _o ^{c wauhsiceh C aolsnog wenaista alss ZoIcKiaVted S wynitdhro GmBeS,. A whdidcihtio innacllluyd,e insfe mcticiornoc oefp phraelgnant} _{3 B7th4e(1r0 b):ir pth.9 d5e1f-e8c;ts D.r (Migglaekrsa,r, R J..W, e.,t e atl. a,l Z.,i Zkaika V Viruirs Associated with Microcephaly. N Engl J Med,} ^y ₂₀ ^a ₁ ⁿ ₆ ^d _{. Straain Abnormalities. us Infection with Prolonged Maternal Viremia and Fetal Mop N Engl J Med, 2016.374(22): p.2142-51; Hennessey, M., M. Fischer, and J.E. rtleasl, W Zikklya R Veirpu,s 2 S0p1r6ea.d 6s5 t(o3 N):e pw. A 55r-e8a;s R - Rasemgiounss oefn th,e S A.Am.,er eitca asl,., May 2015-January 2016. MMWR Morb the Evidence for Causality. N Engl J Med, 2016.374(20): p.1981-7) Z.ika Virus and Birth Defects--Reviewing} ^[ _a ⁰ _n ⁰ _d ^8] _pathoge ^T _n ^h _e ^e _s ^r _is ^e _. ^{are, however, fundamental gaps in the understanding of flaviviruses immunology [ n009] Vaccines are currently available for only yellow fever and Japanese and TBE; however, heavwe v sahcociwnnes t fhoart d felanvgivuieru asneds, W esepstec Niaillley a dreen ingu celin viicrauls tr hiaalss in humans. In recent years, many studies response during the infection (Diamo the ability to inhibit the innate immune} _{30; Jones M, Dav} ^{nd MS (September 2009), J. Interferon Cytokine Res.29 (9): 521–} _{virus has many nidosonnst Aru,ct Huribable prtro Lt,ei ents al t.h (aMta ayllo 2w00 t5h)e. J i.nh Vibiriotli.o 7n9 o (f9) v:a 5ri4o1u4–20). Indeed, the dengue immune system response. Disease diagnosis can be dif s mediators of the innate genetically closely related ficult as all flaviviruses are antigenically and main approach to disease. c Tohnetrreol a irse t nhoro eufgfehc vtiavcec ainnattivioirnal a tnhder vaepciteosr t choantt erxoils.t for any flavivirus so the}

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c ^{xeipno,s tuhree p troes aen fltav diivsicrluossu inre a further relates to an in froommpr thisees su pbrojevcitd.i Tnghe a m bieothloogdic faulr stahmerp cleom frpom the subject, the biological sampl seub cojemctp.r Tishineg m Tet cheollds present disclosure. The m rises contacting the sample with the composition of the} _{cell response, and detectin} ^e _g ^th _t ^o _h ^d _e ^a _p ^l _r ^s _e ^o _se ^c _n ^o _c ^m _e ^p _o ^r _r ^is _a ^e _b ^s _s ^p _e ^r _n ^o _c ^c _e ^es _o ^s _f ⁱⁿ _t ^g _he ^th _T ^e _c ^s _e ^am _ll ^p _re ^le _sp ^t _o ^o _n ^d _se ^e _. ^te _T ^c _h ^t _e ^th _p ^e _re ^p _s ^r _e ^e _n ^se _c ⁿ _e ^c _o ^e _f ^{of a T} _{cell response being indicative that the subject has been infected with or ex the T The method may further include causing a transmission of posed to the flavivirus. information indicative of whether the subject has been infec atend e wleictthro onric ex npootsifeidca ttoio tnhe da fltaav civoinruvse.ying} ^{[ c0o0m15p]uting d Ienvi ocene as nsoocni-altimedit winigth e am pbaordtiicmuleanrt u,s tehre, w elheiccthro cnainc b neot aif micaetdioicnal d eaxtpaer its o trra tnhsem sitted to a some specific practical implementations, the computing device asso ubject. In} _{expert may include a smartphone, a tablet, a ge} ^{ciated with the particular medical} _{computing device and o tthheer e sluecitt neral purpose computer and/or any other suitable message and/or or any arbolneic ele ncotrtiofniciacti moness daagtea. may convey an e-mail message, an SMS} ^{[ m00e1th6]od of i Andsu ecminbg,od eniehdan acnidng b,r ooadly described herein, the present disclosure further relates to a} _{the methioodn o cfo tm} ^{r sustaining an immune response against a flavivirus in a subject,} _{composit hepr pirseinsgent c doinstcaloctsiunrge. T cells of the subject with an effective amount of the} ^[ _a ⁰ _m ⁰¹ _o ⁷ _u ^] _{nt of th} ^In _{e c} ^o _o ⁿ _m ^e _p ⁿ _o ^o _s ⁿ _it-_io ^lim _n ⁱ _t ^t _o ⁱⁿ _t ^g _he ^em _su ^b _b ^o _je ^d _c ⁱ _t ^m _. ^{ent, the contacting includes administrating the effective [ t0h0e18 e]ffective In am onoeun nto onf-li tmheitin cogm empobsoitdioimn,en ant,d th aedm coinntisatcrtaitning includes contacting T cells ex vivo with} _{The method may further comprise expansion o} ^{g the contacted T cells to the subject.} _{contacted T cells to the subject. f the T cells in vitro prior to administrating the} 4

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[0022] In one non-limiting embodiment, the flavivirus is a Dengue virus.

[ ^{e0n0h2a3n]cing, o Irn s tuhseta ciansieng wh aner iem tmheu fnlaev rivesirpuosn isse a a Zgaikinas vtir au fsl,a tvhie herein described method of inducing,} _{symptoms associated with a Zika virus in} ^{virus in a subject may treat or mitigate} _{pain behind the eyes, conjunctivitis, musclefe ocrtio jonin stu pcahin a,s n,a buustea n,o vto lmimitiitnegd, t oor, lo fesvse or,f r aapsphe,t hiteea.dache,} ^[ _o ⁰ _b ⁰ _t ² _a ⁴ _in ^] _{ed by} ^In _any ^on _k ^e _no ⁿ _w ^o _n ^n- _t ^l _e ^im _ch ⁱ _n ^ti _i ⁿ _q ^g _ue ^e _, ^m _fo ^b _r ^o _e ^d _x ^im _am ^en _p ^t _l ^, _e ^t _b ^h _y ^e _d ^h _ra ^e _w ^re _in ⁱⁿ _g, ^d _b ^e _y ^sc _n ^ri _o ^b _n ^e-^d _inv ^b _a ⁱ _s ^o _i ^l _v ^{ogical sample can be} _{sample collections or banks, etc. e techniques, or from} ^{[ o0n0e25 o]r m In one non-limiting embodiment, the composition of the present disclosure may include an acceptoabrele a ccacrerpietarb mleay ca brreie srel seecletecdted fro frmom go tlhde p aacrcteicpletasb,l sete crarriers described herein. For example,} _{buffered solutions. Carriers may inc} ^{ile water, saline, glucose, dextrose, or} _{stabilizers (i lude auxiliary agents including, but not limited to, diluents, buffering age.en.t,s, s vuigsacrossit ayn ednh aamnicniong a acdiddsit)i,ve psr,e csoerlovrasti avnesd, th weet litkineg. agents, emulsifying agents, pH} 5

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e_t ^{sgib exemplification of carriers, modes of administration, dosage forms, etc.,} _{uncd.,er mstaaynd b} ^{ilities from which the carriers, modes of administration, dosage forms,}

,_{e ho sewleecvteerd, th foart a unsye g wiviethn f tohrem purleastieonnt a inndve mntoiodne. o Tfh adomsein oisftr oartdioinna sreyle skill in the art will tested to determine that it achieves the desired results. cted should first be} ^{[ in00tr3a0p]eritone Male,t ihnotdrasm oufs acdumlari,ni ssutrbactuiotna include, but are not limited to, parenteral, e.g., int}

rⁿ _a ^e _d ^o _e ^u _rm ^s, _a ^m _{l r} ^u _o ^c _u ^o _t ^s _e ^a _s ^l _{. A} ^(e _d ^.g _m ^., _in ^o _i ^r _s ^a _tr ^{l, ravenous,} _{intraocular), intrathecal, topical and int at} ⁱ _i ⁿ _o ^t _n ^ra _c ⁿ _a ^a _n ^sa _b ^l, _e ^b _sy ^u _s ^c _t ^c _e ^a _m ^l, _ic ^va _o ^g _r ⁱⁿ _lo ^a _c ^l, _al ^r _. ^{ectal, [ a0}

i^nd0

j^m31

e^citn]

i^{oisntra mtiao T}

y^nhe

b^{e by co}

p^{r ienm}

s^{jeepositions of the present disclosure may be formulated for parenteral nctteiodn i,n e u.gn.i,t d boysa bgoelu fsorm in,je ec.tgio.,n in o arm cpoonutliensu oorus in i mnfuulstiio-dno.se Fo cromntualianteions for an added preservative. The compositions may take such forms as suspension rs, with} _{emulsions in oily or aqueous vehicles, and may contain formulatory a} ^{s, solutions or} _{stabilizing and/or dispersing agenet,s e.. Ag.l,t setrenrailteiv peylyr, gents such as suspending, constitution with a suitable vehicl o tgheen a fcretieve wa integrr,e bdeiefnotre m uasye. be in powder form for} ^{[ o0f03 a2n] inject Fabolre in psrteapnacre, the composition of the present disclosure may be administered in the form suspensions may be foramtiuolna,te sduc ahcco asrd sitnegril teo i tnejcehcntaiqbulees aq kuneoowuns i onr th oelea agrtin uosuinsg su suspiteanbsleio dnis. These or wetting agents and suspending agents. The sterile injectable prepara spersing} _{injectable solutions or suspensions in non-toxic parente} ^{tions may also be sterile} _{may be given parenterally, for examp rally-acceptable diluents or solvents. They injection, by infusion o le intravenously, intramuscularly or sub-cutaneously by amount of each of the crom pepro onse.n Stusi itnab tlhee d coosmagpeossi wtioilnl, v tahrey, d deesipreednd eifnfgec utp (sohnor ftac otro lrosn sgu tcehrm as), t thhee}

7

^r _k ^o _n ^u _o ^t _w ^e _n ^of _in ^ad _th ^m _e ⁱⁿ _ar ^is _t ^t _m ^ra _a ^ti _y ^on _b ^, _e ^t _u ^h _s ^e _ed ^ag _f ^e _or ^an _a ^d _dm ^th _in ^e _i ^w _st ^e _e ⁱ _r ^g _in ^h _g ^{t o} _th ^f _e ^th _c ^e _om ^su _p ^b _o ^je _si ^c _t ^t _io ^t _n ^o _o ^b _f ^e _t ^t _h ^r _e ^ea _p ^te _re ^d _s ^. _e ^A _n ⁿ _t ^y _di ^o _sc ^th _lo ^e _s ^r _u ^m _re ^e _. ^{thods well [}

s ^ly0

t^o03

o^p3

r^ah]

g^{ielize odf b f Toh}

i^ore

l^{m c}

o^g).om

i^{c Falrp}

l^yeoeszi

a^eti

c^-o

t^dn

i^{vr o}

e^yinf

m^{g th}

a^{t (eae}

r^{ls p}

i^aore

l ^nse

b^an

e^mt

c^{ae d}

u^disc

s^{e lylo}

o^os

f^puhr

t^{hieleis ma lta}

o^ioy

w^{n b)e T te ims fo}

p ^orm

e^{frtu e}

a^etnlat

u^{r ud}

e^{se a}

e^{ds f ao drr pyr peosewrvdaetrio (ni.e. a,n ind} _th ^{ypically the liquid antigen is freeze dried in the presence of agen xposure during drying.} _{sue lyophilization process and to yield a cake with desira} ^{ts to protect the antigen during} _{cocmromsoe,n mlya unsneidto flo,r tr cerhyaolopsreo,te ocrtio lanct oofse (present at an inbitlieal p coowndceenrt crahtaioranct oerfis 1t0ic-s2.0 S0u mgagr/sm suLc)h a ares powder characteristics. Ly protein antigens and to yield lyophilized cake with desirable composition. ophilizing the composition theoretically results in a more stable} ^{[ a0}

p^s0

r^{o a34}

d ^l]

u^{iq In certain embodiments, the composition of the present disclosure may be formulated m cuti fdor (e r.egc.o anqsuteitouutsio fnor wmituhla wtioatne)r, o e.rg. o,t ahser sy sruuiptasb oler v suehspicelnes bioenfos,re or us me.ay Su bceh p lriqeuseidnted as a drug suay be prepared by conventional means with pharmaceutically acceptable ad preparations agespnetsnd (ein.gg., ag leecnitthsi (ne.g o.r, s aocrabciitao);l s nyornu-pa,q cueeloluuloss vee dheicrilvatives or hydrogenated edible fadtisti)v;e esmu sulscifhyin ags vegetable oils); and preservatives (e. es (e.g., almond oil, oily esters, or fractionated} _{pharmaceutical compositi} ^{g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The} _{conventional means wions may take the form of, for example, tablets or capsules prepared by pregelatinized maize statrhch, ph paorlmyvainceyult picyarlrlyoli adcocneept oabrle hy edxrcoixpyiepnrotspy slu mcheth asylc beilnludloinsge); ag fiellnetrss ( (ee..gg.., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g , stearate, talc or silica); disintegrants (e.g., potato starch or sodium star ., magnesium (e.g., sodium lauryl sulphate). The tablets may be coated by methods wcehll- gklyncoowlante i)n; o thre w aerttt.ing agents} ^{[ r0e0s3p5ir]atory ( We.gh.e nraesa tlh)e mu cocomspa,o tsyitpioicnally of it t ishe for pmreusleantet disclosure is intended for delivery to the an aerosol or nasal drops, or alternatively, d as an aqueous solution for administration as} _{passage. Compositions for admi} ^{as a dry powder, e.g. for rapid deposition within the nasal} _{type usually included in nistration as nasal drops may contain one or more excipients of the tonicity adjusting agents, su bcuhffe croinmgpo agsietniotsn,s, an fodr t ehxeam likpel.e V pirsecsoesrivtayti avgees,nt vsis ccaonsit bye ad mjuicsrtoincgrys atgaelnlintse,} 8

^{c pe}

e_x ^ol

a^wlu

m^dlo

p^es

l^re,

e_, ^{m c}

m^ah

u^yito

c_o ^asa

a^ls _d ^on,

h_e ^{c s}

s^ota

i_v ^nr

e^tcah

a^ines,

g_en ^{o p}

t^no

s_, ^ely

b ^osa

u^rcc

l_ki ^mh

n_g ^oarri

a^ede

g_e ^es

n^x,

t^{c a}

sⁱⁿ

,^pd

a^ie _n ^{n th}

d^tse

a_g ^{u li}

e^sk

n^ue

t^a.

s^{ll C} _{s t} ^yom

o ⁱ _d ⁿ _e ^cp

l^lo

i^us

v_e ^dit

r^{eidon isn fo surc ahdm coinmisptroastiitoionn ass, d foryr} _{trizeeha clohsaer,a acntedri xsytilcitso.l. Bulking and powder flow and size agents may app inrcolpurdieate m paonwnidtoerl, fl souwcro ansed,} ^{[ e0}

s^{lm03p6lo]y, for I enxa omnpele n,o an d-liipmstiitcinkg, embodiment, the herein described methods and/or kits may skide, a multi-well pla a membrane, a chip, a disk, a test strip, a filter, a microsphere, a usiellded wh iner t te, an optical fiber, and the like, or any other variant available to the person t eh ae s aarmtp wlieth toou bte d teepstaerdtin cgan fr boem ad tdheed p drerosepnwtis deis tcolo asu sarem.p Fleor ap epxalimcaptiloe,n a pa tedst pr setsriepnt m oany t bhe leest strip, and the presence of at least an isolated peptide comprising an amino sequenc e amast 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100 e which is at} _{a en} ⁱ _i ⁿ _m ^o _m ^a _u ^c _n ^id _od ^se _e ^q _te ^u _c ^e _t ⁿ _io ^c _n ^e _m ^se _e ^t _t ^f _h ^o _o ^r _d ^th _w ⁱⁿ _hic ^an _h ^y _d ^o _e ⁿ _te ^e _ct ^o _s ^f _p ^S _r ^E _es ^Q _en ^I _c ^D _{e o} ^N _f ^O _th ^: _e ¹ _a ^t _t ^o _le ^S _a ^E _st ^Q _on ^I _e ^D _su ^N _c ^O _h ^: _p ⁹ _e ^3%

p_t ^{is m idaednetic baals teod t ohne}

_{imarlier in the text, the person of skill will readily understand that su ide. As discussed pepmtiudneo odfet tehceti polnur maleittyho odf t whheic ishol dateetdec ptsep ptriedseen ccoe of a plurality ofc thhe te issotl sattreipd m peapyti mdea,k weh uesree o efac anh different from one another, as describ mprises a respective amino acid sequence which is immunodetection method may include an imemd above with respect to the composition. Such variant thereof, and the like, or any othe unochromatographic test, an ELISA or ELISPOT or without departing from the present disclorsu sruei.table method available to the person skilled in the art} ^{[ d0e0t3e7c]ting age Innt o wnheic nho ins-“lipmaictkinagge edm”.b Aodsi umseendt, he threein h,e threein ter dmesc“rpibacekdag kit may include at least one solid matrix or material such as glass, plastic, paper, fiber, f ed” can refer to the use of a fixed limits the at least one detection reagen oil and the like, capable of holding within include the at least one detecting ag t. Thus, in one non-limiting embodiment, the kit may} _{milligram quantities of the at} ^{ent“packaged” in a glass vial used to contain microgram or} _{may include the at least one detecting agent. In another non-limiting embodiment, the kit microgram quant least one detecting agent“packaged” in a microtiter plate well to which non-li ities of the at least one detecting agent has been operatively affixed. In another micropmairttinicgles e emnbtroadpipmeedn wt,ith thine a k pitor mouasy m inemclubdrae}

9_{ne th oer e amtb leedasdted o inne a d teestetc sttirnipg o arge dniptst cicoka,te edtc. o Inn}

^{a cno o}

p^o

t_ho ^a

e_s ^tt

s^eh i_nib ^der v_iel ^o _int ^{n n}

i_te ^t

i_s ^on- o_{n e} ^alim ._xi ^mi

s_t ^eti

a^mng

n_d ^{br e}

w^amn _il ^{ebodiment, the kit may include the at least one detecting agent directly} l^, _b ^t _e ^es _r ^t _e ^s _a ^t _d ^r _i ⁱ _l ^p _y ^o _re ^r _co ^d _g ^ip _n ^s _i ^t _z ⁱ _e ^c _d ^k, _b ^e _y ^tc _t ^. _h ^w _os ^h _e ^ic _s ^h _ki ^c _ll ^o _e ⁿ _d ^ta _in ^cts _th ^t _i ^h _s ^e _ar ^s _t ^am _wi ^p _th ^le _ou ^fl _t ^ui _d ^d _e ^. _p ^M _ar ^a _ti ⁿ _n ^y _g ^o _fr ^t _o ^h _m ^er

[0038] In one embodiment, the herein described subject can be a mammal, preferably a human. ^{[ n0o0t39 m]utually Al elx fceluatsuivrees c oanf e bxeem copmlabryin eemdb woidthim oennets a which are described in this disclosure and are} _{utilized in the oth} ^{nother. Elements of one embodiment can be} _{present invention werill e bmebcoodmimee anptpsar wenittho tout th fuorsther mention. Other aspects and features of the following description of specific embodiments ine co onrjduinncatrioilyn w skiitlhled the in ac tchoem aprtan uypinogn F riegvuierews. of the} BRIEF DESCRIPTION OF THE DRAWINGS

[ _r ⁰ _e ⁰ _fe ⁴ _r ⁰ _e ^] _{nce to} ^A _th ^d _e ^e _a ^t _c ^a _c ^il _o ^e _m ^d _p ^de _an ^sc _y ^r _i ⁱ _n ^p _g ^ti _d ^o _r ⁿ _aw ^of _in ^s _g ^p _s ^ec _in ^ifi _w ^c _h ^e _i ^x _c ^e _h ^m _: ^{plary embodiments is provided herein below with [} id⁰⁰e⁴n¹ti^]ficatio ^Fn^ig o^.f ¹ ep^Aito ^tp^oes ^F rⁱe^gc^.og ¹n^Cize ^sd^ho b^wy C ⁿD^o8^n+-l T^im c^iteⁱlⁿls^g in ^ch W^arT^ac C^te5^r7^izB^aL^tio/ⁿ6 m ^oi^fce ^Z tr^Ie^Ka^Vt ^{infection and} _{blocking antibody in accordance with an embodiment of the present disclosure;} ed with IFNAR- ^[ _L ⁰ _y ⁰ _s ⁴ _M ^2] _Cre+IF ^F _N ^ig _A ^. _R ² _fl/ ^A _{fl m} ^t _o ^o _use ^F _m ^ig. _ode ² _l ^C _of ^s _Z ^ho _IK ^w _V ^e _in ^xe _fe ^m _ct ^p _io ^la _n ^ry _in ⁿ _a ^o _c ⁿ _c-_o ^li _r ^m _da ^it _n ⁱⁿ _ce ^g _w ^r _it ^e _h ^su _a ^lt _n ^{s obtained with a} _{present disclosure; embodiment of the} ^[ _o ⁰ _f ⁰⁴ _Z ³ _I ^] _{KV ep} ^F _i ⁱ _t ^g _o ^. _p ³

t_e ^A

h_{se r} ^a

p_e ⁿ

r_c ^d

e_osg ^F

e_n ^ig

n_itz ^. _e ³

d_d ^B

i_{sc b} ^s

l_oy ^ho

s _C ^w

u_rD ^e

e_;8 ^x ₊ ^em _T ^p _c ^l _e ^a _l ^r _l ^y _s ⁿ _in ^{on-limiting results obtained from the identification} _{embodiment of LysMCre+IFNARfl/fl mice in accordance with an} ^[ _Z ⁰ _I ^{044] Fig.4A to Fig.4F show exemplary non-limiting results of polyfunctional phenotype of} _{emKboVdim epeintotp oef- tshpeec pirfeicsen CtD di8s+clo Tsur cee;lls in LysMCre+IFNARfl/fl mice in accordance with an}

10

^[ _s ⁰

t_hp ⁰

e_e ⁴ _c ⁵

p_if ^]

r_{iecse CnD} ^F

t _d8 ⁱ

i₊ ^g.

s_{c T} ⁵

l_{os c} ^A

u_erll ^an

e_{; re} ^d _sp ^F _o ⁱ _n ^g. _se ⁵ _i ^B _{n L} ^sh _y ^o _sM ^w _C ^e _r ^x _e ^e ₊ ^m _IF ^p _N ^la _A ^ry _Rf ⁿ _l/ ^o _fl ⁿ _m ^-l _i ⁱ _c ^m _e ^it _i ⁱ _n ^ng _ac ^r _c ^e _o ^s _r ^u _d ^lt _a ^s _nc ^o _e ^f _w ^k _i ⁱ _t ⁿ _h ^et _a ^ic _n ^s _e ^o _m ^f _b ^t _o ^h _d ^e _im ^Z _e ^I _n ^K _t ^V _o-_f ^{[ s0e0r4u6m] of Ly FsiMg.C 6rAe+I tFoN 6ADR sflh/folw tre garteadph wsit thha dtep illluetsitnragte an ntio-Cn-Dlim8i otirn igso lteyvpeels c oofnt irnofle acntitous virus in the} _{and 1 before infection with 105 FFU of MR766 or FSS1} ^{ibody on days 3} _{the present disclosure; 3025 in accordance with an embodiment of} ^[ _e ⁰ _p ⁰ _i ⁴ _to ⁷ _p ^] _{es rec} ^F _o ⁱ _g ^g _n ^. _i ⁷ _z ^A _ed ^to _by ^Fi _C ^g _D ^.7 ₈ ^{E+ s} _T ^{how graphs that illustrate non-limiting results of the identification of} _{in accordance with an embodime cnetll osf in the W pTre Cse5n7tB dLis/c6lo msuicree; treated with IFNAR-blocking antibody} ^[ _p ⁰ _r ⁰ _e ⁴ _d ⁸ _i ^] _{ction a} ^F _p ⁱ _p ^g _r ^. _o ⁸ _ac ^sh _h ^o _es ^w _t ^s _o ^g _i ^r _d ^a _e ^p _n ^h _t ^s _ify ^th _Z ^{at illustrate a non-limiting experimental procedure for the peptide} _{of 15-mer overlapping peptides in acIcKoVrd-adnecrieve wdith ep aitno epmesb roedciomgennizte odf b thye C pDre8s} ⁺ _{en Tt d ceislclslo vsiuar sec;reening} ^[ _A ⁰⁰ _*0 ⁴⁹ ₁ ^] _01-rest ^F _ri ^{ig.9 illustrates a non-limiting screening of ZIKV-derived HLA-B*0702 and HLA-} _{embodiment octfe tdhe e pprietosepnet d ciasncdloidsuartees; in Ifnar-/- HLA transgenic mice in accordance with an} ^F _of ^ig _C ^.1 _D ⁰ ₈ ^{A+ t} _T ^o _c ^F _e ⁱ _l ^g _ls ^.1 _d ⁰ _ir ^E _ec ^s _t ^h _ed ^ow _to ^{s g} _H ^r _L ^ap _A ^h-^s _B ^t _* ^h ₀ ^a ₇ ^t ₀ ⁱ ₂ ^ll-^{ustrate non-limiting results of cytokine secretion pattern} _{ELISPOT in accordance with an embodimen antd of H thLeA p-Are*s0e1n0t1 d-ibscinlodsinurge; epitopes identified via IFNγ} ^[ _t ⁰ _h ⁰ _e ⁵⁰ _im ^] _pact ^F _o ⁱ _f ^g _p ^. _r ¹ _i ¹ _o ^A _{r D} ^to _EN ^Fi _V ^g. ₂ ¹ _i ¹ _n ^E _fec ^s _t ^h _io ^o _n ^ws _on ^gra _th ^{phs that illustrate non-limiting results that demonstrate} _{with an embodiment of the present disclosuree; ZIKV-specific CD8+ T cell response in accordance} ^{[ Z0I0K51V]-speci Ffiicg. a 1n2dA t ZoIK FiVg./D 12EHN sVhow cro gsrsa-prehasct tihvaet i plleupsttirdaete i nmomn-ulinmizitaitnig results that demonstrate} _{response and mediated protection against} ^{on elicited CD8+ T cell} _{disclosure; ZIKV in accordance with an embodiment of the present}

11

^[ _t ⁰

m_h ⁰

e_a ⁵

d_t ²

i _H ^]

a_{tLedA- bB} ^F

y_* ⁱ

C₀ ^g

D₇ ^. ₀ ¹

8₂ ³

+-^A _{a Tn} ^t _d ^o

c_{e H} ^F

l_lsL ^ig

i_A ^{. E}

n- ¹

a_A ³

c_*c0o1 ^s

r_d0 ^h

a₁ ^o

n-^w _rcees ^g _t ^r

w_r ^a _iic ^p

t_ht ^h _e ^s _d ^t

a_{n Z} ^h

e_I ^{at illustrate non-limiting results that demonstrate} m_{KbVod piempteindte o imf tmheun pirzeasteionnt- dmisecdloiastuerde; protection is} ^[ _t ⁰ _h ⁰ _e ⁵³ _id ^] _entific ^F _a ⁱ _t ^g _i ^. _on ^14a _of ^an _Z ^d _IK ^F _V ^ig. _e ¹ _p ⁴ _i ^b _top ^sh _es ^ow _th ^g _a ^r _t ^ap _ar ^h _e ^s _c ^th _ro ^a _s ^t _s- ^il _r ^l _e ^u _a ^s _c ^tr _t ^a _iv ^te _e ^{non-limiting results that demonstrate} _{embodiment of the present disclosure; with DENV in accordance with an} ^[ _s ⁰ _iz ⁰ _e ⁵⁴ _d ^] _uring ^F _m ^ig _a ^. _te ¹ _r ⁵ _n ^A _al ^t _Z ^o _IK ^Fi _V ^g. _i ¹ _n ⁵ _f ^D _ec ^{show graphs that illustrate non-limiting result}

c_{ticoonrd inan Icfnea wr1it} ^-/ _h- _{m ani} ^{s of fetal weight and} _{both CD4+ and CD8+ T cells in a c eem wbiothdi omre wntit ohfo tuhte d perpelseetinotn d oisfcl CoDsu8re} ⁺ _{,; CD4} ⁺ _{, or} ^[ _s ⁰ _iz ⁰ _e ⁵⁵ _a ^] _{nd vira} ^F _l ^ig _b ^. _u ¹ _rd ⁶ _e ^A _n ^t _i ^o _n ^F _If ⁱ _n ^g. _ar ¹ ₁ ⁶-^K _blo ^s _c ^h _k ^o _in ^w _g ^g _A ^ra _b ^p-^h _t ^{s that illustrate non-limiting results of fetal wei}

e_{rseeantetd di WscTlos duarme;s with} ^{ght and} _{in accordance with an embodiment of the pr or without CD8} ⁺ _{T cell depletion} ^[ _I ⁰

a_f ⁰

c_n ⁵

c_a ⁶

o_rr1 ^]

d_-abnloceck ^F

w_i ⁱ _n ^g

i_tg ^.1

h _A ⁷

a_nb ^A _{- et} ^t

m_r ^o _eba ^F _toe ^ig

d_d ^.1

i_{m W} ^7D

e_{nTt o d} ^{show graphs that illustrate non-limiting results of ZIKV burden in} f_{am thse a pnredse tnhtei dris fceltoussuerse; on day 2 and 3 after ZIKV infection in} ^[ _e ⁰ _p ⁰ _i ⁵ _to ⁷ _p ^] _e-spe ^F _ci ⁱ _f ^g _ic ^.1 _C ⁸ _D ^A ₈ ^{+ to} _T ^F _c ⁱ _e ^g _l ^. _{l r} ¹ _e ⁸ _s ^J _po ^s _n ^h _s ^o _e ^w _in ^gr _s ^a _p ^p _le ^h _e ^s _ns ^th _f ^a _r ^t _om ^illu _I ^s _fn ^tr _a ^a _r ^t ₁ ^e _-b ⁿ _l ^o _o ⁿ _c-_k ^l _i ⁱ _n ^m _g ^it _A ⁱⁿ _b ^g _-t ^r _r ^e _e ^s _a ^{ults of cross-reactive} _{3 after ZIKV infection in accordance with an embodiment of the present disclotseudre W;T dams on day} ^[ _e ⁰ _p ⁰ _i ⁵ _to ⁸ _p ^] _e-spe ^F _ci ⁱ _f ^g _ic ^.1 _C ⁹ _D ^A ₈ ^{+ to} _T ^F _c ⁱ _e ^g _l ^. _{l r} ¹ _e ⁹ _s ^I _po ^s _n ^h _s ^o _e ^w _in ^gr _s ^a _p ^p _le ^h _e ^s _ns ^th _f ^a _ro ^t _m ^illu _I ^s _fn ^tr _a ^a _r ^t ₁ ^e _-b ⁿ _l ^o _o ⁿ _ck ^-l _i ⁱ _n ^m _g ^it _A ⁱⁿ _b ^g _-t ^r _r ^e _e ^s _a ^u _te ^lt _d ^s _W ^of _T ^c _d ^ro _am ^ss- _s ^r _o ^ea _n ^ct _d ^iv _a ^e _{7 after ZIKV infection in accordance with an embodiment of the present disclosure; y} ^{[ e0p0i5to9p]e-spe Fciifgic.2 C0DA8+ to T F ciegl.l r 2e0sEpo snhsow graphs that illustrate non-limiting results of cross-reactive} _{Ifnar1 mAb-trea} ^{e in placenta with decidua of non-immune or DENV2-immune} _{of the present distecdlos WurTe; dams seven days after ZIKV infection in accordance with an embodiment} ^[ _f ⁰ _e ⁰ _tu ⁶⁰ _se ^] _{s from} ^Fi _Z ^g. _IK ²¹ _V ^A _-in ^an _fe ^d _ct ^F _e ⁱ _d ^g. _n ² _o ¹ _n ^B _-im ^sh _m ^ow _un ^g _e ^r _a ^a _n ^p _d ^hs _D ^th _E ^a _N ^t _V ^ill ₂ ^u-^s _i ^t _m ^ra _m ^te _u ⁿ _n ^o _e ⁿ _I-_fn ^li _a ^m _r1 ^it-ⁱ _/ ⁿ-^g _da ^r _m ^es _s ^u _t ^lt _h ^s _a ^o _t ^f _w ^a _er ^p _e ^h _d ^e _e ⁿ _p ^o _l ^t _e ^y _t ^p _ed ^{e o} _o ^f _f

12

^C _di ^D _sc ⁸ _lo ⁺ _s ^, _u ^C _re ^D _; ^{4+, or both CD4+ and CD8+ T cells in accordance with an embodiment of the present [} _C ⁰

n_D ⁰⁶

d₄ ¹

D^+] _{,E CNDV8} ^F

2⁺ _, ^ig

-_{i o} ^.

m_r ²

m _c ² _o ^A

u_m ^{to Fig.22L show graphs that illustrate non-limiting results of the effect of} _{a neb Iinfneadr1 C-/-D d4a} ⁺ _{m asn idn C acDco8r} ⁺ _{d Tan cceell w diethpl aentio enm obnod ZimIKenVt v oifra thle bu prrdeesenn itn d nisocnlo-ismurme;une} ^[ _f ⁰ _ro ⁰⁶ _m ^2] _{n Ton c-eilm} ^Fi

l _dm ^g.

e_pu ² _n ³

l_ee ^s

t_{io o} ^h

n_r ^o _D ^w

i_nE ^gr

a_N ^{aphs that illustrate non-limiting results of a phenotype of fetuses at E14.5} _{CD8+ ccVor2d-aimncmeu wnieth W anT e dmambosd tirmeaetnetd o wfi tthhe I pfnreasre1n-btl doicscklionsgu Areb; with or without} ^[ _d ⁰ _e ⁰ _p ⁶ _l ³ _e ^] _{tion in} ^F _W ^ig. _T ² _d ⁴ _am ^sh _s ^o _a ^w _dm ^g _i ^r _n ^a _i ^p _s ^h _te ^s _re ^t _d ^ha _a ^t _nt ⁱ _i ^l-^lu _C ^s _D ^{trate non-limiting results of efficiency of CD8+ T cell} _{disclosure. 8 Ab in accordance with an embodiment of the present} ^{[ e0x0p6r4e]ssly un Idne trhsteoo ddraw thinatgs t,h eexe dmespclrairpyti eomnb aonddim dernatwsin arges il alustrated by way of example. It is to be} _{certainit esm obfo tdhiem inenvtesnt ainodn. are an a} ^{re only for the purpose of illustrating} _{the lim id for understanding. They are not intended to be a definition of} DETAILED DESCRIPTION

[ ^{a0lo06n5g] A detailed description of one or more embodiments of the invention is provided below describ weidth in acc coomnnpeanctyioinng f wigituhres su tchhat e ilmlubstordatimee tnhtes, principles of the invention. The invention is embodiment. The scope of the inve but the invention is not limited to any} _{set f} ^{ntion is limited only by the claims. Numerous specific details are} _{Thesoert dhe itnai tlshe ar feoll porwoivnigde ddes fcorrip tthioen p inur oprodseer t oof provide a thorough understanding of the invention. practiced according to the claims without some o norn a-lllim ofit tinhgese ex sapmecpilfeics d anetdail tsh.e F ionrv tehnetio pnurp moay be clarity, technical material that is kennotiwonn i isn n tohte u tnenchecneicssaalr fiileyld os se of described in detail so that the inv bs rceularteedd. to the invention has not been} ^[ _s ⁰ _u ⁰ _p ⁶ _p ⁶ _o ^] _{rts t} ^{The present application describes experimental results and line of reasoning which} _{approach,h thean de wvhelaotp hmasen btee onf p mreovrieou esflfyec dteivsceri fblaevdi.virus vaccine, diagnosis assay, and/or treatment}

13

^[ _r ⁰ _e ⁰ _la ⁶ _t ⁷ _e ^] _{s to ZI} ^I _K ⁿ _V ^o _. ^{ne embodiment, the flavivirus vaccine, diagnosis assay, and/or treatment approach [} _r ⁰ _e ⁰ _la ⁶ _t ⁸ _e ^] _{s to D} ^I _E ⁿ _N ^o _V ⁿ _. ^{e embodiment, the flavivirus vaccine, diagnosis assay, and/or treatment approach [} _r ⁰ _e ⁰ _la ⁶ _t ⁹ _e ^] _{s to ZI} ^I _K ⁿ _V ^on _a ^e _nd ^em _D ^b _E ^o _N ^di _V ^m _. ^{ent, the flavivirus vaccine, diagnosis assay, and/or treatment approach}

^{i [s0o070] ZIKV is a positive-sense, single-stranded, enveloped RNA flavivirus that was first m} _be ⁱ _e ^lldat

n^,e

i ^sd

d^e _e ^{l i}

n^fn- _t ^l _i ^{i 1}

f^m9

i_e ⁱ⁴

d^t7i _: ⁿ _E ^{g in}

a ^{a U}

sⁿ _t ^dgan

A_f ^s _r ^pd

i_c ^oa

a^r _n ^{a frdo}

,ⁱ _W ^cm

e ^{d a}

s^{is se}

t^e _A ^{ans he}

f^etin

r_ic ^inel

a_n ^{A r}

, _a ^f _n ^ris

d^{cuas macaque, and until recently was known to cause} A ^a _s ⁿ _ia ^d _n ^S ₍ ^o _L ^u _a ^t _n ^h _c ^e _io ^as _tt ^t _i ^A _et ^si _a ^a _l ^. _., ^T ₂ ^h ₀ ^r ₁ ^e ₆ ^e _). ^g _Z ^en _IK ^et _V ^ic _s ^li _t ⁿ _ra ^e _i ^a _n ^ge _M ^s _R ^h ₇ ^a ₆ ^ve _{of the East African lineage was isolated in the 1940s, whereas both West A 6 were discovered in the 1960s. frican and Asian strains} ^{[ o0f07 p1r]otein m DoEleNcuVle cso (nCta,i pnrsM ab aonudt 1 E1),0 t0h0at n fuocrlmeot thidee v birausess p,a wrhtiicclhe c aondde s feovren the three different types} _{molecules (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5) t} ^{other types of protein} _{a driseti rnecqtuioirnesd b feotrw reeepnlic tahteio sn hat are found in infected host cells only and er oofty tphees v airreus b.a Tsehder oen ar thee fiirve an sttirgaeinnisci otyf. the virus (i.e., serotypes), where the} ^{[ t0u0m72o]r cells C.D CD8+8 c+yt Toto cexlilc i Tmm ceullnse pl raeysp ao knesyes ro alere in d trhivee defense against intracellular pathogens and} _{presented by major histocompati} ^{n by the recognition of foreign peptides} _{identifi bility complex class I (MHC I) molecules at the cell surface. The diseasec paatitohnog oenfe tshises aend pe eptitoidloegsy ( aCsD w8e+ll T as c foelrl v eapcictoinpee ds)es iisgn th.erefore important for understanding} ^{[ c0e0ll7s3] in prot Aec ltaiorgne a bnodd pya othfo ligteenraetsuisre d huarisng pr doevnided evidence for a potential dual role for CD8+ T Tang et al., 2015; Weiskopf and Sette, 2014; Zeglulwee vgierurs an (DdE SNhrVes)ta in,f 2e0c1ti4o)n. E (Spcirdeeamtoinolo etgi acl., st 2u0d1i5;} _{indicate that Severe Dengue is most often seen in individuals experien} ^es _{infection after prior seroconversion to at least cing a heterotypic DENV Sangkawibha et al., 1984). Some studies show oneed o cfro thses- orethacetriv tehr CeeD s8er+o Ttyp ceesll (sG aurzem manor eet a alc.t,i 2v0at0e0d;} 14

^{d ( a}

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l^ary

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r^l.,ect

c ^2ro0i0o

s^s6n

-^{)re ( (IM}

a^mo

c^tirn

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c^.,p

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s^{.07 H; et M a}

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t^loh

y^{th a o}

e^{ma snub, 20p0ti5m)a slug Tges cteilnlg p ah penoosstiybplee p} _p ^r _r ^o _ev ^te _io ^ct _u ⁱ _s ^ve _w ^r _o ^o _r ^l _k ^e _o ^fo _n ^r _D ^T _E ^c _N ^el _V ^{ls i} _u ⁿ _si ^D _ng ^E _m ^N _o ^V _us ⁱⁿ _e ^f _m ^ec _o ^ti _d ^o _e ⁿ _ls ^(W _(Pr ^{eiskopf et al., 2013;m Weregiisnkgop lfite erta atlu.,re 20 p15o)i,n atsnd to ou ar} _{al., 2009; Zellweger et al., 2014; Ze estwood et al., 2012b; Yauch et al., 2010; Yauch et 129/Sv mice lacking type I I llweger et al., 2013; Zellweger et al., 2015) in C57BL/6 and A ce1ll2s9., and AG129) has provFidNed re mceuplttioprle (I liFnNesA oRf) e avloidneenc oer i bnodtihca ttyinpge I a a pnrdot IeIct IiFveN ro relece fpotror CsD (A8+B6 T,}

