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Antibodies Elicited by an NS1-Based Vaccine Protect Mice Against Zika Virus

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Antibodies Elicited by an NS1-Based Vaccine Protect Mice Against Zika Virus

Mark J Bailey et al. mBio.

Abstract

Zika virus is a mosquito-borne flavivirus which can cause severe disease in humans, including microcephaly and other congenital malformations in newborns and Guillain-Barré syndrome in adults. There are currently no approved prophylactics or therapeutics for Zika virus; the development of a safe and effective vaccine is an urgent priority. Preclinical studies suggest that the envelope glycoprotein can elicit potently neutralizing antibodies. However, such antibodies are implicated in the phenomenon of antibody-dependent enhancement of disease. We have previously shown that monoclonal antibodies targeting the Zika virus nonstructural NS1 protein are protective without inducing antibody-dependent enhancement of disease. Here, we investigated whether the NS1 protein itself is a viable vaccine target. Wild-type mice were vaccinated with an NS1-expressing DNA plasmid followed by two adjuvanted protein boosters, which elicited high antibody titers. Passive transfer of the immune sera was able to significantly protect STAT2 knockout mice against lethal challenge by Zika virus. In addition, long-lasting NS1-specific IgG responses were detected in serum samples from patients in either the acute or the convalescent phase of Zika virus infection. These NS1-specific antibodies were able to functionally engage Fcγ receptors. In contrast, envelope-specific antibodies did not activate Fc-mediated effector functions on infected cells. Our data suggest that the Zika virus NS1 protein, which is expressed on infected cells, is critical for Fc-dependent cell-mediated immunity. The present study demonstrates that the Zika virus NS1 protein is highly immunogenic and can elicit protective antibodies, underscoring its potential for an effective Zika virus vaccine.IMPORTANCE Zika virus is a global public health threat that causes microcephaly and congenital malformations in newborns and Guillain-Barré syndrome in adults. Currently, no vaccines or treatments are available. While antibodies targeting the envelope glycoprotein can neutralize virus, they carry the risk of antibody-dependent enhancement of disease (ADE). In contrast, antibodies generated against the NS1 protein can be protective without eliciting ADE. The present study demonstrates the effectiveness of an NS1-based vaccine in eliciting high titers of protective antibodies against Zika virus disease in a mouse model. Sera generated by this vaccine can elicit Fc-mediated effector functions against Zika virus-infected cells. Lastly, we provide human data suggesting that the antibody response against the Zika virus NS1 protein is long-lasting and functionally active. Overall, our work will inform the development of a safe and effective Zika virus vaccine.

Keywords: Fc-mediated responses; NS1; Zika virus; antibody-dependent enhancement of disease; flavivirus; nonneutralizing antibodies; vaccine.

