The rVSV constructs were generated and characterized as previously described (29). The viral vector is a recombinant Vesicular Stomatitis virus (rVSV) lacking the viral M protein and containing the Zika virus (Puerto Rico strain 2015) prME transgene inserted 5’ to the viral G gene ( Figure 1A ). Six female pigtail macaques (7- 12 yrs) that were seronegative for ZIKV, West Nile virus (WNV), dengue virus (DENV), chikungunya virus (CHKV), and yellow fever virus (YFV) flaviviruses were enrolled in the study ( Tables S1, S2 ). All animals received two intramuscular injections of 1×10⁷ PFU of either rVSVΔM-ZprME (n=4) or rVSVΔM (n=2) in the quadricep muscle of the leg at weeks 0 and 4, and a higher boost immunization of 2×10⁷ PFU given at week 8 ( Figure 1B ). The intramuscular inoculation route and prime-boost strategy were chosen based on the immunization regimen commonly employed in nonhuman primate models and for direct translation for clinical trials. At 10 weeks post-prime immunization, animals underwent heterologous virus challenge via subcutaneous administration of 1×10⁵ plaque forming units (PFU) of ZIKV FSS13025 (GenBank no. MH368551) in five separate 100 µl injections administered into the bilateral forearm at different sites.

All animals used in this study were housed at the Washington National Primate Research Center (WaNPRC), as accredited by the American Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The University of Washington’s Institutional Animal Care and Use Committee (IACUC) approved all experiments (IACUC Protocol Number 4158-10) and were in compliance with the U.S. Department of Health and Human Services Guide for the Care and Use of Laboratory Animals and Animal Welfare. For timepoints involving experimental procedures, animals were sedated with 10 mg/kg ketamine and dexmeditomidine sedative intramuscular (Ketaset^® Henry Schein).

Animal care and welfare was monitored daily by veterinary staff and animal technicians throughout the course of the study. Animals were weighed and rectal temperatures collected following sedation at each sampling timepoint. On days of immunization, whole blood was collected prior to vaccine administration. Serum, plasma, and peripheral blood mononuclear cells (PBMCs) were isolated from whole blood as previously described (30). Serum chemistry analysis was performed on pre-immunization sera collected 2 weeks before prime, sera collected at 4, 8 and 10 weeks post-prime immunization, pre-challenge, 2 days post-challenge, and sera collected at weekly intervals through the end of study (7-30 dpi). SST samples were spun at 1100 x g for 10 min at room temperature and isolated sera submitted to Research Testing Services at the University of Washington Department of Laboratory Medicine. Samples were run on a chemistry panel with 17 analytes that included electrolytes and a comprehensive metabolic panel (CMP) to monitor liver and kidney function, such as alkaline phosphatase (ALP), alanine transaminase (ALT), and aspartate aminotransferase (AST) ( Table S3 ). At the end of the study the animals were humanely euthanized and a complete postmortem exam was performed. Representative samples from all tissues and organs were preserved in 10% neutral buffered formalin, processed routinely, embedded in paraffin, sectioned at 3-5um, Hematoxylin and eosin stained, and evaluated by light microscopy.

All animals were pre-screened and negative for i) CHV-1 (B virus), ii) SIV, iii) SRV, and iv) STLV prior to assignment to the study. We used a viral-specific diagnostic assay to screen animals for ZIKV, DENV, CHIKV, WNV and YFV IgG to assist with animal study assignment ( Table S2 ). Ultraviolet-inactivated serum collected from naïve animals was run according to the manufacturer’s (Xpress Biosystems, Frederick MD) instructions as previously described (31). Diluted serum samples were added to the wells of the ELISA plate coated with viral antigens and incubated at 37°C for 45 min. Wells were washed 5x and peroxidase conjugate was added to each well and incubated for 45 min at 37°C. Following incubation, the plates were washed 5 times followed by the addition of 2,2’-azino-bis (3-ethylbenzothiazoline-6- sulphonic acid)-peroxidase substrate to each well. The plates were incubated at room temperature for 30 min and the absorbance of the colorimetric reaction in each well was read within 15 min on a plate reader at 405 nm.

