Ethics statement

B6(Cg)-Ifnar1tm1.2Ees/J Ifnar1−/− mice were purchased from Jackson Laboratories and a breeding colony was established. Female C57BL/6 J mice ages 6–8 weeks were purchased from Jackson Laboratory. Mice were housed in Life Sciences Annex building on the University of Nebraska – Lincoln (UNL) campus under the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) guidelines. The protocols were approved by the UNL Institutional Animal Care and Use Committee (IACUC) (Project ID 1448: Viral Vectored Flavivirus Vaccines). All animal experiments were carried out according to the provisions of the Animal Welfare Act, PHS Animal Welfare Policy, the principles of the NIH Guide for the Care and Use of Laboratory Animals, and the policies and procedures of UNL. All immunizations and bleeds were performed under ketamine and xylazine induced anesthesia.

Viruses

Zika virus (ZIKV) strain PRVABC59 (ZIKV-PR; Puerto Rico, 2015) was provided by the Biodefense and Emerging Infections Research Resources Repository (BEI Resources). ZIKV strain Dakar 41525 was adapted for mice by passage in Rag1−/− mice40. Viral stocks of ZIKV strain PRVABC59 were grown on C6/36 cells and collected after 7 days. Viral stocks of mouse-adapted ZIKV strain Dakar 41525 were grown on Vero cells and harvested after 3 days. Supernatant was collected and clarified by centrifugation at 350 g for 10 min. Aliquots were stored at −80 °C. The virus was quantified by plaque assay or focus forming assay (FFA)39.

Recombinant adenovirus type 5 plasmid construction

The full prM-E gene from ZIKV strain PRVABC59 was codon-optimized for human gene expression, and the VSV G signal sequence was added to the N-terminus of the protein. The DNA was synthesized by GenScript and kindly provided by Dr. Asit Pattnaik and cloned in a pCI-Neo mammalian expression vector. Recombinant Adenovirus 5 was created using the AdEasy Adenoviral Vector System (Agilent). Briefly, to introduce unique restriction enzyme sites for downstream cloning, the prM-E region was amplified by PCR from the pCI-Neo DNA plasmid vaccine using the Q5 High-Fidelity 2X Master Mix (NEB). It was cloned into pCR-Blunt using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen) for sequence confirmation. After confirmation, the gene was cloned into the pShuttle-CMV supplied in the AdEasy kit using T4 DNA ligase (NEB). This shuttle contains both a CMV promoter and a SV40 PolyA signal. The pShuttle-CMV-prM-E was linearized and transformed into BJ5183 electrocompetent cells with the pAdEasy-1 vector (Adenovirus type 5) to undergo homologous recombination. This recombination inserts the prM-E expression cassette into the E1 region of the Adenovirus type 5 (Ad5) genome. In addition, this vector is also deleted for the E3 region. Recombination was confirmed by restriction digest and a successful recombinant was transformed into XL1 cells for midiprep using the Qiagen Plasmid Midi Kit (Qiagen).

Recombinant adenovirus type 4 plasmid construction

The Adenovirus type 4 genome was cloned and a shuttle plasmid to replace the E1 region was created as previously described35. Briefly, the complete Adenovirus type 4 (pAd4) genome was cloned into a single low-copy plasmid, and a shuttle plasmid (pAd4-recomb-cloner) to replace the E1 region of Ad4 was created using overlapping PCR products. The CMV-prM-E-PolyA region from the pShuttle-CMV-prM-E plasmid was amplified using the Platinum PCR SuperMix High Fidelity (Invitrogen) and fused to an Frt-Zeo-Frt fragment using overlapping PCR. This fragment was then cloned into pCR8/Gw/TOPO for sequence confirmation (Invitrogen). After sequence confirmation, the shuttle was linearized and transformed with the pAd4 plasmid into BJ5183 electrocompetent cells for homologous recombination. Recombinants were screened on kanamycin and zeocin and confirmed with restriction digest. A successful recombinant was then transformed into XL1 cells, and plasmid DNA was purified using the Qiagen Plasmid Midi Kit (Qiagen).

Recombinant adenovirus virus rescue and purification

The recombinant Adenovirus 5 and 4 genomes with the ZIKV prM-E insert were linearized and buffer exchanged using a Strataprep PCR purification kit (Agilent Technologies). The linearized recombinant gDNA was transfected into 293 cells using the PolyFect Transfection Reagent (Qiagen). After virus rescue was observed via plaque formation, cells were harvested and virus was released by 3 freeze-thaw cycles. Virus was amplified by sequential passages in 293 cells until a final amplification using a Corning 10-cell stack (~6300 cm2). The virus was purified by 2 sequential CsCl ultracentrifuge gradients, desalted using Econo-Pac 10DG Desalting Columns (Bio-Rad), and stored at −80 °C in Ad-tris buffer (20 mM Tris-HCl, 100 mM NaCl 2 , 1 mM MgCl 2 •6H 2 O, 10% glycerol).

