Experimental model and subject details

Outbred, healthy Indian-origin female rhesus monkeys (Macaca mulatta) were housed at Alphagenesis, Yemassee, SC. Animals selected for this study were research naive. Upon arrival and standard quarantine procedures, animals were tested for tuberculosis (TB) at least three times at intervals of two weeks. Animals were also screened for Herpes B, Simian retrovirus (SRV), Simian Immunodeficiency Virus (SIV), Simian T-cell Leukemia Virus (STLV), and Measles. All animals were Herpes B, SRV, SIV, and STLV negative. Study animals were selected based on age. Females were all aged 4- 8 years old with similar age and weight distribution per study group. The study protocol was reviewed and approved by the Alphagenesis Institutional Care and Use Committee (IACUC). All experiments conformed to regulatory standards outlined by the American Veterinary Medical Association (AVMA) and American Association of Laboratory Animal Medicine (AALAM).

Breeding, immunization, and ZIKV challenge

Seven weeks prior to immunization, females were removed from their breeding group. For Group 1 (Ad26.M.Env vaccinated, ZIKV-challenge), nine dams that were not pregnant according to ultrasound were intramuscularly immunized with 1×1011 vp Ad26.M.Env. The nine immunized dams were reintroduced to their breeding groups 17 days later. All animals were provided enrichment according to recommended guidelines. Dams were monitored for pregnancy every 2 weeks by ultrasound until confirmed pregnant. After confirmed pregnancy, dams were monitored by ultrasound every 4 weeks. For Group 1, all nine dams had confirmed pregnancy. For Group 2 (non-vaccinated, ZIKV-challenge controls), 7 dams were included in the study, of which 5 were confirmed pregnant. Pregnant control dams (Group 3, no vaccination, no ZIKV-challenge) were included from the breeding colony and were of similar age and source as vaccinated and non-vaccinated, challenged animals (Groups 1&2).

Approximately six weeks after calculated date of conception (based on ultrasound results), equivalent to human 1st trimester of pregnancy, dams from groups 1 and 2 were challenged with 1 × 106 vp (103 PFU) of ZIKV-BR via the subcutaneous route. During pregnancy, blood and PBMCs were isolated for immunogenicity readouts. Plasma, cerebrospinal fluid (CFS), urine, colorectal biopsies, inguinal/axillary lymph node (LN) biopsies, rectal, vaginal and saliva secretion were taken to monitor ZIKV vRNA. At approximately week 21–23 of pregnancy ( ≈ week 16 following challenge) fetuses of all groups were delivered by C- section except for one (OBE) from the vaccine group that was born naturally due to earlier than predicted delivery. One pregnancy was lost in the vaccine group due to culture confirmed staphylococcal placentitis (539) and this dam/infant pair was removed from the study. Reproductive failure or preterm delivery is significant among primates, and the observations in this study were within the historical control range for the testing facility (AGI), and within expected outcomes for pregnancies in rhesus monkeys.

Five live male infants and three live female infants were delivered among the vaccinated, challenged group. Three live male infants and two live female infants were delivered to the non-vaccinated, challenged group. Two live males and one live female infant were delivered to the control group (non-vaccinated, non-ZIKV challenged; Table 1). Dams and fetuses were euthanized for post-mortem gross pathology, histopathology, and virologic assessments.

Method detailsAd26.M.Env

Complete details about the construction and production of the Ad26.M.Env construct are available15,16,22. Briefly, the vaccine was produced on the human PER.C61 cell line and purified and characterized as described previously in ref. 58. Ad26 particle concentrations were determined by optical density at 260 nm and viral infectivity by TCID50 assay. All vaccine preparations were tested for bioburden and endotoxin levels (MicroSafe, Millipore, Leiden, The Netherlands) and have passed pre-set release criteria for animal experiments.

ZIKV challenge stock preparation

ZIKV-BR (Brazil ZKV2015) was propagated in Vero cells (World Health Organization, NICSC-011038011038) that were maintained in EMEM media supplemented with 10%FBS, 6mM L-glutamine and 1x pen/strep. Cells were passaged twice a week and incubated at 37 °C, 10% CO2.

Ultrasonography

Ultrasounds were performed every 2–4 weeks in the ZIKV-infected pregnant rhesus monkeys as well as in 3 uninfected pregnant rhesus monkeys in the same breeding facility. Animals were sedated with Telazol (5 mg/kg), and a GE Logic E with an 8CRS Micro-convex transducer (FOV 132, 3.6–10 MHz) was used for multiparameter biometric measurements, including biparietal diameter (BPD), occipitofrontal diameter (OFD), head circumference (HC), crown-rump length (CRL), abdominal circumference (AC), and femur length (FL).

