Cutting Edge: Innate Immune Augmenting Vesicular Stomatitis Virus Expressing Zika Virus Proteins Confers Protective Immunity

Zika virus (ZIKV), first isolated in the Zika forest of Uganda in 1947, belongs to the genus Flavivirus and is related to dengue virus, yellow fever virus, Japanese encephalitis virus, and West Nile virus (1). That ZIKV may be involved in causing significant disease was only reported in French Polynesia in 2013, where evidence indicated that this virus may be transmitted perinatally and is potentially associated with Guillain-Barré syndrome, in which the immune system targets peripheral nerves (1, 2). In 2015, substantial outbreaks of ZIKV were reported for the first time in Brazil and were associated with the fetal damage seen in aborted fetuses and in infants born to ZIKV-infected mothers, because ZIKV was retrieved from amniotic fluid and placental and brain tissue in affected fetuses (3, 4). Cases of ZIKV have also now been reported worldwide, including in the United States. There are no accepted therapies or vaccines to treat or prevent ZIKV infection, respectively; thus, the development of such measures are of paramount importance (5, 6). Immunogens based on ZIKV envelope protein (ZENV) may be of use in efficacious vaccine design because this viral protein does not vary significantly in strains isolated from around the world, and it has not changed significantly from the original isolate in 1947. In this study, we evaluated the use of recombinant vesicular stomatitis virus (rVSV) to independently express two versions of ZENV to assess vaccine efficacy in murine models: ZENV containing the precursor to membrane protein (ZprME) and ZENV alone. Mice inoculated with rVSVs generated Ig to ZENV, which was able to neutralize ZIKV infection in vitro, as well as to protect offspring from lethal ZIKV infection. Thus, rVSV-based vectors may be a safe and effective way to provide protection against ZIKV infection and warrant further assessment as a preventive measure against ZIKV infection.

293T cells (human embryonic kidney epithelial cells; American Type Culture Collection [ATCC]), HeLa cells (human cervical adenocarcinoma cells; ATCC), and BHK-21-WI cells (immortalized hamster kidney fibroblasts; generously provided by M.A. Whitt) were maintained in DMEM (Life Technologies/Invitrogen) supplemented with 10% FBS and 5% penicillin-streptomycin. Vero cells (immortalized Cercopithecus aethiops kidney epithelial cells; ATCC) were maintained in Medium 199 (Sigma) supplemented with 10% FBS and 5% penicillin-streptomycin.

Plasmid clones that contain the ZIKV genes for the premembrane and envelope proteins were purchased from GenScript. The sequences contain the premembrane and envelope region together (ZprME) or the envelope region alone (ZENV) and were constructed using ZIKV strain ZikaSPH2015 (accession no. KU321639; https://www.ncbi.nlm.nih.gov/nuccore/KU321639) as a reference. The sequences were made with restriction sites XhoI and NheI flanking the gene and a hemagglutinin (HA) tag (corresponding to aa 98–106 of the human influenza HA) toward the C-terminal region of the gene. The generation of vesicular stomatitis virus (VSV) and VSV with mutated matrix protein (VSVm) vectors containing the ZprME or ZENV proteins was done using restriction digest with XhoI and NheI (NEB) to create compatible ends to ligate into the VSV and VSVm cDNA plasmids using quick T4 DNA ligation (NEB). The ligated product was transfected into DH10B Escherichia coli, and liquid cultures from colonies were grown at 30°C overnight. DNA preps were confirmed using restriction digest and verified by sequencing reactions. Plasmid Midiprep Kits (QIAGEN) were used for transfection to recover infectious virions.

All plasmid transfections were done using Lipofectamine 2000 (Invitrogen), following the manufacturer’s recommended protocol.