Recombinant adenovirus quantification

After CsCl purification and desalting, the virus particle quantity was determined on a NanoDrop Lite spectrophotometer (Thermo Fisher) with an OD260. The infectious unit titer was determined using the QuickTiter Adenovirus Titer Immunoassay Kit (Cell Biolabs, Inc.). Briefly, 293 cells were infected with serial dilutions of Adenovirus stock in triplicate. After incubation for 48 hours, cells were fixed with cold methanol, blocked with PBS with 1% BSA, and then incubated with anti-hexon antibody. Cell were washed, incubated with secondary HRP-conjugated antibody, and developed with DAB. Positive cells were stained brown and counted to determine the infectious units per mL.

Western blotting

For confirmation of protein expression from the recombinant Adenoviruses, confluent 293 cells were infected with Ad5-prM-E or Ad4-prM-E at either 500 vp/cell or an MOI of 1. They were incubated at 37 °C and 5% CO 2 and harvest at 48 hours. Cells were denatured using Laemmli buffer plus 2-mercaptoethanol and boiled at 100 °C for 10 minutes. The sample was then passed through a QIAshredder (Qiagen). Sample was loaded onto a 12.5% SDS-PAGE gel and separated using electrophoresis. Protein was transferred to a nitrocellulose membrane and blocked for 30 minutes with 5% milk in TBST. The membrane was incubated in mouse anti-ZIKV E protein antibody (mAb-0302156; BioFront Technologies) at 1:5000 and mouse anti-GAPDH (sc-47724; Santa Cruz Biotechnology, Inc.) at 1:2000 in TBST 1% milk overnight at 4 °C. After 3 washes in TBST, the membrane was incubated with goat anti-mouse-HRP conjugated antibody (Millipore Sigma) at 1:2000 in TBST 1% milk for 1 hour at room temperature. After incubation, the membrane was washed and developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).

ZIKV plaque assay

Cell supernatants were serially diluted in DMEM 2% FBS and added to confluent Vero cells. After incubation with rocking at 37 °C and 5% CO 2 for 1 h, cells were washed once with PBS and 1 mL of agar/DMEM overlay was added. Plates were incubated for 4 days at 37 °C and 5% CO 2. After 4 days, cells were fixed with 10% formaldehyde, the agar plugs removed, and the cell monolayer was stained using crystal violet. Plaques were counted and plaque forming unit (PFU) per mL were calculated.

ZIKV foci forming unit assay

Virus stocks were titrated by focus forming assay (FFA) on Vero cells as previously described39 and stored in aliquots at −80 °C.

Mouse challenge studies

Female Ifnar1−/− mice (8-9 weeks old) were immunized intramuscularly at day 0 with 1010 vp of Ad4-prM-E (n = 9), Ad5-prM-E (n = 10), or control PBS (n = 10). Two experimental replicates were performed however, after day 4, three mice in the Ad4-prM-E group were lost to unrelated causes due to anesthesia complications. Female C57BL/6 mice were immunized intramuscularly at day 0 with 1010 vp of the indicated Ad vaccine or control PBS. All mice were boosted at week 3 with 1010 vp of indicated Adenovirus or sham vaccine and then challenged at week 7 with 106 FFU of mouse-adapted ZIKV-Dakar 41525 via the intraperitoneal (i.p.) route. C57BL/6 mice were administered 2 mg of anti-mouse Ifnar1 mAb (MAR1-5A3, BioXcell) via the i.p. route one day before ZIKV infection and given 0.5 mg more at day 4 post infection. All mice were monitored daily for weight loss and mice were euthanized at 25% weight loss.

Antibody and T-cell assays

Female C57BL/6 mice were immunized with 1010 vp of indicated Adenovirus or control PBS. All immunizations were performed intramuscularly with a 27-gauge needle into both quadriceps in two 25 µl injections. At week 6, prime/boost animals were boosted with 1010 vp of indicated Adenovirus. At week 8, all animals were terminally bled via a cardiac puncture and spleens were harvested. Sera was isolated from whole blood with a BD Microtainer Blood Collection Tube (Becton Dickinson) and used for further ELISA and neutralization tests. Splenocytes were harvested for T cell immune assays.

