Viruses and cells

Zika virus strain PRVABC59, isolated from a patient serum sample, was provided by the Arboviral Diseases Branch, Division of Vector-Borne Diseases, Centers for Disease Control and Prevention (ADB/DVBD/CDC), Fort Collins, Colorado. The patient was infected in Puerto Rico during the ZIKV outbreak in 2015, and the genome sequence (GenBank: KU501215.1) was generated from the specimen by the Diagnostic and Reference Laboratory/ADB/DVBD using deep sequencing. The virus isolate was made by two passages in Vero cells and one in C6/36 cells (V2/C6-1), and was confirmed free of mycoplasma and Togavirus cross-contamination before shipment from DVBD/CDC. Upon receiving this virus isolate at Takeda, it was amplified once (P1) in Vero cells using serum free Dulbecco’s modified minimal essential medium (DMEM) (Corning; Corning, NY). Additional ZIKV strains (DakAr41524, P6-740, MR766, and R103451) were also provided by the ADB/DVBD/CDC and amplified in Vero cells under serum-free conditions to make working stocks for this project. Vero cells were grown and maintained in DMEM containing penicillin-streptomycin (Hyclone; Logan, UT), L-glutamine (Hyclone; Logan, UT) and 10% FBS sourced from a bovine spongiform encephalopathy (BSE)-negative country (Sigma; St. Louis, MO) (DMEM-10%FBS). Recombinant trypsin (Gibco; Gaithersburg, MD) was used to maintain and dissociate cells.

Virus titration

Plaque titration

Mouse viremia and virus aliquots used for mouse challenge and neutralization assays were measured by plaque titration on freshly confluent monolayers of Vero cells grown in 6-well plates as described57. Briefly, the first 4 mL overlay medium, containing 0.8% agarose in DMEM with 2% FBS (DMEM-2%FBS) or YELAH (0.165% lactalbumin hydrolysate, 0.033% yeast extract, Earle’s balanced salt solution, 25 mg of gentamicin sulfate and 1.0 mg of amphotericin B per liter) with 2% FBS, was added after adsorption of 100 µL/well of serially diluted samples onto Vero cells for 1.5 hrs. Following incubation for 4 days at 37 °C/5% CO 2 , 2 mL of a second agarose overlay medium containing 160 µg/mL of neutral red dye (Sigma; St. Louis, MO) was added. Plaques were analyzed on days 5 and 6.

50% tissue culture infectious dose(TCID50)

To facilitate eventual large-scale vaccine manufacturing, a high-throughput TCID50 assay was established to determine the infectious titer of material used to prepare inactivated vaccine. Virus titers were determined by titration on freshly confluent monolayers of Vero cells grown in 96-well plates. At the time of the assay, frozen virus aliquots were thawed and ten-fold dilution series were made in DMEM-2%FBS, and 100 μL/well of each virus dilution was added in quadruplicate to the Vero cell plates. The plates were incubated under 5% CO 2 for 5 days at 36 °C ± 2°C, before visual observation of the cell monolayer under a microscope for the presence of cytopathic effect (CPE) resulting from viral infection. The TCID 50 was calculated by the Reed\Muench method58 to indicate the maximum dilution level of the virus that resulted in at least 50% of cell infection. The TCID 50 titer is approximately 0.5–1 log 10 higher than the titer measured by plaque titration described above.

Generation and growth kinetics of ZIKV P6 sub-isolates

ZIKV PRVABC59 P1 stock, described above, was used to generate sub-isolates of the virus for producing PIZV candidates. Briefly, P1 virus was titrated on Vero cells as described above for plaque titration, and multiple large plaques were isolated and each picked agarose plug was mixed into 0.5 mL of culture medium and incubated overnight at 37 °C (P2). Three of the P2 isolates were subjected to two additional rounds of plaque purification (P3-4), and six final plaques (P4) were picked and directly amplified in individual Vero cell flasks to generate P5 virus stocks (P5a-P5f). Viral titer of the P5 stocks was determined by TCID 50 , and amplified once more in Vero cells (P6) by infecting Vero cells at 0.01 TCID 50 /cell. Two sequential virus harvests, taken three and five days pi, were pooled for each P6 culture, clarified by centrifugation, stabilized in DMEM containing a final concentration of 18% (w/v) trehalose (Pfanstiehl; Waukegan, IL) and stored at ≤−60 °C (Table 1). Growth kinetics of the P6 sub-isolates was conducted in duplicate flasks of Vero cells. Cells were infected with an MOI of 0.01 TCID 50 /cell of each sub-isolate virus in a serum free growth medium and aliquots of culture fluid were taken daily for titration by TCID 50 assay.

