Zika Virus Infection in Cynomolgus Macaques Fusataka Koide Program Leader, Emerging Pathogens Southern Research

SouthernResearch

• Mosquito-bornevirusbelongingtotheflavivirusgenusoftheFlaviviridaefamily.

•  Dengueviruses,WestNileVirus,St.LouisEncephaliBsvirus

•  SmallviruseswithanRNAgenomethatishighlymutable

•  TransmiFedbetweenvertebratehosts(humansandprimates)byAedesspeciesmosquitoes

Background

SouthernResearch

GlobalSpreadofZikaVirus

SouthernResearch

TransmissionofZikaVirus

SouthernResearch

ZikaInfecBoninHumans

Guillain–Barrésyndrome- Autoimmunedisorder- Peripheralnervoussystemimpairment

hFps://en.wikipedia.org/wiki/Zika_virus_outbreak_(2015present)#Guillain.E2.80.93Barr.C3.A9_syndrome

NoFDAApprovedVaccinesorTherapeuBcsAvailableforZika

SouthernResearch

ZikaAnimalModelsØ  A129MouseModel(lethal)Ø  AG129MouseModel(lethal)Ø  WildtypeMouse(non-lethal,viremiafollowingIVinfecBon)Ø  WildtypeMouse(lethal,typeIIFNblockade)Ø  IndianRhesusMacaques(Viremia,Immunology,Microcephaly)

SouthernResearch

ü  Humanchallengestudymynotbefeasibleü  AnimalmodelrecapitulaBnghumaninfecBonisneeded

WhyMauriBanCynomolgusMacaque(MCM)forZika?

Ø MCMproducedrobust immunogenicitydata inthepastDengueLATDV*studies

Ø Sustainable viremia following infecBon with Dengue,YellowFeverorWestNileviruses

Ø HavelowFlavivirussero-prevalence(<0.6%)*LiveaFenuatedtetravalentdenguevaccine

SouthernResearch

WhyMauriBanCynomolgusMacaque(MCM)forZika?

Ø SmalleranddocilethanIndianrhesusmacaques(IRM)

Ø ReadilyavailablecomparedtoIRM

Ø Lesscostly

Ø ScienBficjusBficaBonsandlogisBcaladvantage

SouthernResearch

ZikaNaturalHistoryStudyinCynomolgusMacaques(N=6)

Group! N Sex! Virus! Challenge strain!(Day 0)! Virus lineage!

Challenge Volume

(SC)!Challenge Dose!

1! 1! M! ZIKV! PRVABC59! Asian 0.5 mL ! 5.0 x105 PFU!2! 1! F! ZIKV! PRVABC59! Asian 0.5 mL ! 1.0 x104 PFU!3! 1! M! ZIKV! FSS13025! Asian 0.5 mL ! 5.0 x105 PFU!4! 1! F! ZIKV! FSS13025! Asian 0.5 mL ! 1.0 x104 PFU!5! 1! M! ZIKV! IBH 30656! African 0.5 mL ! 5.0 x105 PFU!6! 1! F! ZIKV! IBH 30656! African 0.5 mL ! 1.0 x104 PFU!

MainObjecBves:ü Testabilitytosustainviremiaahersubcutaneouschallenge

ü DetecBonofneutralizinganBbody(Nab)postchallenge

SouthernResearch

Day-20123456810143060

ZikaChallenge

Serum,UrineandSalivaCollecBon

Orchiectomy

SerumCollecBon

EuthanasiaandTissueHarvest

ZikaNaturalHistoryStudyinCynomolgusMacaques(N=6)

Serum,UrineandSalivacollecBon SerumcollecBon

Orchiectomy

SouthernResearch

ZikaSerumViralLoadinCynomolgusmacaques

Gen

omecopies/m

L

DaysPostInfec;on

1.E+0

1.E+1

1.E+2

1.E+3

1.E+4

1.E+5

1.E+6

PRVABC59

PRVABC59

FSS13025

FSS13025

IBH30656

IBH30656

LLOQ

12346810143060

InfecBonwithPRVABCandFSSbutnotwithIbHSouthernResearch

ViralLoadintheTestesofZIKVinfectedCynomolgusmacaques

*

*

D4D8D4D8D4D8PRVABC59FSS13025IBH30656

Gen

omecopies/g;ssue

*p-value<0.05

Againconsistentwiththeserumanalysis,noviralloadwasdetectedinthetestesofanimalchallengedwiththeAfricanstrainIBH30656.SouthernResearch

Day Urine OralSwabs PRVABC59 FSS13025 IBH30656 PRVABC59 FSS13025 IBH30656 #5262M #5258F #5260M #5259

F#5261M

#5257F #5262M

#5258F

#5260M

#5259

#5261M

#5257F

1 0 0 0 N/C 0 N/C 0 0 0 1.6 N/T N/T2 265 0 N/C N/C 43 N/C 105 0 0 0 N/T N/T3 0 0 19 N/C N/C N/C 198 0 52 0 N/T N/T4 0 N/C N/C N/C 0 N/C 169 146 0 1 N/T N/T6 0 29 0 N/C 0 N/C 0 0 6.6 2 N/T N/T8 0 0 N/C N/C 0 N/C 138 142 0 81 N/T N/T10 0 87 0 N/C 200 N/C 244 0 12 0 N/T N/T14 0 114 N/C N/C 0 N/C 109 359 0 0 N/T N/T

ViralSheddingfromBiologicalFluids

ViralSheddingUrinewascollecteddirectlyfromanimals(asopposedtocagepans).Detectedsporadicverylow<300copies/mLinurineand<400copies/mLinsaliva.Asexpected,animalsfromtheAfricanlineageIBH30656challenge,exhibitedextremelylowlevelsofvirusshedding.