^{[ Z0I0K74V]’s imm Siugnnoslo ogfi ccli snimicaillar Zitiyka to di DseEasNeV ha hvaes h ailsstoor biceaelnly d boeceunm seimntieladr. B tola ssitg snesar ocfh d reengue fever, and ZIKV and DENV have about 52%-57% amino acid sequence homology. Indeed sseults show that reactivity of these two viruses has probably contributed to misdiagno rologic cross-} _{ZIKV, and cases of concurrent infection with ZIKV and DENV ha} ^{sis and underdiagnosis of} _{Cellular immunity to flaviviruses is also cross-reactive, a ve also been documented. i CnD p8r+ot Tect cieolnls a hnadve pa ntohtog benesis. However, nd cross-reactive T cells may play a dual role siese,n an idde dnetvifeileodp, to date ZIKV epitopes recognized by human CD4+ or immunity and pathogene m anendt t ohfe virac idceinnetsifi acnadtio pnote wnotuialldly a dcicaeglneorastteics in.vestigations of} ^{[ 400 s7e5ro]types E opfidemiologic and laboratory studies from the relatively large body of knowledge on the most commonl DyE wNheVn i pnadtiiceanttes t ahraet i tnhfeec steevder weit ahnd a s peoctoenndtia DllyE fNatVal s feorromtyp oef d afetnegru ienf deicsteioasne b oycc aunrds r aenctoigveenriyc s firno”m su agg feirsstst t hheatter doisloeagsoeus sev DeEritNyV inc sreeraosteysp ien. s Oecnoend hayrpyo itnhfeescitsio dne bemeed“original T cell during the first DENV infection predominate in the subsequent inf cause T cells primed} _{serotype, and these serotype-cross-reactive T cells fai} ^{ection with a different DENV} _{the second DENV serotype. Similar T cell cr l to mount an appropriate immune response to ZIKV and DENV share high amino acid idoss-reactivity may exist between ZIKV and DENV, as studies have revealed cross-r entity. Consistent with this homology, several recent particular, both pla eactivity between ZIKV and DENV at the antibody response level. In potent neutralizings amctaiv aitnyd ag maoinnsotc ZloInKaVl a anntdib coadnie ms iesdoilaatteed an ftriobmody D-dEeNpeVn-deexnpto esendha dnocneomresn cta (nA hDaEve)}

15

^o _A ^f _D ^Z _E ^IK _o ^V _{f D} ⁱⁿ _E ^fe _N ^c _V ^tio _i ⁿ _n ^. _fe ^I _c ⁿ _tio ^fa _n ^c _i ^t _n ^{, m} _vit ^o _ro ^no _an ^cl _d ^on _in ^al _vi ^a _v ⁿ _o ^t _i ⁱ _n ^bo _m ^d _i ⁱ _c ^e _e ^s _. ^{isolated from ZIKV-immune donors can induce [ Z0}

v^eI0

c^K76

t^oV]

r^{s an andd D Ve}

g^Er

e^oNy

g^{V litt}

r^{ap wle}

h^{il i}

i^ls

c^{a c k n}

l^on

d^no

i^{tsiwntruibe, h}

u ^ttoow

i^{o ce}

n^ov

s^-,ce

i^ir

t^{r,c a}

i^ub

s^lao

c^{tut T cell-mediated responses to ZIKV at present. As reiti icnal m taony sta rertgi eoxnpslo orfin thge th weor plrdo dteucetiv toe t vhseir common} _{pathogenic influence of T cells induced by prior DENV exp} ^{. potentially} _{a cboonut the T cell epitopes that are un osure on ZIKV infection. Knowledge esleoqpumenecnet, ar seu nitoabtl ae ique to ZIKV or shared with DENV is lacking. As a dev vai tloabolles. for investigating ZIKV-specific T cell immunity and vaccine} ^[ _p ⁰ _l ⁰ _a ⁷ _y ⁷ _a ^] _{dual r} ^C _o ^e _le ^llu _in ^la _p ^r _r ⁱ _o ^m _te ^m _ct ^u _io ⁿ _n ^ity _an ^t _d ^o _p ^f _a ^la _t ^v _h ⁱ _o ^v _g ^ir _e ^u _n ^s _e ^e _s ^s _is ⁱ _. ^{s also cross-reactive, and cross-reactive T cells may} Definitions

_i ^{m [0078] Unless otherwise defined, all technical and scientific terms used herein have the same} _en ^an

a_v ^e

c_ehn ⁱ

o_t ⁿ _ifo ^g

t_n ^a

h_{e p} ^s _e ^c

f_or ^o _tla ^m

l_oin ^m

w_si. ^o

n _A ^nl

g_s ^y

t_{e u} ^u

r_ms ⁿ _esd ^de

s_{h h} ^rs

a_e ^t

l_r ^o

l_e ^o

h_in ^d

a_, ^b

v_{e an} ^y

t_hd ^a

e _u ^p

d_n ^e

e_lfe ^rs

i_s ^o

n_s ⁿ

i_{t siot} ^o _ant ^f _e ^{ordinary skill in the art to which the present} s_{det o ftohrethrw bieselow or. required otherwise by context,} ^{[ a0s07 a9] chitos“aAndm niannisotpearirntigc”le a)n t eoxpr aess cieolnl v ceocmtoprr,i nseuscle tircan acsidduc minogle,cu trlea,n or a delivery vehicle (such} _{translocating, fusing, phagocytosing} ^{sfecting, electroporation,} _{protei , shooting or ballistic methods, etc., i.e., any means by which a a cell.n or nucleic acid can be transported across a cell membrane and preferably into the nucleus of} ^{[ p0r0o8t0e]in, or v Tehcteor t,er inmdic“aretecsom thbatin tahnet” ce wll,h nenuc ulesiecd ac widit,h pr roetfeeirnen ocre, ve ec.gto.,r, to has a b ceeelln, o mrod niufcieleic acid, introduction of a heterologous nucleic acid or protein or the al d by the} _{protein, or that the cell is derived} ^{teration of a native nucleic acid or} _{express genes that are not found wit fhroinm th ae c nealtliv seo ( mnaotudrifailelyd. o Tcchuursr,in fgo)r f eoxrmam ople, recombinant cells second copy of a native gene that is otherwise norm f the cell or express a not expressed at all. ally or abnormally expressed, under expressed or} 16

^{[ i 81}

nⁿ⁰⁰

u^{c eliet]hoetirde sin“}

a^gN

n^lea-uc

l^{o ole}

g^ric

s ^{d oo a}

r^ucibd

m^l”eo-e

d^{s rtirf}

f^ae

i^enrs

d^{de t}

b^do

a^{c f doe}

k^ro

b^mxy

o^n.r

e ^Tibho

r^enu

e^{s tidecrl}

u^meot

e^{s eidne}

o^cs

r^{om or}

l^{inpka ri}

a^sb

g^so

e^{essnu nculecoletiicde asci adnsd co ponltyamineinrsg t khneorewonf o} _n ^c _u ^c _c ^u _le ^r _i ^r _c ⁱⁿ _a ^g _c ^, _id ^a _, ⁿ _a ^d _nd ⁿ _w ^on _h-_i ⁿ _ch ^atu _ar ^r _e ^all _m ^y _e ^o _ta ^c _b ^c _o ^ur _li ^r _z ⁱ _e ⁿ _d ^g, _i ^{which have similar bind,in wghi pcrhop aerretie ssyn atshe tthice, r nefaeturernalclye} _{of such analogs include, wit n a manner similar to the reference nucleotides. Examples phosph)o.nates, chir hout limitation, phosphorothioates, phosphoramidates, methyl (PNAs al-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids} ^{[ e0n0c8o2m]passes Un cloenssserv oathtievrewlyise mo idnidfiiecdate vda,rian ats p tahretirceuolafr (e. ngu.,cl deeicgen aecriadte s ceoqduoennce also implicitly} _{complementary sequences, as well as the sequence explicitly indi} ^{substitutions) and} _{interchangeably with gene, cDNA, mRNA, oligonucl cated. The term nucleic acid is used sequences are displayed herein in the conventional 5’-3’e ooriteidneta,ti aonnd. polynucleotide. The nucleotide} ^{[ r0e0fe8r3] to a po Tlyhmee trer omfs am“pinooly apceipdt rideesi,d”u“eps.ep Tthidee t”er and“protein” are used interchangeably herein to m} _as ^o _w ^re _el ^a _l ^m _as ^ino _to ^ac _n ⁱ _a ^d _tu ^r _r ^e _a ^s _l ⁱ _l ^d _y ^u _o ^e _c ⁱ _c ^s _u ^a _r ⁿ _ri ^{analog or mimetic omfs a a cpoprrlyes tpoo anmdiinngo n aactidur palolyly omcecrusrr ining wh amichin oon aeci odr,} _{addition of carbohy ng amino acid polymers. Polypeptides can be modified, e.g., by the “protein” include gdlyractoepr roesteidinuse,s a tos f woermll a gslyc noopnr-ogtleyicnosp.r Tohteein tse.rm Tshe“p poolylpypepeptitdide,e” s“epqeupetnidcee”s and displayed herein in the conventional N-terminal to C-terminal orientation. are} ^{[ a0m08in4o] acid T anhaelo tegrsm an“adm aimnoin aocid a”cid ref merism teoti ncastu thraaltly f ouncccutirorning in an ad m syanntnheertic si ammilianro t aoci tdhse, as well as occurring amino acids. Naturally occurring amino acids are those encoded by naturally well as those amino acids that are later modified, e.g., hydroxypr the genetic code, as phosphoserine. The expression“amino acid analogs” oline, carboxyglutamate, and O-} _{chemical structure as a naturally occurri} ^{refers to compounds that have the same basic} _{hydrogen, a carboxyl group, an a ng amino acid, i.e., an .alpha. carbon that is bound to a methionine sulfoxide, meth mino group, and an R group, e.g., homoserine, norleucine, (e.g., norleucine) ionine, and methyl sulfonium. Such analogs have modified R groups naturally occurrin ogr a mmoindoifie adcid p.ep Atmidein boac ackibdon meism, betuicts re rteafienrs th teo s cahmeem bicaaslic co cmhepmoiucnalds str tuhcattu hreav aes a a}

17

^s _a ^tr _m ^u _a ^c _n ^tu _n ^r _e ^e _r ^t _s ^h _im ^at _il ⁱ _a ^s _r ^d _t ⁱ _o ^ffe _a ^r _n ^en _at ^t _u ^f _r ^r _a ^o _ll ^m _{y o} ^th _cc ^e _u ^g _r ^e _ri ⁿ _n ^e _g ^ra _a ^l _m ^c _i ^h _n ^e _o ^m _a ⁱ _c ^c _i ^a _d ^l _. ^{structure of an amino acid, but that functions in [ s0}

t^oe0q8

t^u5

h^e]

o^{nscee ns.u W“C}

c^leito

i^chn

a ^rs

c^ee

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s^ea

w^cttiv

h ^te

i^oly

c^{h pa m}

e^rnto

c^icd

o^ui

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i^{d neu v}

n^ca

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a^can

l ^at

o^cs

r^{i”d es s a}

s^ep

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n^uli

t^iee

a^ns

l^{lcye tso}

i^{, co bontsherv aamtivineoly m acoiddif aiendd va nruiacnletsic re afceirds the nucleic acid does not encode an amino aci dentical amino acid sequences, or where Specifically, degenerate codon d sequence, to essentially identical sequences. t dheiordxyi pnoossiitnioen re o substitutions may be achieved by generating sequences in which the sifdu oense (B oartz mero erte a sl.e,l Necutecdleic (o Arc aidll) R ceos.d 1o9n:s50 i8s1 s (u1b9s9t1it)u;t Oedht wsuitkha e mti axle.,d J-base and/or 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1 . Biol. Chem. of the genetic code, a large number of functionally 994)). Because of the degeneracy} _{For instance, the codons GCA, GC} ^{identical nucleic acids encode any given protein.} _{every position where an alan C, GCG and GCU all encode the amino acid alanine. Thus, at altered to any of t ine is specified by a codon in an amino acid herein, the codon can be S vaurcih he corresponding codons described without altering the encoded polypeptide. ati nouncsl.ei Ecv aecridy v naurcialetiiocn asc aidre s“esqiuleenntc vear hiaetrieoinns, w”h wichhich en acroed oense a sp pecies of conservatively modified possible silent variation of the nucleic acid. On olypeptide also describes every acid (except AUG, which is ordin e of skill will recognize that each codon in a nucleic the only codon for arily the only codon for methionine, and TGG, which is ordinarily According tryptophan) can be modified to yield a functionally identical molecule. described slye,qu eaecnhce s.ilent variation of a nucleic acid which encodes a polypeptide is implicit in each} ^{[ a0d0d8i6ti]ons tha Ats al ttoer, a amdidno or a dciedlet aen ad s ninugclleei acm acid sequences, individual substitutions, deletions or} _{acids or nucleotides in the seq} ^{ino acid or-nucleotide or a small percentage of amino} _{results in the substitution ofu aennc aem cirneoate ac aid“c wonitsher ava cthiveemlyic malolydi sfiiemdil varari aamntin,”o w ahceidre. t Choen asleterrvaattiion substitution tables providing functionally similar amino acids are well known in the art.. ve} ^[ _s ⁰ _u ⁰ _b ⁸ _s ⁷ _t ^] _itutions ^F _f ^o _o ^r _r ^e _o ^x _n ^a _e ^m _a ^p _n ^l _o ^e _t ^, _h ^t _e ^h _r ^e _(s ^f _e ^o _e ^l _, ^lo _e. ^w _g. ⁱ _, ⁿ _C ^g _re ^g _i ^r _g ^o _h ^u _t ^p _o ^s _n, ^e _P ^a _r ^c _o ^h _te ^c _in ^o _s ^nt ₍ ^a ₁ ^{in amino acids that are conservative} _{6-20, for a discussion of amino acid properties): 984) W.H. Freeman, New York, pages}

18

^A _S ^{anine (A), Glycine (G) A} _e ^l

A^s _{r s} ^p _in ^ar _e ^ti ₍ ^c _S) ^a _, ^c _T ^id _hr ^(D _eo ⁾ _n ^, _i ^G _ne ^lu ₍ ^t _T ^am ₎ ^{ic acid (E)}

C_{yparagine (N), Glutamine (Q)} A ^Isrog _st l^ien _ei uⁱ _n cⁿ _e i^e ₍ n^{e (} _C ^E ₎ (^I) _, )^, _M , ^{L Ly} _{e F} ^es _{t )} ^ui _h ⁿ _{i ,} ^ce _on

_T ^{in (}

y^eK _in ⁾ _e ^{, H} _(M ^is ₎ ^{tidine (H)}

P_{henylalanine ( ro} ⁽ _s ^L _in ^), _e ^V _(Y ^al ₎ ⁱⁿ _, ^e _Tr ^(V _yp ⁾ _{tophan (W)}

[ ^{in00 t8h8e] examp Inles lig ohft th oef t phrees pernetse tenxtt d,i tshcelo pseurrseo,n in o pfa srktiicular in view of the experimental data described b} _co ^e _m ^s _p ^ub _ri ^s _s ^t _in ^it _g ^ut _a ^e _n ^{d, ll will readily understand which amino acid may} a_m ^de _i ^l _n ^e _o ^te _a ^d _ci ^o _d ^r _se ^a _q ^d _u ^d _e ^e _n ^d _ce ^to _w ^a _hi ^g _c ⁱ _h ^ve _i ^{n sequence to create a conservatively modified variant} _{at least 98%, or at least 99%, identical to thse at am leainsto a atc liedas ste 9q5u%en,c oer s aett least 96%, or at least 97%, or NO: 1 to SEQ ID NO: 93 without undue effort. forth in any one of SEQ ID} ^{[ s0tr0a8n9d] by n“uPclreimice arcs”id a hreyb isroilated nucleic acids that are annealed to a complementary target DNA s} _P ^{t dization to form a hybrid between the primer and the target DNA} r^r _i ^a _m ⁿ _e ^d _r ^, _p ^th _a ^e _ir ⁿ _s ^e _o ^x _f ^te _th ⁿ _e ^de _p ^d _re ^a _s ^l _e ^o _n ⁿ _t ^g _in ^th _ve ^e _n ^t _t ^{arget DNA strand by a polymerase, e.g., a DNA polymerase.} _{sequence, e.g., by the polymerasieon c rheafienr t roea tchtieoirn u (sPeC fRor) a omrpl oiftication of a target nucleic acid amplification methods, such as qPCR. her conventional nucleic-acid} ^{[ s0e0q9u0e]nce” r Tefheer t pohr aas pehsys“iccoaldi sntgruc steuqrueen ccoem,”pr“issitnrugc atunra olrd seeqrluyen arcrea,n”ge anmden“tst orfuc ntuurcaleli nucleic acid nucleic acids are arranged in a series of nucleic a c acids. The encodes for a sp cid triplets that each form a codon. Each codon} _{nucleic acid sequ} ^e _e ^c _n ⁱ _c ^fi _e ^c _e ^a _n ^m _c ⁱ _o ⁿ _d ^o _e ^a _a ^cid _se ^. _r ^T _ie ^h _s ^u _o ^s _f ^{, the coding sequence, structural sequence, and structural} _{sequence. The coding sequence, structural am seiqnuoen acceid,s an fodrm strinugctu ara protein, polypeptide, or peptide contained within a larger nucleic acid molecule, vector, or thel l nikuec.le Iicn a acdiddit sieoqnu,en thcee m oradyer bley} 19

^a _fi ^r _g ^r _u ^an _re ^g _, ^e _t ^m _ab ^e _l ⁿ _e ^t _{, e} ^o _le ^f _c ⁿ _tr ^u _o ^c _n ^le _i ⁱ _c ^c _m ^ac _e ⁱ _d ^d _i ^s _um ⁱⁿ _, ^t _o ^h _r ^e _t ^s _h ^e _e ^s _l ^e _ik ^q _e ^u _. ^{ences may be depicted in the form of a sequence listing, [ r0}

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c^y _id ^{shiec ph}

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d ^o”

w^rd“

i_t ^en

h^{rulcleic acid sequence,” and“nucleic acid molecule”} i^y _n ^a _a ^rr _l ^a _a ⁿ _rg ^g _e ^e _r ^m _n ^e _u ⁿ _c ^t _le ^o _ic ^{f n} _a ^u _ci ^c _d ^le _m ^ic _o ^a _l ^c _e ⁱ _c ^d _u ^s _l ^. _e ^T _, ^h _ve ^e _c ^D _to ^N _r, ^A _or ^se _th ^quence _{In addition, the orderly arrangemen elte octfro nnucicle mice adciiudms, in or th thesee li ske e like. of a sequence listing, figure, table, eq.uences may be depicted in the form} ^[ _c ⁰ _o ⁰ _r ⁹ _r ² _e ^] _spondin ^T _g ^he _mR ^te _N ^rm _{A a} ^“ _n ^e _d ^xp _tr ^r _a ^e _n ^ss _s ⁱ _l ^{on” refers to the transcription of a gene to produce the} _{(i.e., a peptide, polypeptide, or proatteioinn). of this mRNA to produce the corresponding gene product} ^{[ s0u0b9s3t]antially T ohre e tsesremnti“ailsloyla ftreede” fr roefmers co tomp moanteernitasl, w suhcichh as no ar nmuaclllyeic ac accoidm opran ay p orort ieninte,r wahich is: (1)} _{material as found in its naturally occurring environment or (} ^{ct with the} _{e anndvi/roornm pleacnet,d t ahte a m locautesri ianl t hheas ce bllee onthe arlte 2) if the material is in its natural threadn t bhye l doecluibse nraatteive hu tom tahne m inatetervrieanl.tion to a composition} ^{[ s0y0m94p]toms o Tfh ane i tnefremcti“otnre oartin ag d”ise reafseers ass tooci aat pedro wceitshs a by fla wvihviicruhs an infection or a disease or the} _{eliminated. As used herein, the te} ^{strain are alleviated or completely} _{disease or s rm“preventing” refers to a process by which an infection or a delayed. ymptoms of an infection or a disease associated with a flavivirus are obstructed or} ^[ _c ⁰ _o ⁰ _m ⁹⁵ _p ^] _{ound t} ^T _ha ^h _t ^e _ca ^e _n ^xp _b ^r _e ^e _a ^ss _d ⁱ _m ^on _ini ^“ _st ^a _e ⁿ _red ^ac _t ^c _o ^ep _a ^t _s ^a _u ^b _b ^le _jec ^c _t ^a _w ^rr _i ⁱ _t ^e _h ^r _o ^” _ut ^m _si ^a _g ^y _nif ^r _i ^e _c ^f _a ^e _n ^r _{t a} ^to _dve ^a _rse ^ve _e ^h _f ⁱ _f ^c _e ^l _c ^e _ts. ^{for containing a [ in00v9e6n]tion to As in ucsreedas heer tehien, c thoem tpeormsiti“oand’jsuv imanmt”u mnoegaennsic ait syu.b Tsthanece m aedcdheadni tsom th oef co hmoposition of the} _{operates is not entirely known. Some adjuvants are believ} ^{w an adjuvant} _{(humoral and/or cellular response) by ed to enhance the immune response strongly immunogenic in their own right an sldow arlye b reellieeavseindg to th fuenc atniotinge sny,ne wrghiisletica oltlhy.er adjuvants are}

20

^{[ w0}

c^eh09

l^lisc7

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E ^{taycphic sa T}

p^llh

o^ye

t ^{a tl e}

h^lox

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v^isio

e^un

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h^{e oO}

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r^{)ea o E}

c^fn

t^{i ivnzydmiveid-Luainlk aecdtiv Iamtemdu onro rSepspootn adssinayg p quroavnitditeastiv beot (hth qeu farleitqautievnec (yre ogfar rdesinpgon thdein sgp cecific cytokine or othere se ccerlel.te Tdh iums,m thuene EL mIoSlPeOcuTle) as asnady speaking, in an ELISPOT assay, ells within the test population) information. Generally plate are coated with captu the membrane surfaces in a 96-well PVDF-membrane microtiter D} _se ^u _ed ^ri _e ⁿ _d ^g _i ^t _n ^h _t ^e _o ^c _t ^e _h ^l _e ^{l i re antibody that binds a specific epitope of the cytokine being assayed.}

wⁿ _e ^c _l ^u _ls ^ba _o ^t _f ^io _t ⁿ _he ^an _p ^d _lat ^s _e ^tim _alo ^u _n ^la _g ^ti _w ^on _ith ^st _t ^e _h ^p _e ^{, a} _an ^b _t ⁱ _i ^o _ge ^lo _n ^gi ₍ ^c _w ^a _h ^l _i ^s _c ^a _h ^m _c ^p _a ^l _n ^e _b ^(t _e ^yp _a ^ic _p ^a _e ^ll _p ^y _ti ^c _d ^o _e ⁿ _a ^t _s ^ain _de ⁱⁿ _s ^g _cr ^P _ib ^B _e ^{MCs) is} _{present disclosure), and forms a monolayer on the membrane surface of the well. As d in the specific cells are activated, they release the cytokine, which is captured the antigen- surface by the immobilized antibody. The cytokine is thus“cap directly on the membrane surrounding the secreting cell, before it has a chance to diff tured” in the area directly degraded by proteases and bound by receptors on bystanuse into the culture media, or to be v acistuivaalitzeed t cheell. immobilized cytokine as an ImmunoSpot; essednetrial cleylls th.e Su sbecsreeqtuoernyt fo doettepcrtiniotn of ste thpes}

^{[ u0s0e9d8] herein, T ghee terms“determining,”“measuring,”“evaluating,”“assessing,” and“assaying,” as present or notn ienra ally b rieofleorgi tcoal an saym fporlem. T ohfe mseea tseurrmemse innct,lu adned b inoctlhud qeua dnettietramtivinein agnd if/ aonr e qlueamlietanttiv is} _{determinations, which both require sample processing and transformation steps of th} ^e _{sample. Assessing may be relative or absolute. The phrase“assessing the pr e biological determining the amount of something present, as well as determining whethere iste insc pere osfe”n cta onr i anbcsleundte.} ^{[ s0a0m99p]le that T ishe su esxppercetsesdio onf“ cboimolopgriiscianlg sa am Tpl cee”ll, includes in the present disclosure any biological thereto, blood and fractions thereof, urin such as for example but without being limited} _{fluid, pre-ejaculatory fl} ^{e, excreta, semen, seminal fluid, seminal plasma, prostatic} _{ascites uid (Cowper’s fluid), pleural effusion, tears, saliva, sputum, sweat, biopsy, bronch,i aaml snecioretitcio fnlusi,d b,r leyamstp she,cr veatgioinnasl, s aencdre tthioen lisk,e e.ndometrial secretions, gastrointestinal secretions,} ^[ _r ⁰ _e ⁰ _s ¹ _p ⁰ _o ⁰ _n ^] _{se aga} ^T _in ^h _s ^e _{t a} ^ex _f ^p _la ^r _v ^e _i ^s _v ^s _i ⁱ _r ^o _u ⁿ _{s i} ^“ _n ^t _f ^r _e ^e _c ^a _t ^t _i ^m _on ^en _o ^t” _{r s} ⁱ _y ⁿ _m ^cl _p ^u _t ^d _o ^e _m ^s _s ⁱⁿ _a ^d _ss ^u _o ^c _c ⁱⁿ _ia ^g _t ^, _ed ^en _t ^h _h ^a _e ⁿ _re ^c _t ⁱ _o ⁿ _. ^g, _Fo ^o _r ^r _e ^su _xa ^s _m ^tai _p ⁿ _l ⁱ _e ⁿ _, ^g _th ^a _e ⁿ _tr ⁱ _e ^m _at ^m _m ^u _e ⁿ _n ^e _t

21

^m

p _su ^a

r_b ^y

e_fjeec ⁱⁿ

r_at ^d

b_{l f} ^u

y_o ^c _l ^{e r}

C_lo ^,

D_w ⁱⁿ

8_in ^c

+_g ^ea

T _t ^s

c_h ^e

e_e ^{, p}

l_{ls. tr} ^r _e ^o _a ^m _tm ^o _e ^te _nt ^o _. ^r _F ^s _o ^t _r ^im _e ^u _x ^l _a ^a _m ^te _p ^a _le ⁿ _, ^ti- _th ^fl _e ^avi _i ^v _m ^ir _m ^us _un ^a _e ^cti _s ^v _y ⁱ _s ^t _t ^y _em ^of _c ^im _ell ^m _s ^u _m ^ne _ay ^sy _in ^st _c ^e _l ^m _ude ^ce _T ^lls _c ⁱ _e ⁿ _lls ^a _, ^{[ a0ll0o1w01 t]he co Tmhpeo enxepnrtes osrio cnom“tphoersapeutically effective amount” may include the amount necessary to ca ition to which it refers to perform its immunological role without} _{T ach} ^u

c_e ^sin

o_{r edx} ^g

i_a ^o

n_cgt ^ve

t _a ^r

o_m ^ly

f_o ⁿ

a_cu ^e

t_n ^g

o_t ^a

r_{s o} ^ti

s_f ^ve

u _tch ^e

h_e ^ff

a _c ^e

s_o ^ct

t_m ^s

h_ep ⁱⁿ _{o tyn} ^th

p_een ^e

o_t ^h _s ^o

f _t ^s

c_o ^t

o_{n b} ^to

d_e ^w

i_{t u} ^{hich the component or composition is administered.} i_{osned be oirng th tere caotemdp, tohseiti toynpe to an bde a agdem ofin tihstered will vary treated, the mode of administration, as well as the other ingredients in the compositione. subject to be} EXAMPLES

[ ^{c0e0r1ta0i2n] comp Tohseitio fonlslo twhiantg ar eex damespcrleibsed de hsecrreibine. I stom sheou elxdem bep ulanrdyer mstoodoeds of making and practicing} _{illustrative puripno.ses only and are not me} ^{that these examples are for} _{described here ant to limit the scope of the compositions and methods} Example 1

[00103] Example 1 refers to the results shown in Fig.1A to Fig.8.

[00104] Example 1 can be summarized as follows:

[ ^{m00ic1e05 t]reated H- w2ibth m boluoscek minogd aenlsti o-IfF ZNIKARV i mnfoenctoioclnon reaclen antltyib hoadvye been established in WT C57BL/6 globally lack IFNAR or both IFNAR and and in gene-deficient mice that 2016; Lazea type II IFN receptors (Dowall et al., 2016; Govero et al., responses inr e Ht a-2l.b, 20 m1i6c;e, Ro ass mio etde all., o 2f01 Z6I)K.V To in infevcetsitoignat wea IsFN receptor-competent CD8+ T cell} _{C57BL/6 mice, which lack IFNAR} ^{established in LysMCre+IFNARfl/fl} _{T cells in a subset of myeloid cells but express normal IFNAR levels on LysMC,re B+IF cNelAls,Rf al/nfld C7 mBoLs/t6 d menicderi atnicd c aenltlsi-I (FCNlaAuRsen ant eit al., 1999; Diamond et al., 2011). Both were infected with ZIKV MR body-treated wild-type (WT) C57BL/6 mice cell r 766 and FSS13025 strains and mapped the H-2b-restricted CD8+ T ZIKVes ipnofencsteios.n A indd LiytsioMnCalrlye, a protective role was demonstrated for CD8+}

+_{IFNARfl/fl mice. T cells in controlling}

22

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a ^la0

n^c0

a^k1

lⁱ⁰

yⁿ⁶

s^g]

i^{s t ihdeen t Tyh}

t^ipe

f^{ie C}

e^{d ID}

2 ⁱⁿ⁸

6^{t+e ar T}

n^{fedr coe}

1^{nll r}

5 ^re

r^es

e^cp

a^eo

c^pn

t^itvose

e^{r i}

p ^wn

e^{a Z}

p^sI

t^{i eK}

d^veaV

s^lu-i

f^an

o^tf

r^eedct

Z^{I iend}

K ^{a Ly susMbsCetre o+fIF mNyAelRoifld/fl C ce5ll7sB.L IF/N6γ (H-E-L2bIS)P mOicTe l ainndeag deem stornaisntrsa,t reespectively. Intracellular cytokine staininVg A vfarliicdaanted (M tRhe76 i6d)en atnitdy A osfia tnhe (sFeS eSp1i3to0p25es)} _cel ^{d induction of polyfunctional ZIKV-specific CD8+ T cells. Furthermore, CD8+ T} _{r we ct asiat}

a_dl

s_{usc f}

i_nerdom

c_{re vair i}

s_an

e_lf

d _{beu inredd}

Z_{en m}

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V _we

-_{h m}

i_nere

f_ed

e_{cte de}

d_{edp Cl c}

D_eytti8oo

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i_{c City}

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c_{8 A}

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p_{a ct}

r_ei

e_{lvldse l t m}

t_{erdan tosfe hrig ohfer Z tIiKssVue-i bumrduennes C anDd8 m+o Trt caeliltlys demonstrate that CD8+ T cells protect ag o Wild-type. Collectively, these results and thoroughly characterized H-2b mousea minosdte Zl fIoKrV inv inefsetcigtiaotinng an ZdIK prVo-vsipdeeci afnic i Tm cmeulln roecspoomnpseetse.nt} 1. Materials & Methods for Example 1

1.1 Viral strains and mice

[ ^{f0o0r10 E7m]ergin ZgIK ViVru sstersai annsd M ARr7b66 and FSS13025 were obtained from the World Reference Center sentinel monkey rhesus (766) ino evaisruts Aesfri (cWaR (DCiEckV,A 19).52 M).R S7in66c,e A thfirsic iasonla ltinioena,ge th wea MsR is7o6l6ate isdol fartoem ha a b s weaesn is poalsastaegde idn o 2v0e1r01 f0r0om tim aes C ianm mboicdeia unsin pged iniattrraicce craesbera (lH ineaoncgul eattio alns (Dick, 1952). ZIKV FSS13025 low number of times. MR766 and FSS13025 were cultur ., 2012) and has been passaged a cells as described previously (Prestwoo ed using C6/36 Aedes albopictus mosquito} _{10 days after infect} ^{d et al., 2008). Virus was harvested from cell supernatants 7-} _{ultrac ion, followed by clarification via centrifugation, and concentration via hamsteenrtr kiifdungeaytio (BnH aKs) p-r2e1vi coeulls-lbya dseedsc froibceuds-f (oPrrmesitnwgo aosdsa eyt ( aFlF.,A 2)0.1 Z2aI). Virus was titrated using Baby in Senegal in 1984, was also obtained from WRCEVA, KV strain Dakar 41519, isolated amplified once in Vero cells (African green M passaged four times in RAG-/- mice and al., 2016; Sappara sptruain et an adl onkey Kidney Epithelial Cells) as described (Govero et sequence of each ., th 2e01 ab6s).en Nceex otf a gdenveenrattitioionus s peqautheongceinngs. of ZIKV stocks confirmed the} ^{[ a0}

Wⁿ⁰

a^d108

s_h ^C]

i_n ^D _gt ⁸ _o ^{α Wild type mice were purchased from the Jackson laboratories, and LysMCre+IFNARfl/fl} n^-/- _U ^C _n ⁵ _iv ⁷ _e ^B _r ^L _si ^/ _ty ⁶ _Sc ^m _h ⁱ _o ^ce _{ol o} ^w _f ^er _M ^e _ed ^b _i ^r _c ^e _i ^d _ne ^a _A ^t _n ^L _im ^a _al ^Jo _F ^l _a ^la _cil ^I _it ⁿ _ie ^s _s ^ti _. ^t _W ^ute _ild ^f _t ^o _y ^r _pe ^A ₍ ^l _W ^ler _T ^g ₎ ^y _m ^& _ice ^I _w ^m _e ^m _re ^un _tr ^o _e ^l _a ^o _te ^g _d ^y _w ^a _i ⁿ _th ^d 23

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h^{oorn mmtr aeo ldl e} _p ⁿ

b_e ^d _rfo ^f _r ^e _m ^m _e ^a _d ^le _e ^m _ith ^ic _e ^e _{r r} ^b _e ^e _t ^t _r ^w _o- ^e _o ^e _r ⁿ _bi ⁵ _t-_a ⁷ _{l o} ^w _r ^e _s ^e _u ^k _b ^{s i}

c_u ^o _t ^f _an ^ag _e ^e _ous ^e _i ^r _n ^e _oc ^u _u ^se _la ^d _tio ^itntee

n_s ^th _w ^is _ith ^stu ₂₀ ^d ₀ ^y _µ ^an _l ^d _of ^a _Z ^ll _I ⁱ _K ⁿ _V ^viv _i ^o _n ⁱ ₁ ⁿ ₀ ^fe _% ^ct _F ^io _B ⁿ _S ^s _/ ^w _P ^e _B ^r _S ^e _{r weu}

i_cf

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s _{atr Fc}

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P_{s U}

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S_{.OitCsK (F.F FUo)r o sufr vviirvuasl. st Iund ayl,l m exicpeer wimereent ins,fe mctiecde}

^[ ₁ ⁰⁰ _a ¹ _n ⁰ _d ^9] _{7 as p} ^T _re ^o _vi ^a _o ^s _u ^se _s ^s _ly ^{s t} _d ^h _e ^e _sc ^c _r ^l _i ⁱ _b ⁿ _e ^ic _d ^al _(T ^fe

e_a ^a _n ^t

d_g ^ur

a_{y e} ^e _t ^s,

o_{f a} ^m _{l i.n,} ^ic

f ₂ ^e

e_0c1 ^w

t_i6 ^e

o₎ ^r

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. _W ^ch _ei ^{ecked each day and assigned a score between} _{to the initial weight obtained on th ghts were recorded, reported and compared} 1.2 Titration of virus by FFA

[ ^{3070°1C1,0] 5% C BOH2K ov-2er1n cellts. woere plated at 2 x 105 cells per well in a 24-well plate and incubated at M hoEmMog-aelnpihzaa-tmioned aiunmig}

d ^{th (h}

e^{Innv F}

c^iternoll

t^goew

r^ifni

u^)ng

g^{at in mo}

i^{on pu}

a^rs

t^{ee- 2w p}

0^ee

0^ir

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e^ina

c^{gns s wteeerle h baeravdess,te fdol ilnow 1e mdl b oyf to infect BHK cells following serial dilution, larified supernatant was used} _{every 15 minutes. After i} ^{and cells were infected for 1 hour with gentle shaking} _{T nfection, wells were overlaid with carboxymethyl cellulose (CMC) (Sigma). 1%wo T driatyosn aTMfte Xr i (nSfiegcmtiao)n,, a cnedlls bl woecrkeed fix weidth w 1it0h% 4% FB fSo-rPmBaSl.in V (iFrisher Chemicals), permeabilized with pan-flavivirus anti-envelope (E) antibo al antigen was detected using 4G2, a (HRP)-conjug dy, following by a secondary antibody, horseradish peroxidase substrate (KPLat)e adn gdo water aent cio-munotuesde m IgaGnu (aSlliyg.ma). Foci were revealed after incubation with True Blue} 1.3 Peptide prediction approaches

[ ^{f0ro01m11 t]he NC ABllI k pnroowtenin Z dIaKtaVba pseol iynpr Joatneuinary se 2q0u1e6n.c MesH foCr c Alafsrican and Asian lineages were obtained} _{were performed at the Immune Epitop} ^{s I-peptide binding affinity predictions} _{recommended” method selection, e Database (IEDB) Tools website using“IEDB- affinities for all non-redundant 8-1 a1sm perre pveioputisdlyes d tehsactrib beodun (dKi Hm2- eKtb al a.,nd 20 H122)-.D Pbre wdiecrteed ob btianidniendg.}

24

^F _ra ^o _n ^r _k ^e _a ^a _n ^c _d ^h _r ^a _e ^l _s ^le _tr ^le _ic ^, _t ^t _e ^h _d ^e _to ^lis _t ^t _h ^s _e ^o _t ^f _o ^p _p ^e ₁ ^p _% ^tid _. ^{es obtained above were sorted by increasing consensus percentile [} _e ⁰

w_p ⁰

e_i ¹

r_teo ¹ _p ^2]

d_{eessi ugsnien} ^T

d_g ^h

f _t ^e

r_hoe ^E

m _ov ^p

t_he ^r

e_r ^o _l ^t _a ^e

E_p ⁱ _p ⁿ

p_irn ^f

o_g ^ro

t_{e m} ^m

i_{ne sth} ^Z

e_qo ^I

u_d ^K

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n _(cF ^s

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n ₈ ⁱⁿ

d_). ^s

s _1y5 ^M

n_-tm ^R7

h_ee ^{66 and FSS13025 was selected to identify} s_{riz peedp.tides that overlapped by 11 amino acids} 1.4 Peptide synthesis

[ ^a0

s_p ⁿ⁰

e^d11

c_t ¹³

r^5]

a-_l ^m _a ^e _n ^{r A}

a_l ^pl

y^{elp p}

s_is ^teidp

o^et

f^sid

e ^fe

a^os

c^{r w}

h ^Eer

p^Le

e^I _p ^{S syPn}

t_i ^{OthTes wizeerde b syyn Styhnetshizeetidc B asio cmruodleecu mleast,er Siaanl o Dnie ago 1,-m CAg. s Acallle 9- a,n 1d0- m, 1a1ss-} _{cytome de was performed to validate the synthesis. Peptides for flow were distrsioclv aendaly inse DsM weSrOe s aynndth aelisqizueodte adn.d purified by reverse-phase HPLC to≥ 95% purity. Peptides} 1.5 Ex vivo gamma interferon (IFNγ) ELISPOT

[ ^{G00e1r1m4a]ny). A CD t8+ T cells were isolated by magnetic bead positive selection (Miltenyi Biotec, p} _(I ^r _m ^es _m ^en _o ^t _b ⁱⁿ _il ^g _on ^cTeMll-^sot

P ^(a

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M^{P o}

i^Cf

l_l ^{s 2}

i_p ^{) ×}

o ^a _r ⁿ _e ^{d 10}

, _M ^{150 C}

A ^µD

)^g8

c_o ^o+

a^{f T}

t ⁱ _e ^{n c}

d^dei _w ^vlls

i^{id w}

t_h ^uae

a^lre

n ^Z _ti ^{I s}

-_m ^KtimVu

o_u ^-dla

s_e ^etre

I^idv _F ^{e with 1 × 105 LPS-blast cells as antigen} N^d _γ ^p _m ^ep _o ^t _n ^id _o ^e _cl ⁱ _o ⁿ _n ⁹ _a ⁶ _l- _a ^w _n ^e _t ^l _i ^l _bo ^fl _d ^at _y ^-b _(m ^ot _A ^to _b ^m _{) (} ^p _c ^l _l ^a _o ^t _n ^e _e ^s _{A NNgo1n8o; M eta ablt.,ec 2h0,1 S6)w.e Pdoesnit)iv ine t preipplticidaetes. w IFNγ-ELISPOT was performed as previously detailed (Elong CD8+ T cells≥ 20 and a stimulation indeerxe≥ th 2os beas wedith on a n thuem nbeegrat oivfe sp cootn-tfroorlm (DinMg cSeOlls). (SFC) per 106} 1.7 Flow cytometric analyses

[ ^{ly0s0i1s1 a5n]d r For intracellular cytokine staining (ICS), splenocytes were counted after red blood cell were platedesu anspde sntdimedul iante 1d0% wit FhB 1S µ/RgP oMf iInd miveiddiuuaml p aetp 4t0ide x a 106 cells per ml. Splenocytes (2 x 106)} _{al., 2016). Positive (PMA-Ion} ^{s previously detailed (Elong Ngono et} _{exp omycin) and negative (No stimulation) controls were added for all CDe4r4im (ecnlotns.e C IeMlls7), we arned la abnetil-eCdD w6i2thL a (ncltoi-nCeD M3e (lC-lone 145-2C11), anti-CD8 (clone 53-67), anti- followed by staining with anti-granzyme B (clone1 N4G).Z CBe)l,ls an thtie-InFN wγere (cl foixneed X aMndG p 1e.2rm)e aanbdili aznetdi-,} 25

^T _us ^N _in ^F _g ^α _F ⁽ _lo ^cl _w ^on _Jo ^e _TM ^M _s ^P _o ⁶ _f-_tw ^X _a ^T _r ² _e ² _X ^). ₁ ^S ₀ ^a _.0 ^m _.7 ^pl ₍ ^e _T ^s _re ^w _e ^e _S ^re _tar ^r _, ^ea _A ^d _sh ^o _l ⁿ _an ^a _d ⁿ _{, O} ^L _R ^SR _). ^{II (BD Biosciences) and were analyzed} 1.8 In vivo cytotoxicity assay