Genetic sequencing

The viral genomes of P6 sub-isolates were subjected to Sanger-based sequencing as described previously59. A QIAampViral RNA Mini Spin kit (Qiagen; Hilden, Germany) was used to extract viral RNA from P6 sub-isolate stocks, and 6 cDNA fragments encompassing the entire ZIKV genome for each extracted RNA sample were RT-PCR amplified using a Titan One Tube RT-PCR kit (Roche; Basel, Switzerland). After gel purification of the cDNA fragments using a Qiagen Quick Gel Extraction Kit (Qiagen; Hilden, Germany), each fragment was sequenced by automatic sequencing. Primers for RT-PCR and sequencing are available upon request.

Purification, inactivation and formulation of vaccine candidates

A single laboratory scale preparation of the PIZV candidates (sub-isolates b and e) was generated for this discovery/pre-clinical study at Takeda. Each of the P6-b and P6-e virus stocks was amplified in Vero cells at MOI of 0.01 for 5 days in a 36 °C ± 2°C /5% CO 2 incubator. Virus supernatants were harvested on day 3 and 5 pi, clarified by centrifugation, stabilized in DMEM containing a final concentration of 18% (w/v) trehalose and stored at ≤−60 °C. Pooled, clarified viral supernatants (200 mL each candidate) were rapidly thawed in a 37 °C water bath and treated with 20 U/mL of benzonase (Millipore; Burlington, MA) overnight at 2–8 °C to digest host cell DNA, followed by depth filtration with a 1.2 µM filter (Sartorius; Göttingen, Germany). Filtrate was applied to a Centricon Plus-70, 100 KDa MW centrifugal filter (Millipore; Burlington, MA) for a single round of concentration and buffer exchange into binding buffer (30 mL of 40 mM NaCl, 50 mM potassium glutamate, 10 mM L-Histidine and 10% trehalose, pH 7.5). The concentrated samples were diluted to 200 mL in binding buffer and subjected to Sartobind IEX nano ion-exchange chromatography (Sartorius; Göttingen, Germany). Virus samples were washed with binding buffer and eluted with elution buffer (750 mM NaCl, 50 mM potassium glutamate, 10 mM L-Histidine and 10% trehalose, pH 7.5), diluted to 300 mL in elution buffer lacking NaCl and applied to a second Sartobind IEX nano ion exchange column. Samples were eluted from the second column with buffer containing 2.7 mM KCl, 1.8 mM KH 2 PO4, 10 mM Na 2 HPO 4 , and 250 mM NaCl, pH 7.5, and applied to a Centricon Plus-70 100 KDa MW centrifugal filter for a single round of concentration and buffer exchange (40 mL) into PBS. Sample was then diluted to a final volume of 35 ml with PBS and stored at 2–8 °C.

For viral inactivation, freshly prepared 1% (v/v) formaldehyde (Fisher; Hampton, NH) was added drop-wise to each purified sample to obtain a final formaldehyde concentration of 0.02% and incubated at room temperature (~22 °C) for 14 days with frequent inversion. In a preliminary study, we had determined 7 days was required to completely inactivate virus. Therefore 14 days of formalin treatment was chosen to prepare the PIZV candidates used in this study (double the previously demonstrated duration of inactivation). Formaldehyde was neutralized with 0.04% w/v of sodium metabisulfite (Fisher; Hampton, NH) for 15 minutes at room temperature and sample was subjected to four rounds of concentration and buffer exchange (Centricon Plus-70, 100 KDa MW) with drug substance buffer (50 mL each; 10 mM NaH 2 PO 4 , 50 mM NaCl, 6% sucrose, pH 7.4). Samples were then diluted to 15 mL in drug substance buffer, sterilized through a 0.2 µm syringe filter, aliquoted into sterile stoppered glass vials, and frozen at ≤−60 °C.