VSV expressing ZENV (VSV-ZENV), VSV expressing ZprME (VSV-ZprME), VSVm expressing ZENV (VSVm-ZENV), and VSVm expressing ZprME (VSVm-ZprME) were recovered using established VSV-recovery methods. In brief, 1.5 × 10⁶ 293T cells were seeded into six-well plates and allowed to adhere overnight. The following day, they were infected with vTF7-3 at a multiplicity of infection (MOI) of 2.5 in serum-free medium for 45 min. After the incubation, cells were washed with serum-free medium, and DMEM with 5% low-IgG FBS (Life Technologies) was added to the cells. vTF7-3–infected 293T cells were transfected using Lipofectamine 2000 (Invitrogen) with pBluescript SK+ (pBS) plasmids expressing the VSV proteins nucleocapsid (N), phosphoprotein (P) and large polymerase subunit (L). Each well was transfected with 0.5 μg of pBS VSV-N, 0.83 μg of pBS-VSV-P, 0.17 μg of pBS-VSV-L, and 5 μg of the respective pVSV-ZIKV vector. The transfection mix was prepared in 100 μl of Opti-MEM (Invitrogen) and added to the well. The next day, the media were filtered through a 0.2-μm syringe onto fresh 293T cells to remove vTF7-3. If a cytopathic effect was observed after 24–48 h, the milieu was collected, and VSV virions were plaque isolated and further amplified by ultracentrifugation using a 10% OptiPrep cushion. Virus titers were determined by standard plaque assay using BHK-21-WI cells.

Cells were seeded in 6- or 12-well plates. Adherent cells were allowed to adhere overnight and were 80–90% confluent, unless otherwise indicated. Adherent cells were infected with rVSVs at the indicated MOI in a reduced volume of serum-free DMEM for 1 h, with agitation at 15-min intervals. Subsequently, cells were washed with 1× PBS twice, and complete medium was added back to the cells.

Infected cells were collected and incubated in RIPA lysis buffer with protease inhibitor mixture (Sigma) for 30 min at 4°C with gentle agitation. Cell debris was removed by centrifugation for 10 min at 15,000 × g. Protein concentration was quantitated using Coomassie blue (Thermo Scientific), and the OD was read at 595 nm. Equal amounts of protein (20 μg) were separated using SDS–10% PAGE and transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% milk powder in PBS–0.1% Tween 20 at room temperature and then probed with primary Abs against VSV glycoprotein (1:5,000; Sigma), flavivirus (4G2 mouse anti-flavivirus–specific Ab, 1:1,000; provided by Dr. D. Watkins), β-actin (1:10,000; Sigma), and HA (1:2,000; Sigma). Membranes were then washed with PBS–0.1% Tween 20 and probed with secondary Abs. Images were resolved using an ECL system (Thermo Scientific) and detected by autoradiography (Kodak).

A total of 5 × 10⁵ HeLa cells per well was seeded in a six-well plate. Cells were infected with VSV-GFP, VSVm, VSV-ZENV, VSV-ZprME, VSVm-ZENV, or VSVm-ZprME at an MOI of 0.001 in serum-free DMEM for 1 h, with agitation every 15 min. Next, the cells were washed twice with 1× PBS, and 3 ml of complete medium was added to each well. The culture supernatants were harvested at the indicated times and kept at −80°C until virus titer was measured using a standard plaque assay with BHK-21-WI cells.

HeLa cells seeded on coverslips were infected with rVSV-ZIKV viruses at an MOI of 10 for 6 h. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, blocked with 5% BSA for 1 h, and immunostained with mouse anti-HA Ab (1:500; Sigma), followed by Alexa Fluor 488–conjugated goat anti-mouse IgG (1:1000; Invitrogen). Cells were counterstained with DAPI. Images were taken with a Leica LSM confocal microscope at the Image Core Facility, University of Miami.

Female C57BL/6 and BALB/c mice were purchased from the Jackson Laboratory. All mice were 6–8 wk old. Mice care and study were conducted under the approval of the Institutional Animal Care and Use Committee of the University of Miami.

To determine the efficacy of the rVSV-ZIKV vaccine, 8-wk-old female C57BL/6 mice were vaccinated i.v. with 2 × 10⁶ PFU VSVm, VSV-ZENV, VSV-ZprME, VSVm-ZENV, or VSVm-ZprME (n = 5 per group) and boosted on day 22. Female BALB/c mice were vaccinated i.v. or i.m. with 2 × 10⁶ PFU VSVm, VSV-ZENV, VSV-ZprME, VSVm-ZENV, or VSVm-ZprME (n = 5 per group) and boosted on day 14. Mice were bled periodically using a submandibular bleed method under anesthesia. Serum was isolated from whole blood, and Ab titer was analyzed using ZIKV IgG ELISA analysis and neutralizing Ab assay.