[ ^{S0 sM}

s^te0

i^mv1e1

u^n6]

l^{at dioayns L}

w ^py

i^{tohst a-Ci pnre}

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l^{tiNARfl/fl and WT mice were infected with 104 FFU of MR766 or FSS13025. oofn H,- s2pbl-ernesotcriyctteesd w ZeIKreV h-paervpetisdteeds (P frroMm169- n17a7,ïv Ee297 donor mice, followed by as“Target Cells” or with DMSO for 3 h at 37 -305, NS52783-2792) referred to (} ₍ ^I _H ⁿ _i ^v _g ⁱ _h ^tr ₎ ^o _o ^g _r ^en ₁ ⁾ ₀₀ ⁱⁿ _nM ^PB _C ^S/ _S ⁰ _F ^. _E ^1% _(Lo ^{B °C. The cells were washed and labeled with CSFE} w^SA _{) fo} ^fo _r ^r _un ¹ _s ⁰ _tim ^m _u ⁱⁿ _lat ^a _e ^t _d ³ _c ⁷ _e ^° _l ^C _ls. ^. _A ^T _f ^a _t ^r _e ^g _r ^e _w ^t _a ^c _s ^e _h ^ll _i ^s _ng ^w _, ^e ₁ ^r ₀ ^{e7 l} _o ^a _f ^be _la ^le _b ^d _ele ^w _d ^it _c ^h _el ¹ _ls ^{µM CSFE} _{each population) were injected intravenously into MOCK and inf (5 x 10} ⁶ _{of recipients were harvested 4 (Wild ty ected recipients. Splenocytes from cytometry. The percentage of killing ipse c)a olcrul 1a2ted h a laste forll (oLwyseMd:C 1r0e0+I -F (N%A ZRIflK/fl)V a-pnedp atindaelyzed by flow infected mice / % DMSO-stimulated in in0fe)c.ted mice) / (% ZIK stimulated in / % DMSO-stimulated in naïve mice) x 10 V-peptide stimulated in naïve mice} 1.9 Depletion and adoptive transfer of CD8⁺ T cells

[ ^{in00tr1a1p7e]ritone Aalllly an (it.pib.)od wiietsh f CoDr d8e cpelellt-idoenpl settuindgies (c wloenree Y puTrSch 1a6s9e.d from BioXCell. Mice were injected} _{LTF-2) antibodies on days 3 and 1 prio} ^{4) or rat IgG2 isotype control (clone} _{F deStSe1rm30i2n5e.d O ursginagns B wHeKre-2 h1ar cveells-tbeadse adt d FaFyA 6r}

._{, t 8o o irn 1fe0c atifotenr w iniftehct 1io0n5 F anFdU le ovfel ZsI oKfV infe MctRio7u6s6 v oirrus Z wIKerVe} ^{[ a0f0te1r18 in]fectio ZnIK wV-immune CD8+ T cells were isolated from LysMCre+IFNARfl/fl mice on day 120 kit (Miltenyi Bioittehch 1,05 C FDF8Ua o Lfy- M2)R.776.56 o xr 1 F0S6S C1D3082+5 T us cienlgls m waegrneet trican psofsitive CD8+ T cells selection} _{mice, and recipieunets m wiceere w mereeas cuhraellden ugseindg w BiHth} ^{erred into 5 week-old naïve} _{Viral titers in tiss K e-it2h1er ce Mll-Rb7a6se6d o FrF FASS t1h3r0ee25 da oynse p doasyt- acfhtaelrle cneglle t.ransfer.} 1.10 Statistical analyses

[ _D ⁰⁰ _ie ¹ _g ¹ _o ⁹ _, ^] _CA) ^A _an ^ll _d ^da _e ^t _x ^a _p ^w _re ^e _s ^r _s ^e _ed ^an _a ^a _s ^ly _m ^ze _e ^d _an ^wi _± ^th _S ^P _E ^r _M ^ism _{. S} ^TM _ta ^s _ti ^o _s ^f _t ^t _ic ^w _a ^a _l ^r _s ^e _ig ^v _n ^e _i ^r _f ^s _ic ^io _a ⁿ _nc ⁵ _e ^.0 _w ⁽ _a ^G _s ^r _d ^ap _et ^h _e ^P _rm ^ad _in ^S _e ^o _d ^ft _u ^w _s ^a _in ^re _g ^, _t ^I _h ⁿ _e ^c., _n ^S _o ^a _n ⁿ- 26

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e_rO ^{roups and the Wilcoxon test to compare two} e_{dV aAs s oigrni tfhicean Kt.ruskal-Wallis test was used to} 2. Results

2_b ^. _l ¹ _o ^. _{cking ant} ^C _ib ^h _o ^a _d ^r _y ^{acterization of CD8+ T cell response in WT C57BL/6 mice treated with IFNAR- [ a0}

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e^{0s1R6; o Lra tzreeaarte edt a wl.i,th 20 I1F6N;A MRin-berlo ectki anlg.,} _{mice were treated with an IFNAR-block} ^{ponse in H-2b mice, WT C57BL/6} _{inoculation with ZIKV strains MR766 oirng FS aSn1ti3b0o2d5y a MndAR in1f-e5cAtio3u (sSh veireuhsan pa erttic alle.,s 2006) prior to spleen were measured at days 1 and 3 post-infection. in serum and} ^{[ w0e0r1e21 a]dmin Tistheere fdoll IoFwNinAgR re-bsuloltcsk ainreg w ainthtib roefdeyre (nce to Fig.1A to Fig.1C, where WT C57BL/6 mice F coonctursol F morimcein ingje Uctneidts w (F MAR1-5A3) one day prior to infection with 104 itFhU 1)0% of F ZBIKS/VPB stSra.i Inns F MigR.716A6 l (env=el4s) o ofr in FfSeSct1i3025 (n=4). MOCK represents on day 1 or 3 post-infection were measur ous ZIKV in serum and spleen} _{of total CD8+ T} ^{ed by BHK-21 cell-based FFA. In Fig.1B, the expansion} _{mice (MR766 an cdell FsS aSn1d30 C2D5,44 n+=C4D)6 w2Las- C dDet8e+rm Tin ceedlls o inn M daOyC 7K p gorsotu-ipnf (n=3) or ZIKV-infected percentage of granzyme B produced by infecte ection. In Fig.1C, the Kruskall Wallis test was d (n=4) and MOCK mice (n=4) is represented. compare MOCK vs. each Z uIsKedV- fiinrsftec ttoed c momoupsaere gro alulp g.r Soeuep aslso fo Flloigw.7in.g by Mann-Whitney test to} [00122] The results obtained are the following:

[ ^{t0re0a1t2e3d] with A atnt di-aIyF 1N,A inRfec atniotiubsod ZyIK (FVig w.1aAs) d.e Ttehcrteaebl dea iyns a allft oefr t ihe sera and some spleens from mice} _{serum of mice infected with MR} ^{nfection, the viral load decreased in} _{of infectious virus in both groups7 i6n6cr beuaste ndo att t dhaoyse 3 i cnofmecpteadre wdi ttoh d FaSyS 11.3025. In the spleen, the level}

27

^{[ c0}

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l^{feVcti sotnra.i Tnshe in fr tehqisue mnociuesse o mf toodteall, C thDe8 C+D T8 c+el Tls bexlopcekraiednece rdela CtiDve8+ to T M cOelClK mice (Fig.1B). Infecteds m wiceere c ionnctraeianseedd 2 in-f ionlfdec mteodre m tioceta wl oitrh a InFtNigAenR-} _{mice, respectiv} ^{s than MOCK mice. In addition, ZIKV MR766- and FSS13025-infected} _{c byettowteoexnici Mty)R c7oe}

6_mly

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K_{e gllra renszpyomnese B w (aas m oabrskeerrve odf expansion and activation in WT mice treated with IFNAR-block V induces CD8+ T cell suitable for identifying ZIKV-derived epitopes recognized by CDi8n+g T an cteibllos.dy, and that this model is} ² _C ^. ₅ ² _{7BL/6 m} ^I _ic ^d _e ^en _tr ^t _e ^if _a ⁱ _t ^c _e ^a _d ^tio _w ⁿ _ith ^o _I ^f _FN ^Z _A ^IK _R ^V _-b-_l ^d _o ^e _c ^r _k ^iv _in ^e _g ^d _an ^ep _ti ⁱ _b ^to _o ^p _d ^e _y ^{s recognized by CD8+ T cells from WT [ p0r0o1t2e5o]me of T ZoIK mVap w tahse fi srpstec inifsicpietycte odf f tohre th MeH pCres celnacsse o I–fr peestprticidteeds p CreDd8ic+ted T to ce bll response, the} _{m 20o1l2e)c.ules (Kb and Db) with high affinity using a bioinformatic prediction progr} ⁱ _a ⁿ _m ^{d H} _(K ^-2 _im ^{b c} _e ^la _t ^s _a ^s _l. ^I _, ^{[ t0re0a1t2e6d] with T tyhpee f Iol IlFowNin rgec reepstuolrts-b alroec with respect to Fig.7A to Fig.7E, where WT C57BL/6 mice 104 FFU of ZIKV strai king antibody on day 1 prior to infection were inoculated with (MOCK, n=2). Fig.7An s MR766 (n=3) or FSS13025 (n=3), or injected with 10% FBS/PBS performed using hows results where seven days post-infection, IFNγ-ELISPOT was strains CD8+ T cells isolated from the spleen to screen 244 peptides from both ZIKV (n=8 m thicaet w peerre e pxrpeedriicmteednt t)o a bnidnd tw Ho-2K inbd aenpden Hde-n2Dtb e.x Fpoeurirm inendtespe fnodren FtSS ex1p3e0r2i5me (nnt=s8 fo mri MR766} _{experiment) were performed in triplicate for each peptide. The data are ex} ^{ce per} _{spot forming cells (SFC) per 106 CD8+ T cells. One-way ANOVA w pressed as the mean of each peptide with the control (DMSO). Fig.7B shows th as used to compare the mean of IFNγ upon stimulation with MR766 and FSS1302 e frequency of CD8+ T cells producing cytokine staining (ICS), usi 5-derived peptides, as detected via intracellular or MOCK (n=4) ng splenocytes from mice infected with MR766 (n=5), FSS13025 (n=5), corresponds to th.e P aovoerleadge d vaatalue fro ombtai tnweod i bnyde MpeOnCdeKnt af etxepre srtiimmeunlatstio anre (0 s.h4o3w%n. of Th IFeN dγo+tCteDd8 l+in Te} 28

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s^{steionnc,e f oofll Bowreefdeld biny A stim (BuFlAat)io annd w CithD1107 µag f oofr o (tfop CD p8a+ne Tl) c oelrls F pSroS1d3u0c2in5g-d beorith IFNγ and TNFα uposnes ssteimdu vliaati IoCnS. w Fitihg. M 7CR7 s6h6o-wdser tihveed fr peqeputeindceys} _CD107a ⁺ _IFNγ ⁺ _CD8 ⁺ _{T ce} ^{ved peptides (bottom panel). Fig.7D shows the frequency of} _{F spSlSe1n3o0c2y5te-sde frriovemd ( nbaoïvtteom mi pll}

c_as

e_{n oeba}

t_hl)t

a_{t pien}

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u _Fh

l_io

s_ge.w

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w_{i sf}

t_hhteor

Z_{w s}

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r _{atfotepr p inafneeclt)io onr, NS52783-2792) as“target” were injected into infected and M M169-177; E297-305; shows the percentage of“tarmgeetd c tewllisc”e k ainllde OCK mice 4 hours prior to harvest, and All experiments were perfor d er inro mr bicaers in afreec rteepdr wesiethnt MedR i7n6 S6E (Mn=.2) or FSS13025 (n=3).} [00127] The results obtained are the following:

[ ^{b0o0t1h28 Z]IKV A str toaitnasl, o afn 2d4442 pr sepdeiccitfeidc H fo-r2b F-SbSi1n3d0in2g5. p Aepmtiodes were identified with 202 shared between 2} _w ^K _er ^b _e ^, _t ¹ _e ⁴ _s ⁸ _ted ^fo _i ^r _n ^H _div ^-2 _id ^D _u ^b _al ^, _ly ^an _in ^d _a ^{22 were predicted to bind bontgh t MheHseC p celpatsisde Is, al 9le6le ws.e Nree sxpt,ec ailflic pe fpotrid Hes-} _{either MR766 or FSS n IFNγ-ELISPOT assay using CD8+ T cells from mice infected with panel) and 15 peptides1 w3e0r2e5 p.o Tswitievnety fo-srix ZI pKepVti FdeSsS1 w3e0r2e5 p (Foisgi.ti 7vAe, f boortt ZoImK pVan MelR).766 (Fig.7A, top} ^{[ F0S0S11293]025) o Tfh tehe id 1e0nt ZifiIeKdV Z pIKroVtein espi itnocpluesdin arge st dreurcitvuerdal f proromtein 9s ( pforMr M anRd76 E6,) a anndd no frno-mstru 7ct (ufroar} _p ^l _{CrDot8e+in Ts N ceSll1, re NspSo2nBse (son toly Z MIKR7V66 M),R N76S62A a,nd NS Z3I,K NVS4 FASS,1 N30S245B w anerde N noS5t i (dFeing.tic 7aAl,). th Aelt Eho pugh the derived epitopes predominated in both ZIKV strains. rotein-} ^{[ s0t0130] To validate the identification of these ZIKV-derived peptides, intracellular cytokine fraeiqnuinengcy (IC ofS) IF wNaγs-p preordfuocrminegd C.D Sp8l+en Toc cyetlelss w waesre rep stoimrteudlat feodr e wacithh p aelplt pidoesitive peptides and the} _{(Fig.7B). Five of 26 MR766-derived peptides (top panel) an} ^{for both ZIKV strains} _{(bottom panel) induced a high frequency of d four of 15 FSS13025-derived peptides shared between both ZIKV strains: prM169-1 I77F,N Eγ29-4e-3x0p2,re Es2s9i7n-3g05 c aenldls. N TSh5e27 f83o-2l7l9o2w. Oinngly 4 N pSep31t8id86e-1s874 w weares}

29

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Z^{a+IoFsNitiγv+e d poeupbtildee-pso csoitnivfeirm ceeldls b (Fyig IF.N 7Cγ-I aCndS} _{k 7iEll)in.g Th apespero rxesimulatste dlyem 70o%ns otrfat sepl aen} ^{IKV strains exhibited cytolytic activity by} p_{ooclyytfeusnc lotiaodneadl w phitehn portyMp1e69 o-17f7, Z EI2K97V-305 a anntdige NnS-s5p27e8c3-i2f792 peptides (Fig. upon infection with either ZIKV strain in C57BL/6 mice after IFNAR blockade. ic CD8+ T cells} 2.3 LysMCre⁺IFNAR^fl/fl mice, a novel H-2^b model susceptible to ZIKV infection

[ ^{im00m13u1n]ocom Tpoete inntve mstoigdaetle th tahne m rioclee w oitfh C glDob8a+l IF TNA ceRlls blo dcukriandge, Z LyIKsMVCr ien+fection in a more mice, recently published for utility in studying DENV infe IFNARfl/fl C57BL/6 these mice display normal T and B cell ction (Pinto et al., 2015), were evaluated;} _{subset of myeloid cells. Th} ^{immune responses and lack IFNAR expression only in a} _{granulocy e Ifnar gene deletion is efficient in mature macrophages (83-98%) and D Mi 0%}

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e_{rBeLn/d6rit micic cee wllser (e16 in%fe)ct (eCdla iunsteranve ento aul.s,ly 19 w9it9h; after infection were determined. um, liver, spleen, and brain at 1 and 3 days} ^{[ L0y0s1M32C]re+IF TNhAeR fflo/flllo Cw5i7nBgL/ re6su mltisce ar aet 5 wi wtheek resfe orfen acgee w toere Fi ign.fe 2cAted to wi Fthig 1.026C F,FU wh oerfe M WR7T and FSS13025. Serum, liver, spleen, and brain were harvested at day 1 and 3 post 66 or levels of infectious ZIKV were determined using BHK-21 cell -infection, and the infectious in Fig.2A MR766 virus or in Fig.2B FSS1 -based FFA. The quantities of (white squares) post-infection are s 3025 virus at day 1 (black circles) and day 3} _{weight and clinical sc} ^{hown. Four mice were included in each group. In Fig.2C the} _{unpaired t t ores of infected WT and LysMCre+IFNARfl/fl mice were monitored and Fig.2D, a reesptr wesitehnt Watievlech d’sen csoirtryec ptliootn s whoasw uinsged C tDo4 c4om anpdar CeD th6e2 tLwo ex gprroeusspison at a enadch time point. In frequency of CD3+CD8+ T cells and CD44+CD62L- CD8+ T cells from Ly in Fig.2E, the infected with 104 FFU of ZIKV or MOCK are shown. Kruskall W sMCre+IFNARfl/fl mice compare all groups and the Mann-Whitney test was use allis test was used first to infected group. All error bars correspond to SEM. d to compare MOCK and each ZIKV-}

30

[00133] The results obtained are the following:

[ ^{L0y0s1M34C]re+IF ANtA dRayfl/f 1l m poicset- iinnffeecctteiodn w,i tthhe M inRfe7c6t6io (uFsig v.ir 2uAs) w aansd d FeSteSc1table in all of the tissues tested in} _{u LnysdMetCecrtea+bIlFeN inA WRfTl/fl m miiccee.. A Btas deady 3 on po thste-sin} ^{3025 (Fig.2B), whereas virus was} e_{f reecstuioltns,, i LnfyescMtiCoures+ ZIFIKNVAR w1eflr/efl m stiiclle d,e utectable in tissues of susceptible to ZIKV infection. nlike WT mice, are} ^{[ Z0}

c^{oI0135] To evaluate whether LysMCre+IFNARfl/fl mice demonstrate a clinical phenotype of mKpVar iendf.ec Utisoinng, t ah celin cliicnailca clrit secroiare ssca alne,d it th weas w oebigshets of LysMCre+IFNARfl/fl vs. WT mice were} _{clinical features up to score 3, corresponding to ruf} ^r _f ^v _l ^e _in ^d _g ^th _o ^a _f ^t _t ^L _he ^y _i ^s _r ^M _f ^C _ur ^r _. ^e+ _T ^I _h ^F _e ^N _i ^A _n ^R _fe ^f _c ^l/ _t ^fl _io ^m _n ^ic _a ^e _lso ^de _i ^v _n ^e _d ^lo _u ^p _c ^ed _{weight loss in LysMCre+IFNARfl/fl mice between days 4 and 7 post-inf ed signs of paralysis, a dominant phenotype o ection (Fig.2C). However, no observed. f ZIKV-infected IFNAR-/- mice, and death were} ^{[ L0}

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e^riC]

e^{nre Next, CD8+ T cell expansion and activation following ZIKV infection of c+eIdFN CDAR8fl/fl}

+ ^{T m ceilcle su wbsaest e axsp rleoprreeds.en CteDd4 i4n a thned g CatDin6g2L markers differentiated the antigen- MOCK and ZIKV-infected (MR766 strategy (Fig.2D) for splenocytes from} _{number of tot} ^{and FSS13025) mice. ZIKV infection led to an increase in the} _{cells in mice inafle CctDed8+ w Tith ce MllsR a7n6d6 a apnpdro FxSiSm1a3t0e2ly5, 5 r-e asnpdec 6ti-vfeollyd, e axspansion of CD44+CD62L- CD8+ T 2E). Thus, LysMCre+IFNARfl/fl mice mount a robust CD8+ T cell c roesmppoanrseed to to Z uInKinVfe icntfeedct mioince. (Fig.} ² _L ^. _y ⁴ _sMCre+IF ^I _N ^de _A ⁿ _R ^ti _f ^f _l/ ^ic _fl ^a _m ^ti _i ^o _c ⁿ _e ^{and validation of ZIKV-derived epitopes recognized by CD8+ T cells in [} _in ⁰⁰ _fe ¹ _c ³ _t ⁷ _e ^] _{d mic} ^A _e. ^{ll 244 peptides were tested by IFNγ-ELISPOT assay using CD8+ T cells from ZIKV- [ L0y0s1M38C]re+IF TNhAeR ffol/lfllo wweirneg in refesuctltesd a rreetr wo-iothrb rietaflelryen wciteh to 10 F4i FgF.U 3A o and Fig.3B, where five-week-old} _{3A shows a graph that illustrates the resul} ^{f ZIKV MR766 or FSS13025. Fig.} _{CD8+ T cells isolated from infected mice.t As f trootmal o anf 2 I4F4N pγe-EptLidISesPO froTm w ZhiIcKhV wa sstra pienrsfo prrmedeidct uedsin tog} 31

^{b Zi}

e^xIn

p^Kd

r^{eV H}

s^{s s- et2}

d^{rKai anb a}

s ^(nnd

t^{h=e5 H}

m ^m-2

e^iDceb

a^{n f w}

o^{ofrith e sapc h}

o^high

t ^{e fx a}

o^pf

r^ef

m^riniimit

n^{geyn w}

c^te)e

l ^{wreer secr peeernfoedrm. Tedwo in i tnrdipelpiceantede pnetr e pxeppetridime.e Tnthse f doarta ea acrhe r} _c ^e _o ^p _n ^r _t ^e _r ^s _o ^e _l ⁿ ₍ ^t _D ^ed _M ^a _S ^s _O ^S ₎ ^E ₍ ^M _P ^. _> ^On _0. ^e ₀-₅ ^{way ANOVA wasl usse (SdF tCo) c poemrp 1a0re6 t ChDe8 m+ea Tn c oeflls e,ac ahnd pe eprtriodre w baitrhs t ahree} _{derived e ). Fig.3B shows a graph that illustrates the results to confirm ZIKV- i Tnhtreac deolltutlp}

e_ai

d_{rto cp}

l_iye

n_{teosk r}

c_ienc

o_eog

r_{re stn}

s_ai

p_iz

o_ne

n_{idndg by}

s _{(“ C}

t_{oICDS8”+), T de cteelrlsm iinne Ldys sMevCenre d+IaFysN pAoRsfl/fl mice and IFNγ production via r tihveed a pveepr t-infection with positive peptides. stimulated with positive ZIKV-de atgidees am (0o.1u9n %t o off I IFFNNγγ+C prDod8u+c Ted ce bllys). MOCK mice when} [00139] The results obtained are the following:

[ ^{F0S0S11403]025 (F Fiigf.te 3eAn, p beoptttoidmes p waneerel). s Etaitgishttic aanlldy 7 positive for both MR766 (Fig.3A, top panel) and among these positive peptide proteins of the 10 ZIKV proteins are represented 13% from prM, N s from MR766 and FSS13025, respectively: 40% are from E protein,} _{e ZpIiKtoVpe sstr aarie} ^{S2A, NS3 or NS5, and 6% from NS2B, NS4A, or NS4B for MR766. FSS13025} n_{s si amreila irnlydic reapterdese innt Tedab alse M 1.R F7o6u6r.te Aelnl e ppeiptotipdeess i adreen rtiefcieodgn bizyed IFNγ-ELISPOT for both CD8+ T cells, 3 are specific for FSS13025-prim ceedlls C (DTa8b+le T 1 c only by MR766-primed MR766- and ZIKV FSS13025-primed CD8+ T ,e slelse, n aenxdt t pwaeglev)e. are recognized by both} ^{[ m00o1le4c1u]les (D Pebp atinddes K frbo).m T MheR7 p6o6sit ainodn FSS13025 ZIKV strains were predicted to bind H-2b class I induced a positive T ce s, sequences, and lengths of each of the 29 peptides that sequence co ll response, as determined via IFNγ-ELISPOT assay, are shown. The} _{BLASTP 2.5.} ⁿ ₁ ^se _o ^r _n ^va _N ^ti _C ^on _BI ^a _, ^m _an ^o _d ⁿ ₈ ^g _0% ^mo _o ^r _f ^e _th ^th _es ^a _e ⁿ _st ¹ _r ⁰ _a ⁰ _ins ^Z _r ^I _e ^K _pr ^V _ese ^s _n ^tr _t ^a ₂ ⁱⁿ ₀ ^s ₁₅ ^w _-2 ^a ₀ ^s ₁₆ ^ob _is ^t _o ^a _l ⁱⁿ _at ^e _e ^d _{s f} ^u _ro ^si _m ^{ng the program} _{Singapore, Venezuela, Australia, and Braz Japan, Florida, 100% (Y(100%)) or 80% (Y(80%)) of sequeinl.c Ye id ceonrrtietsyp wonitdhs th toe m hiagjholryity co onfs tehreve pdub pliesphteidde sst,ra sihnasr.ing} ^[ _p ⁰ _r ⁰ _e ¹ _d ⁴ _i ² _c ^] _tio ^{To verify the map of the CD8+ T cell response to ZIKV, the computational epitope} _{that overnlap ap bpyro 11ac ahm winaos a ccoimdsp ianre tdhe to E t phreot oeviner flraopmpin bgot phep ZtIidKeV m setrtahionds). (screening 15-mer peptides}

32 Our Ref.2016-103-03 ^{SE NQ 1O I:D Seq AuAeFnTcFeTKV Len 8gth Db K Xb Protein Start-position End-position Conserved Strains 23 A IASGFAAWTTYLVGYVV 99 X X N PS E}

r^{M2B 1600 607 Y (100%) MR766}

1^{489 1497 Y (100%) MR766 4 MSYECPML 8 X X PrM 14751 11573 Y (80%) MR766 5 PSVRNGNEI 9 X NS3 2186 Y (100%) MR766 67 RQV RMANIWIVYSMSWWLL 181 X X X N NSS55 2989936 318784}

2 ^{20900 Y Y ( (110000%%)) M MRR776666 8 SSIAARGYI 9 X NS3 1795 18002 Y (100%) MR766 9 SSLVNGVVRL 10 X NS5 2839 283 Y (100%) MR766 10 SSWLWKEL 8 X NS5 2899 29408 Y (100%) MR766 11 TGWSNWEEV 9 X NS5 3220 326 Y (100%) MR766 12 TTVSNMAEV 9 X E 338 34268 Y Y ( (100%) MR766 13 VMIFLSTAV 9 X X E 784 792 Y (18000%%) MR766 1} ₁ ⁴ ₅ ^YSL _A ^E _A ^C _F ^D _TF ^P _T ^AV _K ^I _I ¹ ₈ ^{0 X} _{X X} ^N _E ^{S1 9} ₆ ⁶ ₀ ⁹ ₀ ⁹ ₆ ⁷ ₀ ⁸ ₇ ^Y _Y ⁽ ₍ ¹ ₁ ⁰ ₀ ⁰ ₀ ^%)

%^{) MR766} ) ^M _F ^R _S ⁷⁶⁶ 1_{176 SSLINGVVRL 10 X NS5 2839 2848 Y ( (180%) FSSS}

1_{18 TL}

9 _{AT IG}

M_{MM SVYAN EVK CGC PLY MLI 98 X}

9 _{X X N P}

P_SrM

r_{M4A 21}

1₂₅

6₅₀

9_{7 21}

1₂₅

7₆₈

7_{4 Y}

Y _{Y ( (100}

1_000%

0_%)

%_{)) M MRR7 F}

7_{6S 66S 6/FSS 20 CAEAPNMKVI 10 X NS5 2783 2792 Y (100% /FSS 21 IGVSNRDFV 9 X E 294 302 Y (100%) MR766/FSS 22 MAVDMQTLTPV 11 X E 635 645 Y (1 ) MR766/FSS 23 RMAVLGDTA 9 X E 710 718 Y (00%) MR766/FSS 24 RSYCYEASI 9 X E 347 355 Y (100%) MR766/FSS 25 SNRDFVEGM 9 X E 297 305 Y (100%) MR766/FSS 26 SQLTPLTLI 9 X NS4B 2371 2 100%) MR766/FSS 27 SVKKNLPFVM 10 X N 379 Y (100%) MR766/FSS 28 VSFIFRA S2A 1336 1345 Y (100%) MR766/FSS 29 VVIKNGSYNV 89 X X X N NSS23A 11263576 11264644 Y Y ( (110000%%)) M MRR776666//FFSSSS}

3³

Our Ref.2016-103-03 [00143] The results obtained are the following:

[ ^{F0S0S11443]025, r Iensp teocttaivl,ely 1.4 S aixnd of 15 the pe 8pt cidoemsp guetanteiorantaeldly b pyred oivcetreldap M wRe7r6e6 positive for MR766 and} _{c apopmrpouatcahti (oFnigal.ly 8). predicted FSS13025-peptides were identified as positiv} ^p _e ^ep _u ^t _s ⁱ _i ^d _n ^e _g ^s _t ^a _h ⁿ _e ^d _o ⁴ _ve ^o _r ^f _lap ^th _p ^e _in ⁵ _g ^{[ w0e0r1e45 i]nfecte Tdh reet frooll-oowrbinitgal rlyes wulittsh ar 1e x wi 1th04 re FsFpUect o tfo M FiRg.7686, w ohrer FeS sSi1x3-w02eek-old LysMCre+IFNARfl/fl s IpFaNnγn-iEngLI tShPeO ET pr inot teriinpl oicfa bteost.h T MhR766 and FSS13025 were screened and5 a.ll A pe topttiadle osf w 1e2re7 t pesetpetdid beys 106 CD8+ T cell e data are expressed as the mean of Spot Forming Cells (SFC) per} _{106 CD8+ T cell} ^s _s ^a _g ⁿ _re ^d _a ^e _te ^rr _r ^o _t ^r _h ^b _an ^ar ₂ ^s ₀ ^ar _a ^e _n ^r _d ^ep _a ^r _s ^e _t ^s _i ^e _m ⁿ _u ^te _la ^d _ti ^a _o ^s _n ^S _i ^E _nd ^M _e ^. _x ^A _fa ^p _c ^o _to ^s _r ^iti _g ^v _r ^e _ea ^p _t ^e _e ^p _r ^t _t ^{ide induced a SFC value per} _{negative control (DMSO). All peptide sequences pr han 2 in comparison to the are identified in bold. Fou eviously identified by computational approach F thSeS n13eg0a2t5iv (eb coottnotmrol p (apn<e0l).0.5 Or}

)_{.nteee-wnay pe ApNtidOesVA are wa pso usisteivde to fo crom MpRa7re66 the (to mpea pnan oefl) ea acnhd pe fpifttiedeen w fiothr}

^{[ a0n0d14 C6D]8+ T T coell va plridoadtuect tihoen e opitopes identified via the IFNγ-ELISPOT assay, ICS was performed was quantified. Among all pofsi ItFivNeγ p,e TpNtidFeαs, a idnednt CifDie1d07 bay a IfFteNrγ s-tEimLuISlaPtiOonT w aistshay in,d 8iv pidual peptides} _{MR766-infected mice (Fig.3B, top panel) and 4 peptides from FS} ^{eptides from} _{bottom panel) induced a high frequency of IF S13025-infected mice (Fig.3B, prM (25%), E (37%e)r,e N frSo3m (1 p2r% Nγ+ cells. For MR766, epitopes were derived from protein and 16% w M), N anSd2A N,S a5n (d25 N%S5).. For FSS13025, 50% of epitopes were from the E} ^{[ f0re0q14u7e]ncy o Pfo IFlyNfuγn+cTtiNonFaαl+ity an odf CD8+ T cells after peptide stimulation was evaluated based on the} _{MR766-primed (Fig.4A and Fig.} ^C ₄ ^D _C ¹ _, ⁰ _t ⁷ _o ^a _p ^+I _p ^F _a ^N _n ^γ _e ⁺ _{l) a} ⁽ _n ^g _d ^ati _F ⁿ _S ^g _S1 ^st ₃ ^r ₀ ^a ₂ ^te ₅ ^g-^y _p ^, _ri ^F _m ^ig _e ^. _d ⁴ _C ^B _D ^{) d} ₈₊ ^ou _T ^bl _c ^e _e ^-p _lls ^os ₍ ⁱ _F ^ti _i ^v _g ^e _. ^{cells in} _{Fig.4C, bottom panel). 4A and} ^[ _L ⁰ _y ⁰ _s ¹ _M ⁴⁸ _C ^] _re ⁺ _IF ^T _N ^h _A ^e _R ^fofl/lfllo _m ^w _i ⁱ _c ⁿ _e ^g _w ^re _e ^{sults are with reference to Fig.4A to Fig.4F, where splenocytes from} _{(n=5). Fig.4A shows the freqreue innfceyc otefd C wDit8h+ 1 T0} ⁴ _{ce FlFlsU pr oofd ZucIiKngV I sFtNraγin a MndR T76N6F (αn= up4)on or st FimSSu1la3t0io2n5} 3⁴

^{w eth}

s^txi

r^{paetergi MmR}

y^{e un7}

s^t6

e^s6

d ^w-d

t^ee

o^rreiv

s^{e pe}

l^eed

c^{rtfo p}

c^re

e^mpt

l^lseidde

e^{x tswpri (ct}

e^eop

s^{s ainngd pa}

b ^en

o^rerl

t^o)

h^{r a}

C ^bn

D^{adrs a FrSeS r1e3p0r2e5se-dneteridve ind S pEeMpt.id Feisg. ( 4bBot sthoomws p tahneel g).ati Anlgl s sthimowuslat tihoen f writh relevant ZIKV-peptide (E294-302)10 o7ra w ainthd i IrrFeNleγva innt M ZOIKCVK-p aenpdtid inefe (Ect7e1d0-71 m8).ic Feig u.p 4oCn F equency of CD107a+IFNγ+ CD8+ T cells obtained after stimulation with MR766- or} _apn ^S

o_d ^S1

s_{it c} ³

i_vo ⁰

e_r ² _r ^5d

C_es- D_p ^e _o ^r

8_+n ^iv _d ^e

T_e ^d _d ^p

c_{e tlo} ^ep

l_{s ( 0} ^ti

p_. ^d ₀ ^e

a₇ ^s.

n_{e %} ^Th

l _{C f)o} ^e

._r ^b

F _I ^a

i_F ^ck

g_.N ^g

4_γ ^ro

D_+T ^un

s_N ^d

h_oF ^o

w_α ^b

s₊ ^ta

t _(hp ⁱⁿ

e_a ^e _n ^d

p_eel ⁱⁿ

r_{c A} ^M

e_{)nt a} ^O

a_n ^C _d ^K _2.3 ^m ₆ ^ic _% ^{e is} _fo ^re _r ^p _C ^re _D ^se ₁ ⁿ ₀ ^t ₇ ^e _a ^d _+I ^b _F ^y _N ^d _γ ^o ₊ ^tt _d ^ed _ou ^li _b ⁿ _l ^e _e ^s- _{cells from mice infected with MR766 (n=8) or FSS ge of granzyme B produced by CD8+ T shows a representation of In vivo cytotox 13025 ZIKV (n=8) and MOCK (n=4). Fig.4E the percentage of kill icity of target cells in ZIKV-infected mice. Fig.4F shows (n=4). ing was obtained in mice infected with ZIKV (n=4) for 7 days or in MOCK} [00149] The results obtained are the following:

[ ^{im00m15u0n]odom Tinhaent r eepsuitlotsp confirmed that prM169-177, E294-302, E297-305, and NS52783-2792 are the C ZDIK8V+ T str c es. The investigation was expanded by assessing granzyme B expression in aeilnlss. T fohre M pRer7c6e6nt aangeds F oSfS g1r3a0n2z5y,m aend B+ w CerDe81+5 T- a cnedlls 1 i4n-f ionlfdec hteigdh meri,c ree wspere similar for both uninfected animals (Fig.4D). To verify cytolytic activi ectively, relative to} _{vivo cytotoxicity assay was performed} ^{ty of the ZIKV-specific CD8+ T cells, an in} _{(prM169-177; E297-305; NS52783-2792) as targe utssin (Fgig s.p 4leEn)o.c Aytse esx ppuelcsteedd, w ait hhig thhr peeer icmenmtaugneo odofm cyitnant peptides observed in both MR766- and FSS13025-infectedx mhiibciet a (F piogl.yf 4uFn)c.ti Toank otoxicity was demonstrate that the epitope-specific CD8+ T cells e aeln ph teongoettyhpeer,. these results} 2.5 Kinetics of the ZIKV-specific CD8⁺ T cell response in LysMCre⁺IFNAR^fl/fl mice

[ ^{L0y0s1M51C]re+IF TNhAeRfl f/oflll mowicieng wer reesu inltfsect aerde w withith 10 respect to Fig. 5A and Fig. 5B, where Splenocytes were harvested at 3, 7, and 144 FF dU of ZIKV strain MR766 or FSS13025.} _{immunodominant ZIKV-derived peptides} ^{ays post-infection and stimulated with} _{frequency of IFNγ-produc to assess cytokine production by ICS. Fig.5A shows the CD8 ing CD8+ T cells and Fig.5B shows the frequency of CD44+CD62L- + T cells at day 3 (white), 7 (black), and 14 (grey) post-infection. The background production of}

35

^I _c ^F _o ^N _m ^γ _pa ^o _re ^bt _t ^a _h ⁱ _e ^ne _ti ^d _me ⁱⁿ _po ^M _in ^O _ts ^C _f ^K _or ^w _ea ^a _c ^s _h ^s _p ^u _e ^b _p ^t _t ^r _i ^a _d ^c _e ^te ₍ ^d _{P >} ^fro ₀ ^m _.05 ^a _). ^ll _T ^v _h ^a _e ^lu _e ^e _r ^s _r ^. _or ^T _b ^w _a ^o _rs ^-w _co ^ay _rre ^A _sp ^N _o ^O _nd ^V _t ^A _{o S} ^te _E ^s _M ^{t w} _. ^{as used to} [00152] The results obtained are the following:

[ ^{e0 ] The kinetics of the splenic CD8+ T cell response induced by the immunodominant Mp0}

R^i1to53

7^{6p6es o art F dSaSys1330,275. an Tdhe 14 p perocsetn-itnafgeect oiofn IF wNerγe+ C mDea8s+ur Ted ce inlls L wysaMs hCirge+IFNARfl/fl mice infected with} _{14 post-infection for both MR766 and FSS13025-inf} ^{her at day 7 than day 3 or day} _{C deDm44+CD62L- cells in infect ected mice (Fig.5A). Similarly, the frequency of eo pnesatrkaste at t dhaayt m ed mice was higher at day 7 than day 3 (Fig.5B). These results mic , 7 a poosnt-gin tfheecti toimn.e points measured, the CD8+ T cell response in ZIKV-infected} 2.6 CD8⁺ T cells control ZIKV infection in LysMCre⁺IFNAR^fl/fl mice

[ _p ⁰ _e ⁰ _r ¹ _f ⁵ _o ⁴ _r ^] _{ming a} ^T _n ^h _t ^e _ib ^f _o ^o _d ^l _y ^lo _-m ^wi _e ⁿ _d ^g _ia ^r _t ^e _e ^s _d ^ul _d ^ts _ep ^e _l ^x _e ^p _ti ^l _o ^o _n ^re _st ^t _u ^h _d ^e _ie ^ro _s. ^{le of CD8+ T cells in controlling ZIKV infection by [ w0e0r1e55 t]reated T whieth fo dlleopwleintigng re asnutlit-sC aDre8 w oirth is roetsyppect to Fig.6A to Fig.6F, where LysMCre+IFNARfl/fl w poitsht- 1in0f5e FctFioUn. o Tfh MeR le7v6e6ls o orf F iSnSfe1c3 e control antibody on days 3 and 1 before infection t0io2u5s. M viricues w ine trhee sa (Fcriigf.ic 6eAd) a snedru tmiss,u (eFsig h.a 6rBve)s stpedlee ant, 6 (, 8 and 10 days and (Fig.6D) sciatic nerve were quantified using BHK-21 cell Fig.6C) brain, was used to compare the levels of infectious ZI -based FFA. A two-way ANOVA test administered groups for al KV between the isotype and the anti-CD8 antibody- or FSS1 l time points and tissues. Fig.6E, on day 120 after infection with MR766} _challenge ³⁰ _w ²⁵ _it ^, _h ^7. ₁ ⁵ ₀₅ ^x _F ¹ _F ⁰ _U ^{6 C} _o ^D _f ⁸ _M ⁺ _R ^T ₇₆ ^ce ₆ ^lls _or ^w _F ^er _S ^e _S1 ^tr ₃ ^a ₀ ⁿ ₂ ^s ₅ ^f _. ^er _F ^re _o ^d _r ⁱ _c ⁿ _o ^t _n ^o _tr ⁵ _o-_l ^w _s, ^ee _C ^k _D ^-o ₈ ^ld _{+ T} ^nai _c ^v _e ^e _lls ^m _w ^{ice one day before} _{naive LysMCre+IFNARfl/fl mice. Infectious ZIKV was quant ere isolated from compare naïve CD8+ T cells vs. ZIKV-immun ified. Mann-Whitney test was used to CD8α-/- that were treated with 2 e CD8+ T cells. Fig.6F, seven-week-old WT and subcutaneously w mg of IFNAR-blocking antibody at day -1, and then inoculated monitored for 21i dthay 1s0 in5 P bFoUth o gfro muposus aend ad raepptoerdte Dda fokarr W 4T15 (1n9= Z15IK, BVlac stkra siqnu aatre d)a aynd 0. C SDur8vival was Red circle) mice. Pooled data from three independent experiments a -/- (n=11, (Mantel-cox) test was used to compare groups. re represented and the log-rank} 36

[00156] The results obtained are the following:

[ ⁿ⁰

m^{e0 r g.6C) and sciatic} _co ^ir1 n^cv5

e^e7] t_a ^{. (}

i_n ^AF

e^{tig. Levels of infectious virus in serum (Fig.6A), spleen (Fig.6B), brain (Fi} d ^{d 6}

h^aD

i_g ^y)

h_e ^{6 w}

r ^pe

v^oe

i_r ^s _a ^{t a}

l^-si _b ^nse

u^fess

r_d ^cet _e ^id

n^o _s ^{n 6,, 8}

i_n ^{C o}

t^Dr

h_e ⁸¹⁺⁰

s_e ^{T day}

r_um ^cse _, ^{l al} _s ^-f

p^dte

l^er

e^p _e ^{l i}

n^en

,^tfee

b^dcti

r_ai ^mon

n_, ^{ic oef i CnfDec8t+ed T c weiltlh-su MffRic7ie6n6t a onrd F -SdSep13le0t2ed5} _{sufficient control mice. At day 8 post-in and sciatic relative to the CD8} ⁺ _{T cell- controtl a alln odf t ChDe8 ti-sd fection, the amount of virus decreased in all tissues, in both almos suepesle (tFedig. g 6rAou,p Fsi.g. A 6tB d, aanyd 1 F0,ig t.h 6eD l)ev eexlce opft i tnhfee bctriaoiuns (F ZigIK.6VC) w.as undetectable in} ^{[ d0o0n15o8r] mice N inefexct,te mde wmitohry MR C7D686+ o Tr F cSeSll1s30 w2e5re fo ard 1o2p0ti dveaylys. t ZraInKsfVe-rirmedmu frnoem m LemysoMrCre+IFNARfl/fl were transferred to naïve recipient LysMCre+IFNARfl/fl mice one- y CD8+ T cells} _{or FSS13025. Transfer of 7.5 x 106 memceor (yFig C.D 6E8+) i T} ^{day prior to infection with MR766} _{compared to control T cells from naïve mi n t cheells ser ruesmul atendd b inra dinec (rFeiags.e 6dE) Z.IKV burden} ^{[ t0}

c^h0

o^a1

nⁿ⁵⁹

f^{ir 1]}

m^{00 t thim D}

e^{e rosur}

l ^iening

o ^{m i}

f^ots

C^{uDs geen}

8^{+ berraation in the 1940s and 1950s, ZIKV MR766 was passaged serially more Tin cs,el llesad diunrgin tgo Z aI nKeVuro inlofegcictaiollny a udsainpgte ad s veicruosnd (H ZaIdKdoVw st ertai anl., o 2f01 A2f)r.i To lineage as well as another loss-of-function model for CD8+ T cells, a surviv can using mouse-adapted ZIKV strain Dakar 41519 and Cd8a gene al study was performed} _{Survival was monitored in IFNAR-blockin} ^{-deficient mice lacking CD8+ T cells.} _{deficient (CD8-/-) C57BL/6 mice g antibody-treated WT and congenic CD8+ T cell- adapted ZIKV D (Fig.6F). Mice started to die 12 days after infection with mouse- of akar 41519. Eighteen days later, all CD8-/- mice were dead compared to only 25% infe WctiTon m.ic Ceo.l Tlehctuivs,ely a, l tahcekse of re CsuDlts8+ de cmeollsns stirganteifi aca cnrtiltyica inlc rroelaesed susceptibility to lethal ZIKV ZIKV infection and pathogenesis in mice. for CD8+ T cells in controlling} 3. Discussion on Example 1

[ ^{c0e0ll1s6 p0]lay a p Broasteedcti ovne r tohlee r aegsauinltsst o ZbItKaiVne idnf ienct eioxample 1, it is reasonable to conclude that CD8+ T} _{T cells and dendritic cells, and} ^{n in an animal model with IFN receptor-competent} _{ZIKV strains. The present dis tchlaotsu three s ppreocvifidiceisty a of va thlidea CteDd8 m+ Tap ce olfl r tehsepo CnDse8+ va Trie cse slllig rhestlpyo anmseon tog} 37

^{Z MI}

f_o ^oK

u_n ^reV

d^o _a ^{v s}

t_i ^et

o^rr

n^{,ain a} _fo ^{lsl M}

r ^t _i ^hR

n^r _v ^e7

e^e66

s_ti ⁱ _g ^{m a} n_adt ^mnd

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V ^{i CnD tohe Z FISKSV13 M02R57 r6e6sp aonndse F.S ISn1 H30-22b5 DENV dominant epitopes are within NS3, NS4B, 8+ T cell response, whereas for} _{2009). When grouped by protein,} ^{and NS5 (Weiskopf et al., 2013; Yauch et al.,} _{similar for the prM epitope immunodominance between the two ZIKV strains was NS31866-1874. Over,a Ell,, t ahned H N-2Sb5 C eDpi8to+p Tes c.e Hllo rewsepvoenrs,e a to st MroRng7e6r6 r wesapso bnrsoead wears th saenen to fo FrS MR766 especially for NS1, NS3, and NS5 epitopes. S13025,} ^{[ w0h01e6th3]e The contribution of CD8+ T cells to protection vs. pathogenesis in ZIKV infection, and infectiorn cr woisths-r aeac stimiveila arnt filbavoidviieruss or th Tro cueglhls a cnantib wodoyrs-denep tehned course of ZIKV infection following antigenic sin, respectively, remain to b ent enhancement or original T cell} _{evidence of these phenome} ^{e determined (Lazear and Diamond, 2016). However,} _{DENV would indicate thatn vaac (Hcinaleste daedv,el 2o0p0e7r;s M neoendgk tools caopnasyiade ert t ahl.e, 2 ef0f0e3c)ts fr oofm ZI cKasVes v oaf severe recipients subsequently become infected with DENV. Therefore the ccine if subunit vaccine may be a good alternative for ZIKV. use of epitopes to design a}

38

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) ^{dnicsetiaosne o inf l coocmalpizoenden etvse onfts ZI pKrVec’sed cilningica aln pdict fuorlelo.w Dioncgum syesntteamtioicn o inff mecuticoonsal th traatns amreissi tohne b most t,hr ietat isen tihneg intercourse leading to systemic infection is growing (D’Ortenzio et al., y vaginal and anal Foy et al., 2011; Hills et al., 2016; Musso et al., 2015; Venturi e 2016; Deckard et al., 2016;} _{i fneftaelct biornain ha asn bdee dnev eassttaabtliinsghe cdo,n tsraenquspelnacceental infection and tran} ^t _sm ^al _i ^. _s ^, _s ² _io ⁰ _n ¹⁶ _a ^). _llo ^O _w ⁿ _s ^ce _fo ^m _r ^a _in ^te _f ^r _e ⁿ _ct ^a _i ^l _o ^s _n ^ys _o ^t _f ^em _th ^ic _{e Diamond, 2016; Malone et al., 2016 s including microcephaly (Brasil et al., 2016; Lazear and Ventura et al., 2016). Post-s ; Mlakar et al., 2016; Oliveira Melo et al., 2016; Tetro, 2016; (Atkinson et al., 2016; G ystemic infection entry of the virus into semen-producing tissues vector. Finally overo et al., 2016) allows the virus to be transmitted without its mosquito (GBS) c , evidence is also mounting that autoimmune disease such as Guillain-Barré syndrome Malone eatn a flo.,l 2lo0w16 s;y Osteehmleicr e intf aelc.,ti 2o0n14 w).ith ZIKV (Deckard et al., 2016; Lazear and Diamond, 2016;} ^{[ C0D0186+5] T cell- ZdIeKpeVn’sde mnto csltea dreavnacseta otifn ogth celirn niceaulr eoftfreocts result from infection of the fetal brain, and Diamond, 2004). ZIKV burden observed in t pic flaviviruses is well documented (Shrestha and LysMCre+IFNARfl/fl mice is consist he brains of both CD8+ T cell-sufficient and -depleted (Cugola et al., 2016; Dowall et al ent with published evidence of ZIKV’s neurotropism in mice} _{al., 2016; Rossi et al} ^{., 2016; Lazear et al., 2016; Li et al., 2016a; Li et al., 2016b; Miner et} _{in the brain ., 2016). In the latter mice, disproportionately increased levels of ZIKV MR766 brains ( seen at day 6 post-infection may reflect the strain’s passage history through mouse FSS1302D5ick, 1952). This observation in the brains of mice infected with MR766 relative to observed a hti egahrlliigehrt tsim oene po oifnt tshe (d daiyfsfe 1re anncdes 3 b peotswt-eiennfe tchteiosne) t.wo strains, albeit this difference was not} ^{[ I0F0N166 r]espon Tsehe in su mscyeeplotiibdilit cyel of LysMCre+IFNARfl/fl mice to ZIKV indicates that loss of type I consistent with reported p ls is sufficient to permit robust ZIKV infection. This finding is} _{flaviviruses (Mangad} ^{ermissiveness of monocytes and macrophages to replication of other} _{Transplace a et al., 2002; Prestwood et al., 2012a; Shrestha et al., 2008; Yang et al., 2014). response, bnuttal n ZoI mKeVnt tiroanns omfis thsieon sp wecaisfi rcec ceenlltullyar re}

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.^{s B aats deday o 6n-7 C aDfte8r+ in Tfe ccetiloln e,x wpahnicshio cno,r preoslpyfounndcsti toona plea pkhe CnDot8y+p Te o cefll Z IIFNγ epitope-specific CD8+ T cells, and CD8+ T cell-mediated viral clearance, i KV} _{c reosnpcolundsee i tnha thte mseye mloicide. t Typheis I co IFnNclu rseiosnpo isns seim is not necessary for priming an eff} ^t _ici ⁱ _e ^s _nt ^re _C ^as _D ^o ₈ ⁿ ₊ ^ab _T ^le _ce ^to _{ll virus, vesicular stomat ilar to studies of DENV (Yauch et al., 2009), vaccinia IFNAR-deficient micei.ti Fsu vritrhuesr ( cThhaormacptesroiznat eiotn al. o,f 20 th0e6), L aynsMdC Sreen+dIaFiN vAirRusfl/f (lL moopuesz et al., 2006) in provide a platform for studying ZIKV-specific T cell responses e model should candidates. , and for testing vaccine and antiviral} Example 2

[00167] Example 2 refers to the results shown in Fig.9 to Fig.14B.

[00168] Example 2 can be summarized as follows:

[ ^{c0e0ll16 re9s]ponse C tDo8 Z+i Tka c veilrluss p (laZyIK anV i)m ispo asrta ynett t rool be in controlling Flavivirus infection, but the CD8+ T flaviviruses, an understand e defined. Due to sharing of host space with other analysis, the present inventionrgs p orfed circotsesd-r 1ea0c7ti ZveIK imVm puenpittiyde iss t aols boin edss HenLtiAal-.B U*0s7in0g2 c aonmdp 9utational peptides to bind HLA-A*0101, and screened CD8+ T cells for IFNγ response 0 ZIKV interferon (IFN)α/β receptor (Ifnar)-/- HLA-B*0702 and H from ZIKV-infected example 2 identified 37 HLA- B*0702-re LA-A*0101 transgenic mice. The data in} _{using ELISPOT with 1} ^{stricted epitopes and 13 HLA-A*0101-restricted epitopes} _{lineages, respectively. T8w anendty 7-f piveepti HdeLsA c-oBm*0m7o0n2- tboin bdointhg A pferpictaidnes (M aRn7d661) a HndLA A-sAia*n01 (0F1S-Sb13025) peptide were confirmed to stimulate CD8+ T cell IFNγ production by intr inding (ICS). The cross-reactivity of ZIKV epitopes to D acellular cytokine staining ELISPOT and IFNγ-ICS on CD8+ T cells f engue virus (DENV) was tested using IFNγ- B*0701-binding peptides we rom DENV-infected mice, and 5 cross-reactive HLA- in DENV-immune mice expraen iddeednt pifoiestd Z bIyK bVot chh aasllseanygs.e Z anIKdV d/oDmEinNatVed c irnos ssu-breseacqtuiveent C CDD88+}

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r^{Dp8e+s T elic cietelld d aenptliegteionn-ex cpoenrfieirnmceedd} _{ZIKV/DENV cross-reactive epitope} ^{esults identify ZIKV-specific and} _{t ahgeain DsEt ZNIVK-Vim.m Thuensee s reetstuinltgs r healavteiv iem tpos, an}

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o_{oratetecti avne a roltleere fdor im epmitounpoe-dsopmeciinfiacnc CeD p8a+tt Tern cel ilns efforts, and provide a new m ns for ZIKV vaccine development and testing relevance. ouse model for evaluating anti-ZIKV CD8+ T cell responses of human} 4. Materials & Methods for Example 2

4.1 Mice and ethics statement

[ ^{v0i0a1 i7n0t]ercros Isfinnagr-/ o-f H HLALA-B-* B07*00270 a2nd an Idfna Hr-/L-A H-LAA-A*0101 transgenic mice were previously generated b} _A ^r _l ^e _l ^d _ex ^a _p ^t _e ^th _ri ^e _m ^L _e ^a _nt ^J _s ^ol _i ^l _n ^a _v ^I _o ⁿ _l ^s _v ^t _i ⁱ _n ^tu _g ^te _th ^f _e ^o _s ^r _e ^A _m ^lle _ic ^r _e ^gy _w ^a _e ⁿ _r ^d _e ^I _a ^m*

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e^{eicr m staicneda wrdith pa Iftnhaorg-/e- mni fcree2e2. c Monicdeiti woenrse.} _{Committee under protocol #AP028-SS Institutional Animal Care and Use similar studies. Animal experiments wer1e-0 n6o1t5 r.a Snadmomplieze sdize asnd w berlien edsetdim.ated based on experiments in} 4.2 Epitope prediction and peptide synthesis

[ ^{I0E0D17A1]R web Tshitee o HnLlinAe-B s*0702- and HLA-A*0101-binding peptides were predicted using the 2% of all candidates. Oonftewa hruen.d Preepdtid seevse wner HeL cAho-Bse*n07 i0f2 t-hbeiinrd pinregdi acntidve 9 s0co Hres ranked in the top epitope candidates were synthesized by Synthetic LA-A*0101-binding} _{which were confirmed} ^{Biomolecules (San Diego, USA) as crude materials} _{peptides, 5 HLA-A*01 b0y1- mbinasdsin sgpe pcterpotmideetsry an andal aysi Hs.e Spiaxti itmism Cun voirduosm (iHnaCnt HLA-B*0702-binding TPPAYRRPPNAPIL (SEQ ID NO: 80) restricted by mo V)-core helper peptide with a purity of >99% and used for use MHC molecule I-Ab were synthesized concentration of 40 mg/ml and store idm amtu -2n0iz℃in.g mice. All peptides were dissolved in DMSO with a} 4.3 Viral strains and mouse infection

41

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[^{N00 3] Two HLA-B*0702-binding ZIKV-specific peptides (FSS-NS2A133-141}

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42

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2 ^c ₎ ^lo _at ^ne _{3 d} ^Y _a ^T _y ^S _{s a} ¹ _n ⁶ _d ^9. ₁ ⁴⁾ _da ^o _y ^r _b ^is _e ^o _for ^p _e ^e _Z ^c _I ^o _K ⁿ _V ^tro _c ^l _ha ^m _ll ^o _en ⁿ _g ^o _e ^c _. ^lo _M ^na _ic ^l _e ^a _w ^nt _e ⁱ _r ^b _e ^od _in ^y _je ⁽²⁵⁰ _{R.O. with 1×104 FFU ZIKV FSS13025. Three days after infection mice wer cted a anndd b seraruinm w wereere ha ursveedst feodr. Z ICIKSV as tsiatyer asn ind t FisFsuAe,s r wesepreec mtiv e sacrificed, and spleen eealsyu.r Aedft uersin cagr FdiFaAc. perfusion with PBS, liver} 4.6 LPS-blast preparation

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[ ^{(0M01il7te6n]yi Bio CtDec8,+ G Term ceallnsy) w 7ere da iyssol aaftteedr v friroums i snpfleecntioocny.te 2s× u1s0i5ng CD m8a+gn Tet cicell bse wader peo sstiitmivue selection 1×105 LPS-blasts loaded with 10 µg of individual peptide in 96-w lated with (ImmobilonTM-P; Millipore, Bedford, MA) that wer ell flat-bottom plates Mabtech, Stockholm, Sweden) in tr e coated with anti-IFNγ mAb (clone AN18; After 20 hou iplicate. Concanavalin A (ConA) was used as positive control.} _{ABC peroxid} ^r _a ^s _se ^of _(V ⁱⁿ _e ^c _c ^u _to ^b _r ^at _L ^io _a ⁿ _b ^, _o ^b _r ⁱ _a ^o _to ^ti _r ⁿ _i ^y _e ^l _s ^a _, ^te _B ^d _ur ^a _l ⁿ _in ^ti _g-_a ^m _m ^o _e ^u _, ^se _CA ^IF _, ^N _U ^γ _SA ^m ₎ ^A _a ^b _nd ^{(R4-6A2; Mabtech), followed by} _{(Sigma-Aldrich, St. Louis, MO, USA) were then 3-amino-9-ethylcarbazole of IFNγ spot-formi added into the wells. Responses are expressed as number magnitude of responnsge w cealsls > (2S0FC SFs)Cs p,e arn 1d× h1a0d6 a C sDtim8+u Tlati coenlls in adnedx ( wSeI;re rat cioon osifd tered positive if the SFCs) of >2. A peptide inducing a magnitude of >500 SFCs/ est SFCs to control immunodominant peptide. 106 CD8+ T cells was considered as an} 4.8 ICS assay

43

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i^{ethlels c weiltlsh.ou Stpl aennyoc sytitmesula sttiimonul wateerde t whieth ne PgMatiAve-} _{(Clone 145-2C11, TONBO), PE-} ^{PerCpCyTM 5.5-conjugated anti-CD3 mAb} _{eFluorTM 450-conjugated anti-CD4C4y m7TAM-bco (cnlojungeat IeMd7 a,n etBi-iCoDsc8ien mcAe)b, a (ncdlo AnePC 53 e-F67, BD Biosciences), anti-CD62L mAb (clone Mel-14, eBioscience). Ce luor 780-conjugated Cytofix/CytopermTM solut lls were then fixed and permeabilized using I eFBNioγsc mienAcbe) (.c Sloanmep XlesM wGer 1io}

e_.2n

r_{,un T (BODN BBiOos)c aienndce As)P,C f-oclolonwjuegdat beyd s atnatini-iTnNgF wαith mA FIbT (Cc-lconjugated anti- sh ulasnindg, O anR L).SRTM II (BD Bio one MP6-XT22, software X 10.0.7 (Tree Star, A sciences) and analyzed using FlowJoTM} 4.9 Statistical analyses

[ ^{D00ie1g7o8] All data were analyzed with PrismTM software version 6.0 (GraphPad Software, Inc., San} _{of the} ^, _v ^C _a ^A _lu ⁾ _es ^an _is ^d _a ^ex _s ^p _ig ^r _n ^es _if ^s _i ^e _c ^d _an ^a _t ^s _o ^m _u ^e _tl ^a _ie ⁿ _r ^± _fro ^S _m ^EM _th ^. _e ^G _r ^r _e ^u _s ^b _t. ^b _S ^s’ _ta ^t _t ^{est was performed to determine whether one} _{non-parametric Mann-Whitney test to compare two groiusptisc.a Pl s <ign 0i.f0i5ca wnacse c woanssi ddeetreerdm aisne sidgn uisfiincagn tth.e} 5. Results

5.1 Identification of HLA-B*0702- and HLA-A*0101-restricted ZIKV-derived epitopes ^{[ I0fn0a1r7-/9-] mice P wreerveio uusseldy i gnesnteeardate odf i wnil vdiv-oty mpeod meilcse o,f be DcaEuNseV D inEfNecVtio cnan inno HtL bAloc tkra tnyspgeen Iic I Ifnar-/- mice.} _{and replicate in murine cells. Thateio HnLs:A transgenic Ifnar-} ^{FN signaling} _{been validated by several observ /- mouse models of DENV infection have} (^{i) T} _ce ^h _ll ^e _s ^e _(P ^p _B ^ito _M ^p _C ^es _{) f} ^id _ro ^e _m ^nti _D ^fie _E ^d _N ⁱⁿ _V ^m _-ex ^ic _p ^e _o ^w _se ^e _d ^re _hu ^al _m ^so _an ^r _s ^e ₂ ^c ₂ ^o _; ^{gnized by peripheral blood mononuclear}

44

^{(ii) A} _hum ^do _an ^m _s ⁱ _; ^{nance of HLA B*0702-restricted response was observed in both mice and (iii) C} _D ^D _E ⁸ _N ⁺ _V ^T _{3 b} ^c _u ^e _t ^ll _p ^r _r ^e _e ^s _d ^p _o ^o _m ⁿ _i ^s _n ^e _an ^t _t ^a _l ^r _y ^ge _N ^ts _S ^b _p ^o _ro ^th _tein ^st _s ^ru _in ^ctu _th ^ra _e ^l _o ^a _t ⁿ _h ^d _er ^{nonstructural (NS) proteins in} m_{ice and humans; three DENV serotypes in both} (^{iv) C prDot8e+in Ts) c feollllo responses were broad (targeting both structural and nonstructural (NS)} a_{nd humans} ^{wing primary and homotypic secondary DENV infection in both mice} i _{pnro mteiinces2.3 Si amn,}

i_{d wh}

l_{ar ne}

t_ar

o_teua

D_{rsaEl CD}

N _re8

V_in ⁺

,_{f Ze T}

I_{cKt cioel}

V_nl

c_{s re}

a_{n isnpo}

n_{o hn}

t_us

e_mes

v_{aa f}

d_no

e_sllo

t_{y fw}

p_oecinug

I_{s Ie h}

F_det

N _te

-_or

m_wot

e_ay

d_rp

i_di

a_{tsc s}

e_{d tehc}

i_meon

m _cdoa

u_{nr nsy iteyr in}

._{vefedcti NonS} ^[ _w ⁰ _e ⁰ _r ¹ _e ⁸ _, ⁰ _t ^] _herefo ^In _re, ^v _u ^ie _s ^w _ed ^o _t ^f _o ^s _i ^u _d ^c _en ^h _ti ^v _f ^a _y ^li _Z ^d _I ^a _K ^tio _V ⁿ-^, _d ^t _e ^h _ri ^e _ve ^I _d ^fna _H ^r- _L ^/- _A ^H-^L _re ^A _s-_tr ^B _ic ^* _t ⁰ _e ⁷ _d ⁰² _ep ^a _it ⁿ _o ^d _pe ^H _s. ^{LA-A*0101 transgenic mice [ u0s0in18g1 C]D8+ T The ce follsllo iswoilnatged re fsruoltms a Irfena wr-/ith respect to Fig.9, where IFNγ ELISPOT was performed A*0101 transgenic m - HLA-B*0702 transgenic mice (a and b) and Ifnar-/- HLA- Z avIeKraVged str aanind t FhSeS e1r3ro02ic}

r^5e

b^{a o (d}

r^{sr a}

r ^Mn

e^pRd

r^{e7 e}

s⁶⁾

e^{6n.7 et}

t ^{T d}

t^hway

e^os

S^{E i anf}

M^dteer

.^{p Te r}

h^nedr

d^eon-o

a^ttrb

a ^{e axit}

r^pa

e^el

e^{r (}

x^iRm.

p^{reOn.t)s in pfeercfotiromne wdit ihn 1 t×rip1l0ic2a FteFU we oref} _{forming cells (SFC) per 10} ⁶ _CD8 ⁺ _{T cells. The c} ^{essed as the mean number of spot} _{>20, and a stimulation in riteria for positivity were net SFC per 10} ⁶ _{cells of represent dex of >2.0 when compared with the negative control. Dotted lines according t thhee c cuotrorfefsp voanludein.g * Z InIdKicVate psro atei pnos (ictiv aend re fs)p;o wnsheit.e A alnld po bslitive peptides were grouped response of all identifipedtid peoss iintiv tehi pse pprtoidteeisn f.r Co ack bars are the total IFNγ number of positive pe omnA an d ienndoictaetse Cdo pnrocatenianv;a nliunm Ab.ers in parentheses are the} ^{[ r0e0182] One hundred seven HLA-B*0702-binding epitope candidates (8-, 9-, 10-, and 11-mers), Repsroeusercnetin (IgE tDheAR to)p, w 2e%re o cfho casnendid foatres sy pnrtehdeisciste.d Th bey n thuem Ibmermsu onfe p Epitope Database and Analysis} _{NS2A, NS2B, NS3, NS4A, NS4B, and NS5 were 3, 2, 6, 1} ^{eptides in C, prM, M, E, NS1,} _{Seven days after infection of Ifnar 3, 12, 12, 3, 23, 4, 16, 13, respectively. MR766, CD8+ T cells were isolated-/ f-ro HmLA sp-Ble*n0o7c0y2tes tr aanndsg secnriece mneidce b wyi ItFhN ZγIK ELVIS stPrOainT F aSssSa1y3.0 Z2I5K oVr} 45

^M

p ^IfrnR

o^ar7

d^-/6

u^-6

c ^{H in}

i^nLf

g^Aec

C^-DBtio0n

8^{*+7 T0 in2du}

c^{e tlrc}

l^saend

i^{nsg a}

m^{en st}

i^{icro mngiceer a (Fndig. b 9ro,a ade arnd CD c)8.+ T The c ferlleq ruesepnocniesse o tfha pnep ZtiIdKeV-sp FecSiSfi1c30 I2F5N iγn- 1 p2a2ge3) S sFhoCw/1s0 ce infected with ZIKV FSS13025 and ZIKV MR766 ranged from 140- t6h CeD ke8y+ ch Tar caecltlesri asntidcs 9 o8f-1 p3o6s2iti SvFeC p/ep10ti6de CsD. A8+ to Tta cells, respectively. Table 2 (see next from ZIKV FSS13025 and ZIKV MR766, res l of 19 and 36 epitopes were derived} _{iNncSl2uAdi1n33g-14 t1h,e f MollRo-w} ^{pectively. The two ZIKV strains shared 18 epitopes,}

N_{inSg2A 913 im3-1m41,uno FdSoSm/MinaRn-tN pSe2pAti1d48e-s15:5, FSS F/SMSR/M-NRS-2NAS725-B84, FSS/MR-NS2A89-99, FSS- FSS/MR-NS3 68-75}

5_{74-582, and FSS/MR-NS4B426- 4 , FSS/MR-NS3206-215, FSS13025 but not Z 35 (Table 2). The FSS-C25-35 epitope is present in ZIKV NS2A induced the hiIgKheVst M mRa7g6n6it.u Tdehe of N CSD38 p+ro Tte cinell c roenstpaoinnesdes t.h Teh lear mgeasjtor nituymber of epitopes and epitopes was located in NS2A, but the most immunodomi of immunodominant contained in NS4B (Fig.9, a to c). nant epitope, FSS/MR-NS4B426-435, was}

46 ^S Our Ref.2016-103-03 ^{N IEDOQ Pptid Sq HLA p IE}

P^{dDitBi C ti (% SF IFCN/1γ0 C CDD88 T T llll) 3 ktil FSS13025 MR766 SPH2015}

3^{01 F FSSSS/CMRM S G}

E ^{LP KPFSGHSTLRKKRLLPA B B007702 075 Y Y FSS13025 MR766 S221}

(^087092%)

3^{323 F FSSSS/ 02}

/^{MR 04 Y Y Y 235}

M^{RE TPPNTVSPDRIAEELA B B00770 3}

0^{2 145 Y Y Y (01290883%)}

3 ^{51 345 F FSSSS//MMRE SPRAEATL B07022 00285 Y Y Y Y Y Y 167 115058}

3^{367 F FSSSS/ERE T}

M^{RNS1 GP}

G^{PHCWKVNPNAKQEMAALV B B00770022 01375 Y Y Y Y Y 158 (022317%81)} _{38 MR} ^{PQRLPVPV B0702 14 Y Y Y 103}

3_{49 2A VVMILGGFSM B0702 1}

0 _{F NS}

F_{S 6 Y (013076%) 51 NSSSS2//AMMRR RPALLVSFIF B0702 16 Y Y Y (075133%) (250842%) (05888%) 41 N}

M _{FSS2A TPRESMLLAL B0702 015 Y Y Y (085067%) (089253%) 55 42 VPRTDNITL B070 MRSNNSS22AA V 2 03 Y Y (0798%6) (110178%2)}

4_{3 FSRNS2A LPPIRLTADANLIAL B0702 02 Y (065448%) (173796%)}

4_{4 NSS TPL B0702 02 Y}

2_{/AMR TPLARGTL B0702 045 Y Y (052480%)}

4₅

_{F 0 1}

N_{SSSS Y Y Y (0786 S2//BMMRR S LPPRELIVDRVEAALI B B0077002 6 Y 0}

2 _{012 Y Y Y (0925663%%)) ( (0980}

7_975%5)

4_{6 F 17739%) 63}

4⁷

^{4 N3 %}

4^{87 F FNSS3/ A %)) (063036 N %) 49 FSSS/MMRR A FPPDTRSNVVSPAIMEM B B00770022 0122 Y Y Y Y Y Y (07}

3³⁴

3^%9

5^{) ( (0223 SS 07534 NSSS33/MR RVIDSRRCL B0702 1 (054695%)}

5^{50 FS R}

N^{SSS SS3/}

3^{/M G 1 Y Y}

1 ^{F Y}

N^{MR RPPEMAPDVKTHVAAASAI B B0 36%) (0741%8 0770 (0223 02 )}

2^{002555 Y Y Y 140 204 136 5523 FSSMR Y Y Y 157}

N ^FNSSSS3/

3^{/MR K RVPRCWSDMHDAAARLV B B070 6}

7^{022 013 Y Y Y (09799%) (11099%5) (036530%) 54 0 3 Y Y}

5₅₅ ^{MRN AAG}

6 _F ^{KRGAAL B0702 06 Y Y (04}

1³¹

4¹¹

8^{%) 44 NS4AS3}

N _{FSSS SS4//}

N_S4AMR

A_{MR RPYKAAAAQL B0702025 Y Y Y 120 557 SPQDNQMAI B0702 05 Y Y Y 159}

8 _{N F FS}

N_{SSS4/BMR RPASAWAIY}

F_{S LA B0702035 Y Y Y NSSSS4/}

4_/BM

B_{MRR T SPPNLTKLYIWVANISISTA B B007702 (014890%) 59 02105455 Y Y Y (0146%3)}

6_{60 FSSMR Y Y Y 195 49 1 N FSSS4//BMRNS5 R APPGTQAFGCSIAKSVSLL B B007702 03 Y Y Y (212282%3) (213366%2) (0930%) 662 02 03 Y Y Y 98}

3 _{F 47 NS MSS}

R_5/MR

N_{S5 R RPPRAVECGTGKKETEVFM B B00770022 0011 Y Y Y Y 162 (02321} 48 ₍₀₃₇₄₁

_028%

5_7%%))

^{664 F FNSSS5/MMRR}

I ^{VPPYTLGGRKTRTEW B0}

^{6656 NSSS5/ DL B0770022 00945 Y Y Y Y Y Y 178 (012251%) (01584%) FNSSSS5/MRpM V RPTRRTRTGWNAAEYNYI A B0071002 5}

0^{1 0}

1 ^{02 823 (04}

%^{) (062368%) 67 75 Y Y Y Y (032 246561%) 68 F 79 F FSS}

S^{S/C 0 F FSS/M 1 SSS//MR}

M^{MRpEM H YLMDC}

D^{KDNQASTDMTSQYY A A001101 012 Y Y Y 4288}

7_{2 FSS/MR} ^RR _E ^{EE G} _F ^E _S ^TL _D ^D _L ^EF _Y ^S _Y ^DR _L ^LA _T ^YK _M ^{YV A} _A ^A0 ₀ ⁰¹ ₁ ¹⁰ ₀ ^{01 Y}

1¹ ₀ ⁰¹ ₂ ^{235 Y Y Y Y Y 59152 25518}

7 ⁶

7_{34 F FSSSS//MM 5 Y} ^{Y 993 64} 0₅ ^Y _{Y Y} ^{Y 7}

7 _{RRENS1 E DLVDEPPFGDSY A0101 02 Y Y Y 329886 388 75 FNSSS3/MR FTDPASWSIRADARY A A00110011 09255 Y Y Y Y Y 583 260}

7_{6 FNSSS4/AMR MTERFQEAI A0 Y 286}

7_{7 F 101}

N_{SSS4/BMR YLA 115 Y Y Y 266}

7_{8 F}

_{NSSS4/BMR MSAGLASLIY A0101 055 Y Y Y (016604}

1 _{3 Y %6) (113587%4)}

7_{9 FS 1 0}

a_{NSS5/MR YAQ} ^d _{MEWFYQSLYLY A A001100 02 Y Y Y Y Y 337183 225}

a^: _b ^T _br ^h _e ^e _vi ^p _a ^o _ti ^s _o ^it _n ^io _s ⁿ _fo ^o _r ^f _F ^p _S ^e _S ^p ₁ ^t ₃ ⁱ ₀ ^e ₂ ^s ₅ ^w _a ^a _n ^s _d ^d _M ^et _R ^er ₇ ^m ₆₆ ⁱⁿ _, ^e _r ^d _es ^a _p ^c _e ^c _c ^o _t ^r _iv ^d _e ⁱⁿ _ly ^g _; ^{to the amino acid sequence of ZIKV FSS13025. FSS and MR are} b:“Y” means having the same sequence;

c: Peptides are positive based on IFNγ ELISPOT count and/or IFNγ ICS percentage in parenthesis.

49

Our Ref.2016-103-03 ^{[ n0}

1^0u0

,^m18

1^b3

1^e]

,^{rs 3, of N}

3^{, pei}

1^pn

8^te

,^itdy

1^{e,s H}

2 ^inLA

0^{, C- 1,A}

6 ^p*

,^r0M1

r^e,0

s^{p M1- e,b}

c^{t Ein}

i^{v,d NinSg1, ep NitSo2pAe, N caSn2dBid,a NteSs3 w, NerSe4 aAls,o N cSh4oBs,e annd fo NrS s5yn wthereesi 2s., 5 T,h 1e, s Atrso snhgoewr CnD in8+ T Tab cleell 2 r,e tshp ely. In contrast to HLA B*0702 mice, FSS13026 induced a eon fsreeq tuheannci MesR o7f66 p ienpt Iifdnea-rs-/p- HecLifAic- I AF*N0γ1-0p1ro trdauncsignegni CcD m8ic+e T (F cige.lls 9, in d to f).} _{infected with ZIKV FSS13025 and ZIKV MR766 ranged from 286-1646 SFC/10} ⁶ _CD ^mice _{2 T2h5e-1 tw57o4 Z SIFKCV/1 s0tr6a CinDs8 sh+a Tred ce 7lls p,o resistpiveect piveeplty. Thirteen peptides were identified as po8si} ⁺ _{ti Tve c ienlls to atnadl. E159-167, FSS/MR-E195-2 ides, including 4 immunodominant peptides: FSS/MR- largest number of epit0o3p,e FsS aSn/dM iRn-dNucSe1d23- t3h1,e F hSiSgh/MestR m-NaSg4nBitu23d1-e239 o (fT CabDle8+ 2 T). E cel plr roetsepino contained the immunodominant epitope, FSS/MR-NS4B231-239, was contained in NS4B (Fig.9, d to fn).se. The most} 5.2 Epitope confirmation and characterization of cytokine secretion

[ ^w0

p_o ^e0

s^r1

i^e84

t_i ⁱ _v ^s]

e^ola _p ^t _e ^e _p ^{d T}

t_i ^fo

d^r _e ^{o f}

s^murte

, _a ^Zh

n^I _d ^{Kr c}

t^Vh

h^{-a r}

e ^irna

f_r ^{fcetceteizde I tfhnaer- e/p-it HopLeAs- iBd*e0n7ti0f2ied tra vniasg IeFnNicγ m EiLceI,S sPtOimTul aanteadly wsisit,h sp ealecnho ocfyt 3e7s} _{determined by ICS. equency of IFNγ- and/or TNFα-producing CD3+ CD8+ T cells was} ^{[ f0ro01m85] Ifnar-/- T HhLeA fo-Bllo*0w7i0n2g r treasnulstgse anriec w mitihce re (sFpiegc.t 1 t0ob F,ig n.= 170b m tioc Fig.10e, where splenocytes isolated A*0101 transgenic mice (Fig.10d, n=4 mice; Fig e; c, n=6 mice) and Ifnar-/- HLA- 1×102 FFU of ZIKV strain FSS1302 .10e, n=6 mice) 7 days after R.O. infection with} _{B*0702-binding pept} ^{5 or MR766 were stimulated with each of the 37 positive HLA-} _{ELISPOT a ides or 13 positive HLA-A*0101-binding peptides identified via IFNγ gate and then pder tcheennta IgCeSs a osfs IaFyN wγa+s a pnedrf/oorrm TeNd.F Aα+ re cperllesse (Fnitga.ti 1v0ea f)i.g Duraeta sh reopwrses tehe CD3+ CD8+ T cell independent experiments and are expressed as me.an ± SEM. *, nt the average of two Mann-Whitney test. P/I denotes PMA/ionomycin P < 0.05; **, P < 0.01; Two-tailed} [00186] The results obtained are as follows:

[ ^{00.02138%7] to 2.3 T6h%e p oefr tcoetnatla CgeDs3 o+f IFNγ- producing CD8+ T cells ranged from 0.22% to 2.28% and} _{infected mice, respectively (Tab} ^C _le ^D ₂ ⁸ ₎ ⁺ _{. T} ^T _w ^c _e ^e _n ^ll _t ^s _y- ⁱ _f ⁿ _iv ^Z _e ^I

5⁰ _o ^K _f ^V _th ^F _e ^S ₃ ^S ₇ ¹³ _IF ⁰² _N ⁵ _γ ^-in _E ^f _L ^ec _IS ^te _P ^d _O ^m _T ⁱ-^c _p ^e _o ^a _si ⁿ _ti ^d _ve ^Z _p ^IK _ep ^V _tid ^M _es ^R _w ⁷⁶ _e ⁶ _re-

^{c Co}

F^SDnf

S⁸ⁱ

1^+rm

3^{0 Ted}

2^{5 c oe bly}

r^{ls IF}

Z ^sIiN

K^mγ

V^{u IltC.}

M^anS

R^{e7o S}

6^uo

6^sm

.^lye

I^{n s oef}

Z^{c t}

I^rh

K^{eetVe FdS}

F ^bS/

S^oM

S^{1thR-NS2A89-99 and FSS/MR-NS4B426-435 peptide-stimulated 30 I2F5N-inγf aenctded T mNiFceα, i FnSS m-Cic2e5-3 i5n aflescote sdtim wuitlahte edit phreord ZucItKioV o n (FfSS b/oMthR I-FNNS2γA a7n5-d84, T FNSSF/αM (FRi-gN.S 120Ab1)4.8-1 I5n5, F ZSIKS/VMR M-RN7S626-infected mice, four additional peptides simultaneous production B68-75, and FSS/MR-NS3574-582) stimulated s} _T ^p _h ^e _e ^{ci of IFNγ and TNFα (Fig.10c). Collectively, these results define the} e^fi _p ^c _i ⁱ _t ^t _o ^y _p ^o _e ^f _m ^th _a ^e _p ^a _s ⁿ _h ^t _o ^i- _w ^Z _s ^IK _th ^V _at ^C _N ^D _S ⁸ _p ⁺ _r ^T _ote ^c _i ^e _n ^ll _s ^r _N ^es _S ^p ₂ ^o _A ⁿ _, ^se _N ^r _S ^e ₃ ^s _, ^tr _N ^ic _S ^te ₄ ^{d by HLA-B*0702 in this mouse model.} _{HLA-B*0702- restricted CD8+ T cel B, and NS5 are the major targets of the the African li l response to both African and Asian lineage ZIKV, and that than the Asiannea lginee ZagIeKV ZI MKVR76 F6SS c1o3n0t2a5in.e Hdo mwoerveer H,L amA-oBn*g071032-r IeFsNtrγict EedLI CSDP8+ T cell epitopes A*0101-binding peptides, only FSS/MR-NS4B231-239 OT-positive HLA- e ZitIhKeVr F FSSSS1133002255 o-r an MdR Z76I6KV (Fi was confirmed by IFNγ ICS in mice infected with}

M_{g.R 1706d6 a-inndfe 1c0teed). I Tfnhaer-/ p-e HrcLeAn-tAag*e0s1 o0f1 I tFrNanγs-gpernoidcu mcinig CD8+ T cells in 1.38%, respectively (Table 2). ce were 0.6% and} 5.3 Cross-reactivity of ZIKV epitopes with DENV

[ ^{e0p0i1to8p8]es wit Tho DE evNalVua,t CeD p8otential cross-reactivity of the HLA-B*0702-restricted ZIKV-derived} _with ^{+ T cells from spleens of Ifnar-/- HLA-B*0702 transgenic mice infected} _{ELIS DPOETNV an2al sytsriasi.n S221 were stimulated by each of 37 ZIKV-derived epitopes identified by IFNγ} ^{[B0*0017890]2 tran Tsgheeni fcol mloiwcein wge rreesu inltfsec atreed w Rit.Oh. re wsipthect 2× to10 F4ig F.F 1U4a of an DdE FNigV.214 sbtr,ai wnh Se2r2e1 If fnoarr-/ 7- H dLaA- CD8+ T cells isolated from splenocytes were stimulated with each of ys.} _{identified via IFNγ ELISPOT to perform (Fig.14a) IF} ^{the 37 ZIKV epitopes} _{represent the average of two in Nγ ELISPOT and (Fig.14b) ICS assay. Data experiment dependent IFNγ ELISPOT experiments and two independent ICS value. *, P <s ( 0n.0=57. mice) and are expressed as mean ± SEM. Dotted line corresponds to the cutoff} [00190] The results obtained are as follows: 51