Complete virus inactivation of the prepared PIZV candidates was determined by two sequential Vero cell culture amplifications of purified and inactivated sample aliquots. Sample aliquots taken on days 1, 11 and 14 were diluted (1:10 in DMEM-2%) to infect 8 replicate wells of a 96-well Vero plate. The plates were incubated and observed for CPE by TCID 50 as described for 6 days. On day 6 pi, total supernatants from 4 replicates/sample of the 96-well plate were transferred individually into 4 wells of a 6-well confluent Vero plate for a second culture amplification and CPE observation for 8 days.

Protein concentration was determined by Bradford Protein Assay (BioRad; Hercules, CA) before each sample was formulated to 1 μg/mL or 10 μg/mL in PBS with or without dropwise addition of 0.5 mg/mL of alum (Alhydrogel, Brenntag; Essen, Germany) under aseptic conditions and incubated overnight at 2–8 °C with gentle agitation. The resulting drug product lots were then aliquoted into sterile stoppered glass vials and stored at 2–8 °C until use.

SDS-PAGE and Immunoblotting

Samples were analyzed on 4–12% NuPage polyacrylamide gels (ThermoFisher; Franklin, MA) with a ZIKV recombinant sE protein control (from insect cells; MyBiosource,San Diego, CA). For total protein staining, gels were stained with SyproRuby (ThermoFisher; Franklin, MA) according to manufacturer protocol. For immunoblotting, protein was transferred to nitrocellulose membrane, blocked with 8% milk in wash buffer (TBS with 0.05% Tween-20) for 30 minutes, and followed by overnight 4°C incubation with a rabbit anti-ZIKV E polyclonal antibody (IBT Bioservices; Rockville, MD) diluted to 1:1000 in wash buffer. Membranes were washed, incubated with 1:1000 diluted alkaline phosphatase-conjugated goat α-rabbit antibody (KPL; Gaithersburg, MD) for 1 hour at room temperature, and then washed again prior to colorimetric development with BCIP/NBT substrate (KPL; Gaithersburg, MD) according to manufacturer protocol.

Sandwich ELISA

96-well Immulon II HB plates (ThermoFisher; Franklin, MA) were coated with 50 µL/well of human mAb, EDE C10 or EDE C8 (Absolute Antibody; Upper Heyford, UK) diluted 1 µg/mL in coating buffer (15 mM Na 2 CO 3 , 35 mM NaHCO 3 , pH 9.6) overnight at 4 °C. Following overnight incubation, plates were blocked for 60 min at 37 °C with blocking buffer (5% milk/PBS/0.1% Tween-20). After plate washing, 10-fold serially diluted ZIKV antigen (live ZIKV PRVABC59, PIZV-e candidate, or recombinant sE) in PBS/0.05% Tween-20 was added (50 µL/well) to plates, and incubated for 60 min at 37 °C. The secondary mouse mAb, flavivirus cross-reactive 4G2 (Absolute Antibody; Upper Heyford, UK) or anti-Zika sE 1176–56 (BioFront; Tallahassee, FL), diluted to 1 µg/mL in PBS/0.05% Tween-20 was added (50 µL/well) for 60 min at 37 °C before plate washing. A goat anti-mouse IgG conjugated with alkaline phosphatase (KPL; Gaithersburg, MD), diluted to 0.67 µg/mL in blocking buffer, was added and incubated for 60 min at room temperature. After removal of the conjugated antibody and plate washing, 100 µL/well of p-nitrophenyl phosphate substrate (Sigma; St. Louis MO) was added at room temperature for 20 min before addition of 20 µl/well of 3 M NaOH stop solution. The optical density at 405 nm was read on a BioTek plate reader, (Winooski, VT, USA).

Electron microscopy

Cryo transmission electron microscopy (cryoTEM) imaging was performed at NanoImaging Services, Inc (San Diego, CA USA). Briefly, samples were imaged undiluted and preserved in vitrified ice supported by holey carbon films on 400-mesh copper grids. Electron microscopy was performed using an FEI Tecnai T12 electron microscope, operating at 120 keV equipped with an FEI Eagle 4k × 4k CCD camera. Vitreous ice grids were transferred into the electron microscope using a cryostage. Images of each grid were acquired at multiple scales to assess the overall specimen distribution. The images were acquired at a nominal underfocus of −5 µm to −3 µm and electron doses of 10–25 e−/Å2.

