Zika Virus Infection in Cynomolgus Macaques
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1 Zika Virus Infection in Cynomolgus Macaques Fusataka Koide Program Leader, Emerging Pathogens
2 Background Mosquito-borne virus belonging to the flavivirus genus of the Flaviviridae family. Dengue viruses, West Nile Virus, St. Louis EncephaliBs virus Small viruses with an RNA genome that is highly mutable TransmiFed between vertebrate hosts (humans and primates) by Aedes species mosquitoes
3 Global Spread of Zika Virus
4 Transmission of Zika Virus
5 Zika InfecBon in Humans Guillain Barré syndrome - Autoimmune disorder - Peripheral nervous system impairment No FDA Approved Vaccines or TherapeuBcs Available for Zika hfps://en.wikipedia.org/wiki/zika_virus_outbreak_(2015 present)#guillain.e barr.c3.a9_syndrome
6 Zika Animal Models Ø A129 Mouse Model (lethal) Ø AG129 Mouse Model (lethal) Ø Wild type Mouse (non-lethal, viremia following IV infecbon) Ø Wild type Mouse (lethal, type I IFN blockade) Ø Indian Rhesus Macaques (Viremia, Immunology, Microcephaly ) ü Human challenge study my not be feasible ü Animal model recapitulabng human infecbon is needed
7 Why MauriBan Cynomolgus Macaque (MCM) for Zika? Ø MCM produced robust immunogenicity data in the past Dengue LATDV* studies Ø Sustainable viremia following infecbon with Dengue, Yellow Fever or West Nile viruses Ø Have low Flavivirus sero-prevalence (<0.6%) *Live afenuated tetravalent dengue vaccine
8 Why MauriBan Cynomolgus Macaque (MCM) for Zika? Ø Smaller and docile than Indian rhesus macaques (IRM) Ø Readily available compared to IRM Ø Less costly Ø ScienBfic jusbficabons and logisbcal advantage
9 Zika Natural History Study in Cynomolgus Macaques (N=6) Main ObjecBves: ü Test ability to sustain viremia aher subcutaneous challenge ü DetecBon of neutralizing anbbody (Nab) post challenge 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 x10 5 PFU! 2! 1! F! ZIKV! PRVABC59! Asian 0.5 ml! 1.0 x10 4 PFU! 3! 1! M! ZIKV! FSS13025! Asian 0.5 ml! 5.0 x10 5 PFU! 4! 1! F! ZIKV! FSS13025! Asian 0.5 ml! 1.0 x10 4 PFU! 5! 1! M! ZIKV! IBH 30656! African 0.5 ml! 5.0 x10 5 PFU! 6! 1! F! ZIKV! IBH 30656! African 0.5 ml! 1.0 x10 4 PFU!
10 Zika Natural History Study in Cynomolgus Macaques (N=6) Serum, Urine and Saliva collecbon Serum collecbon Day Zika Challenge Orchiectomy Serum, Urine and Saliva CollecBon Euthanasia and Tissue Harvest Serum CollecBon Orchiectomy
11 Zika Serum Viral Load in Cynomolgus macaques 1.E+6 Genome copies/ml 1.E+5 1.E+4 1.E+3 1.E+2 1.E+1 PRVABC59 PRVABC59 FSS13025 FSS13025 IBH30656 IBH30656 LLOQ 1.E Days Post Infec;on InfecBon with PRVABC and FSS but not with IbH
12 Viral Load in the Testes of ZIKV infected Cynomolgus macaques * Genome copies/g ;ssue * *p-value < 0.05 D4 D8 D4 D8 D4 D8 PRVABC59 FSS13025 IBH30656 Again consistent with the serum analysis, no viral load was detected in the testes of animal challenged with the African strain IBH30656.