Amniocentesis

Animals were sedated with Telazol HCL (4–7 mg/kg IM). The area on the abdomen was clipped and sterilely prepped with triple alternating applications of betadine and alcohol. Using sterile technique, a 22-gauge 3.34-inch needle on a 3-cc syringe was inserted into the ventral abdomen to the amniotic sac with ultrasound guidance. 2 cc of amniotic fluid was collected and frozen immediately.

RT-PCR

RT-PCR assays were utilized to monitor viral loads in plasma, CSF, lymph node biopsies, colorectal biopsies, colorectal weck samples, and urine longitudinally every 2–4 weeks as indicated in the experimental design (see Fig. 1a) and amniotic fluid collected by amniocentesis at day 14 post-ZIKV infection, and from tissues collected at necropsy, essentially as previously described in refs. 15,16,26,33. RNA was extracted with a QIAcube HT (Qiagen, Germany). Liquid samples were extracted using the Qiacube 96 Cador pathogen HT, and tissue samples were lysed in Qiazol, using the Tissuelyser II (Qiagen, Germany), chloroform treated and extracted with the Qiacube 96 RNeasy HT kit. The wildtype ZIKV BeH815744 Cap gene was utilized as a standard. RNA standards were generated using the AmpliCap-Max T 7 High Yield Message Maker Kit (Cell Script) and purified with RNA clean and concentrator kit (Zymo Research, CA, USA). RNA quality and concentration was assessed by the BIDMC Molecular Core Facility. Log dilutions of the RNA standard were reverse transcribed and included with each RT-PCR assay. Viral loads were calculated as virus particles (VP) per microgram of total RNA as measured on the NanoDrop (Thermo Scientific, Waltham, MA, USA) or as VP per million cells, as shown in Figs. 3 and 4. Assay sensitivity was >100 copies/mL, >100 copies per million cells, and >3 copies/mg total RNA.

Neutralization Assays

ZIKV-specific neutralizing antibodies were measured by fold reduction neutralization (FRNT) and microneutralization (MN) assays, as previously described in refs. 15,16,26,33. For FRNT assay Vero cells were seeded at a concentration of 2 × 104 cells/well in 96-well plates 24 h prior to the assay initiation. Heat inactivated serum samples were serially diluted prior to being mixed and incubated with input virus ZIKV-PR (PRVABC59) for 1 hour at 37 °C. Cell-seeded 96-well plates were infected with 100 μL of the virus/serum mixtures for 1 hour before the addition of overlay media. Each serum dilution was tested in triplicate wells. Approximately 24 h after infection, ZIKV foci were detected using an antiflavivirus detection antibody, a horseradish peroxidase (HRP)-conjugated secondary antibody and True-Blue peroxidase substrate. ZIKV foci were visualized and counted using an ImmunoSpot analyzer and software. Each assay run included virus input and media-only control wells, as well as negative and positive control serum samples. Neutralizing antibody titers were reported as the inverse of the serum dilution estimated to reduce the number of input virus by 50% (FRNT50) as shown in Fig. 1.

Microneutralization assays were performed at WRAIR. Serum samples were serially diluted three-fold in 96-well micro-plates, in a total volume of 100 uL. 102 PFU ZIKV-PR (PRVABC59) in a total volume of 100uL was added and incubated at 35 °C for 2 h. Serum/virus mixtures were then transferred to microtiter plates containing confluent Vero cell monolayers (World Health Organization, NICSC-011038011038). After incubation for 4 days, cells were fixed with absolute ethanol: methanol for 1 h at –20 °C and washed three times with PBS. The pan-flavivirus monoclonal antibody 6B6-C1 conjugated to HRP (6B6-C1 was a gift from JT Roehrig, CDC) was then added to each well, incubated at 35 °C for 2 h, and washed with PBS. Plates were washed, developed with 3,3’,5,5’–tetramethylbenzidine (TMB) for 50 min at room temperature, stopped with 1:25 phosphoric acid, and absorbance was read at 450 nm. For a valid assay, the average absorbance at 450 nm of three non-infected control wells had to be % 0.5, and virus-only control wells had to be R 0.9. Normalized absorbance values were calculated, the MN50 titer was determined by a log mid-point linear regression model. The MN50 titer was calculated as the reciprocal of the serum dilution that neutralized R 50% of ZIKV, and seropositivity was defined as a titer R10, with the maximum measurable titer 7290, as shown in Supplementary Fig. 2.

