ZIKV epidemiology, diagnostics and pathogenesis from studies of blood donors and recipients Graham Simmons, PhD Blood Systems Research Institute University of California, San Francisco 

Statement of potential conflicts Blood Systems Research Institute has received funding for research from Grifols for ZIKA related research, and BSRI and Creative Testing Solutions are performing supplemental testing for Investigational New Drug Applications from both Roche Molecular Systems and Grifols This project has been funded in whole or in part with Federal funds from the Biomedical Advanced Research and Development Authority, Office of the Assistant Secretary for Preparedness and Response, Office of the Secretary, Department of Health and Human Services, under Contract #HHSO100201600010C This project has been funded in whole or in part with Federal funds from the Biomedical Advanced Research and Development Authority, Office of the Assistant Secretary for Preparedness and Response, Office of the Secretary, Department of Health and Human Services, under Contract #HHSO100201600024C Statement of potential conflicts

BSRI Blinded ZIKV Panel Study management 25 member blinded panel was prepared by BSRI Panel members were coded and shipped to participants Results were sent back to BSRI for decoding and summary Blinded Panel 2014 Polynesia culture isolate and 2015 Brazilian donor plasma Serial half log dilution in defibrinated human serum Negative controls Participants CDC Fort Collins, CO (Laniotti) and CDC San Juan Puerto Rico (Munoz) BSRI Roche Molecular Systems Grifols/Hologic, Inc. Others (EFS, Tahiti, Brazil, UC Davis, FDA) In order to determine specificity and sensitivity of assays, we produced a dilution panel of Zika RNA positive samples, together with negatives using a tissue-culture isolate and RNA reactive plasma from a Brazilian blood donor. BSRI Blinded ZIKV Panel

ZIKV NAT detection assays The two commercial blood screening assays from Roche and Grifols, shown in red, performed very similarly, and clearly outperformed versions of the CDC RT-PCR assay. Stone et al. 2017 Transfusion ZIKV NAT detection assays

Example of sensitive NAT detection increasing detection window Assay Day post infection 1 2 3 4 5 6 7 8 9 10 11 12 CDC RT-PCR - + NT Infectivity ID-NAT platform As a practical example of the enhanced sensitivity, in macaques experimentally infected with Zika, the standard CDC assay could detect viremia for 5 days after infection, while using the Hologic platform the detection window was extended by an additional five days. Coffey et al. 2017 PLoS One. Studies performed at CNPRC Example of sensitive NAT detection increasing detection window Presentation Title | Presenter’s Name | Date | BUSINESS USE ONLY

Donors Screened under Roche and Hologic IND = CTS Labs = CTS Clients 1,860,103 Donors Screened under Roche and Hologic IND 423 Reactive 396 Confirmed Positive 91 False Positive 1,859,680 Nonreactive Using both the Roche and Grifols platforms Creative testing Solutions have tested near two million donor samples since the assays were introduced under IND. The majority of reactive donations were in Puerto Rico, while the majority of continental US reactive donations were associated with known travel to epidemic regions. US 1,790,843 PR 69,260 US 58 PR 365 Zika RNA reactives

Weekly Detection Rate of ZIKV RNA in Blood Donated in Puerto Rico Screening under IND using the Roche Molecular Systems test in Puerto Rico has demonstrated a high proportion of reactive blood donors – peaking at nearly 2% in July. The data presented here are preliminary and have not been reviewed by FDA. Weekly Detection Rate of ZIKV RNA in Blood Donated in Puerto Rico

ZIKV vs CHIKV epidemics in PR Compared to the chikungunya epidemic in 2014, similar peak levels of reactive donors was seen, although the ZIKV epidemic occurred earlier in the year. ZIKV vs CHIKV epidemics in PR

Shifting PR epidemic 70% 71% 68% 74% 50% 44% As the epidemic in Puerto Rico progressed, the balance of NAT yields shifted from mostly seronegative acute stage donations, to a more even balance of seronegative and seropositive tail-end donations – suggesting that the rate of new infections was falling. 50% 44% Shifting PR epidemic

Majority of donations in PR were simulated minipool positive % ID-NAT only: 7 26 25 36 25 42 56 36 57 % IgM positive: 33 37 27 27 25 50 61 36 86 We see a similar shift as the epidemic progressed looking at ID-NAT only reactive – ie they were non-reactive in simulated MP. ID-NAT only peaked in Oct to Dec, suggesting a waning of the epidemic and a majority of the donations were tail-end infections. The proportion of ID-NAT only and seronegative donations that represent early ramp-up infections peaked in July at almost 9%. Majority of donations in PR were simulated minipool positive