13 Viral Shedding from Biological Fluids Day Urine Oral Swabs PRVABC59 FSS13025 IBH30656 PRVABC59 FSS13025 IBH30656 #5262M #5258F #5260M #5259 F #5261 M #5257F #5262 M #5258 F #5260 M #525 9 #5261 M #5257 F N/C 0 N/C N/T N/T N/C N/C 43 N/C N/T N/T N/C N/C N/C N/T N/T 4 0 N/C N/C N/C 0 N/C N/T N/T N/C 0 N/C N/T N/T N/C N/C 0 N/C N/T N/T N/C 200 N/C N/T N/T N/C N/C 0 N/C N/T N/T Viral Shedding Urine was collected directly from animals (as opposed to cage pans). Detected sporadic very low < 300 copies/ml in urine and < 400 copies/ml in saliva. As expected, animals from the African lineage IBH30656 challenge, exhibited extremely low levels of virus shedding.
14 ZIKV InfecBon in Cynomolgus Macaques Viral Load by qrt-pcr (urine and saliva) Challenge Strain ZKV Puerto Rico PRVABC #5262 Urine #5262 Saliva Copies/mL Urine or Saliva Days Post InoculaBon Prolonged ZIKV shedding observed in the saliva of infected MCM
15 Neutralizing AnBbody Response PRNT 50 Titer to PRVABC PRNT50 Titer Day 14 Day 30 Day 60 Animal ID
16 Neutralizing AnBbody Response to PRVABC 59 Animal ID Challenge Strain Dose (PFU) PRNT 50 Titer D-2 D14 D30 D M PRVABC x 10 5 < F PRVABC x 10 4 < M FSS x 10 5 < F FSS x 10 4 < M IBH x 10 5 < 10 < 10 < 10 < F IBH x 10 4 < 10 < 10 < 10 < 10 ü The monkeys inoculated with the PRV and FSS achieved high Nab Bter to PRV by day 14. ü The IBH challenged monkeys (5261and5257)that exhibited very low viral load throughout study period also failed to produce cross reacbve Nab to PRVABC59.
17 Development of 96-well FRNT assay to accelerate development of Zika countermeasures Cells fixed and stained only 24 hours aher infecbon combined with automated counbng of foci increases turnaround Bme compared to standard PRNT assay
18 Cytokine and Chemokine Analysis in the Serum P R (M ) F S S (M ) IB H (M ) P R (M ) F S S (M ) IB H (M ) P R (F ) F S S (F ) IB H (F ) P R (F ) F S S (F ) IB H (F ) IL -1 R a (p g /m l) IL -7 (p g /m l) T im e P o s t-in fe c tio n (D a y s ) T im e P o s t-in fe c tio n (D a y s ) P R (M ) F S S (M ) IB H (M ) P R (M ) F S S (M ) IB H (M ) P R (F ) F S S (F ) IB H (F ) P R (F ) F S S (F ) IB H (F ) IL -8 (p g /m l) IF N -a (p g /m l) T im e P o s t-in fe c tio n (D a y s ) T im e P o s t-in fe c tio n (D a y s ) P R (M ) F S S (M ) IB H (M ) P R (M ) F S S (M ) IB H (M ) P R (F ) F S S (F ) IB H (F ) P R (F ) F S S (F ) IB H (F ) IF N -g(p g /m l) 4 0 M C P -1 (p g /m l) T im e P o s t-in fe c tio n (D a y s ) T im e P o s t-in fe c tio n (D a y s )
19 UBlity of Cynomolgus Macaques in Zika Vaccine Development Ø Immunogenicity and Efficacy Ø Vaccine Safety -Neurovirulence -Tissue Tropism Ø Dose-ranging and Immune DuraBon study Ø Cross-protecBon study
20 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 10 3 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
21 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. Funding and ContribuBng ScienBsts Institute, Frederick, MD. (IR&D) Scott Goebel Beth Snyder Kevin Walters Alison Gast Kimberly Hagelin Anju Singh Timothy J. Sellati Raj Kalkeri Jonathan Rayner
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