Plasma was isolated from whole blood collected at -5, 1, 2, 4, 6, 7, 14, and 21 days post-challenge, and at necropsy (days 27-30). Viral RNA load was assessed in plasma using a ZIKV-specific RT-qPCR assay, as previously described (30). RNA was isolated using the QIAamp Viral RNA Mini Kit (Qiagen). Plasma RNAs were concentrated using the RNA Clean & Concentrator Kit (Zymo Research) and eluted in 12 µl nuclease-free water. The iScript Select cDNA Synthesis Kit (Bio-Rad) was used for gene-specific cDNA synthesis with an input of 10 µl RNA sample. Viral RNA was quantified using the TaqMan Universal PCR Master Mix (Applied Biosystems) and a 7300 Real-Time PCR System (Applied Biosystems) using a ZIKV prM-specific primer/probe set.

NHP sera collected at baseline (-2 weeks), and weeks 2, 4, 6, 8 and 10 post-prime immunization were tested in PRNT assay for neutralizing antibody production. The highest serum dilution reducing plaque numbers by 50% (PRNT₅₀) were determined with a limit of detection (LOD) of 1:50. The assay was repeated three times. ZIKV strain used was ZIKV FSS13025 (Cambodia, 2010).

NHP sera and/or plasma was assessed for anti-VSV Glycoprotein (G) and anti-ZIKV envelope (E) IgG titers by an Enzyme-Linked Immunosorbent Assay (ELISA) and for anti-ZIKV E IgM titers using a ZIKA Detect™ 2.0 IgM Capture ELISA Kit (InBios, Lot# BM6156) by the WaNPRC Pathogen Detection Services Laboratory (PDSL) (32). For anti-VSV G and anti-ZIKV E IgG, samples were diluted 1:100 and/or 1:200 in a blocking buffer (5% w/v nonfat dried milk (Bio-Rad Laboratories) and 0.5% v/v Tween-20) and added in triplicate to a high-binding 96-well plate (Costar) pre-coated with either recombinant VSV G (Alpha Diagnostics VSIG15-R) or ZIKV E (Fitzgerald Industries International, 30-1932). Plasma IgG specific for VSV G or ZIKV E protein were detected by a Horseradish peroxidase-linked antibody (ThermoFisher) via a color change reaction upon addition of the SureBlue Reserve substrate (KPL). Reaction was stopped after 30 minutes with 1N HCl (VWR) and absorbance at 450nm was measured on a EMax plate reader (Molecular Devices). A standard curve (MyBioSource) was to calculate anti-VSV G IgG levels via a linear interpolation performed in Microsoft Excel. For anti-ZIKV IgM, UV-inactivated NHP sera and/or plasma was diluted 1:100 and 50 µl added to the three assay wells, ZIKV antigen (Zika Ag), Cross-reactive Control Antigen (CCA) and Normal Cell Antigen (NCA), per sample.

Antigen-specific T-cells secreting IFN-γ in the PBMC were detected using a Human IFN-γ Single-Color ELISPOT (ImmunoSpot, Shaker Heights, Cleveland, OH), per the manufacturer’s protocol. Briefly, cryopreserved PBMC cells were thawed, and 1 x 10⁵ cells were stimulated for 24 hours in duplicate with 8 Zika Envelope peptide pools (15-mers with 12 amino acid overlap) (NR-50553, BEI Resources, Manassas, VA) at a concentration of 1 μg/mL per peptide. DMSO was used as a negative control and Phorbol 12-myristate 13-acetate (PMA) and Ionomycin (Sigma-Aldrich, St. Louis, MO) were used as positive controls. Spots were counted on an Immunospot Analyzer with CTL Immunospot Profession Software (Cellular Technology Ltd., Shaker Heights, Cleveland, OH). Spot forming cells (SFC) were computed following DMSO subtraction and were considered positive if the number of SFC was > 20 SFC per 1 x 10⁶ cells.