Majority of donations in PR were seronegative at index Overall, approximately only 4% of reactive donors were in the ramp-up stage of ID-NAT only and seronegative that are most likely to be missed by testing due to low viral loads and transmissable due to a lack of neutralizing antibodies. Majority of donations in PR were seronegative at index

ZIKV NAT reactive donors seroconvert in follow-up sampling Limited to people with 2 followups Virtually Everyone coverts IgM 3/88 donors do not go above equivocal DENV IgG negative donors ZIKV NAT reactive donors seroconvert in follow-up sampling

Follow-up study of Zika NAT reactive blood donors Extended Follow-up Characterization of humoral and cellular immunity Discriminate recent vs remote infections Month 9 Month 12 6 Follow-up study of Zika NAT reactive blood donors

ZIKV RNA persists in whole blood, primarily associated with RBCs In the follow-up samples after blood donors were found to be RNA positive at donation, we more reliably detected viral RNA in both whole blood and packed red blood cells. In this instance we used a modified version of the CDC RT-PCR assay that allows for greater input volumes. The presence of virus associated with RBC is important for extending the detection window of acute viremia for diagnosis of clinical cases and monitoring pregnant women and travelers. The implications on infectivity of blood and cellular products is under study using tissue culture and murine and macaque infection models. However, as these donors are likely to be ZIKV seropositive and are producing neutralizing antibodies, at this point it is not clear how infectious these samples may be, and hence it is premature to consider the need to screen donors using whole blood instead of plasma and for duration of deferral following detection of a donor as ZIKV infected. Surprisingly, about 10% of donors do not develop the red cell persistence finding. ZIKV RNA persists in whole blood, primarily associated with RBCs

  Pre-seroconversion the majority of NAT reactive donors had higher viral loads in plasma compared to RBCs. However, after IgM seroconversion plasma loads dropped rapidly, but viral loads in RBCs remained higher. This suggests antibody may play a role in RBC association, but we have yet to identify a firm mechanism. Partitioning of ZIKV in index plasma and LR-pRBC samples from the pre- vs post-SC

ZIKV RNA persists in other components and matrices Other components and matrices showed extended persistence, but not to the extent of RBCs ZIKV RNA persists in other components and matrices

The use of whole blood extends the window of diagnosis Impact on donation policy: to extend deferral period or consider NAT testing whole blood? Does the virus continue to replicate IS RBC-ASSOCIATED VIRUS INFECTIOUS? Summary

Calculation of minimal infectious dose Mouse model – Interferon α/β receptor knock-out (IFNα/βR -/-) mice. See prolonged peak viremia Virus source Genome copies/ml 50% Infectious dose/ml in mice RNA copies/ID50 IgM -ve human plasma 3.0 x 105 1.4 x 104 21 We have begum to address this in animal models. Firstly we are using immunodeficient mice to determine the minimal infectious dose – firstly with plasma, but then subsequently hopefully with tail-end whole blood or RBC components Preliminary data from one plasma sample suggests ID50 of IgM-/IgG- plasma in mice (21 RNA copies) below LOD of CDC RT-PCR assay Calculation of minimal infectious dose

NAT assays are ultrasensitive and outperform existing CDC RT-PCR assays. A significant proportion of NAT reactive donations were non-reactive upon simulated minipooling. Only 4% of ID-NAT-only donations were seronegative. Significant persistence of ZIKV RNA in packed RBC at least 3 months after acute infection – diagnostic and TT potential? Only 21 RNA copies in plasma is infectious in a mouse model, highlighting the risk of TT and the need for sensitive testing. Summary

Acknowledgements Roche REDS-III Central Laboratory, BSRI Susan Galel Lisa Lee Pate Hologic/Grifols Jeff Linen Kui Gao CTS Phillip Williamson UC Davis Koen Van Rompay Lark Coffee REDS-III Chair Steve Kleinman REDS-III Central Laboratory, BSRI Michael Busch Mars Stone Philip Norris Sonia Bakkoor Tzong-Hae Lee Brian Custer Marion Lanteri Thema Gonzalez REDS-III Data Coordinating center, RTI International Don Brambilla Chris McClure NHLBI Simone Glynn Acknowledgements