Ninety-six–well polyvinyl chloride microtiter plates were coated with recombinant ZENV (MyBioSource) at 0.25 μg/ml overnight at 4°C. After washing with PBS, plates were blocked with 5% BSA for 1 h, incubated with appropriately diluted serum drawn from vaccinated or control mice for 2 h, and incubated with HRP-conjugated anti-mouse IgG (1:5000) for 1 h. The HRP signal was developed with TMB for 30 min at room temperature, and the reaction was stopped with 1 M HCl. OD was read at 450 nm on a plate reader. A serial 4G2 Ab dilution was used as the standard to quantitate the anti-serum.

Vero cells were plated at 2.5 × 10⁵ cells per well in 12-well plates and allowed to adhere overnight. Serum was heat inactivated at 56°C for 30 min, diluted 20-fold in the first tube, and then serial 2-fold dilutions were made. ZIKV was diluted to 50 PFU per well, mixed with the serial diluted serum samples, and incubated for 3 h at 37°C. Next, 100 μl of inoculum was added to Vero cells and incubated for 1 h, with agitation every 10–15 min. After the adsorption period, the inoculum was discarded and replaced with 2 ml of Medium 199 with 10% FBS and 0.8% high-viscosity carboxymethyl cellulose, and the plates were incubated for 4 d. After the incubation period, the monolayers were fixed with 10% formalin for 1 h and stained with 1% Crystal violet in ethanol for 30 s. Plates were rinsed with a gentle H₂O wash and air dried, and plaques were counted. The percentage of neutralization was calculated using virus control without serum. Plaque reduction neutralization titration (PRNT)50 was calculated using probit analysis, as described by Cutchins et al. (7).

Vero cells were plated at 2.5 × 10⁵ cells per well in 12-well plates and allowed to adhere overnight. A total of 0.2 g of brain tissue from euthanized mice was homogenized in 500 μl of Medium 199. After three cycles of freezing at −80°C and thawing, lysate was cleared by centrifugation. Serial 10-fold dilutions were made in Medium 199. A total of 100 μl of serially diluted tissue lysate was added to Vero cells and incubated for 1 h, with agitation every 10–15 min. After the adsorption period, the inoculum was discarded and replaced with 2 ml of Medium 199 with 10% FBS and 0.8% high-viscosity carboxymethyl cellulose, and the plates were incubated for 4 d. After the incubation period, the monolayers were fixed with 10% formalin for 1 h and stained with 1% Crystal violet in ethanol for 30 s. Plates were rinsed with a gentle H₂O wash and air dried. Plaques were counted, and viral titer was calculated as PFU/g of tissue.

The ZIKV-specific CTL response was assessed using splenocytes isolated from vaccinated mice. Cells were stimulated with 2 μg/ml of overlapping 15-aa peptides covering the ZprME region of ZIKV (custom synthesized by GenScript). For ELISA analysis, cell media were collected 72 h poststimulation, and the IFN-γ level was assessed using a mouse IFN-γ ELISA Kit (R&D Systems), following the manufacturer’s instructions. For flow cytometry, cells were stimulated for 6 or 72 h. Brefeldin A (3 μg/ml) was added to the cells 6 h before analysis. Cells were then washed, stained with cell surface marker, permeabilized with Cytofix/Cytoperm (BD Biosciences), and stained with IFN-γ. Data were acquired using an LSR II flow cytometer.

Total RNA was extracted and reverse transcribed using a QuantiTect Reverse Transcription Kit (QIAGEN). Real-time PCR was performed with SYBR Green reagent (Thermo Scientific). Primers used for the ZIKV MR766 strain were forward: 5′-ACTAGCTCGAGGCACACTGC-3′ and reverse: 5′-AGGTTCTTCTTCACACTACC-3′.

All statistical analyses were performed using the Student t test, unless specified. The data were considered to be significantly different at p < 0.05.