ELISA

Immunolon 4 HBX microtiter 96-well strips (VWR) were coated with 150 ng of recombinant ZIKV E protein (Cat #:MBS596088; MyBioSource) in bicarbonate/carbonate coating buffer overnight at 4 °C. The plates were blocked with 2.0% BSA in PBS for 2 hours at room temperature (RT). Sera was diluted in 1.0% BSA in PBS and incubated for 2 hours at RT. The plates were washed 6X with PBST and incubated with goat anti-mouse-HRP antibody (1:2000; Thermo Fisher) in 1.0% BSA in PBS for 1 hour at RT. After washing 4X with PBST and 2X with PBS, the plate was developed with 1-Step Ultra TMB-ELISA (Thermo Fisher), and the reaction was stopped with 2 M sulfuric acid. The OD450 was detected using a SpectraMax i3x Multi-Mode microplate reader (Molecular Devices), and the endpoint titer was determined as signal that was two times background values.

ELISPOT

The T-cell epitopes were mapped using a peptide array of the ZIKV strain PRVABC59 E protein from BEI resources (Catalog No. NR-50553). This 164-peptide array spans the entire E region and consists of 15-mers with 12 amino acid overlap. Potential immunogenic peptides were identified using a matrix of peptides pools, and the epitopes were confirmed using individual peptides. Splenocytes were isolated from mice using a 40 μm Nylon cell strainer (BD Labware). Red blood cells were lysed using ACK lysis buffer and the splenocytes were resuspended in cRPMI at a concentration of 106 splenocytes/mL. Ninety-six well polyvinylidene difluoride-backed plates (MultiScreen-IP, Millipore) coated with 50 μl of anti-mouse IFN-γ mAb AN18 (5 µg/ml; Mabtech) overnight at 4 °C. Plates were washed and blocked with RPMI at 37 °C for 1 hour. Equal volumes (50 µL) of the single-cell suspension splenocytes and peptide (5ug/mL) were added to the wells in duplicate. Plates were incubated overnight at 37 °C with 5% CO 2 . The plates were washed 6X with PBS and incubated with 100 μl of biotinylated anti-mouse IFN-γ mAb (1:1000 dilution; Mabtech) diluted in PBS with 1.0% FBS for 1 hour at RT. Plates were washed 6X with PBS and incubated with 100 µl of streptavidin-alkaline phosphatase conjugate (1:1000 dilution; Mabtech) diluted in PBS 1.0% FBS. After 1 hour at RT, the plates were washed 6x with PBS. To develop, 100 µl of BCIP/NBT (Plus) alkaline phosphatase substrate (Thermo Fisher) was added to each well and development was stopped by washing several times in dH 2 O. The plates were air dried and spots were counted using an automated ELISpot plate reader (AID iSpot Reader Spectrum). Results are expressed as spot-forming cells (SFC) per 106 splenocytes.

qPCR for viral load quantification

Challenged mice were bled from the submandibular vein at day 4 post infection and sera was isolated. RNA from sera was extracted using the PureLink Viral RNA/DNA Mini Kit according to manufacturer’s instructions (Invitrogen). Real time RT-qPCR was performed using the Luna Universal Probe One-Step RT-qPCR Kit (NEB). It was run on a QuantStudio 3 Real-Time PCR System (Applied Biosystems) using the Luna kit cycling conditions (55 °C for 10 min, 95 °C for 1 min, and 40 cycles of 95 °C for 10 s and 60 °C for 1 min). Results were compared to a standard curve created using ZIKV RNA extracted from a known quantity of infectious virus. Unamplified samples were set at the limit of detection (100 FFU equivalent/mL). The following primer probe set was used: 1183 F: 50-CCACCAATGTTCTCTTGCAGACATATTG-30; 1268 R: 50-TTCGGACAGCCGTTGTCCAACACAAG-30; and probes (1213 F): 5′FAM/AGCCTACCTTGACAAGCAGTC/BHQ1–3′22.

Plaque reduction neutralization assay

Mouse sera was heat-inactivated at 56 °C for 30 min. Sera then was diluted 2-fold in DMEM 2% FBS and incubated with 50 PFU of ZIKV strain PRVABC59 for 2 hours at 37 °C and 5% CO 2. This was then added to a 24-well plate of confluent Vero cells. The plaque assay was continued as described above.

Statistical analysis

GraphPad Prism software was used to analyze all data. Data are expressed as the mean with standard error (SEM). Survival curves were analyzed using the log rank test and weight loss was analyzed using two-way ANOVA multiple comparison test. ELISA, PRNT50, T-cell data, and viral burden were analyzed using one-way ANOVA with Bonferroni multiple comparisons. A p value < 0.05 was considered statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).