Whole blood was assessed for viability with a live/dead stain (Life Technologies) and stained with a panel of antibodies in brilliant stain buffer (BD Biosciences) to identify immune cells, as described previously (30): Beckman Coulter: NKG2A (Z199); Biolegend: CD16 (3G8), CD20 (2H7), CD4 (OKT4), HLA-DR (L243), CD14 (M5E2), CD28 (CD28.2), CD40 (5C3), CD69 (FN50); BD Biosciences: CD11b (ICRF44), CD3 (Sp34-2), CD11c (S-HCL-3), CD45 (D058-1283); and eBioscience: CD123 (6H6), CD8 (RPA-T8), CD95 (DX2). Cells were then re-suspended in 1% paraformaldehyde and samples were acquired on a LSRII (BD Biosciences) using FACS Diva software (version 8). Samples were analyzed using FlowJo software version 9.9.4 (FlowJo, LLC). All events were first gated on FSC singlets, CD45⁺ leukocytes, live, and then mononuclear cells according to FSC-A and SSC-A profiles. CD3⁺ T-cells were gated in CD4+ and CD8+ populations and into naïve (CD95^-CD28⁺), central memory (CM) (CD95⁺CD28⁺) and effector memory (EM) (CD95⁺CD28^-). Cellular activation of T-cells were measured using CD69 that met a minimum threshold of ≥100 cells/gate.

Vaccine safety assessment was performed to evaluate the overall health of the animals following vaccination and through the entirety of the study. All animals remained clinically normal with unremarkable bloodwork throughout the course of the study. There were no abnormal changes in serum blood chemistries measured that included a metabolic panel to evaluate liver and kidney function ( Figures 2A–C ; Table S2 ). A single animal (R10117) had elevated blood AST levels at 2 days post-ZIKV infection corresponding to peak viremia ( Figure S2 ). Vaccinated rVSVΔM-ZprME and rVSVΔM control animals had stable weight throughout the study and did not show signs of fever following immunization, with rectal temperatures ranging from 98.2°F to 102.6°F (average 100.3°F vaccine phase) ( Figures 2D, E ). Body temperature became slightly elevated, but within a normal range, post-ZIKV challenge (average 101.2°F challenge phase) for both rVSVΔM-ZprME and rVSVΔM control animals, with temperature fluctuations among individual animals. A complete gross exam and histologic evaluation of representative samples from all tissues and organ systems was performed at necropsy. In all animals, typical pre-existing lesions that are present in most captive macaques and consistent with inflammatory bowel disease (IBD) were identified; the diagnosis was mild to moderate lymphoplasmacytic, histiocytic and eosinophilic gastro-entero-colitis with enteric villar blunting and fusion (data not shown). Additionally, all animals had commensal infections with gastric spiral bacteria and large intestinal spirochetosis. Finally, Z08064 had pre-existing, moderate and subclinical, hepatic secondary amyloidosis; secondary to chronic inflammation from IBD with mis-metabolism of acute-phase reactive proteins and subsequent hepatic sinusoidal deposition. Secondary amyloidosis is commonly seen in captive pigtail macaques, the disease is slowly progressive, and in this animal, it was relatively early in the disease course. Thus overall, the vaccine was well tolerated in the animals and displayed a normal safety profile and there was no gross or histologic evidence of changes due to the viral challenge.