rVSV constructs were successfully generated using the G-L foreign gene expression site engineered into two VSV vectors: VSV and VSVm (Fig. 1A). The VSVm construct has a mutated matrix protein (aa 52–54: DTY to AAA), such that, following infection, it cannot block host cell mRNA export and, thus, is considerably attenuated as the result of innate immune proteins, including type I IFN, being translated (8, 9) (Supplemental Fig. 1A). ZENV and ZprME genes with a C-terminal HA tag were synthesized and inserted into a pcDNA3.1 vector. Expression was confirmed in 293T cells using Abs against HA and a flavivirus cross-reactive Ab 4G2 that binds to a conserved epitope on flavivirus E proteins (Fig. 1B). Two versions of ZENV were designed because it was not clear whether inclusion of the precursor to membrane protein region would better enhance expression and/or immunogenicity (10, 11). After expression was confirmed, the two ZENV constructs were ligated into VSV and VSVm backbones, infectious rVSV was recovered, and virus concentrations were determined by plaque assay (8, 12). HeLa cells were infected at an MOI of 10, and Western blot was performed 6 h postinfection to confirm ZENV and VSV glycoprotein expression (Fig. 1C). Immunofluorescent microscopy was also performed using rVSV-infected HeLa cells to further establish expression of ZENV postinfection. These data indicated, using 4G2, as well as anti-HA, Ab, that ZENV and ZprME are efficiently expressed by rVSV in vitro (Fig. 1D). The insertion of foreign genes into VSV can produce unintended consequences to the replication of rVSVs and drastically affect the viral lifecycle (13). Thus, a multicycle growth kinetics assay was performed using HeLa cells to verify that the incorporation of ZENV or ZprME into either VSV backbone (VSV or VSVm) did not significantly affect the replicative capacity of VSV. HeLa cells were infected at an MOI of 0.001, and the viral titer was analyzed at various time points. All of the rVSVs displayed robust growth kinetics, with a slight deviation in the kinetics; nonetheless, they were fully replication competent (Fig. 1E). Therefore, incorporation of ZENV or ZprME into rVSVs does not significantly affect replication.

VSV has routinely displayed a well-tolerated safety profile (14). Nevertheless, to confirm that the newly created rVSV-ZIKV constructs are safe, we injected concentrations of rVSVs that were several logs higher than normally used in vaccination studies (i.e., 2 × 10⁶ PFU) into naive BALB/c mice. Mice injected with 5 × 10⁷ or 1 × 10⁸ PFU rVSVs expressing ZENV or ZprME did not display any overt malaise or general adverse effects. No deaths of inoculated mice were observed 1 mo postinoculation (Supplemental Fig. 1B). Also, no deaths were observed in C57BL/6 mice injected i.v. with 1 × 10⁷ PFU rVSVm (Supplemental Fig. 2A).

To assess the efficacy of various rVSVs as an effective vaccine vector, 6-wk-old female C57BL/6 mice were injected i.v. with 2 × 10⁶ PFU rVSVs and boosted again with the same dose. Pre- or postvaccination sera were collected to assess Ab titers (Fig. 2A). ELISA was used to measure the Ab titer (IgG) using solid-phase recombinant ZENV produced from insect cells. ELISA results indicated that the VSVm-ZprME construct was the most effective at generating ZENV Ab using this immunization regimen, followed by the VSV-ZprME construct. Less robust responses were seen using the VSV-ZENV and VSVm-ZENV constructs for reasons that remain unclear (Fig. 2B). To complement this approach, a PRNT assay was performed to preliminarily evaluate whether the Abs generated to ZENV were able to neutralize ZIKV infection in vitro. The results indicated that rVSVs expressing ZprME were able to generate neutralizing Ab, and most mice in the group inoculated with VSVm-ZprME generated a high titer of neutralization Ab, with PRNT50 factor > 1000 (Fig. 2C). We also assessed the effects of the different rVSVs through different inoculation routes (i.v. and i.m.) in naive BALB/c mice (Supplemental Fig. 1C). Similar effects were observed using ELISA (Supplemental Fig. 1D, 1E). Intravenously injected VSV-ZprME and VSVm-ZprME generated anti-ZENV Ab at much higher titers compared with i.m. administration of the same type of virus (Supplemental Fig. 1D, 1E). Similar to i.m. inoculation, less robust responses were seen using VSV-ZENV and VSVm ZENV constructs (Supplemental Fig. 1D, 1E). Collectively, our rVSV-based ZIKV vaccine constructs are able to generate protective serum IgG against ZENV. To our surprise, VSVm-ZprME generated the highest Ab titer among all of the constructs, suggesting the possibility that an activated cellular innate immune response facilitates Ag presentation and the establishment of adaptive immunity (Supplemental Fig. 1A). ZIKV is also known to infect individuals for long periods, indicating that the generation of effective CTL activity may be important in helping to eliminate infection in the presence of Ab (1). Thus, we further evaluated the ability of our vaccines to generate CTLs. High amounts of IFN-γ were produced following stimulation of retrieved splenocytes with the synthetic ZprME peptide pool (15 mer), as shown by ELISA analysis (Fig. 2D). Further, flow cytometry analysis showed significant increases in CD8⁺/CD44^high/IFN-γ⁺ T cell populations (Fig. 2E). Taken together, our data indicate that rVSV expressing ZprME could provide an effective approach to create immune responses that may prevent ZIKV infection.