Tissue Collection and Histopathology

Within 14 days of estimated term gestation (26 weeks), dams and fetuses were euthanized with intravenous sodium pentobarbital, and delivery was by caesarian section. Complete necropsies were performed by a veterinarian (A.J.M) on fetuses immediately following euthanasia, utilizing standard necropsy procedures with standard sterile surgical grade necropsy instruments and dissection blades. Briefly, peripheral lymphoid tissues were collected, followed by the gastrointestinal tract and abdominal organs. The pleural cavity was opened and the tongue, pharynx, trachea, esophagus, heart, and lungs (“pluck”) were removed en masse. Reproductive organs were collected, followed by brain, spinal cord, and eyes. Ruskin-Liston bone cutting forceps were used to expose the spinal cord to the level of the cauda equina. Limited necropsies were performed on dams for tissues previously shown to harbor viral RNA including reproductive organs, lymphoid tissues, spleen, and placenta. Fresh tissues were collected utilizing sterile blades for viral RT-PCR in RNAlater (Ambion). Frozen tissue for histopathology was prepared by trimming tissue, placing tissue samples into cryomolds with optimal cutting temperature medium (OCT, Tissue-Tek), and flash freezing on-site. Additional tissues were fixed in 10% neutral buffered formalin (NBF) for histopathology. Formalin-fixed tissues were trimmed, processed, and embedded in paraffin, sectioned, and stained with hematoxylin and eosin, and evaluated independently by two blinded veterinary pathologists (A.J.M., R.B.). Placenta was evalualated by a blinded gynecologic pathologist (J.L.H).

Immunohistochemistry and in situ hybridization

Immunohistochemistry and in situ hybridization (RNAscopeTM) were performed as previously described in ref. 33. Briefly, tissue sections were deparaffinized in xylene and rehydrated through graded ethanol solutions to distilled water. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide followed by heat induced epitope retrieval (HIER) in citrate buffer (Vector Labs) using a slide steamer (IHC World). Tissues were treated for nonspecific protein binding (Protein Block, DAKO) followed by application of mouse-anti ZIKV envelope (BioFront Technologies; BF-1176-56, 1:200) for 30 min at room temperature. A biotin-free polymer-based alkaline phosphatase kit with Permanent Red was used to detect antigen-antibody complexes (Polink-1 AP, Golden Bridge International Labs; #D18-18). In situ detection of ZIKV RNA was performed using RNAscope (ACDBio) technology. The ZIKV Asian probe (formerly O4, #468361) and red detection kit were used according to the manufacturer’s instructions.

ELISPOT

ZIKV-specific cellular immune responses were assessed by IFN-γ ELISPOT assays shown in Fig. 2 using pools of over- lapping 15-amino-acid peptides covering the prM and Env proteins (JPT, Berlin, Germany), essentially as we previously described in ref. 16. 96-well multiscreen plates (Millipore, MA, USA) were coated overnight with 100 mL/well of 5 mg/ml anti-human interferon-g (BD Biosciences, CA, USA; BD #554699) in endotoxin-free Dulbecco’s PBS (D-PBS). The plates were then washed three times with D-PBS containing 0.25% Tween 20 (D-PBS-Tween), blocked for 1–4 h with D-PBS containing 5% FBS at 37 °C, and incubated with 2 mg/ml of each peptide and 2 × 105 monkey PBMC in triplicate in 100 mL reaction mixture volumes. Following an 18–24 h incubation at 37 °C, the plates were washed nine times with PBS-Tween and incubated for 3 min with distilled water. The plates were then incubated with 1 mg/ml biotinylated anti-human interferon-g (U-Cytech Biosciences, UT, NETH) for 2 h at room temperature, washed six times with PBS-Tween, and incubated for 2 h with streptavidin-alkaline phosphatase (Southern Biotechnology Associates, AL, USA). Following five washes with PBS-Tween and one with PBS, the plates were developed with nitroblue tetrazolium-5- bromo-4-chloro-3-indolyl-phosphate chromogen (Pierce, IL, USA), stopped by washing with tap water, air-dried, and read using an ELISPOT reader (Cellular Technology Ltd., OH, USA). The numbers of spot-forming cells (SFU) per 106 cells were calculated. The medium background levels were typically < 15 SFU per 106 cells. SFU per 106 PBMCs of unstimulated PBMCs was subtracted from specific responses of corresponding individual macaques. Specific responses that were at or below zero after background subtraction were set to 1.

ELISA

Monkey ZIKV NS1 ELISA kits (Alpha Diagnostic International, TX, USA) were used to determine endpoint binding antibody titers using a modified protocol16. 96-well plates coated with ZIKV NS1 protein (RV-403310-1 Alpha Diagnostics) were first equilibrated at room temperature with 300 ml of kit working wash buffer for 5 min. 6 ml of monkey serum was added to the top row, and 3-fold serial dilutions were tested in the remaining rows. Serum samples were incubated at room temperature for 1 hr, and plates washed 4 times. 100 mL of anti-monkey IgG HRP-conjugate working solution was then added to each well and incubated for 30 min at room temperature. Plates were washed 5 times, developed for 15 min at room temperature with 100 ml of TMB substrate, and stopped by the addition of 100 ml of stop solution. Plates were analyzed at 450 nm/550 nm on a VersaMax microplate reader using Softmax Pro 6.0 software (Molecular Devices, CA, USA). ELISA endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance > 2-fold over background values and plotted as Log10 endpoint titer.