Mouse studies

All animal experiments were conducted in accordance with IACUC guidelines and regulations.

Immunogenicity study in CD-1 mice

The experiments in Swiss-ICR CD-1 mice (Charles River; Wilmington, MA) were carried out at the animal facility at Takeda (Cambridge, MA) under animal protocol 16–06–175. Six groups of 6-week old male and female CD-1 mice (n = 10/group) were immunized with 0.1 mL of vaccine or PBS placebo by the intramuscular (i.m.) route (2 × 0.05 mL injections) on days 0, 28 and 56. Blood samples were collected on days −1 (pre-immune), 27, 42 and 70 for NAb measurement.

Protective efficacy study in AG129 mice

The experiments in AG129 mice were carried out at the DVBD/CDC (Fort Collins, CO) under animal protocol 16–017 approved by CDC IACUC. Groups of 4-week old male and female AG129 mice (n = 5/group) were immunized with 0.1 mL of vaccine or PBS placebo by i.m. injections (2 × 0.05 mL) on day 0, and boosted once on day 28. On day 56, mice were intraperitoneally (i.p.) challenged with 104 pfu of ZIKV PRVABC59. The challenge dose used was based on a prior dosing study showing mice morbidity rates (n = 5/dose group) were 0%, 40%, 60%, 100%, and 100% when i.p. challenged with 1, 10, 102, 103, and 104 pfu of the virus, respectively. By Reed-Muench method, the LD 50 of the virus to these adult mice was 31.6 pfu. Although both 103 and 104 pfu challenge resulted in 100% morbidity, the 104 pfu dose resulted in a shorter average survival time (13.6 ± 1.2 days) than the 103 pfu dose (18.6 ± 3.5 days). The 104 pfu was chosen over 103 pfu for a more stringent evaluation of vaccine efficacy.

Blood samples were collected from the tail vein on days −1, 27, 55, 58 and 79 for NAb titration or viremia analysis. Following challenge, mice were weighed and monitored daily for signs of illness for 21 days. Mice were humanely euthanized according to animal protocol upon signs of illness, including ruffled fur, breathing changes, anti-social behavior, hunched posture, twitching, paralysis, or loss of 20% original weight (whichever came first).

Passive immunization study in AG129 mice

Groups (n = 9/group or 6/group) of 14-week old AG129 mice were i.p. immunized with 0.1 mL of pooled day 79 sera (from protective efficacy AG129 study mice) that had been serially diluted 4-fold with PBS. Following passive immunization (16–19 hours later), blood was collected from each mouse and serum was separated by centrifugation prior to virus challenge for determination of circulating neutralizing antibody titer. Twenty-four hours following passive transfer, mice were i.p. challenged with 103 pfu of ZIKV strain PRVABC59. Animals were bled 2, 4 and 6 days post challenge (N = 2–3/group/day) for analysis of post challenge viremia by plaque titration. Following challenge, mice were weighed and monitored daily for signs of illness for 28 days. Mice showing signs of illness as described in the protective efficacy study above were humanely euthanized and counted as non-survivors.

Neutralization assays

Plaque reduction neutralization test (PRNT)

Mouse serum samples were tested to compare PRNT 50 titers against various strains of ZIKVs by a PRNT assay57. Briefly, a target of 100–200 pfu of ZIKV DakAr41524, P6-740, PRVABC59, MR766 or FSS13025 was incubated with equal volume of 2-fold serially diluted heat-inactivated serum samples for 1 hour at 37 °C . Six-well plates of Vero cells were inoculated with 100 µL of the serum/virus mixtures and incubated at 37 °C/5% CO 2 for 1.5 hours. Plates were then treated as described in the plaque titration method section. The neutralizing antibody titer was identified as the log 10 reciprocal serum dilution that reduced virus input plaques (based on back titration of the input virus) by at least 50%. Back titration of each virus was conducted in 6 replicates of 2-fold serial dilutions of the input virus.