SouthernResearch

ZIKVInfecBoninCynomolgusMacaquesViralLoadbyqRT-PCR(urineandsaliva)

0

50

100

150

200

250

300

0 2 4 6 8 10 12 14 16

Copies/m

LUrin

eorSaliva

DaysPostInoculaBon

ChallengeStrainZKVPuertoRicoPRVABC59

#5262Urine#5262Saliva

ProlongedZIKVsheddingobservedinthesalivaofinfectedMCM

SouthernResearch

NeutralizingAnBbodyResponse

1

10

100

1000

10000

5262 5258 5260 5259

PRNT50Titer

AnimalID

PRNT50TitertoPRVABC59

Day14 Day30 Day60

SouthernResearch

NeutralizingAnBbodyResponsetoPRVABC59

Animal ID Challenge

Strain Dose

(PFU) PRNT50 Titer D-2 D14 D30 D60

5262 M PRVABC59 5.0 x 105 < 10 7920 2380 1071 5258 F PRVABC59 1.0 x 104 < 10 2438 875 740 5260 M FSS13025 5.0 x 105 < 10 1053 797 528 5259 F FSS13025 1.0 x 104 < 10 3131 2019 666 5261 M IBH 30656 5.0 x 105 < 10 < 10 < 10 < 10 5257 F IBH 30656 1.0 x 104 < 10 < 10 < 10 < 10

SouthernResearch

ü  ThemonkeysinoculatedwiththePRVandFSSachievedhighNabBtertoPRVbyday14.

ü  TheIBHchallengedmonkeys(5261and5257)thatexhibitedverylowviralloadthroughoutstudyperiodalsofailedtoproducecrossreacBveNabtoPRVABC59.

Developmentof96-wellFRNTassaytoacceleratedevelopmentofZikacountermeasures

Cellsfixedandstainedonly24hoursaherinfecBoncombinedwithautomatedcounBngoffociincreasesturnaroundBmecomparedtostandardPRNTassaySouthernResearch

CytokineandChemokineAnalysisintheSerum

0 1 2 3 4 6 8 1 0 1 4 3 0 6 00

5 0

1 0 0

1 5 0

2 0 04 0 0

5 0 0

6 0 0

7 0 0

8 0 0

T im e P o s t- In fe c tio n (D a y s )

IL-1

Ra

(p

g/m

l)

P R (M )

P R (F )

F S S (M )

F S S (F )

IB H (M )

IB H (F )

0 1 2 3 4 6 8 1 0 1 4 3 0 6 00

1 0

2 0

3 0

T im e P o s t- In fe c tio n (D a y s )

IL-7

(p

g/m

l)

P R (M )

P R (F )

F S S (M )

F S S (F )

IB H (M )

IB H (F )

0 1 2 3 4 6 8 1 0 1 4 3 00

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

T im e P o s t- In fe c tio n (D a y s )

IL-8

(p

g/m

l)

P R (M )

P R (F )

F S S (M )

F S S (F )

IB H (M )

IB H (F )

0 1 2 3 4 6 8 1 0 1 4 3 0 6 00

1 0

2 0

3 0

T im e P o s t- In fe c tio n (D a y s )

IFN

- a (

pg

/ml)

P R (M )

P R (F )

F S S (M )

F S S (F )

IB H (M )

IB H (F )

0 1 2 3 4 6 8 1 0 1 4 3 0 6 00

2 0

4 0

6 0

8 0

T im e P o s t- In fe c tio n (D a y s )

IFN

- g (

pg

/ml)

P R (M )

P R (F )

F S S (M )

F S S (F )

IB H (M )

IB H (F )

0 1 2 3 4 6 8 1 0 1 4 3 0 6 00

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

T im e P o s t- In fe c tio n (D a y s )

MC

P-1

(p

g/m

l)

P R (M )

P R (F )

F S S (M )

F S S (F )

IB H (M )

IB H (F )

SouthernResearch

UBlityofCynomolgusMacaquesinZikaVaccineDevelopmentØ ImmunogenicityandEfficacy

Ø VaccineSafety

-Neurovirulence

-TissueTropism

Ø Dose-rangingandImmuneDuraBonstudy

Ø Cross-protecBonstudy

SouthernResearch

Summary

ü Cynomolgus macaques are susceptible to Zika infection ü  PRVABC59 induced viremia detectable up to day 10, with peak

viral load at 2–3 days p.i. An intermittent recurrent viremia spike was observed on day 30 with titers reaching 2.5 x 103 genomes/mL

ü  The Nab responses were found to be robust with the PRVABC59 strain which correlates with the observation made with serum viral loads

ü We found high levels as compared to the control of PDGF, IP-10, VEGF, RANTES, MIP1A, MIP1B, MCP-1, IL-1Ra, IFN-alpha with the higher dose of the PRVABC 59 strain

MCM could serve as an alternative model to study Zika infection and vaccine development

SouthernResearch

Acknowledgements

We would like to acknowledge the source of our ZIKV FSS13025 stock received from UTMB, Galveston National Laboratory, 301 University Boulevard, Galveston, TX 77550, USA; and ZIKV PRVABC59 from the CDC, 3156 Rampart Road, Fort Collins, CO 80526, USA.

FundingandContribuBngScienBsts

Southern Research Institute, Frederick, MD. (IR&D)

Scott Goebel Beth Snyder Kevin Walters Alison Gast Kimberly Hagelin Anju Singh Timothy J. Sellati Raj Kalkeri Jonathan Rayner

SouthernResearch