As antibodies are important for protection from ZIKV infection (33), we first evaluated humoral immune responses elicited by the rVSVΔM-ZprME vaccine. Pre- and post-immunization sera/plasma were analyzed for IgM and IgG binding antibodies against the ZIKV envelope (Env) protein by ELISA. Three immunizations with rVSVΔM-ZprME were insufficient at inducing IgM or IgG binding antibodies to ZIKV Env ( Figures 3A, B ). Plaque reduction neutralization test (PRNT) was next used to assess the production of ZIKV-specific neutralizing antibodies (NAb) in sera following vaccination. As expected, sera from animals that received the control rVSVΔM vector did not exhibit ZIKV neutralization. One of the four animals vaccinated with the rVSVΔM-ZprME vector (R10117) had detectable NAb (200) following the second boost immunization ( Figure 3C ). We next evaluated ZIKV Env-specific cellular responses in peripheral blood mononuclear cells (PBMC) collected prior to vaccination and after the 3^rd immunization, as measured by IFN-γ activity by ELISPOT. No ZIKV-specific T-cells were induced in response to rVSVΔM-ZprME vaccination following the second boost immunization ( Table S4 ). Collectively, these results demonstrate that rVSVΔM-ZprME did not robustly induce anti-ZIKV humoral or cellular immunity in all animals.

To ensure the vaccine was successfully delivered to the macaques and able to induce an immune response we further evaluated anti-vector IgG responses against fusion glycoprotein (G protein) of VSV and found all control rVSVΔM and rVSVΔM-ZprME vaccinated animals developed an anti-VSV-G-specific IgG response to the vaccine vector after a single immunization ( Figure S1 ). In the absence of ZIKV-specific cellular responses, we next evaluated non-specific responses to determine if the vaccine similarly stimulated T cells in whole blood. Activation of CD8+ T cells was detected in 3/6 animals (1 rVSVΔM and 2 rVSVΔM-ZprME vaccinated animals) 4 weeks after prime immunization and to a greater extent 4 weeks after the boost immunization ( Figure S2 ) and suggests non-specific activation of memory CD8+ T-cells. These results show that overall both rVSVΔM and rVSVΔM-ZprME constructs induced cellular and humoral responses to the VSV vaccine construct; however, the rVSVΔM-ZprME vaccine only induced weak humoral immunity against ZIKV.

To assess the efficacy of the vaccine, all animals were challenged subcutaneously with heterologous ZIKV FSS13025 14 days after the second boost immunization (Week 8) and monitored over a 28-day period. This virus strain causes less robust infection in macaques compared to America lineage isolates (34) and was selected as we have previously shown that infection in a pigtail macaque model of pregnancy results in severe fetal brain lesions (31). The relative efficacy of the vaccine was evaluated by measuring the ZIKV load in plasma. In animals receiving the rVSVΔM control vector, ZIKV RNA was detected in the plasma in both animals at day 2 and in 1 of the animals at day 4 post-challenge ( Figure 4A ), a result that is consistent with our previous findings (31). In contrast, only 1/4 of the animals vaccinated with the rVSVΔM-ZprME vector had detectable levels of plasma viremia (R10074) at days 1 and 2 post-challenge and at levels that were lower compared to the rVSVΔM control animals ( Figure 4A ). Analysis of anti-ZIKV IgM responses post-challenge revealed that all vaccinated and control animals developed responses to the ZIKV infection starting 7-14 days after challenge ( Figure 4B ), indicative of a primary and/or secondary immune response to infection. Analysis of anti-ZIKV IgG antibodies revealed that 3/4 rVSVΔM-ZprME vaccinated and 0/2 rVSVΔM control animals mounted a response 14-21 days post challenge ( Figure 4C ) and is suggestive of a recall antibody response to the rVSVΔM-ZprME vaccine. Anti-ZIKV Env-specific cellular IFN-γ activity was not detected in any of the rVSVΔM control or rVSVΔM-ZprME vaccine animals 14 days post-challenge ( Table S4 ), nor was there robust non-specific cellular activation post-challenge ( Figure S2 ). These results demonstrate that the low levels of immunogenicity elicited in some animals by rVSVΔM-ZprME was capable of reducing ZIKV viremia following heterologous virus challenge.