Live reporter virus microfocus reduction neutralization test (R-mFRNT)

A live reporter chimeric virus, R-WN/ZIKV, expressing prM/E of ZIKV PRVABC59 and a ZsGreen reporter protein on a WNV replication vector (containing the entire WNV genome except its prM/E genes) developed in house at DVBD/CDC laboratory was used for the R-mFRNT. The ZsGreen with a 2 A self-cleavage peptide was inserted in the chimeric WN/ZIKV60 at the beginning of the C gene, similar to the previously described strategy of a DENV-2 reporter construct, DENV-GFP61. The R-mFRNT assay with reporter virus is similar to a previously established micro-immunofocus reduction neutralization test (m-FRNT)62,63 but the fluorescent reporter protein expressed by the chimeric virus eliminates the requirement of immuno-staining and also allows for virus foci detection within 24 hrs pi. The integrity of the reporter gene in the reporter virus lot used was confirmed by RT-PCR, full genome sequencing, and dual-color (viral antigen and reporter) flow cytometry. The R-mFRNT assay using R-WN/ZIKV results in similar NAb titers as compared to m-FRNT and/or PRNT using wild type virus by comparison of the 2 tests with a panel of 8 human control sera samples (within 2-fold difference; ZIKV by PRNT: R-WN/ZIKV by R-mFRNT = 1.3 ± 0.3 fold).

Briefly, serum samples were diluted 10-fold in BA-1 diluent (M199 medium, 1% bovine albumin, penicillin/streptomycin, sodium bicarbonate and Tris buffered, pH 7.4) and heat inactivated in a 56 °C water bath for 30 minutes. Further two-fold dilution series of heat-inactivated samples were made in BA-1 diluent and mixed with an equal volume of R-WN/ZIKV (100–200 focus forming unit, ffu) for 1 hr at 37 °C/5% CO 2 . Each serum/virus mixture (30 µL/well) was added to a confluent Vero cell plate in triplicate for 90 min adsorption at 37 °C/5% CO 2 , followed by addition of 150 µL/well of Gibco FluoroBrite DMEM (ThermoFisher; Franklin, MA) without FBS. After incubation at 37 °C ± 2 °C/5% CO 2 for 24–26 hours, fluorescent virus microfoci were directly live-imaged and analyzed by Celigo image cytometer (Nexcelom; Lawrence, MA). Some experiments were measured by an EliSpot reader (model ELR0681FL) (Autoimmun Diagnostika GmbH; Strassberg, Germany) after cell fixation with 4% formaldehyde for 10 min. Triplicate results were averaged, and NAb titers were expressed as the log 10 transformed reciprocal serum dilution that reduced input virus foci (based on back titration of the input R-WN/ZIKV in the same assay) by 50% or more.

Reporter virus particle (RVP) neutralization assay

NAb titers from the CD-1 mouse study were analyzed by titration of serum samples with a constant amount of Zika RVPs (Integral Molecular; Philadelphia, PA) in Vero cells. Briefly, sera were heat inactivated at 56 °C for 30 min, serially diluted in assay media containing Opti-MEM (Thermo Fisher; Waltham, MA), 1% Penicillin/Streptomycin (Mediatech) and 10% FBS (HyClone; Logan, UT) and then incubated at 37 °C ± 2 °C/5% CO 2 with RVPs (input amount empirically determined for each RVP lot) for 1 hour in a 384-well plate format. The serum/RVP mixture was then mixed with trypsinized Vero cells (4,625 cells/well) and incubated for 72 hours at 37 °C ± 2 °C/5% CO 2 . After addition of Renilla Glo luciferase substrate (Promega; Madison, WI) to each well and incubation for 15 min at room temperature, luciferase intensity was read on an EnSpire plate reader (Perkin Elmer; Waltham, MA). Data was analyzed using JMP11 non-linear 4 parameter analysis, normalized to a positive tracking control (anti-ZIKV rabbit serum) and effective dose 50% (EC50) was reported as neutralization antibody titers resulting in 50% reduction of luminescent signal intensity generated by input RVP.

Statistical analysis

One way ANOVA was used to compare differences of NAb titers between test groups of each animal study. Correlation between circulating NAb titers and peak viremia post challenge from the passive immunization study was assessed by Spearman rank-correlation test. Comparison of geometric mean NAb titers between protected and non-protected mice was performed using a Wilcoxon rank sum test. All statistical analysis was performed using GraphPad Prism v7 (GraphPad Software; Carlsbad, CA), while R v3.3.3 software was used to perform logistic regression analysis to determine an immune correlate of protection.