^{[ 10}

t^h40

e^a19

f ^a1

r^n]

e^{dqu Feingc. C}

y ^1r4o

o^bs

f^)s

e^.-r

p ^Te

i^tha

o^icrt

p^ti

e^eveit

s^ny

p^{e p w}

c^ea

i^ps

f^{itcid d}

I^ee

F^ste

N ^wrm

γ^e+riene

C^{D pdo8s u}

+^istii

T^vneg

c^{, b asot dhet IeFrmNiγne EdL vIiSaP IOFNTγ a EndLI ISFPNOγT IC (FSig a.ss 1a4yas), (F anigd. (pTroabdlueci 2n)g. C SDix8 p+e Tpt cidelelss r waere positive based one IFllsN rγan IgCeSd f arsosmay, 44 an tdo 6 th0e6 S pFeCrc/e1n0ta6g CesD o8+f T IF cNelγls- positive pepti nged from 0.18% to750.63% (Fig.14b and Table 2). Of these 6 IFNγ ICS-} _{N (TSa4bBle4262-4)3.5, T ahn} ^d

e_d ^es,

s_{e F} ⁵ _{S reS} ^e

s_/ ^p

u_M ^ito

l_tsR ^p-^e

t_N ^s

h_uS ⁽

s₅ ^F ₅ ^S

i_3d9 ^S _-e5 ^/ _4n6 ^M ₎ ^R

t_{if w}- i_ee ^N

d_re ^S2A

a _{pto lseiat} ^-8

s_i ⁴ _v ^,

t_e ^F

5 _i ^S _n ^S/

H _b ^M

L_oAt ^R _h ^-N

-_{B I*F} ^S

0_N ³²

7_0γ ^06- _E ²¹⁵ _L ^, _I ^F _S ^S _P ^S _O ^/ _T ^MR _an ^-N _d ^S _I ³ _F ⁵ _N ^74- _γ ⁵⁸² _I ^, _C ^F _S ^SS _a ^/ _ss ^M _ay ^R _{s reactive epitopes with as many as 13 additional H 2-restricted ZIKV/DENV cross- cross-reactive as determined b LA B*0702-restricted epitopes possibly being binding peptides y the IFNγ ELISPOT assay alone. Thirteen positive HLA-A*0101- positive peptide w waser feou tensdte bdy in IF DNE γN ICVS2 (d Sa2t2a1 n-iontfe schtoewdn I)fn.ar-/- HLA-A*0101 transgenic mice but no} ⁵ _D ^.4 _ENV-imm ^I _u ^m _n ^m _e ^u _m ⁿ _i ^o _ce ^{dominance of cross-reactive memory CD8+ T cells during ZIKV infection of [ h0a0v1e92 p]reviou Tshlye b meaejnor eitxypo osfed pe toopl DeE inN tVh3e6. L Taotin in Avemsteirgiactaen h coowun ptrriieosr w exitphos ruecrent ZIKV outbreaks ZIKV-specific CD8+ T cell response, Ifnar-/- HLA- B*0702 trans e to DENV impacts DENV2 strain S221 for 4 weeks, followed by challen genic mice were infected with} _{FSS13025. On day 3 post-ZIKV infection (a t} ^{ge of these DENV-immune mice with ZIKV} _{specific naïve CD8+ T cell re ime point that is too early for development of DENV- DENV2-immun sponse in mouse models37, 38), splenocytes from mock-infected and ICS. e mice were stimulated by each of 23 ZIKV epitopes that were identified by IFNγ} ^{[B0*0017903]2 tran Tsgheenic fo mlloicwein wger rees iunlotscu alareted wi It.hP. r wesitphec 2t× t1o03 F FigF.U 11 of to DE FiNg.V 121e st, where Ifnar-/- HLA- Naive mice (n=5) and DENV2 strain S221-immune mice (n=5) we rain S221 for 4 weeks. FU of ZIKV FSS13025 for three days, and the percent re challenged R.O. with 1×104} _{TNFα+ CD8+ T cells were detected by ICS (Fi} ^{ages of peptide-specific IFNγ+ and/or} _{naive mice (n=5) and DENV2 s g.11a and Fig.11b). In addition, separate groups of FFU of ZIKV FSS1302 train S221-immune mice (n=5) were challenged R.O. with 1×104 TNFα+ CD8+ T cells we5re fo drete secvteedn b dyay IsC,S an asdsa tyhe (F pige.rc 1e1ncta agneds F ofig. pe 1p1tdi)d.e D-spateaci wficere IF eNxpγ+res asnedd/o asr} 52

^m

p ^spe

e^ea

p^cn

tⁱ _i ^f _d ^{i ±c} _es ^{r S}

w^eEs _e ^pM

r_e ^{o.n *}

g^s _r ^{e, P}

o_u ^{w <}

p^h _ed ^{ile 0.0}

a_c ^b5

c^o;

o^l

m_r ^{d **,} b_dei ^“ _n ^{* P} r_g ^{” <} o_{f th} ^r

p_e ^{e 0p.0} o _Z ^re1 s_itIiK ^{s;e T} v_eV ^ntwso p _sep ^Z-t p_etic ^{IaKileVd/ aDnEn-NWVhit cnroeyss t-eresta.c Btivlaeck re“s*p”on insde.ic Aatlels p ZoIsKitiVve-} _{parentheses indicate the nu difeisci itny t ohfis im grmouupn.e response (Fig.11e). Numbers in} [00194] The results obtained are as follows:

[ ^{D00E1N95V]2-imm Inu nnaeiv mei mcei,ce I,F nNoγ- seigxnpirfeicsasnintg ep CitDo8p+e-s Tpe cceiflilcs C diDr8+ T cells were induced (Fig.11a). In r 1e1abc)t.iv Teh pee ppetridceenst (aMgeRs- oNfS t2A31-40, FSS/MR-NS2A75-84, and FeScSt/eMdR to-N tSh3e5743-582 Z)I wKeVre/D dEetNecVted cr (oFsisg-. 0.44±0 hese 3 epitope-specific IFNγ-producing CD8+ T cells were 0.42±0.12%, T cells. w24er%e, d aonudbl 2e.-6p1o±s1it.i1v9e% w,it rhes epxepcrteivsseiloyn. F oSfS b/oMthR I-FNNS2γA an75d-84 T and FSS/MR-NS3574-582 specific CD8+ ZIKV infection revealed that 8 ZIKV-speci NFα. Similar analysis on day 7 post- responses were induced in naive fic and 4 ZIKV/DENV cross-reactive CD8+ T cell ZIKV/DENV cross-reacti mice (Fig.11c); in comparison, 2 ZIKV-specific and 5} _{1re1adc)t.iv Fei,g a.n 1d1e} ^{ve CD8+ T cell responses were elicited in DENV2-immune mice (Fig.}

t_{o staulm empiatoripzee-ssp theceif firceq (buoetnhcy ZI aKndV- mspaegcniiftiucd aend of Z ZIKIKVV/-DspEeNciVfic, cr ZoIsKV/DENV cross- cell responses in naïve vs. DENV-immune mice at 3 and 7 days f s-reactive) CD8+ T results demonstrate that, upon ZIKV challenge of ollowing ZIKV infection. The cross-reactive CD8+ T cells are activate DENV-immune mice, memory ZIKV/DENV response to ZIKV infection d and the immunodominance pattern of the CD8+ T cell naïve mice is broad and inc ilsud aletser reedco reglnatitivioen to o nfa bïvoeth m ZicIeK. TVh-sep aenctifi-icZI aKndV c CrDos8s+- T rea ccetlilv response in whereas the CD8+ T cell response to ZIKV infection in DENV-immune m e epitopes, reactive epitopes. ice is directed to cross-} ⁵ _Z ^. _I ⁵ _KV/DEN ^P _V ^ro _c ^te _ro ^ct _s ⁱ _s ^v-^e _rea ^im _ct ^m _ive ^un _p ⁱ _e ^t _p ^y _ti ^c _d ^o _e ⁿ _s ^{ferred by immunization of mice with ZIKV-specific and [} _s ⁰ _a ⁰ _m ¹ _p ⁹⁶ _le ^] _{s that} ^B _i ^a _m ^se _p ^d _lic ^o _a ⁿ _te ⁱⁿ _a ^c _p ^re _ro ^as _te ⁱⁿ _c ^g _tiv ⁿ _e ^u _r ^m _o ^b _le ^er _f ^s _or ^of _se ^r _r ^e _o ^c _t ^e _y ⁿ _p ^t _e- ^s _c ^t _r ^u _o ^d _s ⁱ _s ^e-^s _re ^u _a ^s _c ⁱ _t ⁿ _iv ^g _e ^m _C ^o _D ^us ₈ ^e ₊ ^m _T ^o _c ^d _e ^e _ll ^l _s ^s _a ^a _g ⁿ _a ^d _in ^h _st ^um _D ^a _E ⁿ _N ^d _V ^o ₁ ⁿ _3, ^o ₂₇ ^r _, ³ _a ⁹ _g ^{, 4} _a ⁰ _i ^, _n ⁱ _s ^t _t ^w _Z ^a _I ^s _K ⁿ _V ^ex _i ^t _n ^h _fe ^y _c ^p _t ^o _io ^th _n ^e _. ^s _T ^iz _o ^ed _di ^t _r ^h _e ^a _c ^t _tl ^Z _y ^I _a ^K _dd ^V _r ^/ _e ^D _ss ^E _th ^N _e ^V _ro ^c _l ^r _e ^os _o ^s _f ^-r _Z ^ea _I ^c _K ^ti _V ^ve _e ^C _p ^D _ito ⁸ _p ⁺ _e- ^T _sp ^c _e ^e _c ^ll _i ^s _fic ^pl _a ^a _n ^y _d ^a _Z ^p _I ^ro _K ^t _V ^ec _/ ^ti _D ^ve _E ^r _N ^o _V ^le 53

^{c mro me}

F_S ^ics

S^es- /_M ^wreea

R^rc

7^eti

6 ^iv

6-^{m C}

N^uD

Sⁿ

S_24B ^{i8z+ed T w citehlls a c inoc pkrtoaitle octfio enith aegra 3in ZstIK ZVIK iVmm inufneoctdioonm,in Ifannatr-/ p-e HpLtiAde-sB* (F0S70S2-N tSra2nAs1g3e3-n14i1c} B_68-75

4_{26-,435 a,n FdSS F/SMS} ^, _{(FSS/MR766-N /RM7R667-6N6-SN2AS475B426-435}

-_{84, FS)S/ oMrR 47 Z66IK-NVS/3D206E-2N15,V an cdro FsSs-Sr/eMactRiv7e66 p-eNpSti3d5e74s- 582}), followed by challenge with ZIKV FSS13025.

[ ^H00

m^L1

i^cA97

e^)-B]

(^{F* e}

i^{0g7.0 T}

1^2h

2^{c ter aann fo}

d^{slgloe Fnw}

i^gicing

.^{1 m2di rce}

)^esu

a ^wlt

n^{desre ar}

m ^{doicv w}

k^idit

(^eh

7^{d r}

m ^iens

i^ctpoec

e^{) ft}

v^{os.u tor ZI g F}

K^riog.

V^{u/p 1}

D^s2:a

E ^{m to}

N^{oVc 1k2f}

c ^{(r5, w}

o^{s mhiecree) v fsiv.e Z-wIKeeVk- poeldpt Iidfnear (-/5- (Fig.12e and Fig.12f). Peptide groups received s-reactive peptide (8 mice) in Materials and Methods corresponding peptide immunizations as described} _{3 days. CD} ^{. All groups were challenged R.O. with 1×104 FFU of ZIKV FSS13025 for} _{TNFα+ cells3+ in C aD r8e+pr CesDen4t4a+tiv CeD m6o2uLs-e T fro cmells m woecrke (F gaigte.d 12 aan)d an thde p pepertcideent (aFgiegs.1 o2fb I)FNγ+ and/or determined by ICS. The levels of infectious ZIKV in sera and brain groups were w Wehrietn eexyp treessts.ed as mean ± SEM. *, P < 0.05; **, P < 0.01; ***,s P we <re 0 m.0e0a1s.u Trewdo v-itaai FleFdA M. Dananta-} [00198] The results obtained are as follows:

[ ^{e0x0a1m99in]ed via O InCS da aynd 3 v airfatler tit veirrsal in c thhaelle snergae, an edpit borpae-specific CD8+ T cells in the spleen were} _{virus measurement was focu} ^{in assessed via focus-forming assay. Infectious} _{ZIKV infection, and the braisned ap inpe tahress teo t bweo a t missaujoesr t baercgaeuts oef v ZirIeKmVia in is b ao dthef fientianlg an fedat audrue of human settings. lt infection} ^{[ m00o2c0k0-]immu Anisze edxp mecicteed (,F nigo. s 1i2gan,if 1ic2acn,t a epitope-specific CD8+ T cell responses were detected in individual epitope- nd 12f). In comparison, a significant frequency of each immunodom specific CD8+ T cells was observed in mice immunized with the 3 ZIKV} _{(CD44+ CD} ⁱ ₆ ⁿ ₂ ^a _L ⁿ-^t ₎ ^e _C ^p _D ^ito ₈ ^p ₊ ^es _T ^{; th} _c ^e _ell ^p _s ^er _d ^ce _ir ⁿ _e ^t _c ^a _t ^g _e ^e _d ^{s o} _to ^{f I} _t ^F _h ^N _e ^γ- _F ^p _S ^r _S ^o-^d _N ^u _S ^c ₂ ⁱⁿ _A ^g ₁₃₃ ^c-^e ₁₄ ^l ₁ ^l _, ^{s a} _F ^m _SS ^o _/ ⁿ _M ^g _R ^{antigen experienced} _{FSS/MR766-NS4B426-435 epitopes were 0.86%±0.71%, 0.6 766-NS2B68-75, and (Fig.12c). In addition, the levels o 1±0.47%, and 1.63±0.76%, respectively these 3 ZIKV immunodominant epfit inofpeecstio wuesre vi sriugsni ifnica thnetly se lroaw aenrd th barnain th oofse m oicfe m imocmk-uinmizmeudn wiziethd}

54

^m

d_re ⁱ

e_a ^c

t_c ^e

e_tcitv ^(F

a_e ^ig

b_{l p} ^.

e_e:p ¹

T_t ²

h_i ^d _dee ^a _s ⁿ

p_, ^d

e _arcn ¹

e_tni ² _g ^e

t_e ⁾

a_n ^{. S}

g_{e e} ⁱ

s_x ^m

o_p ⁱ

f_e ^la _{r Fi} ^r _e ^ly

S_n ^,

S_c/e ⁱⁿ

M_d ^m

R _(C ^ic

7_6D ^e

6₄ ^t

-_N4 ^h+at

S_{4 C} ^w

B_D ^e

4₂₆ ^r

6_-2 ^e

4_3L ^i-m

5_{,) F C} ^m

S_SD ^un

/₈ ⁱ

M^+ze

R _T ^d

7_{6 c} ^w

6_e ⁱ

-_l ^t _lNs ^h

S _r ^t

2_e ^h _c ^{e 4 ZIKV/DENV cross-} A_{o7g5-n84i,z FinSgS/ 4M epRi7to6p6-eNsS w3e2r06e-} ^{201.58,4± an0d.39 F%SS,/ rMesRp7e6ct6i-vNS3574-582 specific cells were 1.52±0.42%, 3.27±1.91%, 0.66±0.24%, and} _{immunized wit} ^{ely (Fig.12f). Infectious ZIKV in both sera and brains of these mice} _{mock-immunizehd t mheice 4 (F ZiIgK.1V2/gD anEdN 1V2h c)r.oss-reactive peptides were significantly lower relative to} ^{[ t0h0e2031] ZIKV Ta ikmemnu tongoedthoemr,in thaese results demonstrate that both peptide immunization protocols (i.e., antigen-experi nt and the 4 ZIKV/DENV cross-reactive epitope cocktails) elicit} _{ZIKV titers in} ^en _t ^c _i ^e _s ^d _su ^, _e ^e _s ^p _. ⁱ _I ^t _m ^op _p ^e _o ^-s _r ^p _ta ^e _n ^c _t ⁱ _l ^f _y ^ic _{, t} ^C _he ^D _y ⁸ _i ⁺ _nd ^T _ica ^c _t ^e _e ^lls _th ^u _a ^p _t ^o _C ⁿ _D ^Z ₈₊ ^IK _T ^V _ce ^c _l ^h _ls ^all _r ^e _e ⁿ _c ^g _o ^e _gn ^a _i ⁿ _zi ^d _ng ^re _n ^d _o ^{uce infectious} _{immunodominant epitopes but also ZIKV/DENV cross-reactive epitopes t only ZIKV reduction in infectious ZIKV titers in vivo. can contribute to 5.6 CD8} ⁺ _{T cell depletion confirms epitope-specific CD8} ⁺ _{T cell-mediated protection}

[ ^p0

H^{r0202] To confirm the protective role of epitope-specific CD8+ T cells in ZIKV infection, the} L^es _A ^en _-B ^t _* ⁱⁿ ₀₇ ^v ₀ ^en ₂ ^t-^o _re ^r _s ^s _tr ^f _i ⁱ _c ^r _t ^s _e ^tl _d ^{y i} _e ^m _p ^m _ito ^u _p ⁿ _e ⁱ _s ^zed _an ^I _d ^fna ₅ ^r-/- _H ^H _L ^L _A ^A _-A ^{- B} _*0 ^* ₁ ⁰ ₀ ⁷ ₁ ⁰-² _re ^a _s ⁿ _tr ^d _ict ^H _ed ^LA _e-_p ^A _it ^{*0101 transgenic mice with 6} _{treated the peptide-immunized mice with anti-CD8 antibody to depleteo CpDes8,+ r Tes cpeelclst.ively; and then} ^{[ H00L2A03-B]*070 T2h trea fnoslgloewniicng m riecseu wltser are with respect to Fig.13a to Fig.13n, where five-week-old Ifnar-/- vs. mock+anti-CD e divided into four groups: mock+isotype antibody (Ab) (9 mice) immunized+anti-CD88 A Abb ( (1100 m miiccee)) (F vsig.. p 1e3pati tdoe- Fimig.m 1u3nei)z.e Pde+pitsiodtey-pimem Aubniz (e1d0 If mnairc-/e-) H vLs.A- pBe*p0tide-} _{transgenic mice were immunized with a cocktail of 6 peptides as described in Ma} ⁷⁰² _{Methods. Five-week-old Ifnar-/- HLA-A*0101 transgenic mice were di terials and mock+isotype Ab (8 mice) vs. mock+anti-CD8 Aeb) ( (F7ig m.i 1c3eh) v tso. vided into four groups: mice) vs. peptide-immunized+anti-CD8 Ab (7 mic F piegp.t 1id3en-)i.m Pmeputnidizee-dim+misoutnyipzeed A Ibfna (r8-} ^{/ M-a HteLrAial-sA a*n0d10 M1e ttrhaondsgenic mice were immunized with a cocktail of 5 peptides as described in} _{isotype control Ab an} ^s _d ^{. M} _an ^o _t ^c _i- ^k _m ^-i _o ^m _u ^m _se ^u _C ⁿⁱ _D ^ze ₈ ^d _A ^m _b ^ice ₃ ^a _d ⁿ _a ^d _ys ^p _a ^e _n ^p _d ^tid ₁ ^e- _d ^im _ay ^m _b ^u _e ⁿ _fo ^iz _r ^e _e ^d _Z ^m _I ⁱ _K ^ce _V ^w _c ^e _h ^r _a ^e _ll ⁱ _e ⁿ _n ^je _g ^c _e ^t _. ^e _A ^d _l ^I _l ^.P _gr ^. _o ^w _u ⁱ _p ^th _s

55

^{w Te}

w_e ^cr

r^ee

e^{ll c}

d^sh

e ^wal

t_e ^ele

r^rn

m^eg

i ^ge

n^ad

e^t _d ^{e Rd.O}

b ^a _y ^n.

I^{d w}

C ^tit

S^hh

.^{e 1}

T ^p×

h^e _e ^r1c0

l^e4

e^{n F}

v^t _e ^aF

l_s ^gUe _o ^{s o}

f ^of

i^{f Z}

n_f ^II

e^FK

c^NV

t_io ^γ _u ^{+ F}

s ^aSnS

Z^d1

I^/3

K^o0

V^{r25 TN foFrα 3+ d caeylsls. C inD m3+oc CkD a8n+d C pDep4t4id+e C gDro6u2pLs-} _{wtaeilreed e Mxparnens-sWedh aitsn meye taenst ±. SEM. *, P < 0.05; **, P < 0.01 i;n * t*i*s,su Pes < w 0e.0re01 m;*e*a*s*u,r Ped < vi 0a.0 F0F0A1;. T Dwaota-} [00204] The results obtained are as follows:

[^{im00m20u5n]ized m Asice e (xFpiegcs.te 1d3,a, a 1ntigen-experienced CD8+ T cell responses were absent in mock- 1 si3gjn).ifi Icnaf 3b, 13h and 13i) but present in peptide-immunized mice (Figs.13c and netclytio luoswe ZrIK thVan le thvoelsse i inn t mheoc ske-rimumm,un liivzeerd, a mnidce b (rIasionty opfe p peeppttiiddee-immunized mice were mock in Figs.13e-13g and Fig.13l). When CD8+ T cell-d -immunized vs. Isotype administered to mice, the majority of peptide epleting anti-CD8 antibody was} _{and infectious ZIKV leve} ^{-specific CD8+ T cells were absent (Fig.13d and 13k)} _{immunized mi ls in the serum, liver, and brain of CD8+ T cell-depleted peptide- s CigDn8if+ic Tant cleyll h-siugche}

f_{feicr w}

i _tehe

n_are

t_{n (A l teh}

n_{veetil-s sa}

C _inm

D_{8 ieso a}

p_teys

p_{pte in}

i_{d ceo a}

-_nn

i_mttri

m_{o-Clu aD}

n_n8

i_{tib aondtyib-toredayt-etdre pateepdtid me-oimckm-imunmizuendiz medice m thicaet, w beuret 13g, and Fig.13l). Collectiv zed vs. Isotype peptide-immunized in Figs.13e- peptide immunization was meelyd,ia ttheedse by re CsuDlt8s+ c Ton cfeilrlms.ed that protective immunity induced by ZIKV} 6. Discussion on example 2

[ ^{Z0I0K20V6]/DEN TVhe cr goosasl-srea ocftiv exea CmDpl8e+ 2 T w ceelrle ep tiotop deesfin theat sp aerecif riecsittryic atendd b ryol ceom ofm ZoIKV-specific and using the Ifnar-/- HLA-B*0702 and HLA-A*0101 transgenic mou n HLA molecules the value of using these mouse models to inv se models. Prior studies have shown responses. Therefore, in estigate DENV epitopes of relevance to human T cell} _{cell e} ^{a first step, HLA-B*0702-restricted and HLA- A*0101-restricted CD8+ T} _{transgpenitiocp mesice w ienrfeec itdeedn wtiiftihed eit thheart th wee Arefri rceacnog onriz Aesdian in lin Iefnagare-/ Z- HIKLA-B*0702 and HLA-A*0101 conserved in not only ZIKV FSS13025 and ZIKV M V. Most identified epitopes are SPH2015. The majority of HLA-B R766, but also in the Brazilian outbreak strain located in nonstructural proteins.*0 I7n02 c-o rnetsrtarsictt,ed few CDer8+ HL TA c-ell A e*p0i1to01p-erses itdreicntteifdied ep inito ZpIeKsV we arree}

56

ⁱ _E ^d

p_rL ^en

o_ItS ^ti

e_P ^fi

i_nO ^ed

E_T.) ⁽ _, ¹ _a ³ _nd ^H _t ^L _h ^A _e- _m ^A _a ^* _j ⁰ _o ¹ _r ⁰ _it ¹ _y ^-r _o ^e _f ^str _H ^ic _L ^te _A ^d _-A ^v _* ^s ₀ ^. ₁₀ ³ ₁ ⁷ _-re ^H _st ^L _ri ^A _ct-_e ^B _d ^*0 _Z ⁷ _I ⁰ _K ^2- _V ^res _e ^t _p ^ri _i ^c _to ^te _p ^d _e ^, _{s r} ^a _e ^s _sid ^id _e ^e _d ^nt _i ⁱ _n ^fie _t ^d _he ^v _s ⁱ _t ^a _ruc ^I _t ^F _u ^N _ra ^γ _l ^{[ b0}

D^e0

E^t2w0

N^e7

V^{e]n2-i ZnI T}

f^eKo

c^{V as}

t^{ed asne}

m^dss

i^{ce D th}

.^Ee

T^{N m}

h^eVre,ag

w ^Zni

e^ItKud

r^eVe

3^{/7D an}

I^Ed

F^{N f}

N^Vun

γ ^{E cc}

L^rtoio

I^sn

S^sP-arl

O^{ea q}

T^cu

-^tciavli

o^ety

n^{fi erp o}

m^{ifteo c}

d^preo

H^sss

L ^w-r

A^ee

-^ra

B^ecti

*^{0 nv}

7^ee

0^{x2t T}

- ^{id ceenlltif rieedspo unsisnegs epitopes tested in DENV2-infected mice and 14 peptides reactive with DENVrestricted ZIKV and/or ICS assays were identified, whereas none of the 13 HLA-A*0101-restrict 2 in ELISPOT w epeirteop cersos asn-d re thacetiirv DeE wNithV D2 vEaNriaVn2t.s T hhaveese 01-84 a HmLinAo-B a*c0id70 s2u-brsetsittruitcitoends Z (sIKV/DEeNdV ZI cKroVss- erpeiatoctpivees} _{is noteworthy that 5 DENV2 variants (RPTFAAGLLL, APTRV} ^e _V ^e _A ^T _A ^a _E ^bl _M ^e _, ^{3 i} _K ⁿ _P ^{next page). It} _{TPRMCTREEF, LPAIVREAI) had been identified as HLA-B*0702-restricted epitopReWsLDARI, mouse models and humans22. Three ZIKV peptides and the corresponding DENV2 varian int both the same C-terminal amino acid residue, suggesting these ZIKV peptides are probabl s have epitopes as well. Of these 5 peptides, FSS/MR766-NS3206-215 (APTRVVAAEM) is conse y human many Flaviviruses, including ZIKV, four DENV serotypes, West Nile Virus, Japan rved among Virus, Usutu Virus, Murray Valley Encephalitis Virus, and Kunjin Virus. ese Encephalitis}

57

^{F FSSSS//MMV2R P}

R^N776p

6^S6tid

6^{2ANNSS2A SE Table 3}

N^{Q 389O ID RPA Sq C ti Rf}

D^{EN 1 RPTFLALVASGFLILFL 20% (22)}

F ^{DENV2NS33 47 AP}

F^{S R766S6 5 RVVAAEM 100% (22) DSESSN//MMV2RN763NS T}

N^{S34B 822 K}

D ^{KPPRRWWMLDDAARRIV 78% (22) F 0 R NPTGQAFCIKV FSENV2NS4B 683 L}

T^{GSFRRCTTITKT FSSN/MVR2 RPT VWL 6808%}

D ^%

D^{E W} D^SES E^{SN/NNSS55 6844 V VP}

/^{MVR27N66S5NS5 6825 TPPPER}

A^RIVMC I^V _V ^CRT R^TEK E^{RAEEIE 7}

I^EFF ₈₉ ⁰ _% ^{% (22)} A _{(22) D M F FSERN SSN7} ^M _V ^R _2N ^N _S ^S ₃ ^{3 4} ₈ ⁶ ₆ ^L _LP

/_{6V62NNS2SA2A 3887 V VVTLMIITLGGNGMFSSFM 40%}

D_{EN/MMVR2NNSS22A 0 T}

S_{MVR2NNSS33A 4 R BB 580 T 9 GP}

%

D_{SESNNVV2 99 G TSPKRE 3}

F ₅

N _{APPNME MKPS PL YVM VM WTL TMHL HT NAA SSSSSL 0%}

D_{ES 88 AAI 90}

F_ESN/

2_NN

N_SS

S₄₄

5 _{601 S}

9_{1 TKPPTGQQRTGFGWSANSTSLTTIAA 36%}

D_/MRNS5

F_{SS/M 2 50%}

D_{ENVR2EE 392 K KPPTVSASSM}

T_{LDDFIEL FSS/MRNS EL 75%}

_{DENV2NS33 5933 R RVIYCSSDDPHLAAALL 56%}

_in ^{ZIKV pptid i bld piti dtidi bth IFNγELISPOTd ICS ys b} _{DENV2-infected mice.} c Underlined amino acid residues are conserved between ZIKV peptide and DENV2 variant. % shared amino acids between ZIKV and DENV2. 58

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l^{een C in}

g^{eDfe8ction on an+d T cells dominated in the later CD8+ T cell response to ZIKV. Moreover, both ZIKV-specific th anen Z d} _of ^IK _bo ^V _t ^/ _h ^D _m ^E _a ^N _gn ^V _itu ^cr _d ^o _e ^ss _a-_n ^re _d ^ac _b ^t _r ^iv _ea ^e _d ^C _th ^D _t ⁸ _h ⁺ _a ^T _n ^c _re ^e _s ^ll _p ^r _o ^e _n ^sp _se ^o _s ⁿ _i ^s _n ^es _p ⁱⁿ _rim ^D _a ^E _ry ^N _Z ^V _I ² _K ^-i _V ^mm _in ^u _f ⁿ _ec ^e _ti ^m _on ^ic _. ^e _T ^w _h ^e _e ^r _s ^e _e ^w _re ^e _s ^a _u ^k _l ^e _t ^r _s ⁱ _i ⁿ _nd ^terms _{that prior DENV immunity can affect both the specificity and magnitude of CD8+ T cell icate t noat ZurIaKl rVe.in Tfehcistio pnhse inno hmuemnoanns, w imasp alylsinog o tbhsaetr ZveIKdV du inrifnegct hioente irnot DypEicN DV-EimNmVun inefe pcetoion in m reiscpeo annsde similarly as heterotypic DENV infection. ple may behave} ^{[ t0h0e2 f0e9t]al neu Irno hnualm staenms, congenital microcephaly and additional birth defects result from infection of mouse neural cells. The data in the present disclosure show that ZIKV can also infect adult addition to m pinriomgeizninitgor v cireellms,ia r,es ZulItKinVg in vac recdinuece cdan cdeildla ptreoslif sehroautilodn p arnodte ccetll fr doemath b.r Taihner ienffoerce, in (ZIKV encephalitis). In the present study, 6 immunodominant HLA-B*0702-restricte tion w EeLrIeSP sOeleTct aendd f IoCrS p aespsatiydse fo imr bmouthni ZzaItKioVn F bSeSca1u3s0e25 (i a)n tdhe ZsIeK pVep MtiRde7s66 w ienrfeec ptioosnit;i (v d epitopes iei) a in both IFNγ were conserved in both ZIKV FSS13025 and ZIKV SPH2015 while five peptides wll six peptides by ZIKV MR766; and (iii) four peptides were cross-reactive with DENV2 as co ere also shared ELISPOT and ICS assays. These peptides were then divided into two groups fnofirmed by IFNγ} _{ZIKV peptide group (two ZIKV-specific peptides and one ZIKV/DENV cross-} ^{r immunization:} _{and ZIKV/DENV cross-reactive peptide group (four ZIKV/DENV cross-re reactive peptide) expected, both ZIKV peptide and ZIKV/DENV cross-reactive peptide iactive peptides). As significant CD8+ T cell responses and reduced infectious ZIKV leve mmunization elicited revealing the potential of these epitopes for preventing ZIKV ls in mouse sera and brains, assays in mice immunized with 6 HLA-B*0702-restrict encephalitis. CD8+ T cell depletion epitopes further confirmed epitope-specific CD ed epitopes or 5 HLA-A*0101-restricted reactive peptides, combined with pr 8+ T cell-mediated protection. The finding of cross- raise the possibility of otection against ZIKV seen in the data of the present disclosure, of ZIKV a developing a single vaccine that can confer protection against multiple strains inactivatedn ZdI DKEVN sVtr.a Ainlstho purogvhid reescen ptro stteucdtiieosn h,a avneti dbeomdyo-ndsetpraetnedde tnhtat en vahcacninceamtioennt w (itAhD suEb)u mnaity an bde}

59

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p^{h0e2s1e0] viruse Asm sohnarge th ae h pigahtho lgenic humman flaviviruses, ZIKV is most closely related to DENV and exreasmenptle in imvepnlitcoartse’ a rec pernotte DctiEe}

v^Nve

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s^-/r

p^-d

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t^{eicgel4y0,, s reimsuillatsr o tof t thhies research, no study to date has provided direct evidence suppor several decades of during DENV infection. Instead, consistent wit ting a pathogenic role for T cells recent studies have beg h the present inventors’ mouse findings21, 37-40, 48-50,} _particu

d _tlar ^{un to support a protective role for DENV-specific T cells in humans. In} _{ass , the magnitude and breadth of DENV-specific CD8+ and CD4+ T cell responses are asisseo}

o_ac

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o _{d aA}

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)_{01 c}

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V_{cittihve slyu,sc reepptribeisleitnyt v Ds.E rNesVist-apnrcoetec totiv deen (gi.ue.e, in the present disclosure of -susceptible alleles27, 29, 51. The identification than HLA-A*01 a greater number of ZIKV-derived HLA-B*0702-restricted epitopes B* 01-restricted epitopes and identification of ZIKV/DENV cross-reactive HLA- CD087+02 T-r ceesltlr ricetsepdon bsuet to no ZtIK HVLA m-aAy* b0e10 H1L-rAes-tlirnickteedd. epitopes suggest that, similar to DENV, the} ^{[ a0n0d21 H1]LA-A I*n01 su0m1m epary, the ZIKV T cell immunity data in example 2 has identified HLA-B*0702 DENV se itopes which are conserved between ZIKV lineages and cross-reactive with a existing DrEoNtypVe. im Thmeu HniLtyA m troadnusglaetnesic Z mIoKuVse-s mpeocdifeicl r CesDul8t+s i Tn t cheell p rreesspeonnts deis dcelovseulre show that pre-} _{ZIKV-specific and ZIKV/DENV cross-reactive CD8} ⁺ _{T cells play a p} ^{opment, and that} _{The results on T cell mediated protection in mice in th rotective role against ZIKV. human infection, as the same mouse model h e present example are likely relevant to independent observations. These r as been validated in DENV infection by several the induction of both ZIKV-specifiecsu anltds s ZuIgKgeVs/tD thEatN ZVIK crVoss v-arcecaicntaivtieon CD ap8p+r Toa ccehlels re sshpoounlsdes i.nclude} Example 3 60

[00212] Example 3 refers to the results shown in Fig.15A to Fig.24B.

[00213] Example 3 can be summarized as follows:

[

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Z^{n]eIK pVre Ags}

i^{nna Z}

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i^{aaltdeemic areas, cases of ZIKV infection in DENV- D hEoNwV pri aonrd D ZEINKVV im inmfeucntiiotyn a mffeocdtesls maternal and Fetuses in ZIKV-infected DENV-immune dams were of were used. o} _o ^b _f ^s _Z ^er _I ^v _K ^e _V ^d- ⁱⁿ _inf ⁿ _e ^o _c ⁿ _te-_d ^im _D ^m _E ^un _N ^e _V ^{d normal size, whereas fetal demise was}

-^a _im ^m _m ^s. _u ^M _n ^o _e ^re _th ^o _a ^v _n ^er, _n ^l _o ^e _n ^ss _-i ^Z _m ^I _m ^K _u ^V _ne ^RN _da ^A _m ^w _s. ^as _D ^{detected in the placenta and fetuses} _{expanded in the maternal spleen and decidua of ZIKV-infectedEN daVms c,ro anssd-r tehaecitriv deep ClDet8io} ⁺ _{n T led cel tls increased ZIKV infection in the placenta and fetus, and feta o immunity can protect agains l demise. Thus, in mice, prior DENV t DhEisN crVo-sesn-pdreomteicct aiorena.s T ahnids f tihnt}

e_{d Zi dnIK}

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p_{sing pregnancy, and CD8+ T cells are necessary for tim foarl u ZnIdKerVst vanacdcininges th.e natural history of ZIKV in} 7. Materials & Methods for Example 3

7.1 Ethics statement

[ _U ⁰⁰ _se ²¹ _C ^{5] This study was performed following the guidelines of the Instituti}

t_{oiomnm, ainttdee al uln edffeorrt psro wteorceo ml #ade A tPo0 m28i-nSimS1} ^{onal Animal Care and} _{inhala i-z0e6 p1a5i.n I.noculations were performed under isoflurane} 7.2 Viruses

[ ^{R0e0f2e1r6e]nce C ZeInKteVr A fosrian Em lineeraggineg st Vraiirnus FSS13025 (Cambodia, 2010) was obtained from the World DENV2 es and Arboviruses (Galveston, TX). The mouse-adapted} _{et al., 200} ^s ₉ ^t ₎ ^r _. ^a _Z ⁱⁿ _IK ^S2 _V ²¹ _an ^is _d ^a _D ^b _E ^io _N ^lo _V ^g ₂ ^ic _w ^al _e ^c _r ^l _e ^o _p ⁿ _r ^e _o ^d _p ^{erived from DENV2 D2S10 (Shresta et al., 2006; Yauch} _{measured by focus forming assay (FFA) witahga btaebdy in ha Cm6/st3e6r A kidedneesy albopictus cells, and viral titers were al., 2017) or by qRT-PCR (Lanciotti et al., 2008). (BHK)-21 cells (Elong Ngono et} 7.3 Mouse experiments and virus infections 61

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T ^{inlefse,c #tio0n00 w66e4re) a ulssoed w:e (1re)} _{receptor (Ifnar1) signaling in WT females was trans males. The type I IFN blocking monoclonal Ab (mAb), as described below. iently blocked via treatment with an Ifnar1-} ^{[ 10x0120138] FFU T oof e DstEabNliVsh2 D viEaN I.VP. im rmunity, 5-week-old Ifnar1-/- female mice were inoculated with DENV2 via R.O. rou oute or WT female mice were inoculated with 1x104 FFU of determined by the presteen.c Aet o 8f a to va 1g0ina wle pelkusg o inf a thgee, m feomrnailneg, m ainced w emerbery monaitced d.ev Perleognancy was estimated as gestational age E0.5. Pregnant female mice were pment was detection. At E7.5, the females were separated from male mice after plug} _{10% FBS or mock-infe} ^{inoculated with 1x104 FFU of ZIKV in 200 µL of PBS with} _{a (st cted with 200 µL of PBS with 10% FBS via R.O. route. Mice were sacrificed er Eu1m0,.5 br oarin E a1n4d.5 sp dleeepne)n,d pinlagce onnta t ahned e fxeptaelri tmisseunetal design. Viral burden in the maternal tissues and size were measured using an analytical balance (sC (ahteaalodg a nnudm bboedry:) S9 w4e7r9e0 qAu,a Fnitsihfieerd. Sc Fieetnutsifi weight Digital Caliper (Model number: 700-113-10, Mitutoyo), respectively. c) and} 7.4 Ifnar1-blocking and T cell-depleting antibodies

[ ^{m00ic2e19 w]ere t Arelalte andt wibiothdie 2s m (Agb Ifsn)a wr1e-rbelo pcukricnhga mseAdb fr MomAR B1i-o5XAC3e vllia (U I.SPA.). For Ifnar1 blocking, WT with ZIKV or DENV2 (WT mice only). To deplet route 1 day before infection anti-mouse CD8 mAb (300 µg/ e CD8+ T cells, mice were injected with either} _{µg/mouse, rat IgG2b, clone LTF-2} ^m ₎ ^o _v ^u _ia ^se _R ^, _.O ^ra _. ^t _ro ^I _u ^g _t ^G _e ² _o ^b _n ^, _d ^c _a ^lo _ys ^ne _{3 a} ² _n ^.4 _d ³ ₁ ⁾ _p ^o _r ^r _ior ^is _t ^o _o ^ty _i ^p _n ^e _fec ^c _t ^o _io ⁿ _n ^tro _an ^l _d ^m _e ^A _v ^{b (300} _{days after ZIKV infection. The same protocol was used for CD4+ T c ery two clone GK1.5) or both CD4+ and CD8+ T cell dep ell depletion (300 µg/mouse, depletion in tissues after treatment using flow cytomleettioryn.. All mice were monitored for CD8+ T cell} 7.5 qRT-PCR analysis of viral burdens

62

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N^{Affer for 3 min using Tissue lyserTM II (QIAGENTM) and ea frsoyTmM t Missiunei s Kaimtp (tleisss and serum obtained from ZIKV-} ( _{RsNerAum,) re (aQl-ItAimGeE qNR)T, r-ePsCpRect wivaesly p. Aerlflo RrmNeAd s uamsiples were stored at} ^u -^e ₈ ^s ₀ ⁾ _oC ^an _. ^d _Fo ^V _r ⁱ _q ^ra _u ^l _an ^R _t ^N _ifi ^A _cat ^M _ion ⁱⁿ _o ^iT _f ^M _v ^K _ira ^it _{l BioScienc ng the qScript One-StepTM qRT-PCR Kit (Quanta publishede psr)im ate trh see Ct [F7X4]9 w6a Tso uuscehdTM to r deaelt-etcimt ZeI PKCVR R dNetAec:tion system (Bio-Rad CFX Manager 3.1). A} Sequence SEQ ID NO Fd 5’TTGGTCATGATACTGCTGATTGC3’ 94 R 5CCTTCCACAAAGTCCCTATTGC3 95 Pb 56FAMCGGCATACAGCATCAGGTGCATAGGAGT Q3 96

^{[ 50 , 95C f 15 i, fll d by} R⁰⁰²

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w ¹ _a ⁵ _s ^s _d ^ec _ete ^a _r ⁿ _m ^d _in ⁶⁰ _ed ^C _b ^f _a ^o _s ^r _ed ¹⁵ _o ^s _n ^ec _an ^{and a final extension of 72C for 30 min. Viral} d_{ilutions of an in vitro transcribed RNA based on ZIKV in stterraninal F sStSan13d0a2rd5. curve composed of serial} 7.6 Peptide Synthesis

[ ^{p0e0p2t2id2]es use Pdep fotirde fslo wwer ceyt poumrcehtrayse wde frreom syn Styhnetshizeetdic a Bniomolecules (A&A Labs). The 9- and 10-mer} 9_{5%nt pituiersity to. P aevpotii} ^{d purified by reverse-phase HPLC up to≥} q_{ua dde fsre wezeere-th staowre dd at -20°C after being dissolved in DMSO and aliquoted into small been published (Wen et al., 2017aa).mage. The sequence and characteristics of all peptides used have} 7.7 Cell isolation and flow cytometric analyses

[ _b ⁰ _e ⁰ _f ² _o ² _r ³ _e ^] _proce ^F _s ^o _si ^r _ng ^ea _a ^c _s ^h _fo ^p _l ^r _lo ^e _w ^gn _s ^a _. ⁿ _B ^t _ri ^m _ef ^o _ly ^u _, ^s _p ^e _l ^, _ac ^p _e ^la _n ^c _t ^e _a ⁿ _s ^t _w ^as _ith ^w _o ^e _u ^r _t ^e _s ^h _ep ^ar _a ^v _ra ^e _t ^s _i ^t _o ^e _n ^d _o ⁱⁿ _{f m} ¹ _a ⁰ _t ^% _ern ^F _a ^B _{l d} ^S _e ^/ _c ^R _id ^P _u ^M _a ^I _w ^a _e ⁿ _re ^d _c ^p _u ^o _t ^o _in ^le _to ^d 63