Although ZIKV does not usually cause disease in weaned wild-type (WT) mice (>3 wk old), suckling WT mice (∼1 wk old) are susceptible to ZIKV infection. Therefore, we assessed the protection efficacy of our rVSV-based ZIKV vaccine through maternal Ab transmission. Naive or VSVm-ZprME–vaccinated female mice were bred to WT C57BL/6 males 11 wk after the first vaccine inoculation. Delivery occurred at 14.5 wk postvaccination, and neonatal mice were challenged with ZIKV 7 d after birth. Neonatal mice were shown to be susceptible to ZIKV infection (strain MR766, 96.5% identical in the ZprME region to Brazil isolates (isolated from Paraiba, Brazil in 2015) (Supplemental Fig. 2B). Neonatal mice born to VSVm-ZprME–vaccinated or naive female C57BL/6 mice were infected i.p. with high-dose ZIKV (MR766) (7 × 10⁵ PFU; average mosquito bite ∼ 1 × 10⁴) and monitored daily for signs of disease and lethality (Fig. 3A). All mice born to naive females developed signs of neurologic disease, including hindlimb paralysis, before succumbing to infection by day 10 (Fig. 3B, left panel, 3C). In comparison, most mice born to VSVm-ZprME–vaccinated females exhibited no morbidity or mortality (Fig. 3B, right panel, 3C). We further evaluated ZIKV replication in progeny of VSVm-ZprME–vaccinated female mice. Neonatal mice born to naive or vaccinated females (18.5 wk after first inoculation) were similarly challenged with ZIKV (MR766) at 7 d after birth and euthanized 7 d postinfection. High copy numbers of ZIKV were detected in brain tissue of mice born to naive females; however, ZIKV was not able to replicate in progeny of VSVm-ZprME–vaccinated mothers (Fig. 3D, 3E). Therefore, the data suggested that our VSVm-ZprME vaccine has the potential to protect from ZIKV infection during prenatal and neonatal development, likely through the transmission of maternal IgG. Follow-up experiments using passive Ab transfer to naive infant mice may help to clarify the importance of neutralization Ab generated by our rVSV-ZIKV constructs.

Mosquito-borne ZIKV has been implicated as the cause of microcephaly in gestating fetuses and is now a major public health concern worldwide (2, 3). There is an urgent need for effective vaccines that prevent ZIKV infection. In this article, we demonstrated that rVSVs can be successfully generated to carry and express ZENVs that may have the capacity to produce protective neutralizing Ab against ZIKV infection. We observed that the ZprME constructs generated the most anti-ZENV IgG, as well as the most neutralizing Ab. Greater vaccine efficacy was seen in the VSVm-ZprME group following our inoculation regimen. This analysis indicates that it is not easy to predict which ZENV product will be the most efficacious at generating protective Ab to ZENV. We further showed that progeny of VSV-ZprME–vaccinated females are largely protected from lethal ZIKV infection, even when the mothers were vaccinated 4.5 mo earlier. This suggests that our vaccine has the potential to provide long-term protection against ZIKV. Long-term follow-up experiments of memory T cell analysis will help to further examine the protection property of our rVSV-ZIKV constructs. Our data also indicate that the rVSV with a mutated matrix gene provided greater Ab responses, suggesting that, because this virus cannot block host cell mRNA export, including type I IFNs, host defense countermeasures may rapidly eliminate this virus, as well as facilitate the establishment of adaptive immunity (9, 15), which provides an additional safety measure for using this attenuated version as a vaccine. Further evaluation of the minimum dose necessary to produce protective neutralization Ab through various inoculation routes will be performed. Possible hurdles, such as Ab-dependent enhancement of the infection, will also need to be evaluated carefully (e.g., whether the maternal acquired low-dose ZIKV Abs will predispose neonatal mice to ZIKV or other flavivirus infection). Overall, our data indicate that rVSV may be a suitable platform for the development of effective vaccines against ZIKV; it warrants further evaluation as candidate vaccines in nonhuman primates, in which ZIKV was first isolated, or directly in phase I trials in human subjects.

We thank Delia Gutman and Auristela Rivera for mice breeding and maintaining.

The authors have no financial conflicts of interest.