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d ^{m 1ec mhagn/imcalll oyf d tiyspsoec Iia ctoeldla,g feilntearseed (W thorortuhgihng otovner) f ao 7r06-0µm mi cne allt s 3t7ra°iCn.er A aftnedr} _{p sulascpeedns uionnde} ^{in 44% PercollTM (GE Healthcare). Another layer of 67% Percoll was} w_{rnaesa itshol bateefdor bee ctweneternifu thgeat dioifnfe arten 3t76 de xns giti aets r oofo Pmer tceomllp aerature for 20 min. The cell layer Cells were counted after erythrocyte lysis using a cell counter (Vi-cenllTdM w XaRsh 2e.d04 th, Breeeck timmaens w Ciothult PeBr)S..} ^{[w0e0l2l2 U4]-botto Fmor p IlCatSe,s i.s Colealtlesd w seprleen soticmytuelsat ferdom wi athll m 1i µcge w ofer iend pilvaitdeuda als Z 2IxK1V06 p spelpetniocytes/well in 96- presence of Brefeldin A (GolgiPlug; BD Biosciences) during t des for 6 h in the [41]. Cells from placenta/maternal decid he last 4 h, as previously described peptides following the same conditionsua as w seprelen polactyetdes a.n Pdo sstiitmivuela cteodnt wroitlhs a us minigxtu are ce ollf a stllim 5u ZlaItKioV cocktail (commercial PMA-Ionomycin-500X, eBiosciences) and negative co n} _{w 45e5re U aVdd (eIdnv initr eoagcehn) p ilnate P.B CSe.ll Asl wlere washed after stimulation and labeled w} ⁿ _i ^t _th ^ro _v ^ls _ia ⁽ _b ¹ _i ⁰ _li ^% _ty ^F _d ^B _ye ^S/ _ef ^R _lu ^P _o ^M _r ^TIM) _{anti-CD8 B cells were stained with anti-CD3 PerCpCyTM 5.5 (Clone 145-2C11), Mel-14), folVlo5w1e0d (c blyon fiexa 5t3io-6n7) a,n adnt pi-eCrmDe4a4bi BliVza7ti8o5n (c ulsoinnge I thMe7 B),D an Cti-yCtoDfi62L APC eFluor 780 (clone staining with a combination of anti–IFNg FITC (clo x/CytopermTM kit and then (clone MP6-XT22) and granzyme B PE-Cy7 ne XMG 1.2), anti-TNF AlexaTM Fluor 700 Fortessa (BD Biosciences) and analyzed using FTMlow (cJlooTnMe s NofGtwZaBre). X S 1am0.0p.l7es (T wreeere St aacrq).uired on LSR-} 7.8 Statistical analysis

[ ^{I0C0S225 a]nd vir Aalll b duatrade wner deat aan,al ayz tewdo w-tiatihled Pri MsmanTMn-W sohftiwtnaerye, t veesrtsion 7.0 (GraphPad Software). For morphological measurements, data were compar was used. For viral burden and} _{comparison test. Percentages of inf} ^{ed by one-way ANOVA with Tukey’s multiple} _{two-sided Fisher’s exact test. All daetcati woner ien e pxlparceesnstead w aisth m deeacnid ±ua S aEnMd, fe atnadl t piss <u0e.s05 we wraes a cssoensssed via as a significant difference. idered} 8. Results

8 _b ^. _u ¹ _{rden in Ifn} ^D _ar ^E _1- ^N _/- ^V _da ² _m ^-e _s ^{licited CD8+ T cells prevent fetal growth restriction and control ZIKV} 64

^[p0

2 _fo ^0r0o2 l^1t2 l_o ^7e6 w^{ac] It has been recently demonstrated that DENV-elicited CD8+ T cells mediated cross- )t.io Pnrev agioauinsslyt, s fuebtasle gqruoewntth Z rIeKstVric itniofnec atinodn d inem aidseul hta mveale be aennd o fbesmeravleed If innar I1fn-/a-r m1-/ic- pere (Wgneannt e mt aicl.e,} _{o utfil pizreioing ZIKV infection (Miner et al., 2016; Yockey et al., 2016). To begin to evaluate the influence dr th DeEiNV immunity on subsequent ZIKV infection during pregnancy, the present inventors with DENVr2 p sutbraliisnh Sed22 m1o fodrel 3 o0f d saeyqsu perniotiral to D ZEINKVV a cnhdal ZleInKgeV (W infeenct eiotn al. i,n 2 w01h7ica)h. mice were primed} ^{[ D00}

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n^{s 15A to Fig.15D, where non-immune and toi-tCypDe4 c+oCntrol Ab (Isotype), anti-CD4 Ab retro-orbital route (R.O.) at embryonic day 7.5 (E7.5) with 103 FFDU8 o)f a ZndIK were inoculated via a FBS-PBS as Mock. To generate DENV2-immune mice, mice were inoculatedV via F aSnS1 in3t0r2ap5e or 10%} _{route (I.P.) with 104 FFU of DENV2 strain S221 for 30 days prior to mating. Fetal body weig} ^r _h ^it _t ^o ₍ ⁿ _F ^eal _{15Al alnodws F:ig.15C) and size (Fig.15B and Fig.15D ig. as fo ) were measured at E14.5. The populations were}

_{3 n fetuses Feature}

3_{84 f ftt f f 54pptt th Ni Mk} 4^{3 ft f 5pt} _{th Ni ZIKV+ityp} 1^{5 ft f 3p th Ni ZIKV+AtiCD8}

2_{2 ft f 3p} ^t _t ^t _t ^h _h ^{DENV2i Mk 25 ft f 3} _{DENV2i ZIKV+ityp} 2^{6 ft f 4pptt tthh D NENiV2i ZIKZVI+KAV+tiACDtiCD8}

3_{6 ft f 4pt th Ni ZIKV+Ati} ^{4 35 ft f 4pt th DENV2} _{CD4+CD8 38 ft f 5pt th DENV2} ⁱ _i ^Z _Z ^I _I ^K _K ^V _V ⁺ ₊ ^A _A ^t _t ⁱ _i ^C _C ^D _D ⁴ _4+CD8

65

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m_b ^]

u_sletripve ^W

l_{ed co D} ^igh

m_apt ^t _a ^an

a_{r w} ^d

i_soer ^s

n_e ^ize

s _{p wo} ^w

a_o ^e

s_{l ue} ^re _d ^d

s_{ed f} ^e _r ^t

,_o ^e

a_m ^rm

n_{d t} ⁱ

d_w ^ne

a_o ^d

t_{a i} ⁱ

a_n ⁿ

r_d ^d

e_e ^iv

e_p ⁱ

x_e ^d

p_n ^u

r_d ^a

e_e ^ll

s_n ^y

s_et ^o

d _e ⁿ

a_x ^{the residual placenta if fetal resorption} s_{pe mriemanen +ts S.E TMuk.e *y*’*s* op<ne0-.w00a0y1 A.NOVA} [00229] The results obtained are as follows:

[ ^{f0o0r2m3i0n]g unit Nsa (FïvFeU a)n odf Z DIEKNVV F-SimS1m30u2n5e (2 If0n1a0r1 C-/-am prbegondaiannt is moilcaet were inoculated with 104 focus sacrificed 7 days later (E14.5). In the non-i e) on embryonic day 7.5 (E7.5) and i Fnifge.c 1ti5oBn). in D aelclr meaisceed r fe mmune group, fetal resorption was observed after ZIKV- egtaarld wleesisgh otf ( tFriega.tm 15eAn)t a wnidth si azne i (sFoitgy.p 1e5B co)n attro El1 o4r.5 an wtei-rCeD co8n Asib (Fig.15A and} _{in the ZIKV-infected non-immune group. Remarkably, DENV-i} ^{stently observed} _{control Ab had normal fetuses that were similar mmune dams treated with isotype resorption was observed in DE in size to uninfected, naive control dams. Fetal and Fig.15B, a NV2-immune mice only in the anti-CD8 Ab-treated group (Fig.15A r imesmorupntieo-nme inddiautcn}

e_eddd

p_{r b F}

o_yig

t_{e Z.}

c_{tI 2}

i_K1A

o_nV).

a _{ign T}

a_fieh

n_ce

s_ts

t_ioe

Z_{n re}

I_{K dsuu}

V_rlit

i_ns

n_{g inic}

I_{f pd}

n_arer1ga

-_nte

/_{-a pn t}

r_ch

e_ya

g_,t

n _{a E}

a_{n p}

n_drio

t _{m CrD D}

i_{ce8.+N TV ce ilmlsm counntirtiybu ptreev toen DtsE fNetVal}

^{[ r0e0s2p3o1n]se (Sw Aasin C eDt a4l+., 2 T01 c2e)ll, t hheelp pr mesaeynt b ienv reenqtuoirrsed ex faomrin deedve tlhoepirm reont of an optimal CD8+ T cell protection against ZIKV during pregnancy. Non-imm le in DENV immune-mediated depleted of CD4+ T cells or both CD4+ and CD8+ T ce une and DENV2-immune dams were or anti-CD4 plus anti-CD8 Abs lls via treatment with cell-depleting anti-CD4 immune groups treated with ant.i F-CetDus4e As ubn adloerngeo oinrg b roetshor apnttioi-nCD we4re an sdee annt ini-C ZDIK8V A-bchsa (lFleign.g 1ed5C, n aon-} _{Fig.15D). However, with prior DENV2 immunity, an intermediate phenotype w} ^nd _{fetuses was found in mice treated with anti-CD4 Ab alone as com ith 47% of viable in the group treated with both anti-CD4 and a pared with nearly 100% resorption These results suggest that cro nti-CD8 Abs (Fig.15C and Fig.15D, and Fig.24B). roles, respectively, in mediatingss D-rEeaNctViv iem CmDu4n+e a pnrodte CcDtio8n+ a Tga cinesllts Z hIaKveV- siunbdourcdeidna fteeta aln ddam daogme.inant} ^[ _b ⁰ _u ⁰ _r ² _d ³ _e ² _n ^] _{in m} ^T _at ^h _e ^e _rn ^p _a ^r _l ^e _t ^s _is ^e _s ⁿ _u ^t _e ⁱ _s ⁿ _s ^v _e ^e _v ⁿ _e ^t _n ^or _d ^s _a ⁿ _ys ^ex _a ^t _ft ^d _e ^e _r ^t _i ^e _n ^r _o ^m _c ⁱ _u ⁿ _l ^e _a ^d _tio ^th _n ^e _at ^im _E ^p ₁ ^a ₄ ^c _.5 ^t _. ^{of prior DENV immunity on ZIKV}

66

^[

( ^{D00 1AE2 0%nN33}

t^{i-V] FC2}

B^D-i

S^-8m

P^{)m Th B wue Sern following results are with respect to Fig.22A to Fig.22L, where non-immune and aee}

s ^{i Mn WoT}

o^{ccukl d}

.^aatm

A^eds

l^{l m R th.iOat were administered isotype control Ab (Isotype) or anti-CD8 Ab ce. w ate erem ibnrjyeoctneidc I d.aPy. w 7.5 (E7.5) with 104 FFU of ZIKV FSS13025 or t} _D ^o _E ^Z _N ^IK _V2 ^V _st ^c _r ^h _a ^a _in ^lle _S ⁿ ₂ ^g ₂ ^e ₁ ^{. D} _fo ^E _r ^N ₃₀ ^V _d ² _a-_y ^im _{s p} ^m _r ^u _io ⁿ _r ^e _to ^m _m ^ice _ati ^w _n ^e _g ^r _. ^e _Fi ^g _g ^e _. ^nit

2^eh

2^r _A ^{a Itfar}

,^end _fet ^a1

u^f- s^tbe _w ^rlo

e ^Rck

i_g ^{.Oing Ab on E6.5, one day prior} h_t ^. _a ⁱ _n ⁿ _d ^fe ₍ ^c _F ^ti _i ^o _g ⁿ _.2 ^w _2B ^{ith 104 FFU of} _{were recorded. Fig.22C to Fig.22H, ZIK ) size at E14.5 collec V RNA levels were measured by qRT-PCR of tissues at E14te.5d. f Friogm. dams (serum, brain, and spleen), placentas with decidua, and fetuses (head and body) and fetal bodie 2s2 aIt t Eo1 F4i.g5. w 22eKre, c paelrccuelantteadg.e Tsh oef Z poIKpuVlat iniofnecst wioenre in as pl faoclelonwtass: with decidua, fetal heads, n fetuses Feature}

34 ft f 6 p t th N i Mk

35 ft f 5 p t th N i ZIKV

31 ft f 4 p t th DENV2i M k

43 ft f 5 p t th DENV2i ZIKV+ityp

39 ft f 4 p t th DENV2i ZIKV+AtiCD8 ^[

a^{0r0e23 in4]dicated T atbolve eabch bafr. th Da ftat wlered p pololedt flromit twobt inidedpe fndent exhpe drime ints. Dhat garoup} _{expressed as meanp + SEM. *p<0.05, **p<0.01, ***p< ukey’s one-w} ^a _a ^re _{AN y exacOtV teAst w foars u msuedlti 0.001, ****p<0.0001. T}

f_{oler F coigm.2p2aIri tsoon Fsig w.2a2sK u.sed for Fig.22A to Fig.22H, while two-sided Fisher’s} [00235] The results obtained are as follows:

[ ^s0e0r2u3m6]

u_n ^, _it ^b _y ^ra _re ^{in In}

s_u ^, _lt ^{a a}

e^nll

d^{d ca}

i_n ^sspe

d^ls

e^e,

c^e _r ^{n dam}

e_as ^c _e ^{os re}

d^{m t}

v^p _ir ^aa

a^rtee

l^dd

R_N ^{w w C}

A^{iitthh a}

l_ev ^t _e ^hn

l^oti

s^s- c^eD

o_m ^tr8

p^{ea A}

a^t _r ^eb

e^{d h}

d _w ^wad

iⁱ _t ^t _h ^{h inc}

t_h ^isr

e^oe

n^tays

o^pe

n^{ed Z}

-_im ^coI

m^nK

u^tVr _n ^{o RNA levels in the} _{imm e} ^l _g ^A _ro ^b _u ^, _p ^a ₍ ⁿ _F ^d _ig ^D ₂₂ ^E _A ^N _t ^V _o

67

^F

A ^isiog

b^t.

-^{y 2}

t^rp2

e^eC

a^{t c)}

e^o.

d^{n Itn}

g^{rro c}

o^lo

u ^Antr

p^bs-a

a^tlrs

s^et,

o^{at d}

r^ea

e^dm

v^{e as}

a^{nli t}

e^mre

d^aa

t^lt

h^sed

a ^{(tF w}

a^igi

n^.th

t ^{22 aDnt tio-C FDig4.2 A2bF) h.a Cdo smimpialarirso mna otefrn anatli- tCissDu4e v veirraslu bsu arndtie-nCsD a8s l gervoeulsp i tnre mateadter wniathl t aisnstui-eCs i-CD8 but not anti-CD4 Ab treatment affected ZIKV RNA}

D ^{(F8ig A.b 22 aGlon teo v Ferigs.us 22 thI)a.t F trineaatlleyd, w noith di bffoetrhen acneti- wCaDs4 ob ansedrv aendti- bCeDtw8e Aen the} _{the brain and spleen of both non-immune and DENV-immune mice and serum of non} ^{bs in} _{m boicthe, a wnthie-CreDas4 a an hdigh anerti- ZCIDK8V A RbNsA tha bnur inde tnho wseas tr oebasteedrve wdith in a DntEi-CNDV8-im Ambune mice adm-iinmismteurnede 22L). Collectively, these results show a key role for CD8+ alone (Fig.22J to Fig. for CD4+ T cells, in DENV immune-mediate T cells, with a more limited requirement tissues from Ifnar1-/- mice. d cross-protection of ZIKV infection in maternal} ⁸ _C ^. _D ² _{8+ T cells} ^{DENV2 immunity prevents fetal growth restriction in Ifnar1 mAb-treated WT mice via [ p0u0b23li7sh]ed m Todoel co onff ZirImKV an vder etixctaeln tdran thsemi fsinsidoinng ins W repTorted so far, the present inventors utilized a a flla.,vi 2v0ir1u6s)e.s P tore rtreepalticmateent in w WithT m thiece If mice with transient Ifnar1 blockade (Miner et wniathr1o-ubtlo scigkninifgica Anbtly M imApRa1c-t5inAg3 C (DSh8e+e Tha cnell et di affl.e,re 2n0t0ia6t)io anllo inwts effector and memory cells (Pinto et al., 2011). WT C57BL/6 female mice were admi o} _{Ifnar1 Ab one day before infection with DENV2, as DENV cannot inhi} ^{nistered anti-} _{production and signaling in mouse cells, unlike in human ce bit type I interferon 2017). Thirty days after DENV2 priming, mice lls (Aguirre and Fernandez-Sesma, DENV2-immune and non-immun were mated with male sires, followed by treatment of on E7.5. Seven days lat e WT dams with anti-Ifnar1 Ab one day prior to ZIKV challenge maternal and fetal tissueser w,e arte d haayrv Ees1t4e.d5., fetal weight, size, and characteristics were recorded, and} ^{[ W00T23 d8a]ms w Terhee tr foealltoewdi wngith re asnutlti-s are with respect to Fig.16A to Fig.16D, where DENV2-immune 15A to Fig.15K. Mice Ifnar1 mAb and challenged with ZIKV at E7.5 as described in Fig} _{15A to} ^{were administered isotype control or anti-CD8 Ab, also as described in Fig} _{m Fig.15K. On days 2 and 3 after ZIKV challenge (E9.5 and E10.5), ZIKV RNA levels in fetautesr+npallace (Fntiga.+d 1e6cAid)ua s weerurem m,ea (Fsuigre.d 1 b6yB q)RT b-rPaCinR,. T anhde po (Fpiugl.atio 16nC w)as s apsle feonllow ans:d (Fig.16D)} 68

n fetuses Feature

19 fetuses from 3 separate mothers ZIKV+isotype

35 fetuses from 4 separate mothers ZIKV+Anti-CD8 at E9.5

n = 46 fetuses from 6 separate mothers ZIKV+isotype

48 fetuses from 6 separate mothers ZIKV+Anti-CD8 at E10.5

[ _S ⁰ _E ⁰² _M ³⁹ _. ^] _*p<0.0 ^D ₅ ^a _, ^t _* ^a _*p ^w _< ^e ₀ ^re _.01 ^p _, ^o _* ^o _* ^l _* ^e _* ^d _p< ^fr ₀ ^o _. ^m ₀₀₀ ^tw ₀₁ ^o _{. T} ⁱⁿ _w ^de _o ^p _-t ^e _a ⁿ _il ^d _e ^e _d ⁿ _M ^{t e} _a ^x _n ^p _n ^e _W ^rim _h ^e _it ⁿ _n ^t _e ^s _y ^. _t ^D _es ^a _t ^ta _wa ^ar _s ^e _u ^e _se ^x _d ^p _. ^{ressed as mean +} [00240] The results obtained are as follows:

[ ^{Z0I0K24V1] infec Atesd se nenon i-nim thmeu Ifnar1-/- mouse model, decreased fetal weight and size was observed in restriction with the fetal sizene a mice, whereas prior DENV immunity prevented fetal growth} a _{trnedat Fedig. w 1i6thB) a.n Atig-CaiDn,8 fe Atabl grow} ⁿ _t ^d _h ^w _re ^e _s ⁱ _t ^g _r ^h _ic ^t _ti ^c _o ^o _n ^m _a ^p _n ^a _d ^ra _r ^b _e ^l _s ^e _or ^to _pti ^t _o ^h _n ^e _w ^m _e ^o _r ^c _e ^k _o ^-i _b ⁿ _s ^f _e ^e _r ^c _v ^te _e ^d _{d i} ^c _n ^on _D ^tr _E ^o _N ^{l g} _V ^r-^o _im ^up _m ⁽ _u ^F _n ^ig _e ^. _m ¹⁶ _ic ^A _{e WT mice with transient I (Fig 16A and Fig.16B, and Fig.23). These results demonstrate that, in induced fetal growth restrifcntaior1n b inlo ack CaDde8,+ p Tri coerll D-dEepNeVnd iemntm munanitnye arf.fords protection against ZIKV-} [^{b0l0o2c4k2in]g Ab I wne Freig. ch 2a3l,le nnogned-im wmithun ZeIK orV D atE EN7V.52- aism dmesucnreib WeT dams that were treated with Ifnar1-} p_{ost-infection at E14.5. Representative images of fetu} ^{d in A-K. Tissues were harvested 7 days} D_{ENV2-immune dams with or without anti-CD8 Ab adsmesini asntrd placentas from non-immune or used are as follows: ation are shown. The populations} n mothers Feature

6 p t th N i Mk

5 p t th N i ZIKV

69

4 separate mothers DENV2-immune-Mock

5 separate mothers DENV2-immune-ZIKV+isotype

4 separate mothers DENV2-immune-ZIKV+Anti-CD8 ^[ _r ⁰ _e ⁰ _s ² _p ⁴ _e ³ _c ^] _tively. ^{Blue and red arrows indicate the presence of fetal growth restriction and resorption, [ s0p0le2e4n4]s, plac Aennatlayssis w oitfh v direaclid buuard,e annd rev feeataleld b tohdaites Z,I wKiV RNA was consistently present in maternal relative to non-immune dams. Administration of anti-tChD r8ed Aubced abr leovgealtsed in th DeE pNroVte-cimtivmeu enfefec mtic oe} _D ^f _{tisEsuNesV c iommmpuanreitdy, to wit ishot syigpneif cicoannttrloyl h Aigher viral RNA levels detected in both maternal and fetal efficiency of anti-CD8 Ab-mediated CDb8-+tr Tea cteedll d DepElNetVio-nim inm tuhnee sp dlaemens a (nFdig d.e 1c6iC-Fig.16H). The >95% (Fig.24A and Fig.24B). dua/placenta were}

^{t Z [00245] In Fig.24, pregnant non-immune or DENV2-immune WT mice were challenged with} _p ^hI

l_o ^eK

t ^pV

s^{re a}

r_e ^set

p^{n E}

r_e ^c7

s^e.5

e ^o _nt ^{f as}

e ^{C d}

d^De

w^8s

e^+cr

r_e ^Tibe

f ^cd

i^e _rs ^{ll i}

t^{sn b F}

g_a ^yig

t_e ^fl.

d^{o 1w6A}

o_n ^{cy t}

C^{too Fig.16K. In Fig.24A, the gating strategy used to analyze} D^m ₃ ^e ₊ ^try _ce ⁱ _l ⁿ _ls. ^th _I ^e _n ^sp _F ^l _i ^e _g ^e _. ⁿ ₂ ^a ₄ ⁿ _B ^d _, ^d _th ^ec _e ^id _p ^u _e ^a _r ^/ _c ^p _e ^{lacenta is illustrated. All} _{depletion inth sp nloenen-im anmdu dneeci adnuda/ DpElaNceVn-t2a a imre ntages of CD8+ T cell dams in bo m reupnrees gernotuepds. for isotype control or anti-CD8 Ab-treated} ^{[ Z0I0K24V6] In DENV-immune dams, anti-CD8 Ab treatment resulted in a greater percentage of Ab trea itnmfeecntito,n wi itnh p 1l0ac0e%nt vaser wsuitsh 7 d4e%cid inua pl aand fetal heads and bodies compared with isotype control} _{82% versus 44% in fetal bodies. The diffe} ^c _r ^e _e ⁿ _n ^ta _c ^s _es ^w _b ^it _e ^h _tw ^de _e ^c _e ⁱ _n ^du _t ^a _h ^, _e ⁶⁹ _is ^% _oty ^v _p ^e _e ^rsu _an ^s _d ³⁷ _a ^% _nti ⁱ-ⁿ _C ^{fetal heads, and} _{DENV immune miceor w pelraece snigtnaif wicitahnt d feocird aulal, th pr <ee0. t0is1su D8 Ab treated exact test: p <0.001 f foers f (eFtiagl. h 1e6aId t,o an Fdig p.1 <60K.0)0 (1T fwoor- fseitdaeld bo Fdisyh)e.r’s} ^[ _f ⁰ _e ⁰ _ta ² _l ⁴ _i ⁷ _n ^] _terfac ^A _e, ^s _th ^de _e ^c _p ^re _re ^a _s ^s _e ^e _n ^d _t ^m _in ^a _v ^t _e ^e _n ^rn _to ^al _rs ^Z _c ^I _o ^K _m ^V _pa ^v _r ⁱ _e ^re _d ^m _v ⁱ _i ^a _ra ^m _{l b} ^a _u ^y _rd ^le _e ^a _n ^d _i ^t _n ^o _m ^lo _a ^w _te ^e _r ^r _na ^Z _l ^I _a ^K _nd ^V _f ^l _e ^e _t ^v _a ^e _l ^l _t ^s _is ⁱ _s ⁿ _ue ^th _s ^e _of ^m _D ^at _E ^er _N ⁿ _V ^al-- 70

ⁱ _c ^m _ha ^m _ll ^u _en ⁿ _g ^e _e. ^{mice treated with isotype control Ab versus anti-CD8 at early time points after ZIKV [ W00}

1^6T24

A ^d8

t^{oa]m Fsig w T}

.^{e 1rh}

6^ee

K ^{t.r per}

M^aets

i^ee

c^dn

e ^wt

w^{i r}

e^te

r^{hsults are with respect to Fig.17A to Fig.17E, where DENV2-immune e a andtmi-Iifnnisatre1re mdA ib and challenged with ZIKV at E7.5 as described in Fig.} 1 _{m6aAter tnoa Flig (.F 1ig6.K.1 O7An) da syesru 2m a,nd (F 3ig a.fte 1r7 Z} ^s

B_I ^o

)_K ^ty _V ^pe

b_{ra cih} ^co

n_a,l ⁿ _le ^tro

a_nng ^l

d_e ^o ₍ ^r _E ^a

(_F9 ⁿ

i_. ^{ti-CD8 Ab, also as described in Fig.} g_{5. an 1d7C E)10 s.5p)l,ee ZnIK aVnd RN (FAig l.eve 1l7s in fetus+placenta+decidua were measured by qRT-PCR. The populations were as follows: D)} n fetuses Feature

19 ft f 3 p t th ZIKV+ityp

35 ft f 4 p t th ZIKV+AtiCD8t E95

46 ft f 6 p t th ZIKV+ityp

48 ft f 6 p t th ZIKV+AtiCD8t E105

^[ _S ⁰ _E ⁰² _M ⁴⁹ _. ^] _p<0.0 ^D _5, ^t _p<0.01 ^p _, ^ld _p< ^f _0.000 ^t _{01. T} ⁱ _w ^d _o ^p _-tail ^d _{ed M} ^t _an ^p _{n W} ⁱ _hitn ^t _ey ^. _t ^D _est ^t _{was used} ^p _. ^{d +} [00250] The results obtained are as follows:

[ ^{o0f02 Z5I1K]V RN AtA E w9e.5re an pdre Ese1n0t.5 in (2 th aend m 3at deranyasl after ZIKV challenge of E7.5 mothers), similar levels contro serum of DENV-immune mice treated with isotype maternlal A bbra oinr a anndti- sCpDlee8n (Fig.17A). In contrast, higher ZIKV RNA levels were detected in the ant} _s ⁱ _e ^-C _rv ^D _ed ^{8 A} _in ^b- _t ^t _h ^r _e ^ea _p ^te _e ^d _rc ^t _e ^h _n ^a _t ⁿ _ag ^{i ast}

e^{o o y}

s^tyn

o^pl

f^e _in ^{c Eo1}

f_e ⁿ⁰

c^t.

t^r5

i^{o and in tahtee placenta with decidua at both E9.5 and E10.5 in} o_{b o} ^l _n ^A _o ^b _f ^-t _t ^r _h ^e _{e p} ^d _la ^{mice (Fig.17B to Fig.17D). No difference was} d_{espeite higher levels of viral RNA in the placenta withce dnetcaisdu wait fhro decidua between the two groups Takunn titoyge ctohnetrr,o tlshe ZseIK dVata in sfuecgtgieosnt i tnha bto itnh W mTate mrnicael a wnidth fe ttraaln tsiisesnm}

u_{t t}

e_{s Ihf vne}

i_{a a}

a_rn1ti

D _b-C

E_{lNoDc8 group (Fig.17E). imm Vka-edxep,o psreiodr m DeEmNorVy}

71

^{C TD}

i_nt ^c _e ^e8

r_f ^l+l _a ^{s T}

c^; _e ^{o c}

,^ne

D^clles. t_E ^{b H} i_sN ^loo s_uV ^ow e_s- ^de ._e ^-v

l_i ^be

c^or

i^{r,n eear ZlyIK duVrin spgr ienafdesct aionnd, r ZeIpKlicVat leesve inls d inis ctairlc suiltaetsi,on inc alrued ninogt i tmhepa mctaetder bnyal t-hfeetsael} _{infection of ted memory CD8+ T cells assume an important role in limiting ZIKV} ⁸ _im ^.3 _{mune WT} ^P _m ^ol _i ^y _c ^f _e ^un _w ^c _it ^t _h ^ion _tr ^a _a ^l _n ^it _s ^y _ien ^o _t ^f _I ^c _fn ^ro _a ^s _r ^s ₁ ^-r _b ^e _l ^a _o ^c _c ^t _k ^iv _a ^e _de ^{CD8+ T cells in the maternal spleen of DENV- [ N00}

r^eaS2

c^352]

t^{iv6e-18 w74it a F}

h^nidve

t^{h N H}

o^Sse5-2b

2^{7 i8n3d- r27e}

u^9s2ti

c^)r

e^{d hcat}

i^ved

n^{e D r ZecI}

E^eK

N^{nVtVl-yd bereievned id CenDti8fi+ed T b cye tllhe ep pirteospeenst ( inpvrMen1t6o9-1r7s7, th Ea2t94 w-30e2,re E c2r9o7-3s0s5 186 -,} _{contribution of DENV} ^{2-infected mice (Wen et al., 2017a). To understand the} _{pregnancy in DENV-im2m-eulinceite WdT CD m8ic} ⁺ _{e T wi cthells tra tnosie pnrtot Iefcntaiorn1 b algoacinksatde Z,I tKheV p irnefseecnttio innv denuring assessed the quantity and phenotype of cross-realycstiisv.e CD8+ T cells in t tors performing intracellular cytokine staining (ICS) ana he maternal spleen by} ^{[ D00}

i^njE2

e^cN53

t^eV]

d^{2- 1im0%m Thue}

F^{nBe fo}

S ^Wllo

-^PTw

B^{S dinag}

(^{m r}

M^se

O ^wsu

C^il

K^tths

) ^{t a}

a^rrae

s^{n ds wieeit}

s^{nh r}

c^{tri Ie}

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i^{1ct b tlooc Fkiagd.e 1 w8Aere to ch Faigll.en 1g8eId, w whitehre Z nIKonV-im atm Eu7n.e5 o orr infection (E14.5), mice were sac n Fig.16A to Fig.16K. Seven days after ZIKV percentages (Fig rificed, and spleens were processed for ICS analysis. The pro .18A and Fig.18B) and numbers (Fig.18C) of CD44highCD62Llow CD8+ T cells} _CD ^d ₄ ^u ₄ ^hciignh _C ^g _D ^IF ₆ ^N _2L ^{glow ar} _C ^e _D ^sh ₈ ^o+w _T ^n. _ce ^T _lls ^he _pr ^p _o ^e _d ^r _u ^ce _c ⁿ _in ^ta _g ^g _b ^es _ot ⁽ _h ^Fi _I ^g _F ^. _N ¹⁸ _g ^D _an ^a _d ^nd _TN ^Fi _F ^g. _a ¹ _r ⁸ _e ^E _r ⁾ _ep ^a _r ^{nd numbers (Fig.18F) of} _{(Fig.18G and Fig.18H) and numbers (Fig.18I) of CD esented. The percentages granzyme B are shown. The following n 44highCD62Llow CD8+ T cells expressing and n = 5 ZIKV) and DENV2-immunumbers of dams were used: Non-immune (n = 6 MOCK two independent experiments e (n = 4 MOCK and n = 5 ZIKV). Data were pooled from MannV-W2-himitnmey and are expressed as mean ± SEM. *p<0.05, **p<0.01. Two-tailed DEN un tees mti wceas fo urs eeadch to sti cmoumlaptaioren c MoOndCitKion ve inrs Fuisg. Z 1I8KBV to-in Ffiegc.t 1e8dJ. or non-immune versus} [00254] The results obtained are as follows:

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m_{mg.u 1n8eE- dFaimg.s 1.8 TGh)e,s oer r gersaunltzsym ie B (Fig.18H-Fig. amemso wriyth C trDan8 ndicated that prior spleen during subsequent ZIKV infection of WT d +sie Tnt c Ieflnla rre1sp boloncskeasde in. the maternal} ^{[ D00E2N56V]2-imm Thuene fo WlloTw dinagm rse wsuiltths t arraens wieitnh respect to Fig.19A to Fig.19I, where non-immune or injected 10% FBS-PBS (MOCK t Ifnar1 blockade were challenged with ZIKV at E7.5 or infection (E14.5), mic ) as described in Fig.16A to Fig.16K. Seven days after ZIKV Fig.19C) The percenteag weesre (F siga.cr 1if9iAced a,n adn Fdig s.pl 1e9eBns) a wnedre n purmobceesrssed (F figo.r 1 I9CCS) a onfal CysDis4.4 (hFigihgC.D 196A to C} _(F ^D _ig ^{8 2Llow} .⁺ ₁₉ ^T _F) ^ce _o ^ll _f ^s _C ^p _D ^ro ₄ ^d ₄ ^uhcighin _C ^g _D ^I ₆ ^F ₂ ^N _L ^{lgow a} _C ^re _D ^s ₈ ^h+o _T ^wn _c ^. _e ^T _lls ^he _pr ^p _o ^e _d ^r _u ^c _c ^e _i ⁿ _n ^t _g ^ag _b ^e _o ^s _th ^(F _I ^ig _F ^. _N ¹ _g ^9D _an ^{and Fig.19E) and numbers} _{percentages (Fig.19G and Fig.19H) and numbers (Fig.1 d TNF are represented. The expressing granzyme B are shown. The followi 9I) of CD44highCD62Llow CD8+ T cells MOCK and n = 5 ZIKV) and DENV2-immnugn neumbers of dams were used: Non-immune (n = 6 pooled from two independent experiments and ar (n = 4 MOCK and n = 5 ZIKV). Data were Two-tailed Mann-Whitney test fo wras ea u e expressed as mean ± SEM. *p<0.05, **p<0.01. versus DENV2-immune mice cshed sti tmou cloamtiopnar ceo MndOitiCoKn i vne Frsiug.s 1 Z9IAK tVo- Finifge.c 1t9eId. or non-immune} [00257] The results obtained are as follows:

[ ^{f0ro02m58] non-im Nmexutn,e th vee 5rs cursos Ds-EreNacVti-viem empuitnoepe d-sapmescif oicn CD8+ T cell responses in the maternal spleen compared, when the primary CD8+ T cell resp day 7 after ZIKV challenge (E14.5) were} _{should peak (Elong Ngono et al., 201} ^{onse to ZIKV infection in non-immune animals} _{specific CD44highCD62Ll 7). The frequencies but not numbers of 3 of the 5 epitope- in DENV-immune thano nwo enff-eimctmoru mneem moicrey ( aFnidg. e 1f9feActo tor C FiDg.81+9 TC c).el Ilns p croomdupcairnisgon IF,N thge w freerqeu henigchieesr} 73

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[ ^{e0p0i2g6e2n]etic si Alelnthcionugg ohf d keecyid cuhaelm Toki cneells ge wneesre th raatre pr ceovemnpta irnefdlux to of sp Tle cneilcls T to c thelels d,e pceidrhuaap (sNa due to al., 2012), the present inventors detected polyfunctional effecto ncy et} _{in the decidua/placenta after restimula} ^{r memory and effector CD8+ T cells} _{Although CD8+ T cells producing IFNtiogn a wlointhe a o} 7_{r m}

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[ ^{C0o02n6g4e]nital Z AIsK tVhe Sy nnudmrobmere o lifke ZlyIK wVill o incfceucrt.io Anss in pregnant women increases, more cases of endemic regions, there is an urge many of these infections will occur in DENV- ZIKV. A major qu ncy to understand the effect of pre-existing DENV immunity on against or pathogeneesstiiso onf in ZI tKheV fi ienlfdec itsio wnh detuhreinrg p priroergn DanEcNy.V T iom amddurneitsys t choinstributes to protection inventors adapted established mouse models of ZIKV in question, the present a pcrqesueirnetd in ovre genetic deficiencies of type I IFN signaling (Minefrec ettio anl., d 2u01ri6n;g Y porcekgenya entc ayl., th 2a0t16 r)e.ly T ohne} _i ^{ntors challenged DENV-immune dams with ZIKV to model sequential DENV-ZIKV} _{fneftaelct tiiossnu.e Isn, i DncEluNdiVn-gim thmeu dneeci mduicae/,p iltac wenasta o,b ansedrv aend in ac rreedasuection of ZIKV burden in maternal and to non-immune mice. Depletion of CD8+ of fetal viability and growth compared role for CD8+ T cel T cells abrogated this effect, demonstrating an essential immunity. Cross-realcst iinve p,r pootelycftuionnct aiognaianls Ct ZDI8K+V T d cuerlilnsg d purreinggna pnrceygn inan thcye c moanyte hxt of prior DENV overcome other pathogenic immune elements associated wi ave the ability to ADE (Bardina et al., 2017). Indeed, it has b th prior DENV exposure, including cells can prto atle.,c 2t0 m15ic).e eve een previously reported that DENV-reactive CD8+ T Zellweger e n under ADE conditions (Wen et al., 2017a; Zellweger et al., 2014;} ^{[ a0b0r2o6g5a]ted D AEtN aVn- eimarmlyu tnime-em peodiiantted aft perro ZteIcKtioVn in infe mctaion of pregnant dams, CD8+ T cell depletion} _{(i.e. decidua/placenta) despite having no effect on mat} ^t _e ^e _r ^r _n ⁿ _a ^a _l ^l _v ^t _i ⁱ _r ^s _e ^s _m ^ue _i ^s _a, ^a _s ⁿ _u ^d _gg ^t _e ^h _s ^e _ti ^m _ng ^at _t ^e _h ^r _a ⁿ _t ^a _D ^l-f _E ^et _N ^al _V ⁱⁿ _-e ^te _li ^r _c ^f _i ^a _te ^c _d ^e 75

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a^{ghean rev ineale cdirc tuhlaattio thne. i enxp DoEsuNreV e-ilmicimteudne cr tohsasn-re naocnti-vimem CDun8e+ d Tam cse.lls Th wuisth, a gtr eeaartleyr s ftuangect oiofn ZalIK acV infencdtio pno,ly pfruionrct DioEnaNlitVy expanded during primary ZIKV infection. Recent studies using bloo tivity compared to those individuals have identified cross-reactive CD8+ T cells in hum d samples from non-pregnant P} _in ^r _f ^o _lu ^u _e ^lx _nc ^e _e ^t _d ^a _t ^l _h ^., _e ² _m ⁰¹ _a ⁷ _g ⁾ _n ^. _i ^O _tu ⁿ _d ^e _{e a} ^o _n ^f _d ^th _q ^e _u ^s _a ^e _lit ^s _y ^tu _o ^d _f ^ie _t ^s _he ^sh _C ^o _D ^we ₈ ^d+ _T ^th _c ^a _e ^t _ll ^D _re ^E _s ^N _po ^{V eaxnpso (sGurreifo pnriio ert to al. Z,I 2K01V7; in Pfaeqcutiionn-} _{that prior DENV immunity may shape the anti-ZIKV CD8+nse (Grifoni et al., 2017), suggesting human primates suggested that prior DENV exposure may co Tnf cell response. A study with non- infection (Pantoja et al., 2017), although a second study er cross-protection against ZIKV effect of previous DENV exposure during subseq reported neither protective nor pathogenic Notably, non-human primates in these studies uent ZIKV infection (McCracken et al., 2017). as compared to our challenge of mice on da were challenged 1-2 years following DENV exposure, d ceeltla rileesdpo envaselusa atgioanins otf Z thIKeV du irnafteioctnio onf d curroisnsg-p pry 30 after DENV priming. Going forward, a more roetgencatinocny m ise ndeieadteedd. by prior DENV-induced CD8+ T} ^{[r0e0a2c6ti6v]e antig Ceonn-sspisetecniftic w CitDh8 t+h Te l coecllasl a elsffoec wte oref d DeEteNctVed-e inlic tihteed d CecD8+ T cells in each tissue, cross- mice. As CD8+ T cell are one of the abundant cell types present iidua/placenta of DENV-immune Lissauer et al., 2017; van Egmond et al., 2016), the T cells n the decidua (Crespo et al., 2017; maternal origin. Future studies using CD45.1 C57BL detected are likely decidual and thus of should confirm the maternal versus fetal origin of /6 female mice with congenic CD45.2 sires origin of CD8+ T cells and the mechanisms these cells. At present, the precise specificity and} _{fetus and antiviral immunity at the mate} ^{by which these cells balance immune tolerance of the} _{specific and fetal antigen-sp rnal-fetal interface are presently unclear, but both virus- (Constantin et al., 2007; Crecific CD8+ T cells have been detected in human and mouse decidua Powell et al., 2017; Til espo et al., 2017; Lissauer et al., 2017; Nancy and Erlebacher, 2014; cells in humans are pburgs and Strominger, 2013; van Egmond et al., 2016). The decidual CD8+ T B compared to p rimarily of effector memory phenotype and express reduced levels of granzyme et al., 2016). Conersiipstheenrtal w biltoho tdh CisD o8b+se Trv caetlilosn (,Po crwoeslsl-r eeta aclt.,iv 2e01 a7n;ti Tgeilnb-usrpgesc eiftic al. C,D 2081+0; T va cnel Elsg imno tnhde}

76

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I_{nK-pVre ignnfeacnttio mnic ine w thiteh p ZlaIcKenVta in afnedcti foentu.s G (iJvaegnge trha ett g ael.s,ta 2tional stage influences the also to evaluate the temporal component of the anti-Z 017), it will be important stages of pregnancy. IKV T cell response through the different} ^{[ a0n0d26 s8u]fficien Tth teo p prreosteenctt a ingvaeinnsttor ssys hteamveic p ZreIvKioVus clhya dlleemngoen isntra btoetdh t nhaaïtve C aDn8d+ D TE cNelVls-i amrem necessary pregnant mice (Elong Ngono et al., 2017; Wen et al., 2017a; Wen et al., 20 une non- data suggests a similar requirement for CD8+ T cells in pr 17b). Here, the present pregnancy and prior DENV exposure. The p otection against ZIKV in the context of} _{CD4+ T cells, suggesti} ^{resent data also suggests a partially protective role for} _{T cell response duringn ZgI tKhaVt C inDfe4c+tio Tn c oelfl D mEedNiaVte-dim-hmelupn mea pyre sghnaapnet a fnem opalteims.a All ctreornssa-triveaective CD8+ cells may exert their effect by regulating humoral immunity or CD4+ re ly, CD4+ T minimize pathology at the maternal-fetal interface. The gulatory T cells could investigating the precise role of CD4 present data thus sets the foundation for humans and animal models. + and CD8+ T cells in ZIKV infection during pregnancy in}

77

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g^r _n ^{s o tfo pr thoetesceti qoune isntidouncsed lik beyly cr woislsl-r peraocvtiidvee} _{inform the feasibility of developing combin ancy in DENV-endemic areas and also DENV. As CD8+ T cell responses i ation vaccines that protect against both ZIKV and cross-reacted with ZIK nduced by a tetravalent live attenuated DENV vaccine also confer protection againsVt Z epIKitoVpe insf (eGctriiofnon ini e ptr aelg.,n 2an01cy7.), M fuorrtehoevre br,o ZoIsKtinVg v oafc Tcin ceesll th responses could to induce optimal T cell responses in addition to Absur minagy p breeg mnoanrecy e.f at are designed solely on Ab responses for protection against ZIKV d fective than those that focus} ^[ _t ⁰ _e ⁰ _a ² _c ⁷ _h ⁰ _in ^] _{gs of} ^O _th ^t _e ^he _p ^r _re ^e _s ^x _e ^a _n ^m _t ^p _d ^l _e ^e _s ^s _cr ^o _ip ^f _t ⁱ _i ^m _on ^pl _a ^e _n ^m _d ^e _a ⁿ _s ^ta _s ^t _u ^io _ch ⁿ _, ^s _w ^w _il ⁱ _l ^ll _n ^b _o ^e _t ^c _b ^o _e ^m _f ^e _ur ^a _t ^p _h ^p _er ^ar _d ^e _e ⁿ _s ^t _cr ^to _ibe ^th _d ^e _h ^r _e ^e _r ^a _e ^d _. ^{er in view of the [ c0o0n2v7e1n]ience N oofte a r tehaadter t,it bleust o inr n soub wtiatyles the msay be used throughout the present disclosure for certain theories may be prop e should limit the scope of the invention. Moreover,} _{right or wron} ^{osed and disclosed herein; however, in no way they, whether they are} _{according to thge, p srheoseunldt d liimsciltos tuhree w sciothpoeut o rfeg thared i fnovre anntiyo pnar stoicu lolanrg th aesor tyh oer i sncvheenmtieon of i asct pioranc.ticed} ^[ _t ⁰ _h ⁰ _e ² _ir ⁷² _e ^] _ntirety ^A _f ^l _o ^l _r ^re _a ^f _l ^e _l ^r _p ^e _u ⁿ _r ^c _p ^e _o ^s _s ^c _e ⁱ _s ^t _. ^{ed throughout the specification are hereby incorporated by reference in [ s0p0e2c7if3i]cation I,t th wei tllerm be“a u”n udseerdsto boefdor bey a t tehromse en ocofm spkialslse ins em thbeod arimte tnhtast co thnrtoaiungihnogu otne th oer m present what the term refers. It will also be understood by those of skill in the art t ore to} _{present specification, the term“comprising”, which is synon} ^{hat throughout the} _{or“characterized by,” is inclusive or o ymous with“including,”“containing,” elements or method steps. pen-ended and does not exclude additional, un-recited} 78

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f^{lyerwise defined, all on technical and scientific terms used herein have the same} c_o ^u _n ⁿ _f ^d _li ^e _c ^r _t ^s _, ^t _t ^o _h ^o _e ^d _pr ^b _e ^y _sent ^e _do ^o _c ^f _um ^o _e ^rd _n ⁱ _t ⁿ _, ^a _in ^ry _clu ^s _d ^ki _i ^l _n ^l _g ⁱⁿ _def ^th _in ^e _iti ^a _o ^r _n ^t _s ^t _w ^o _il ^w _{l c} ^h _o ^ic _n ^h _tro ^t _l ^h _. ^{is invention [ g0}

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n^{itdhi inn t thhee p errerosern mt darisgcinlos guernee,r tahlley te arcmcesp“teadro iunnd th”e,“ aarbt.ou Hte”n ocre,“a npupmroerxiicmalat qeuly” shall} c_{earnal blye i innfcelrurdede if su ncoht e exrprorerss mlya srtgai} ^antities _{“approximately” tned s.uch that the terms“around”,“about” or} ^{[ w0i0l2l7 b6e] app Aarletnhtou tgoh t vhaorsioeu ssk eilmledbo idnim tehnts of the disclosure have been described and illustrated, it} _modi ^{e art in light of the present description that numerous} _{in thefi acpatpioenndse adn cdla vimarisa.tions can be made. The scope of the invention is defined more particularly}

79

References

I– Example 1

A _T. ^t _J ^k _., ⁱⁿ _a ^s _n ^o _d ⁿ _H ^{, B} _e ^. _w ^{, H} _so ^e _n ^a _, ^r _R ^n, _. ^P ₍₂ ^., ₀ ^A ₁₆ ^f ₎ ^r _. ^o _D ^ug _e ^h _te ^, _c ^B _ti ^., _o ^L _n ^u _o ^m _f ^l _Z ^ey _ik ^, _a ^S. _V ^{, C} _iru ^ar _s ^te _in ^r, _S ^D _em ^{., A} _en ^a _. ^ro _E ⁿ _m ^s, _er ^E _g ^.J _I ^. _n ^, _f ^S _e ^im _ct ^p _D ^so _is ⁿ ₂ ^, ₂ ^A _, ^. ₉ ^J. ₄ ^, ₀ ^B _. ^{rooks, B Craarsvial,lh Po., d Peer Seeiqrau,e Ji.rPa,., P Jr.,., M Raacja Gabaglia, C., Damasceno, L., Wakimoto, M., Ribeiro Nogueira, R.M.,} _{a Ml.e (d2.016). Zika Virus Infection} ^ha _in ^do _P ^S _re ^iq _g ^u _n ^e _a ⁱ _n ^r _t ^a, _W ^A _o ^., _m ^A _e ^b _n ^re _in ^u _R ^de _io ^C _d ^a _e ^rv _J ^a _a ^l _n ^h _e ^o _ir ^, _o ^L. -^M _P ^. _r ^, _e ^C _lim ^ot _i ^r _n ^im _ary ^d _R ^a _e ^C _p ^u _o ⁿ _r ^h _t. ^a _N ^{, D} _E ^., _n ^e _g ^t _{l J} ^C _ta ^l _r ^a _g ^u _e ^s _t ^e _in ⁿ _g ^{, B} _in ^.E _m ^., _a ^B _cr ^u _o ^r _p ^k _h ^h _a ^a _g ^rd _e ^t _s ^, _a ^C _n ^. _d ^{, R} _g ^e _ra ^it _n ^h _u ^, _l ^W _oc ^. _y ^, _t ^R _es ^en _u ^k _s ^a _in ^w _g ^it _L ^z, _ys ^R _M ^., _c ^a _r ⁿ _e ^d _m ^F _ic ^o _e ^r _. ^st _T ^e _r ^r, _an ^I. _sg ⁽¹ _e ⁹ _n ⁹ _ic ⁹⁾ _r ^. _e ^C _se ^o _a ⁿ _rc ^d _h ^iti ₈ ^o _, ⁿ ₂ ^a ₆ ^l ₅ ^g-^e ₂ ⁿ ₇ ^e _7. ^C _C ^u _., ^g _A ^o _l ^l _m ^a, _e ^F _id ^.R _ats, ^., _N ^F

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n ₍ ^{B.C., Dias, J.L.M., Guimarães, K.P., Benazzato,} _{birth defec 2ce01 o6n)l.in Tehe pu Bbrlaiczailtiiaonn Z.ika virus strain causes} ^D _D ^’ _e ^O _sc ^r _a ^t _m ^en _p ^z _s ^io _, ^, _D ^E _., ^., _D ^M _am ^ath _o ^e _n ^r _d ^o _, ⁿ _F ^, _. ^S _, ^. _Y ^{, d} _az ^e _d ^L _a ^a _n ^m _pa ^b _n ^a _a ^ll _h ^e _, ^ri _Y ^e, _., ^X _a ^. _n ^, _d ^H _L ^u _e ^b _p ^e _a ^r _r ^t, _c- ^B _G ^., _o ^P _f ⁱ _f ^o _a ^r _r ^k _t, ^o _I ^w _{. (} ^s ₂ ^k ₀ ^i, ₁ ^G ₆₎ ^{., Maquart, M.,}

T_{ransmission of Zika Virus. N Engl J Med. . Evidence of Sexual} ^D _M ^e _e ^c _a ^k _d ^a _, ^r _P ^d _. ^, ₍ ^D ₂₀ ^.T ₁₆ ^., ₎ ^C _{. M} ^hu _a ⁿ _le ^g-^, _to ^W _-M ^.M _a ^. _l ^, _e ^B _S ^r _e ^o _x ^o _u ^k _a ^s _l ^, _T ^J. _r ^T _an ^., _s ^S _m ^m _is ⁱ _s ^th _io ^, _n ^J.C _of ^., _Z ^W _ik ^o _a ^ld _V ^a _i ⁱ _r ^, _u ^S _s ^., - ^H _T ^e _e ⁿ _x ⁿ _a ^e _s, ^ss _J ^e _a ^{y, M., Kwit, N., and} _{Morb Mortal Wkly Rep 65, 372-374. nuary 2016. MMWR} ^{D H.i,am Arotnhdu,r, M C..SD.,. K, Winhdietre,, M J.M.,. M, Katasluinshkeit,a U, H.,. e,t M ala.s (h2a0y1e1k)h.i T,y Mp.e, D I iunntenr,f Gero.Pn., i As sreclheacmtbault, J.M., Lee,} _{d0en03d.ritic cells for immune rejection of} ^{ively required by} _{2 tumors. The Journal of experimental medicine 208, 1989-} ^D ₄₆ ⁱ _, ^c ₅ ^k ₂ ^, ₁ ^G _-5 ^.W ₃₄ ^. _. ^{(1952). Zika virus. II. Pathogenicity and physical properties. Trans R Soc Trop Med Hyg}

80

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5_{.8 (} ^{., Atkinson, B., Hall, G., Watson, R.J., Bosworth, A., Bonney,} ._{2016). A Susceptible Mouse Model for Zika Virus Infection.} ^E _an ^lo _d ⁿ _S ^g _h ^N _re ^g _st ^o _a ⁿ _, ^o _S ^, _. ^A ₍₂ ^. ₀ ^, ₁ ^C ₆ ^h ₎ ^e _. ⁿ _P ^, _ro ^H _t ^. _e ^W _ct ^. _i ^, _v ^T _e ^a _R ⁿ _o ^g _l ^, _e ^W _of ^.W _C ^. _r ^, _o ^J _s ^o _s ^o _-R ^{, Y} _ea ^. _c ^, _t ^K _iv ⁱ _e ^ng _C ^, _D ^K ₈ ^., _T ^W _C ^ei _e ^s _l ^k _ls ^op _A ^f _g ^, _a ^D _in ^. _s ^, _t ^S _D ^id _e ⁿ _n ^e _g ^y, _u ^J _e ^., _V ^S _i ^e _r ^{tte, A.,} _{Infection. EBioMedicine 13, 284-293. us} ^F _L ^o _an ^y, _ci ^B _o ^. _t ^D _ti, ^., _R ^K _.S ^o _., ^b _a ^y _n ^lin _d ^s _T ^ki _e ^, _s ^K _h, ^.C _R ^. _. ^, _B ^C _. ^h ₍₂ ^il ₀ ^s ₁ ^o ₁ ⁿ _). ^F _P ^{oy, J.L., Blitvich, B.J., Travassos da Rosa, A., Haddow, A}

8_{8ro0b-8a8b2le. non-vector-b} ^.D., _{Colorado, USA. Emerg Infect Dis 17, orne transmission of Zika virus,} ^G _J.M ^ov _., ^e _C ^ro _a ^, _in ^J. _e ^, _, ^E _E ^s _. ^a _A ^k _. ^k _, ^y _S ^, _a ^P _la ^. _z ^, _a ^S _r ^c _, ^h _V ^e _. ^a _, ^f _e ^fe _t ^r _a ^, _l ^S _. ^. ₍ ^M ₂₀ ^. ₁ ^, ₆ ^F ₎ ^e _. ^r _Z ^na _ik ⁿ _a ^d _v ^e _i ^z _r ^, _u ^E _s ^. _i ^, _n ^D _fe ^r _c ^u _t ^r _io ^y, _n ^A _d ^. _a ^, _m ^Pl _a ^a _g ^t _e ^t, _s ^D _th ^.J _e ^., _t ^G _es ^o _te ^r _s ^m _in ^an _m ^{, M} _ice ^.J _. ^., _N ^R _a ⁱ _t ^c _u ^h _r ⁿ _e. ^{er, G} _S.B ^uz

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7_g ^{., Valdes, L., Bravo, J., Alvarez, M., Vazques, S., Delgado, I., and Halstead,} _{epi 9ic9; s dtuisdciuesss oionn D 8e0n4g.ue in Santiago de Cuba, 1997. American journal of} ^H _R. ^a _B ^{ddow, A.D., Schuh, A.J., Yasuda, C.Y., Kasper, M.R., Heang, V., Huy, R., Guzman, H., Tesh,} _{of th.,e an Adsia Wne lainveear,g Se..C P.L (o20S1 N2)e.g Gl Tenroetpic D cihsa 6r,ac et1e4r7iz7a.tion of Zika virus strains: geographic expansion} Halstead, S.B. (2007). Dengue. Lancet (London, England) 370, 1644-1652.

H _Ka ^e _s ^a _p ⁿ _e ^g _r ^, _, ^V _M ^., _.R ^Y _. ^a ₍ ^s ₂ ^u ₀ ^d ₁ ^a ₂ ^, ₎ ^C _. ^. _Z ^Y _i ^. _k ^, _a ^S _v ^o _i ^v _r ^a _u ⁿ _s ⁿ _in ^, _f ^L _e ^. _c ^, _t ^H _io ^a _n ^d _, ^d _C ^o _a ^w _m ^, _b ^A _o ^.D _dia ^., _, ^T ₂₀ ^ra ₁ ^v ₀ ^a _. ^s _E ^so _m ^s _e ^d _r ^a _g ^R _In ^o _f ^s _e ^a _c ^, _t ^A _D ^.P _is ^., ₁ ^T ₈ ^e _, ^s ₃ ^h ₄ ^, ₉ ^R _-3 ^.B ₅₁ ^., _. ^{and H} _Tr ⁱ _a ^ll _n ^s, _sm ^S.L _is ^. _s ^, _io ^R _n ^us _o ^s _f ^el _Z ^l, _i ^K _ka ^., _V ^H _ir ^e _u ⁿ _s ⁿ _T ^es _h ^s _r ^e _o ^y _u ^, _g ^M _h ^., _S ^W _ex ⁱ _u ^ll _a ^ia _l ^m _Co ^{s, C., Oster, A.M., Fischer, M., and Mead, P. (2016).} _{Transmission - Continental United States, 2016.n MtaMctW wiRth M Torarbve Mlerosrt taol A Wrkelays R oefp O 6n5g,o 2i1n5g-216.}

I _D ^m _if ^r _f ⁱ _e ^e _r ^, _e ^A _n ^. _t ^, _ia ^M _{l f} ^e _u ^e _n ^k _c ^s, _ti ^J _o ^., _n ^G _al ^u _a ^r _v ^a _i ^r _d ^y _i ^, _ty ^A _o ^., _f ^S _d ^u _e ^k _n ^h _g ^b _u ^a _e ^ta _v ^a _i ^r _r ^, _u ^M _s- ^{., Kitsutani, P., Effler, P., and Zhao, Z. (2007).}

h_{eterologous and previously encountered serotsyppeecsi.fi Jc V Ti-rcoelll 81 cl,o 1n0e0s8 f1o-r10 v0a9ri1a.nt peptides representing} 81

^I _Z ^o _i ^o _ka ^s, _v ^S _ir ^., _u ^M _{s e} ^a _p ^lle _id ^t, _em ^H _i ^. _o ^P _l ^. _o ^{, L} _gy ^ep _a ^a _n ^r _d ^c _r ^G _ec ^o _e ^f _n ^fa _t ^r _e ^t _p ^{, I} _id ^., _e ^G _m ^a _ic ^u _s ^t _. ^h _M ^ier _e ^, _d ^V _ec ^., _in ^C _e ^ar _e ^d _t ^o _m ^so _al ^, _a ^T _di ^. _e ^, _s ^an _in ^d _fe ^H _c ^e _ti ^r _e ⁱ _u ^d _s ^a _e ^, _s ^M ₄ ^. ₄ ⁽ _, ² ₃ ⁰ ₀ ¹ ₂ ⁴-⁾ ₃ ^. ₀ ^C ₇ ^u _. ^rrent _A ^{Kim, Y., Ponomarenko, J., Zhu, Z., Tamang, D., Wang, P., Greenba}

r_{e.s,e Laurcnhd, 4 O0,. W, B5o2u5r-n5e3,0 P..E., et al. (2012). Immune epitope database a} ^u _n ^m _aly ^, _s ^J _i ^. _s ^{, L} _re ^u _s ⁿ _o ^d _u ^e _r ^g _c ^a _e ^a _. ^r _N ^d, _u ^C _cl ^. _e ^, _ic ^Se _a ^t _c ^te _id ^, _s ^L _Z ^a _ik ⁿ _a ^ci _V ^ot _ir ^ti _u ^, _s ^R _i ^. _n ^S. _W ^{, L} _e ^a _s ^m _te ^b _rn ^er _H ^t, _e ^A _m ^.J _i ^. _s ^, _p ^H _he ^o _r ^lo _e, ^d ₂ ⁿ ₀ ^iy ₁ ^, ₅ ^M _{. E} ^., _m ^Sa _e ^a _rg ^ve _I ^d _n ^r _f ^a _e ^, _c ^S _t ^. _D ^{, a} _i ⁿ _s ^d ₂₂ ^S _, ^ig ₉ ⁿ ₃ ^o ₃ ^r _-9 ^L ₃ ^d ₅ ^e _. ^{l, C. (2016). Phylogeny of L} _th ^a _e ^ze _W ^ar _e ^, _s ^H _te ^. _r ^M _n ^. _H ^{, a} _e ⁿ _m ^d _is ^D _p ⁱ _h ^a _e ^m _re ^o _. ⁿ _J ^d _V ^{, M} _iro ^.S _l. ^{. (2016). Zika Virus: New Clinical Syndromes and its Emergence in L} _an ^a _d ^ze _D ^ar _i ^, _a ^H _m ^e _o ^l _n ^en _d, ^M _M ^., _ic ^G _ha ^o _e ^v _l ^e _S ^r _. ^o ₍ ^, ₂ ^J ₀ ^., ₁ ^S ₆ ^m _). ⁱ _A ^th, _M ^A _o ^m _us ^b _e ^er _M ^M _od ^., _e ^P _l ^l _o ^at _f ^t _Z ^{, D} _ik ^e _a ^r _V ^ek _ir ^J _u ^., _s ^F _P ^e _a ^r _t ⁿ _h ^a _o ⁿ _g ^d _e ^e _n ^z _e ^, _s ^E _is ^. _. ^, _C ^M _e ⁱ _l ⁿ _{l H} ^er _o ^{, J} _s ^o _t ⁿ _& ^at _M ^ha _i ⁿ _cr ^J _o ^. _b ^, _e. ^L _Z ⁱ

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l_ln ^{Tang, W., Chai, G., J.A., R.-N., Sheets, N., A.V., T., S., S., and J.G., G.} _{pro In in P ardesusl.t brain shows tropism for neural progenitors and alters} ^L _ef ^o _fi ^p _c ^e _ie ^z _n ^, _t ^C _a ^.B _da ^., _p ^Y _ti ^o _ve ^un _im ^t, _m ^J.S _u ^. _n ^, _i ^H _ty ^e _i ^r _n ^m _d ^e _e ^s _p ^h _e ^, _n ^T _d ^. _e ^, _n ^a _t ⁿ _ly ^d _o ^M _f ^o _ty ^r _p ^an _e ^, _I ^T _in ^.M _te ^. _rf ⁽² _er ⁰ _o ⁰ _n ⁶ _s ⁾ _. ^. _J ^Se _V ⁿ _i ^d _ro ^ai _{l 8} ^v ₀ ^ir _, ^u ₄ ^s ₅ ⁱ ₃ ⁿ ₈ ^fe _-4 ^ct ₅ ⁱ ₄ ^o ₅ ⁿ _. ^{induces M} _Zi ^a _m ^lo _le ⁿ _r ^e _, ^, _R ^R _., ^.W _Ta ^., _lt ^H _on ^o _, ^m _J. ^a _, ⁿ _C ^, _o ^J. _b ^, _b ^C _, ^a _R ^lla _.R ^h _. ^a _, ⁿ _R ^, _u ^M _zi ^. _c ^V _, ^. _I ^, _. ^G _{, e} ^l _t ^as _a ^s _l. ^p ₍ ^o ₂ ^o ₀ ^l ₁ ^-M _6). ^al _Z ^o _i ⁿ _k ^e _a ^, _V ^J., _ir ^D _us ^a _: ^m _M ^o _e ^d _d ^{aran, L., Schneider Ade, B.,} _{Development Challenges. PLoS Negl Trop Dis 10, e0004530. ical Countermeasure} ^{M Enannigsa,d Fa.,A M.,. aMnd., E Rontdhym, Tan.P,. A, N.Li.sa (2la0k0,2 A).., D Cehnugnuseu-tstpiwecaitf,ic S T., V cealulg rhesnp,o Dn.sWes., in Li pberraitpyh,e Dra.H., Green, S.,} _{mononuclear cells obtained prior to secondary dengue viru} ^{l blood} _{Infect Dis 185, 1697-1703. s infections in Thai schoolchildren. J} 82

^M _ce ^a _ll ⁿ _s ^g _t ^a _o ^d _h ^a _e ^, _t ^M _er ^. _o ^M _lo ^., _g ^a _o ⁿ _u ^d _s ^R _se ^o _r ^t _o ^h _t ^m _yp ^a _e ⁿ _s ^, _. ^A _{J I} ^.L _m ^. _m ⁽² _u ⁰ _n ⁰ _o ⁵ _l ^). ₁ ^A ₇₅ ^lt _, ^e ₂ ^re ₆ ^d ₇₆ ^c-^y ₂ ^t ₆ ^o ₈ ^k ₃ ⁱⁿ _. ^{e responses of dengue-specific CD4+ T M} _{P Kll} ⁱ

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D_., ^{Cao, B., Govero, J., Smith, A.M., Fernandez, E., Cabrera, O.H., Garber, C., Noll, M.,} a _{Nmoagguec ahni,d K F.Keta.,l e Dt aelm. (i2se0.1 C6)e.ll Z 1i6k5a, V 1i0r8u1s- I1n0f9e1c.tion during Pregnancy in Mice Causes} ^M _{M K.} ^l

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M_. ^, _, ^{N., Popovic, M., Poljsak-Prijatelj, M., Mraz, J., Kolenc, M., Resman Rus,} e _{Fda 3b7ja4n, 9 V5o1d-9u5se8k., V., et al. (2016). Zika Virus Associated with} ^{M Choanirguknoslrsia,p Aay.,a S,a Jw.,a Dsdeijvnoirrantt,is Sa.,i, D Wu.a,n Xguch, Xin.dNa,., T V.,a Dsaonnawg,a Tth.,an Rao,w Sl.a,n Tdangthawornchaikul, N.,} _{Original antigenic sin and apoptosis in} ^{-Jones, S., et al. (2003).}

9_{21-927. the pathogenesis of dengue hemorrhagic fever. Nat Med 9,} ^M

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^Z _pa ^e _t ^l _h ^lw _o ^e _g ^g _e ^e _n ^r _e ^, _s ^R _is ^. _. ^M _F ^. _r ^, _o ^a _n ⁿ _t ^d _Im ^Sh _m ^re _u ^s _n ^ta _o ^, _l ^S ₅ ^. _, ⁽ ₁ ² ₅ ⁰ ₁ ¹ _. ^{4). Mouse models to study dengue virus immunology and Z} _Ce ^e _l ^l _l ^l _s ^w _C ^eg _a ^e _n ^r, _M ^R _e ^.M _dia ^., _te ^Ta _S ⁿ _h ^g _o ^, _r ^W _t-T ^.W _erm ^{., E} _P ^d _r ^d _o ^y _te ^, _c ^W _tio ^.E _n ^., _a ^K _ga ⁱ _i ⁿ _n ^g _s ^, _t ^K _H ^. _e ^, _t ^S _e ^a _r ⁿ _o ^c _ty ^h _p ^e _i ^z _c ^, _D ^M _e ^.C _ng ^., _u ^a _e ⁿ _V ^d _i ^S _ru ^h _s ^re _R ^st _e ^a _i ^, _n ^S _fe ^. _c ⁽ _t ² _i ⁰ _o ¹ _n ⁵ _i ⁾ _n ^{. C} _M ^D _i ^{8+ T} _{Virol 89, 6494-6505. ce. J} II– Example 2 _E ^{1 Lazear, H. M. & Diamond, M. S. Zika Virus: New Clinical Syndromes and}

1_{6m (e2r0g1e6n)c.e in the Western Hemisphere. Journal of virology, doi:JVI.00252-16 [pii] 10.1128} ⁱ _/ ^ts _JVI.00252- ² _{478, 467472} ^C ₍ ^h ₂ ^o ₀ ^u ₁ ^m _5). ^{et, V. & Despres, P. Dengue and other flavivirus infections. Rev Sci Tech 34, 473- 3} _{century. PLo} ^F _S ^a _n ^y _e ^e _g ^, _le ^O _cte ^. _d ^et _tr ^a _o ^l _p ^. _i ^M _cal ^o _d ^le _is ^c _e ^u _as ^l _e ^a _s ^r ₈ ^e _, ^v _e ^o ₂ ^lu ₆ ^t ₃ ⁱ ₆ ^o _, ⁿ _d ^o _o ^f _i:1 ^Z ₀ ^ik _.1 ^a ₃ ^v ₇ ⁱ ₁ ^r _/ ^u _j ^s _o ^d _u ^u _rn ^ri _a ⁿ _l ^g _.p ⁱ _n ^ts _td ^e _. ^m ₀₀ ^e ₀ ^r ₂ ^g ₆ ^e ₃ ⁿ ₆ ^ce ₍₂ ⁱⁿ ₀₁ ^t ₄ ^h ₎ ^e _. ^{20(th) 4} _{models. Nat} ^C _ur ^u _e ^g ₅ ^o ₃ ^l ₄ ^a _, ^, ₂ ^F ₆ ^. ₇ ^R _-2 ^. ₇ ^et ₁ ^a _, ^l _d ^. _o ^T _i ^h _:1 ^e ₀ ^B _.1 ^r ₀ ^a ₃ ^z ₈ ^il _/ ^ia _n ⁿ _at ^Z _u ⁱ _r ^k _e ^a ₁ ^v ₈ ⁱ ₂ ^r ₉ ^u ₆ ^s ₍ ^s ₂ ^tr ₀ ^a ₁ ⁱⁿ ₆₎ ^c _. ^{auses birth defects in experimental 5} _{Damage and} ^M _F ⁱ _e ⁿ _t ^e _a ^r _l ^, _D ^J. _e ^J _m ^{. e} _i ^t _se ^a _. ^l. _C ^Z _el ⁱ _l ^k ₁ ^a ₆ ^V _5, ^ir ₁ ^u ₀ ^s ₈ ^I ₁ ⁿ-^f ₁ ^e ₀ ^c ₉ ^ti ₁ ^o _, ⁿ _do ^du _i:1 ^ri ₀ ⁿ _. ^g ₁₀ ^P ₁ ^r ₆ ^e _/ ^g _j ⁿ _.c ^a _e ⁿ _l ^c _l. ^y ₂₀ ⁱⁿ ₁₆ ^M _.0 ⁱ ₅ ^c _. ^e ₀₀ ^C ₈ ^au ₍₂ ^se ₀ ^s ₁₆ ^P ₎ ^l _. ^{acental 6} _Microcephal ^L _y ⁱ _i ^, _n ^C _M ^{. e} _i ^t _c ^a _e ^l _. ^. _C ^Z _e ⁱ _l ^k _l ^a _ste ^V _m ^ir _c ^u _el ^s _l ^D ₁₉ ⁱ _, ^sr ₁ ^u ₂ ^p ₀ ^t-^s ₁₂ ^N ₆ ^e _, ^u _d ^r _o ^a _i ^l _:1 ^P ₀ ^r _. ^o ₁ ^g ₀ ^e ₁ ⁿ ₆ ⁱ _/ ^to _j. ^r _st ^D _em ^ev _.2 ^e ₀ ^lo ₁ ^p ₆ ^m _.0 ^e ₄ ⁿ _.0 ^t ₁ ^a ₇ ⁿ ₍ ^d ₂ ^L ₀₁ ^e ₆ ^ad _). ^{s to 7} _{report, Frenc} ^O _h ^e _P ^hl _o ^e _l ^r _y ^, _n ^E _es ^. _i ^e _a ^t _, ^a _D ^l. _e ^Z _c ⁱ _e ^k _m ^a _b ^v _e ^ir _r ^u ₂ ^s ₀ ⁱ ₁ ⁿ ₃ ^fe _. ^c _E ^ti _u ^o _r ⁿ _{o s} ^c _u ^o _r ^m _vei ^p _ll ^l _a ⁱ _n ^c _c ^a _e ^te _: ^d _bu ^b _ll ^y _et ^G _in ^{uillain-Barre syndrome--case} _{transmissibles = European communicable disease bulletin 19 (2014). Europeen sur les maladies} ⁸ _doi:10.1056/ ^D _N ^’O _EJ ^r _M ^ten _c1 ^z ₆ ^io ₀ ^, ₄ ^E ₄₄ ^. ₉ ^et ₍ ^a ₂ ^l. ₀ ^E ₁₆ ^v ₎ ⁱ _. ^{dence of Sexual Transmission of Zika Virus. N Engl J Med,}

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1₃ ^. _{84(9954): p} ^C _. ^a ₁ ^o ₅-₇ ^L ₁ ^o-^r ₂ ^m _. ^{eau, V.M. and D. Musso, Emerging arboviruses in the Pacific. Lancet, 2014. 2} _M ^. _{ed, 2009.} ^D ₃₆ ^u ₀ ^f ₍ ^f ₂ ^y ₄ ^, ₎ ^M _{: p} ^.R _.2 ^., ₅ ^e ₃ ^t ₆ ^a-^l ₄ ^., ₃ ^Z _. ^{ika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J 3} _in ^. _{French Pol} ^C _yn ^a _e ^o _si-_a ^L _: ^o _a ^r _c ^m _as ^e _e- ^a _c ^u _on ^, _t ^V _ro ^. _l ^M _st ^. _u ^, _d ^e _y ^t _. ^a _L ^l. _a ^, _n ^G _ce ^u _t ^il _, ^la ₂ ⁱ ₀ ⁿ ₁ ^-B ₆ ^a _. ^r ₃ ^r ₈ ^e ₇ ^S ₍ ^y ₁ ⁿ ₀ ^d ₀ ^ro ₂ ^m ₇₎ ^e _: ^o _p ^u _. ^tb ₁ ^r ₅ ^ea ₃ ^k _1- ^a ₁ ^s ₅ ^s ₃ ^oc ₉ ^ia _. ^{ted with Zika virus infection 4} ₉ ^. _51-8. ^{Mlakar, J., et al., Zika Virus Associated with Microcephaly. N Engl J Med, 2016.374(10): p. 5} _A ^. _bnormalities ^D _{. N} ^rigg _E ^e _n ^r _g ^s, _{l J} ^R _M ^.W _e ^. _d ^, _, ^e ₂ ^t ₀ ^a ₁ ^l ₆ ^., _. ^Z ₃₇ ^ik ₄ ^a ₍₂ ^V ₂₎ ⁱ _: ^ru _p ^s _.2 ^In ₁ ^f ₄ ^ec ₂ ^ti-^o ₅ ⁿ _1. ^{with Prolonged Maternal Viremia and Fetal Brain 6} _A ^. _{mericas, Ma} ^H _y ^e ₂ ⁿ ₀ ⁿ ₁ ^e ₅ ^s-^s _J ^e _a ^y _n ^, _u ^M _ary ^., ₂ ^M ₀₁ ^. ₆ ^F _. ^is _M ^ch _M ^er _W ^{, a} _R ^nd _M ^J _o ^.E _rb ^{. S} _M ^ta _o ^p _r ^l _t ^e _a ^s _l ^, _W ^Zi _k ^k _ly ^a _R ^V _e ⁱ _p ^ru _, ^s ₂ ^S ₀ ^p ₁ ^r ₆ ^e _. ^ad ₆ ^s ₅ ^t ₍ ^o ₃₎ ^N _{: p} ^ew _.5 ^A _5- ^r ₈ ^ea _. ^{s - Region of the 7} _E ^. _{ngl J Med,} ^R ₂ ^a ₀ ^s ₁ ^m ₆ ^u _. ^s ₃ ^s ₇ ^e ₄ ⁿ ₍ ^, ₂ ^S ₀ ^. ₎ ^A _{: p} ^., _. ^e ₁ ^t ₉ ^a ₈ ^l ₁ ^., _-7 ^Z _. ^{ika Virus and Birth Defects--Reviewing the Evidence for Causality. N 8} _A ^. _utoimmun ^M _{, 2} ^u ₀ ⁿ ₁ ^o ₂ ^z _.- ₃ ^S ₈ ^u ₍ ^a ₂ ⁿ-^o ₃ ^, _): ^A _p ^. _. ^, _J ^e ₁ ^t ₀ ^a ₃ ^l-^., _8. ^{Regulatory T cells protect from autoimmune arthritis during pregnancy. J 9} _pr ^. _{imary infect} ^C _io ^o _n ⁿ _d ^s _u ^t _r ^a _in ⁿ _g ^ti _p ⁿ _r ^, _eg ^C _na ^.M _nc ^. _y ^, _. ^e _J ^t _I ^a _m ^l. _m ^{, N} _un ^or _o ^m _l, ^a ₂ ^l ₀ ^es ₀ ^t ₇ ^ab _. ^l ₁ ^is ₇ ^h ₉ ^m ₍ ^e ₇ ⁿ ₎ ^t _{: p} ^of _. ^v ₄ ⁱ ₃ ^ru ₈ ^s ₃ ^-s-^p ₉ ^e _. ^{cific memory CD8 T cell pool following} 92

¹ ₂ ⁰ ₀ ^. _13.13(1): ^E _p ^r _. ^le ₂ ^b ₃ ^a _-3 ^ch _3. ^{er, A., Mechanisms of T cell tolerance towards the allogeneic fetus. Nat Rev Immunol, 1} ₂ ¹ ₀ ^. _17.21(5): ^A _p ^r _. ^o ₅ ^r ₆ ^a ₁ ^,- ^N ₅₆ ^., ₇ ^e _. ^{t al., Microbial Vertical Transmission during Human Pregnancy. Cell Host Microbe, 1} _P ² _re ^. _{gnancy. Tr} ^K _en ⁱⁿ _d ^g _s ^, _M ^N _i ^. _c ^J. _r ^C _o ^. _b ^, _i ^M _ol, ^.M ₂₀ ^. ₁ ^T ₇ ^e _. ⁱ ₂ ^x ₅ ^ei ₍ ^r ₉ ^a ₎ ^, _: ^a _p ⁿ _. ^d ₇₀ ^S ₁ ^.- ^M ₇₀ ^a ₂ ^h _. ^{alingam, Zika Virus: Mechanisms of Infection During 1} _m ³ _ic ^. _{robial path} ^A _og ⁿ _e ^d _ns ^er _b ^s _y ^, _d ^A _ec ^. _i ^P _du ^., _al ^e _s ^t _tr ^a _o ^l _m ^., _a ^C _{l c} ^u _e ^r _l ^r _ls ^e _. ⁿ _A ^t _m ^con _J ^ce _R ^pt _e ^s _p ⁱ _r ⁿ _od ^ma _Im ^ter _m ^na _u ^l- _n ^fe _o ^ta _l, ^l ₂ ^im ₀₁ ^m ₇ ^u _. ⁿ ₇ ^o ₇ ^lo ₍ ^g ₃ ^y ₎ ^: _. ^{Recognition and response to 1} _F ⁴ _et ^. _{al Demise.} ^M _C ⁱⁿ _el ^e _l, ^r, ₂₀ ^J. ₁ ^J. ₆ ^, _. ^e ₁ ^t ₆ ^a ₅ ^l ₍ ^. ₅ ^, ₎ ^Z _: ⁱ _p ^k _. ^a ₁₀ ^V ₈ ⁱ ₁ ^ru _-9 ^s ₁ ^I _. ^{nfection during Pregnancy in Mice Causes Placental Damage and 1} _In ⁵ _f ^. _{ection. Cel} ^Y _l, ^o ₂ ^c ₀ ^k ₁ ^e ₆ ^y _. ^, ₁ ^L ₆₆ ^.J ₍ ^. ₅ ^, ₎ ^e _: ^t _p. ^al ₁ ^., ₂₄ ^V ₇ ^a _-1 ^gi ₂ ⁿ ₅ ^a ₆ ^{l E} _e4 ^x _. ^{posure to Zika Virus during Pregnancy Leads to Fetal Brain 1} _N ⁶ _a ^. _{ture, 201} ^C _6. ^u ₅ ^g ₃ ^o ₄ ^la ₍₇ ^, ₆ ^F ₀ ^.R ₆₎ ^. _: ^, _p ^e _. ^t ₂ ^a ₆ ^l. ₇ ^,- ^T ₇₁ ^he _. ^{Brazilian Zika virus strain causes birth defects in experimental models. 1} ₂ ⁷ ₀ ^. _16.20(1): ^Q _p ^u _. ⁱ ₈ ^c ₃ ^k-^e ₉ ^, ₀ ^K _. ^{.M., et al., Zika Virus Infects Human Placental Macrophages. Cell Host Microbe, 1} _In ⁸ _s ^. _{ight, 201} ^J ₆ ^u _. ^r ₁ ^a ₍ ^d ₁ ^o ₃ ^, _). ^{K.A., et al., Zika virus productively infects primary human placenta-specific macrophages. JCI 1} _M ⁹ _e ^. _{d, 2016.} ^M ₃₇ ^y ₅ ^s ₍ ^o ₅ ^r ₎ ^e _: ^k _p ^a _. ^r ₄ ^, ₈ ^I ₁ ^.U _-4 ^. _. ^{and M.S. Diamond, Modeling Zika Virus Infection in Pregnancy. N Engl J 2} _M ⁰ _i ^. _{ce. Cell St} ^L _em ^{i, C} _C ^. _e ^, _l ^e _l, ^t ₂ ^a ₀ ^l. ₁ ^, ₆ ^Z _. ⁱ ₁ ^k ₉ ^a ₍₁ ^V _): ⁱ _p ^ru _. ^s ₁ ^D ₂₀ ^is-^r ₆ ^u _. ^{pts Neural Progenitor Development and Leads to Microcephaly in 2} _de ¹ _v ^. _{elopment o} ^W _{f of} ^u _fs ^, _p ^K _rin ^. _g ^Y _m ^., _ic ^e _e ^t _{. C} ^al _e ^., _ll ^V _R ^e _e ^r _s ^ti _, ^ca ₂ ^l ₀₁ ^tr ₆ ^a _. ⁿ ₂ ^sm ₆₍ ^is ₆ ^s ₎ ⁱ _: ^on _p. ^o ₆ ^f ₄ ^Z ₅ ⁱ-^k ₅ ^a _4. ^{virus targeting the radial glial cells affects cortex}

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² _p ² _. ^. _476-81. ^{Halstead, S.B., Pathogenesis of dengue: challenges to molecular biology. Science, 1988.239(4839): 2} _an ³ _d ^. _{in vivo an} ^G _d ^o _st ⁿ _ra ^c _t ^a _e ^l _g ^v _ie ^e _s ^z _f ^, _o ^A _{r p} ^.P _re ^. _v ^, _e ^e _n ^t _ti ^a _on ^l. _. ^, _P ^M _r ^o _o ⁿ _c ^oc _N ^lo _a ⁿ _t ^a _l ^l _A ^an _c ^t _a ^ib _d ^od _S ^y _c-_i ^m _U ^edi _S ^at _A ^ed _, ^e ₂ ⁿ ₀ ^h ₀ ^a ₇ ⁿ _. ^ce ₁ ^m ₀ ^e ₄ ⁿ ₍ ^t ₂ ^o ₂ ^f _): ^de _p ⁿ _. ^gu ₉ ^e ₄₂ ^vi ₂ ^r-^u ₇ ^s _. ^{infection in vitro 2} _m ⁴ _o ^. _{dification. P} ^B _L ^al _o ^si _S ^tis _P ^, _at ^S _h ^.J _o ^., _g, ^e ₂ ^t ₀ ^a ₁ ^l ₀ ^., _.6 ^L ₍ ^e ₂ ^t ₎ ^h _: ^a _p ^l _. ^a _e ⁿ ₁ ^ti ₀ ^b ₀ ^o ₀ ^dy ₇₉ ^e ₀ ⁿ _. ^{hancement of dengue disease in mice is prevented by Fc 2} _en ⁵ _d ^. _{othelial cel} ^Z _ls ^e _i ^l _n ^lw _a ^eg _m ^e _o ^r _u ^, _se ^R _m ^.M _od ^., _el ^T _of ^.R _a ^. _nti ^P _b ^r _o ^e _d ^s _y- ^t _i ^w _nd ^o _u ^o _c ^d _ed ^, _s ^a _e ^{nd S. Shresta, Enhanced infection of liver sinusoidal} _{128-39. vere dengue disease. Cell Host Microbe, 2010.7(2): p.} ² _S ⁶ _ci ^. _{ence, 201} ^K _7. ^{atzelnick, L.C., et al., Antibody-dependent enhancement of severe dengue disease in humans. 2} _lin ⁷ _k ^. _{ed protecti} ^W _ve ^e _r ⁱ _o ^s _l ^k _e ^o _fo ^p _r ^f _C ^{, D} _D8 ^., ₊ ^et _T ^a _c ^l. _e ^, _lls ^C _. ^o _P ^m _r ^p _o ^r _c ^eh _N ^en _a ^s _t ^iv _l ^e _A ^a _c ⁿ _a ^a _d ^ly _S ^si _c ^s _i ^o _U ^{f d} _S ^en _A ^gu _, ^e ₂₀ ^vi ₁ ^r ₃ ^us _.- ₁ ^sp ₁ ^e ₀ ^ci ₍ ^f ₂ ^ic ₂₎ ^r _: ^es _p ^p _. ^on _E ^se ₂ ^s ₀₄ ^su ₆ ^p-^p ₅ ^o ₃ ^r _. ^{ts an HLA- 2} _A ⁸ _s ^. _{sociated W} ^W _ith ^ei _D ^sk _is ^o _t ^p _in ^f _c ^, _t ^D _Pa ^., _tt ^e _er ^t _n ^a _s ^l. _o ^, _f ^H _Pr ^u _o ^m _te ^a _in ⁿ _T ^C _a ^D _rg ⁸ _et ⁺ _s. ^T _J- _I ^C _n ^e _f ^l _e ^l _c ^R _t ^e _D ^sp _i ^o _s ⁿ _, ^s ₂ ^e ₀ ^s ₁ ^A ₅ ^g _. ^a ₂ ⁱⁿ ₁ ^s ₂ ^t ₍₁ ^th ₁ ^e _): ⁴ _p ^D _.1 ^en ₇ ^g ₄ ^u ₃ ^e- ^V ₅₁ ⁱ _. ^{rus Serotypes Are 2} _A ⁹ _s ^. _{sociated wi} ^d _th ^e _a ^A _M ^lw _em ^is _o ^, _ry ^R _P ^., _D ^e _-1 ^t ₊ ^al _P ^., _he ^I _n ^m _ot ^m _yp ^u _e ⁿ _. ^o _J ^do _V ^m _i ⁱ _r ⁿ _o ^a _l ⁿ _, ^t ₂₀ ^D ₁₆ ^en _. ^g ₉ ^u ₀ ^e ₍₉ ^V _): ^ir _p ^u _. ^s- ₄ ^S ₇ ^p ₇ ^e ₁ ^ci-^fi ₉ ^c _. ^{CD8+ T Cell Responses Are 3} _ce ⁰ _ll ^. _{s associated} ^W _w ^e _it ^is _h ^k _p ^o _r ^p _ot ^f _e ^, _ct ^D _ive ^., _im ^et _m ^a _u ^l. _n ^, _it ^D _y. ^e _P ^ng _r ^u _o ^e _c ^v _N ^iru _a ^s _tl ⁱⁿ _A ^fe _c ^ct _a ⁱ _d ^on _S ^e _c ^l _i ^ic _U ^its _S ^hi _A ^gh _, ^ly ₂₀ ^p ₁ ^ol ₅ ^a _. ^ri ₁ ^z ₁ ^ed ₂₍ ^C ₃₁ ^X _): ³ _p ^C _. ^R _E ¹ ₄ ⁺ ₂₅ ^cy ₆ ^t-^o ₆ ^to ₃ ^x _. ^{ic CD4+ T 3} _pr ¹ _o ^. _{tect against} ^S _c ^im _lin ^o _ic ⁿ _al ^-L _de ^o _n ^r _g ⁱ _u ^e _e ^r _. ^e _S ^, _c ^E _i ^. _T ^, _r ^e _a ^t _ns ^a _l ^l. _M ^{, I} _e ⁿ _d ^c _, ^re ₂ ^a ₀ ^se ₁ ^d _7. ^a ₉ ^d ₍ ^a ₄ ^p ₀ ^ti ₅ ^ve _). ^{immune responses and proper feedback regulation 3} _T ² _r ^. _{ansl Med,} ^R ₂ ^iv ₀ ⁱ ₁ ⁿ ₅ ^o _. ^, ₇ ^L ₍₂ ^., ₇ ^e ₈ ^t _): ^a _p ^l., _. ^V ₂₇ ⁱ ₈ ^ru _ra ^s- ₃ ^s ₅ ^pe _. ^{cific T lymphocytes home to the skin during natural dengue infection. Sci 3} _In ³ _f ^. _{ection. EB} ^E _io ^l _M ^on _e ^g _di ^N _cin ^go _e, ⁿ ₂ ^o ₀ ^, ₁ ^A _6. ^., ₁ ^e ₃ ^t _: ^a _p ^l _. ^., ₂ ^P ₈ ^r ₄ ^o-^t ₂ ^ec ₉ ^ti ₃ ^v _. ^{e Role of Cross-Reactive CD8 T Cells Against Dengue Virus} 94

³ _D ⁴ _e ^. _{ngue Virus} ^Z _R ^el _e ^l _i ^w _nf ^e _e ^g _ct ^e _io ^r _n ^{, R} _in ^.M _M ^. _i ^, _ce ^e _. ^t _J ^a _V ^l. _i ^, _ro ^C _l ^D _{, 2} ⁸ ₀ ⁺ ₁₅ ^T _.8 ^C ₉₍ ^e ₁ ^ll ₂ ^s ₎ ^C _{: p} ^an _.6 ^M ₄₉ ^e ₄ ^d-^ia ₅ ^t ₀ ^e ₅ ^S _. ^{hort-Term Protection against Heterotypic 3} ₁ ⁵ ₈ ^. _{2(8): p.48} ^Y ₆ ^a ₅ ^u-^c ₇ ^h ₃ ^, _. ^{L.E., et al., A protective role for dengue virus-specific CD8+ T cells. J Immunol, 2009. 3} _ce ⁶ _ll ^. _{or antibod} ^Y _y ^a _re ^u _s ^c _p ^h _on ^, _s ^L _es ^.E _bu ^., _t ^e _c ^t _on ^a _t ^l _r ^. _i ^, _b ^C _ut ^D _e ⁴ _to ⁺ _pr ^T _ote ^c _c ^e _t ^l _i ^l _o ^s _n ^ar _a ^e _ft ⁿ _er ^ot _va ^re _c ^q _ci ^u _n ⁱ _a ^r _t ^e _i ^d _on ^fo _. ^r _J ^t _I ^h _m ^{e i} _m ^nd _u ^u _n ^ct _o ^io _l ⁿ _{, 2} ^o ₀ ^f ₁ ^d ₀ ^en _. ^g ₁ ^u ₈ ^e ₅ ^v ₍ ⁱ ₉ ^ru _): ^s- _p ^sp _. ^e ₅ ^ci ₄ ^fi ₀ ^c ₅ ^C _-1 ^D ₆ ⁸ _. ^{+ T 3} _re ⁷ _p ^. _{lication and} ^Pr _p ^e _r ^s _ev ^t _e ^w _n ^o _ts ^o _p ^d _a ^, _ra ^T _ly ^. _s ^R _is ^., _in ^et _IF ^a _N ^l., _-a ^G _lp ^a _h ^m _a ^m _/b ^a _et ⁱ _a ^nt _r ^e _e ^r _c ^f _e ^e _p ^r _t ^o _o ⁿ _r-d ^(I _ef ^F _ic ^N _ien ^-g _t ^a _m ^m _i ^m _ce ^a _. ⁾ _{J V} ^rec _ir ^ep _o ^t _l ^o _, ^r ₂₀ ^re ₁ ^st ₂ ^r _. ^ic ₈ ^ts ₆₍ ^s ₂ ^y ₃ ^st ₎ ^e _: ^m _p ^ic _.1 ^d ₂ ^en ₅ ^g ₆ ^u ₁ ^e _-7 ^vi ₀ ^r _. ^{us 3} _de ⁸ _n ^. _{gue disease} ^Z _in ^ell _m ^w _ic ^e _e ^g _. ^e _J ^r _I ^, _m ^R _m ^.M _u ^. _n ^, _o ^e _l ^t _, ^a ₂ ^l ₀ ^., ₁ ^C _4. ^D ₁ ⁸ ₉ ⁺ ₃₍₈ ^T _): ^c _p ^el _. ^ls ₄₁ ^p ₁ ^re ₇ ^v-^e ₂ ⁿ ₄ ^t _. ^{antigen-induced antibody-dependent enhancement of 3} _va ⁹ _c ^. _{cine candid} ^Z _at ^e _e ^l _. ^lw _PL ^eg _o ^e _S ^r, _P ^R _at ^. _h ^M _o ^. _g ^, _, ^e ₂ ^t ₀ ^a ₁ ^l ₃ ^., _. ^R ₉₍ ^o ₁ ^le ₀₎ ^o _: ^f _p ^h _. ^u _e ^m ₁₀ ^or ₀ ^a ₃ ^l ₇ ^v ₂ ^e ₃ ^rs _. ^{us cellular responses induced by a protective dengue 4} _C ⁰ _o ^. _{mmun, 2} ^D ₀₁ ^u ₆ ^d _. ^le ₇ ^y _: ^, _p. ^D ₁ ^. ₂ ^M ₂₀ ^., _4. ^{et al., A rhesus macaque model of Asian-lineage Zika virus infection. Nat 4} _V ¹ _i ^. _{rus Infectio} ^E _n ^l _i ^o _n ⁿ _M ^g _i ^N _ce. ^go _C ⁿ _e ^o _ll ^, _H ^A _o ^., _s ^e _t ^t _M ^al _i ^. _c ^, _r ^M _ob ^a _e ^p _, ^p ₂ ⁱⁿ ₀ ^g ₁ ^a ₇ ⁿ _. ^d ₂₁ ^R ₍ ^o ₁ ^l ₎ ^e _: ^o _p ^f _. ^t ₃ ^h ₅ ^e- ^C ₄ ^D _6. ^{8+ T Cell Response During Primary Zika 4} _to ² _Z ^. _{ika virus.} ^P _P ^ri _r ^y _o ^a _c ^m _N ^va _a ^d _tl ^a _A ^{, L} _c ^. _a ^, _d ^et _S ^a _c ^l _i ^., _U ^H _S ^um _A ^a _, ⁿ ₂₀ ^a ₁ ^nt ₆ ^ib _. ^o ₁ ^d ₁ ^y ₃ ^r ₍ ^e ₂ ^s ₈ ^po _): ^ns _p ^e _. ^s ₇ ^a ₈ ^ft ₅ ^er _2- ^d ₇ ^en _. ^{gue virus infection are highly cross-reactive 4} _in ³ _fe ^. _{ction. Scie} ^S _n ^t _c ^e _e ^tt _, ^le ₂ ^r ₀ ^, ₁₆ ^K _. ^. ₃ ^, ₅ ^e ₃ ^t ₍₆ ^a ₃ ^l. ₀ ^, ₁ ^S _): ^pe _p ^ci _. ^fi ₈ ^ci ₂ ^t ₃ ^y, _-6 ^c _. ^{ross-reactivity, and function of antibodies elicited by Zika virus 4} _S ⁴ _ci ^. _{ence, 201} ^B ₇ ^a _. ^r ₃ ^d ₅ ⁱⁿ ₆₍ ^a ₆ ^, ₃ ^S ₃ ^.V ₄₎ ^. _: ^, _p ^et _. ^a ₁ ^l ₇ ^., ₅ ^E _-1 ⁿ ₈ ^h ₀ ^a _. ^{ncement of Zika virus pathogenesis by preexisting antiflavivirus immunity. 4} _in ⁵ _fe ^. _{ction with} ^D _zi ^e _k ^j _a ⁿⁱ _v ^r _i ^a _ru ^tt _s ⁱ _. ^s _N ^ai, _at ^W _Im ^., _m ^et _u ^a _n ^l. _o ^, _l ^D _{, 2} ^en ₀ ^g ₁ ^u ₆ ^e _. ^v ₁ ⁱ ₇ ^ru ₍₉ ^s ₎ ^s _: ^e _p ^ro _. ^-c ₁ ^r ₁ ^os ₀ ^s ₂ ^-r-^e ₈ ^a _. ^{ctivity drives antibody-dependent enhancement of}

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⁴ _E ⁶ _n ^. _{hance Zik} ^C _a ^h _In ^a _f ^r _e ^l _c ^e _ti ^s _o ^, _n ^A _In ^.S _V ^{. a} _it ⁿ _ro ^d _{. P} ^R _L ^.C _oS ^{. C} _C ^h _u ^r _r ⁱ _r ^s _, ^to ₂₀ ^ff ₁ ^e ₆ ^r _. ^so ₈ ⁿ _. ^{, Utility of a Dengue-Derived Monoclonal Antibody to 4} _fr ⁷ _om ^. _Dengue ^S _P ^w _at ^a _ie ⁿ _n ^s _ts ^tr _A ^om _re ^, _P ^J _r ^. _o ^A _te ^. _c ^, _ti ^e _v ^t _{e a} ^a _g ^l. _a ^, _in ^D _st ^en _Z ^g _i ^u _k ^e _a ^V _V ⁱ _i ^r _r ^u _u ^s _s. ^E _M ^nv _B ^e _i ^l _o ^op _, ^e ₂ ^D ₀₁ ⁱ ₆ ^m _. ^er ₇₍ ^E ₄₎ ^p _. ^{itope Monoclonal Antibodies Isolated 4} _D ⁸ _e ^. _{ngue Virus} ^K _S ^a _e ^w _ro ⁱ _t ^e _y ^c _p ^k _e ⁱ ₂ ^{, A} _Re ^.B _pl ^. _ica ^a _t ⁿ _io ^d _{n I} ^R _n ^.C _V ^. _it ^C _ro ^h _. ^r _J ^is _I ^t _n ^o _f ^f _e ^f _c ^e _t ^rs _D ^on _is ^, _{, 2} ^Z ₀ ^ik ₁₆ ^a _. ^V ₂₁ ⁱ ₄ ^ru ₍₉ ^s- ₎ ^I _: ⁿ _p ^du _. ^c ₁ ^e ₃ ^d ₅ ^A _7- ⁿ ₁ ^t ₃ ^ib ₆ ^o ₀ ^d _. ^{y Response Enhances 4} _de ⁹ _n ^. _{gue virus c} ^W _ros ^e _s- ⁿ _r ^, _ea ^J _c ^. _t ^, _iv ^e _e ^t _C ^a _D ^l., ₈ ^I ₊ ^den _T ^tif _c ⁱ _e ^c _l ^a _ls ^t _. ^io _N ⁿ _a ^o _t ^f _M ^Zi _i ^k _cr ^a _o ^v _b ^ir _io ^us _l, ^e ₂ ^p ₀ ^it ₁ ^o ₇ ^pe _. ^s ₂ ^r _: ^e _p ^ve _. ^a ₁ ^ls ₇₀ ^im ₃ ^m _6. ^{unodominant and protective roles for 5} _V ⁰ _ir ^. _{ol, 2017.} ^{Grifoni, A., et al., Prior Dengue virus exposure shapes T cell immunity to Zika virus in humans. J 5} _V ¹ _a ^. _{ccinated A} ^P _g ^a _a ^q _in ^u _st ⁱⁿ _D ^-P _en ^ro _gu ^u _e ^lx _V ^, _i ^D _ru ^. _s ^, _. ^e _P ^t _a ^a _th ^l. _o ^, _g ^T- _I ^c _m ^ell _m ^R _u ^e _n ^sp _, ^o ₂ ⁿ ₀ ^se ₁ ^s ₇ ⁱ _. ⁿ ₂ ^I ₍ ⁿ ₂ ^d _): ^iv _p ^id _. ^u ₂ ^a ₇ ^ls _4- ^I ₂ ⁿ ₉ ^fe ₂ ^ct _. ^{ed with Zika Virus and in Those 5} _vi ² _ru ^. _{s challenge} ^W _{. N} ^en _a ^, _t ^J _C ^., _o ^e _m ^{t a} _m ^l. _u ^, _n ^D _, ^e ₂ ⁿ ₀ ^gu ₁ ^e _7. ^vi ₈ ^ru ₍₁ ^s- ₎ ^r _: ^ea _p ^c _. ^ti ₁ ^v ₄ ^e ₅ ^C ₉ ^D _. ^{8+ T cells mediate cross-protection against subsequent Zika 5} _to ³ _d ^. _{engue viru} ^P _s. ^a _N ^nt _a ^o _t ^ja _C ^, _o ^P _m ^., _m ^et _u ^a _n ^l. _, ^, ₂ ^Z ₀ ⁱ ₁ ^k ₇ ^a _. ^v ₈ ⁱ _: ^ru _p ^s _. ^p ₁ ^a ₅ ^t ₆ ^ho ₇ ^g ₄ ^e _. ^{nesis in rhesus macaques is unaffected by pre-existing immunity 5} _m ⁴ _a ^. _{caques. PL} ^M _oS ^cC _P ^r _a ^a _t ^c _h ^k _o ^e _g ⁿ _, ^, ₂ ^M ₀₁ ^. ₇ ^K _. ^. ₁ ^, ₃ ^e ₍ ^t ₈₎ ^a _: ^l. _p ^, _. ^I _e ^m ₁ ^p ₀ ^a ₀ ^ct ₆₄ ^o ₈ ^f ₇ ^p _. ^{rior flavivirus immunity on Zika virus infection in rhesus 5} _In ⁵ _f ^. _{ection. J V} ^H _iro ^u _l ^a _, ⁿ ₂ ^g ₀ ^, ₁ ^H _7. ^{., et al., CD8+ T Cell Immune Response in Immunocompetent Mice during Zika Virus 5} _im ⁶ _m ^. _{unity to v} ^S _ir ^w _us ^a _e ⁱ _s ⁿ _. ^, _N ^S _a ^. _t ^L _R ^., _ev ^K _I ^.K _m ^. _m ^M _un ^c _o ^K _l, ⁱⁿ ₂ ^s ₀ ^t ₁ ^ry ₂ ^, _.1 ^a ₂ ⁿ ₍ ^d _2): ^T _p ^.M _.1 ^. ₃₆ ^S-^t ₄ ^ru ₈ ^t _. ^{t, Expanding roles for CD4(+) T cells in 5} _su ⁷ _b ^. _{unit 1 (} ^{Sheehan, K.C., et al., Blocking monoclonal antibodies specific for mouse IFN-alpha/beta receptor} _{2006.26(1I1F):N pA.8R0-41-)1 f9ro.m mice immunized by in vivo hydrodynamic transfection. J Interferon Cytokine Res,} 96

⁵ _ac ⁸ _u ^. _{te West N} ^P _il ⁱ _e ⁿ _v ^t _i ^o _ru ^, _s ^A _in ^.K _fec ^. _t ^, _io ^e _n ^t _. ^a _P ^l., _L ^A _oS ^te _P ^m _a ^p _th ^or _o ^a _g ^l _, ^ro ₂ ^l ₀ ^e ₁ ^o ₁ ^f _. ^ty ₇ ^p ₍ ^e ₁₂ ^I ₎ ⁱ _: ⁿ _p ^te _. ^rf _e ^e ₁ ^ro ₀ ⁿ ₀₂ ^si ₄ ^gn ₀ ^a ₇ ^l _. ^{ing in CD8+ T cell maturation during 5} _S ⁹ _en ^. _{se of DNA} ^Ag _b ^u _y ^ir _c ^r _G ^e _A ^{, S} _S ^. _. ^a _J ⁿ _V ^d _i ^A _ro ^. _l, ^F ₂ ^e ₀ ^rn ₁ ^a ₇ ⁿ _. ^d ₉ ^e ₁ ^z ₍₁ ^-S ₄ ^e _). ^{sma, Collateral Damage during Dengue Virus Infection: Making 6} _in ⁰ _fe ^. _{ctions. J R} ^C _ep ^re _r ^s _o ^p _d ^o _I ^, _m ^A _m ^.C _u ^. _n ^, _o ^e _l ^t _{, 2} ^al ₀ ^., ₁₇ ^C _. ^y ₁ ^to ₁ ^t ₉ ^ox _: ⁱ _p ^c _. ^p ₈ ^o ₅ ^te-ⁿ ₉ ^t ₀ ^ia _. ^{l of decidual NK cells and CD8+ T cells awakened by 6} _m ¹ _a ^. _ternal-fetal ^N _in ^a _t ⁿ _er ^c _fa ^y _c ^, _e. ^P _S ^., _ci ^e _e ^t _nc ^a _e ^l. _, ^, ₂ ^C ₀₁ ^h ₂ ^em _. ^o ₃ ^k ₃ ⁱ ₆ ⁿ ₍ ^e ₆₀ ^ge ₈ ⁿ ₆ ^e _): ^s _p ^ile _. ⁿ ₁ ^c ₃ ⁱⁿ ₁ ^g _7- ⁱⁿ ₂₁ ^d _. ^{ecidual stromal cells limits T cell access to the 6} _of ² _R ^. _{ecife, Bra} ^B _zi ^r _l. ^a _A ^ga _c ^, _t ^C _a ^. _T ^, _r ^e _o ^t _p ^a _, ^l., ₂₀ ^S ₁ ^er ₀ ^o _. ^pr ₁ ^e ₁ ^v ₃ ^al ₍ ^e ₃ ⁿ ₎ ^c _: ^e _p ^a _. ⁿ ₂ ^d ₃ ^r ₄ ^is-^k ₄₀ ^fa _. ^{ctors for dengue infection in socio-economically distinct areas 6} _fa ³ _ct ^. _{ors in Bra} ^d _zi ^e _l: ^A _fin ^r _a ^a _l ^u _r ^j _e ^o _p ^, _or ^T _t ^. _o ^V _f ^. _a ^B _c ^. _a ^, _s ^e _e- ^t _co ^a _n ^l _t ^., _ro ^A _{l s} ^s _t ^s _u ^o _d ^c _y ^ia _. ^t _L ^io _a ⁿ _nc ^b _e ^et _t ^w _I ^e _n ^en _fe ^m _ct ^ic _D ^roc _is ^ep _, ^h ₂ ^a ₀ ^l ₁ ^y, _7. ^{Zika virus infection, and other risk 6} ₄ ⁴ _. ^{. Johansson, M.A., et al., Zika and the Risk of Microcephaly. N Engl J Med, 2016.375(1): p.1- 6} _J ⁵ _R ^. _{eprod Im} ^v _m ^an _un ^E _o ^g _l ^m _{, 2} ^o ₀ ⁿ ₁ ^d ₆ ^, _. ^A ₁₁ ^., ₃ ^e _: ^t _p ^a _. ^l. ₁ ^,- ^T ₈ ^h _. ^{e possible role of virus-specific CD8(+) memory T cells in decidual tissue. 6} _im ⁶ _m ^. _{une respo} ^L _ns ⁱ _e ^ss _t ^a _o ^u _p ^e _r ^r _eg ^, _n ^D _an ^. _c ^, _y ^M _{? P} ^.D _la ^. _ce ^K _n ⁱ _t ^l _a ^b _, ^y ₂ ^, ₀ ^a ₁ ⁿ ₇ ^d _. ^{P. Moss, Maternal effector T cells within decidua: The adaptive 6} _fe ⁷ _ta ^. _{l tolerance} ^T _a ⁱ _n ^l _d ^bu _a ^r _n ^g _ti ^s _v ^, _ir ^T _al ^. _i ^a _m ⁿ _m ^d _u ^J _n ^. _i ^L _ty ^. _. ^S _A ^tr _m ^om _J ⁱ _R ⁿ _e ^g _p ^er _r ^, _o ^C _d ^D _Im ⁸⁺ _mu ^ef _n ^fe _o ^ct _l ^o _, ^r ₂₀ ^T ₁₃ ^ce _. ^ll ₆ ^s ₉ ^a ₍ ^t ₄₎ ^th _: ^e _p. ^fe ₃ ^ta ₉ ^l ₅ ^-m _-4 ^a ₀ ^te ₇ ^rn _. ^{al interface, balancing 6} ₂ ⁸ ₀ ^. _14.58(2-4 ^N _): ^a _p ⁿ _. ^c ₁ ^y, ₈₉ ^P-^. ₉₈ ^an _. ^{d A. Erlebacher, T cell behavior at the maternal-fetal interface. Int J Dev Biol, 6} _P ⁹ _ot ^. _{ential Feta} ^P _l ^o _S ^w _pe ^e _c ^l _i ^l _f ^, _ic ^R _it ^. _y ^M _an ^., _d ^et _a ^a _S ^l. _t ^, _ro ^D _ng ^ec _T ^id _r ^u _a ^a _n ^l _s ^T _crip ^C _ti ^e _o ^ll _n ^{s Exhibit a Highly Differentiated Phenotype and Demonstrate} _{3417. al Response to IFN. J Immunol, 2017.199(10): p.3406-} 97

⁷ _un ⁰ _i ^. _{que proper} ^T _tie ⁱ _s ^lb _. ^u _J ^r _I ^g _m ^s, _m ^T _u ^., _n ^e _o ^t _l, ^a ₂ ^l. ₀ ^, ₁ ^H ₀ ^u _. ^m ₁₈ ^an ₅₍ ^d ₇ ^e ₎ ^c _: ^id _p ^u _. ^a ₄ ^l ₄ ^ti ₇ ^s ₀ ^su-^e _7. ^{contains differentiated CD8+ effector-memory T cells with 7} _V ¹ _i ^. _{rus Infectio} ^W _{n a} ⁱⁿ _n ^k _d ^le _P ^r _r ^, _ev ^C _en ^.W _tin ^. _g ^, _I ^e _n ^t _fe ^a _c ^l _t ^. _i ^, _on ^A _i ^d _n ^a _B ^pt _r ⁱ _a ^v _i ^e _n ^I _a ^m _n ^m _d ^u _T ⁿ _e ^e _st ^R _es. ^es _J ^po _I ⁿ _m ^se _m ^s _u ^to _no ^Z _l ⁱ _, ^k ₂ ^a ₀₁ ^V ₇ ⁱ _. ^ru ₁ ^s ₉₈ ^A ₍₉ ^re _): ^I _p ^m _. ^p ₃ ^o ₅ ^rt ₂ ^a ₆ ⁿ-^t ₃ ^f ₅ ^or ₃₅ ^C _. ^{ontrolling 7} _U ² _t ^. _{ero. Cell H} ^Ja _o ^g _s ^g _t ^e _M ^r, _ic ^B _r ^. _o ^W _be ^., _, ^e ₂ ^t ₀₁ ^a ₇ ^l. _. ^, ₂ ^G ₂ ^e ₍ ^s ₃ ^t ₎ ^a _: ^ti _p ^on _. ^a ₃ ^l ₆₆ ^S-^t ₃ ^ag ₇ ^e ₆ ^a _e ⁿ ₃ ^d _. ^{IFN-lambda Signaling Regulate ZIKV Infection In 7} _pe ³ _r ^. _{meability. J} ^Sh _V ^r _i ^e _r ^s _o ^t _l ^a _, ^, ₂₀ ^S ₀ ^., ₆ ^e _. ^t ₈₀ ^a ₍ ^l ₂ ^., ₀ ^M _{): p} ^ur _. ⁱⁿ ₁ ^e ₀₂ ^m ₀ ^o ₈ ^d-^e ₁ ^l ₇ ^f _. ^{or dengue virus-induced lethal disease with increased vascular 7} _S ⁴ _ta ^. _{te, Micron} ^L _es ^a _ia ⁿ _, ^c ₂ ^io ₀ ^t ₀ ^t ₇ ^i, _. ^R _E ^. _m ^S., _e ^e _rg ^{t a} _I ^l _n ^., _fe ^G _c ^e _t ⁿ _D ^eti _i ^c _s, ^an ₂ ^d ₀₀ ^s ₈ ^er _. ^o ₁ ^lo ₄ ^gi ₍ ^c ₈₎ ^p _: ^ro _p ^p _. ^e ₁ ^rt ₂ ^ie ₃ ^s ₂ ^o-^f ₉ ^Z _. ^{ika virus associated with an epidemic, Yap}

98

Claims

^{WHAT IS CLAIMED IS:}

1^{. A}

t _thoe ^co

t_{h a} ^m

e_{t l} ^p

a_e ^o

m_as ^s

i_t ^it

n_{o o} ^io _n ⁿ

a_e ^c

c_{i p} ^o

d_e ^m

s_pet ^p

q_id ^ri

u_e ^sin

e_{n cco} ^g

e_m ^at

s_p ^l

e_tr ^e _{i fs} ^a

o_i ^s _n ^t

r_tg ^o

h _a ⁿ

i_n ^e

n _a ^is

a_m ^ol

n_yi ^a _n ^t

o_o ^ed

n_{e ac} ^p

o_i ^e _d ^p

f _{S s} ^t

E_e ^id _q ^en

Q_u ^a _{e In} ^d

D_ce ^a

N _w ⁿ

O_h ^a

:_i ^c _c ^c

1_h ^ep

t_{o is} ^ta

S _a ^b

E_t ^le

Q _le ^c _a ^a

I_s ^r

D_t ^r ₉ ^ie

N₅ ^r _% ^or

O_{: id} ^diluent, 9_e3n.tical ^{2. T} _se ^h _q ^e _ue ^c _n ^o _c ^m _e ^p _s ^o _et ^si _f ^t _o ^io _r ⁿ _th ^o _in ^f _a ^c _n ^la _y ^im _on ¹ _e ^, _o ^w _f ^h _S ^e _E ^r _Q ^ein _ID ^the _N ^a _O ^t _: ^l ₁ ^ea _t ^s _o ^t _S ^o _E ⁿ _Q ^{e p} _I ^e _D ^pt _N ^id _O ^e _: ^c ₉ ^o ₃ ^m _. ^{prises the amino acid 3. T} _se ^h _q ^e _ue ^c _n ^o _c ^m _e ^p _w ^o _h ^s _i ⁱ _c ^ti _h ^on _is ^o _a ^f _{t l} ^c _e ^l _a ^a _s ^im _{t 95} ¹ _% ^{, w} _id ^h _e ^e _n ^re _t ⁱ _i ⁿ _cal ^th _to ^e _t ^a _h ^t _e ^le _a ^a _m ^st _in ^o _o ⁿ _a ^e _ci ^p _d ^ep _se ^ti _q ^d _u ^e _en ^co _ce ^ns _s ⁱ _e ^s _t ^ts _fo ^o _r ^f _th ^an _in ^a _a ^m _ny ⁱⁿ _o ^o _n ^a _e ^c _o ^id S_{EQ ID NO: 1 to SEQ ID NO: 93. f} ^{4. T} _se ^h _q ^e _ue ^c _n ^o _c ^m _e ^p _s ^o _et ^si _f ^t _o ^io _r ⁿ _th ^o _in ^{f c} _an ^la _y ^im _on ¹ _e ^, _o ^w _f ^h _S ^e _E ^re _Q ⁱⁿ _ID ^the _N ^a _O ^t _: ^le ₁ ^as _to ^{t o} _S ⁿ _E ^e _Q ^p _I ^e _D ^pti _N ^de _O ^c _: ^o ₉ ⁿ ₃ ^s _. ^{ists of the amino acid} 5. The composition of any one of claims 1 to 4, in lyophilized form.

6. The composition of any one of claims 1 to 4, in frozen form.

7. The composition of any one of claims 1 to 4, in the form of an injectable preparation. 8. The composition of any one of claims 1 to 7, further comprising an adjuvant.

9^{. T} _Z ^h _ik ^e _a ^c _T ^om _ce ^p _ll ^o _e ^s _p ^it _i ⁱ _t ^o _o ⁿ _pe ^o _. ^{f any one of claims 1 to 8, wherein the at least one peptide includes a 10. T} _D ^h _e ^e _ng ^c _u ^o _e ^m _T ^po _ce ^si _l ^t _l ^io _ep ⁿ _it ^o _o ^f _pe ^a _. ^{ny one of claims 1 to 8, wherein the at least one peptide includes a 11. T} _Z ^h _ik ^e _a ^c _T ^om _ce ^p _ll ^o _e ^s _p ^it _i ⁱ _t ^o _o ⁿ _pe ^o _a ^f _n ^a _d ^ny _{a D} ^on _e ^e _ng ^o _u ^f _e ^c _T ^lai _c ^m _e ^s _ll ¹ _ep ^t _i ^o _to ⁸ _p ^, _e. ^{wherein the at least one peptide includes a 12. T} _ep ^h _i ^e _to ^c _p ^o _e ^m _. ^{position of any one of claims 9 to 11, wherein the T cell epitope is a CD8 T cell} 13. The composition of any one of claims 1 to 12, wherein the at least one peptide has a length

99

of at least 10 amino acids.

1^{4. T} _am ^he _in ^c _o ^o _a ^m _ci ^p _d ^o _s ^s _. ^{ition of claim 13, wherein the at least one peptide has a length of at most 100 15. The composition of any of claims 1 to 14, wherein the at least one peptide is selected from:}

•^{• FS/ (S • FS M- FSSSS/-NMR SR2-N ANSS22AA75-84}

8^{9-99 EQ ID 133-141 (SE (QSE IDQ N ID NO O N: 4O: 1): 3 , 490)),,}

^{•• F M} • _F ^SR S^S- S^/N /^MS M^R2 R^-A13 -^N _N ^S3- S²¹⁴ 2^A1 B^{1 (4S8E-15Q5}

6_{8-75 (S} ⁽ _E ^{S IED} Q^{Q N} I_D ^{IOD: 4} N ^N2 O^O), :^{: 44),} • _{FSS/MR-NS3206-215 (SEQ ID 45), • F FS MR-NS3574-582 (SE NO: 47), •• FSSS/ SS//MMRR--NES4B426- 435 Q ID NO: 52), 159-1 (SEQ ID NO: 60), • FSS/MR-E 67}

1_{95- (SEQ ID NO: 70), • FSS/MR-N , • FSS/MR-NSS1203 423-31 (SEQ ID NO: 71) B231- (2S39E (QSE IQD I NDO N:O 74:) 7,7 a)n.d} ^{16. A thne i mne vtithrood m ceothmopdri fsoinrg d:etecting an infection with or an exposure to a flavivirus in a subject, • p} _ce ^r _l ^o _ls ^vi _f ^d _r ⁱ _o ⁿ _m ^{g a} _th ^b _e ⁱ _s ^o _u ^lo _b ^g _je ^ic _c ^a _t, ^{l sample from the subject, the biological sample comprising T} • _{contacting the sample with the composition of any one of claims 1 to 15, • processing the sample to detect the presence of a T cell response, and}

100

^{• d} _T ^e

f_{la c} ^te

v_e ^c

i_vll ^ti

i_{r r} ⁿ

u_e ^g

s_s.p ^th _o ^e _ns ^p _e ^re _is ^se _in ⁿ _d ^ce _ica ^o _t ^r _iv ^a _e ^bs _th ^en _at ^ce _th ^o _e ^f _s ^t _u ^h _b ^e _je ^T _ct ^ce _h ^l _a ^l _s ^re _b ^s _e ^p _e ^o _n ⁿ _i ^s _n ^e _f ^, _e ^w _ct ^h _e ^e _d ^re _w ⁱⁿ _ith ^th _o ^e _r ^p _e ^r _x ^e _p ^se _o ⁿ _se ^c _d ^{e o} _to ^{f t} _t ^h _h ^e _e ^{17. T} _T ^h _c ^e _el ^m _ls ^e _p ^t _r ^h _i ^o _o ^d _{r t} ^o _o ^f _c ^c _o ^la _n ⁱ _t ^m _act ¹ _i ⁶ _n ^, _g ^f _t ^u _h ^r _e ^th _T ^er _c ^c _e ^o _lls ^m _w ^p _i ^r _t ⁱ _h ^sin _th ^g _e ^p _c ^r _o ^o _m ^ce _p ^s _o ^si _s ⁿ _it ^g _io ^t _n ^h _. ^{e biological sample to enrich the 18. T} _of ^h _t ^e _h ^m _{e T} ^eth _c ^o _el ^d _ls. ^{of claim 16 or 17, wherein the response includes an expansion and activation} 19. The method of any one of claims 16 to 18, wherein the T cells include CD8⁺ T cells.

2^{0. T} _m ^h _a ^e _rke ^m _rs ^et _t ^h _o ^o _d ^d _if ^o _fe ^f _re ^c _n ^la _ti ⁱ _a ^m _te ¹ _a ⁹ _n ^, _tig ^w _e ^h _n ^e-^r _e ^e _x ⁱ _p ⁿ _er ^t _i ^h _e ^e _nc ^p _e ^r _d ^o _C ^ce _D ^ss ₈ ⁱⁿ ₊ ^g _T ⁱ _c ⁿ _e ^c _l ^l _l ^u _s. ^{des using CD44 and CD62L as 21. T} _T ^h _N ^e _Fα ^m _, ^e _a ^t _n ^h _d ^od _CD ^of ₁₀ ^c ₇ ^la _a ^im _{as a} ¹⁹ _m ^, _a ^w _rk ^h _e ^e _r ^r _t ^e _o ⁱⁿ _di ^t _f ^h _fe ^e _re ^p _n ^r _t ^o _ia ^c _t ^e _e ^ss _a ⁱ _n ⁿ _t ^g _ige ⁱⁿ _n ^c-^l _e ^u _x ^d _p ^e _e ^s _ri ^u _en ^si _c ⁿ _e ^g _d ^a _C ^t _D ^le ₈ ^a ₊ ^s _T ^{t o} _c ⁿ _el ^e _ls. ^{of IFNγ, 22. T} _de ^h _t ^e _ec ^m _t ^e _a ^t _n ^h _d ^o _/ ^d _o ^o _r ^f _q ^c _u ^la _a ⁱ _n ^m _tif ¹ _y ^9, _p ^{wherein the processing includes implementing an immunoassay to} c_{ells. roduction of the marker to detect antigen-experienced CD8+ T} ^{23. T} _(E ^h _L ^e _IS ^m _P ^e _O ^th _T ^o ₎ ^d _as ^o _s ^f _ay ^cl _a ^a _n ^im _d/o ² _r ^2, _an ^wh _in ^e _t ^r _r ^e _a ⁱ _c ⁿ _el ^t _lu ^h _l ^e _ar ^im _cy ^m _to ^u _k ⁿ _in ^o _e ^as _s ^s _t ^a _a ^y _ini ⁱ _n ^s _g ^an _(IC ^E _S ⁿ ₎ ^z _. ^{yme-Linked ImmunoSpot} 24. The method of any one of claims 16 to 23, wherein the flavivirus is a Zika virus.

25. The method of any one of claims 16 to 23, wherein the flavivirus is a Dengue virus.

26. The method of any one of claims 16 to 25, wherein the subject is a mammal.

27. The method of claim 26, wherein the subject is a human.

2^{8. A} _{a s} ^m _ub ^e _j ^t _e ^h _c ^o _t, ^d _th ^o _e ^f _m ^ind _et ^u _h ^c _o ⁱⁿ _d ^g _c ^, _o ^e _m ^nh _p ^a _r ⁿ _is ^{cing, or sustaining an immune response against a flavivirus in} o_{f the composition of any onein ogf c cloanimtasct 1in tgo T 15 c.ells of the subject with an effective amount} 101

^{29. T} _am ^he _ou ^m _nt ^et _o ^h _f ^o _t ^d _he ^o _c ^f _om ^cla _p ⁱ _o ^m _sit ² _io ⁸ _n ^{, w} _to ^h _t ^e _h ^r _e ^ei _s ⁿ _ub ^s _j ^a _e ^id _ct. ^{contacting includes administrating the effective 30. T}

s _euf ^h _fbe ^e

j_c ^m

e_tciv ^e

t_.e ^tho _am ^d _o ^o _u ^f _n ^c _t ^lai _o ^m _f ² _th ^8, _e ^w _c ^h _o ^e _m ^re _p ⁱⁿ _os ^s _i ^a _ti ⁱ _o ^d _n ^c _, ^on _an ^ta _d ^ct _a ⁱⁿ _d ^g _m ⁱⁿ _in ^c _i ^l _s ^u _tr ^d _a ^e _t ^s _in ^c _g ^on _th ^ta _e ^ct _c ⁱⁿ _o ^g _n ^T _tac ^c _t ^e _e ^l _d ^{ls e} _T ^{x v} _c ⁱ _e ^v _l ^o _ls ^w _t ^it _o ^{h t} _t ^h _h ^e _e ^{31. T} _ad ^h _m ^e _in ^m _is ^e _t ^t _r ^h _a ^o _ti ^d _ng ^o _t ^f _he ^cl _c ^a _o ^im _nta ³ _c ⁰ _te ^, _d ^fu _T ^rt _c ^h _e ^e _ll ^r _s ^c _to ^om _th ^p _e ^ri _s ^s _u ⁱⁿ _b ^g _jec ^e _t ^x _. ^{pansion of the T cells in vitro prior to} 32. The method of any one of claims 28 to 31, wherein the T cells include CD8⁺ T cells.

33. The method of any one of claims 28 to 32, wherein the flavivirus is a Zika virus.

34. The method of any one of claims 28 to 32, wherein the flavivirus is a Dengue virus.

3^{5. T} _ag ^h _a ^e _in ^m _st ^e _t ^t _h ^h _e ^o _f ^d _la ^o _vi ^f _vi ^a _r ⁿ _u ^y _s. ^{one of claims 28 to 34, which is for eliciting the immune response 36. T} _ag ^h _a ^e _in ^m _st ^e _t ^t _h ^h _e ^od _fla ^o _v ^f _iv ^a _ir ⁿ _u ^y _s. ^{one of claims 28 to 34, which is for enhancing the immune response} 37. The method of any one of claims 28 to 36, wherein the subject is a mammal.

38. The method of claim 37, wherein the subject is a human.

102