Figures

FIG 1
FIG 1
Generation of expression plasmids encoding ZIKV NS1. (A) Human-codon-optimized NS1 of ZIKV PRVABC59 was subcloned into a mammalian expression vector, pCAGGS, which includes the last 24 amino acids of the envelope protein at the amino terminus followed by the NS1 coding region (pCAGGS NS1). Of note, the first amino acid of the NS1 coding region is indicated by a bold, red aspartic acid residue. (B) A second version (pCAGGS NS1-His) also encodes ZIKV PRVABC59 NS1, followed by a PreScission Protease cleavage site (LEVLFNGPG; blue region) and a hexahistidine motif (HHHHHH; orange region) at the carboxy terminus. (C) HEK 293T cells were transfected with pCAGGS NS1 or pCAGGS NS1-His or not transfected (mock). At 24 h posttransfection, the cells were fixed with 0.5% paraformaldehyde, and the surface expression of NS1 was detected using an anti-ZIKV NS1 monoclonal antibody, AA12, or a polyclonal anti-histidine antibody. Secondary antibodies conjugated to Alexa Fluor 488 were used to visualize binding using a Celigo imaging cytometer. The scale bars are equal to 500 μm. (D) HEK 293F cells were transfected with pCAGGS NS1-His, and 4 days posttransfection, soluble NS1 from the supernatant and cell lysates was collected and purified over an NI-NTA column. Soluble NS1 proteins from the supernatant and lysates were resolved in an SDS-PAGE gel and detected using a polyclonal anti-histidine antibody in a Western blot assay. BSA was used as a negative control, and a His-tagged soluble hemagglutinin of A/Perth/16/09 (H3N2) was used as a positive control.
FIG 2
FIG 2
Zika virus NS1 vaccine induces a robust and functional antibody response in mice. (A) Schematic outlining the vaccination strategy, where mice were prime immunized with 80 µg of the pCAGGS NS1 DNA plasmid via electroporation, followed by two booster immunizations of soluble NS1 proteins. All vaccinations were administered intramuscularly. (B) Groups of mice vaccinated with each adjuvant used for the protein components. (C to E) The antibody response to NS1 (PRVABC59 ZIKV) was measured by ELISA. Each data point denotes an individual animal, while each color represents one group of mice. The time points are after the DNA priming at day 21, after the protein boost at day 42, or at day 84 (sera from the terminal bleed). ELISA data were run in duplicate and are shown as areas under the curve (AUCs). A nonparametric multiple-comparison Kruskal-Wallis test was used to determine statistical significance at each time point. Asterisks indicates statistical significance of a group (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) in a comparison with naive serum. No significance was observed between control groups and the naive group. FA, Freund's adjuvant. (F and G) To examine the ability of NS1-specific antibodies to activate Fc-mediated effector functions, Vero cells were infected with PRVABC59 ZIKV or HEK 293T cells were transfected with an NS1 expression plasmid (pCAGGS-NS1). Infected Vero cells or transfected HEK 293T cells were used as targets for measuring antibody-mediated effector functions with a genetically modified Jurkat cell line expressing murine FcRγ IV with an inducible luciferase reporter gene. Fold induction was measured in relative light units and calculated by subtracting background signal from wells without effector cells and then dividing wells with sera by wells with no sera added. All sera were tested at a starting dilution of 1:75 and were serially diluted 3-fold in duplicate. A nonlinear regression best-fit curve was generated for each data set using GraphPad Prism 6. Error bars represent SEM.
FIG 3
FIG 3
Passive transfer of serum from vaccinated mice protects against lethal challenge. (A to C) Groups of 4 to 5 male and female B6.129-Stat2−/− mice were injected intraperitoneally with 200 μl of pooled serum before a challenge with 10 LD50s (158 PFU per mouse) of MR766 ZIKV intradermally. (D to F) Groups of 4 male and female B6.129-Stat2−/− mice were injected intraperitoneally with 200 μl of pooled serum before a challenge with 1,000 PFU of PRVABC59 ZIKV intradermally. Weight loss was monitored daily. Clinical scoring was conducted using the predefined criteria, with a maximum possible score of 7: impact on walking (1), unresponsiveness (1), left hind leg paralyzed (1), right hind leg paralyzed (1), left front leg paralyzed (1), and right front leg paralyzed (1). Deceased animals were awarded a score of 7. The ratios in the figures indicate the number of animals that survived challenge over the total number of animals per group. The Mantel-Cox and Gehan-Breslow-Wilcoxon tests were used to analyze statistical significance of survival between two groups. A multiple t test and the Holm-Šidák method were used to determine statistical significance at each time point for the weight curve and the clinical score. Asterisks indicate statistical significance of a group (*, P < 0.05) in a comparison with mice vaccinated with BSA.
FIG 4
FIG 4
NS1-specific antibodies are long-lived and functional in ZIKV infected humans. ELISA data of individual human sera against recombinant NS1 protein from PRVABC59 ZIKV. ELISA data were run in duplicate, and values represent AUCs. (B) ELISA data of select human samples with repeat blood draws. Each color represents an individual patient. UTMB, patient designation. (C to F). To test the ability of human sera to activate Fc-mediated effector functions, Vero cells were infected with PRVABC59 ZIKV and an Fc-FcγR reporter assay was performed as previously shown. The legend includes the patient identifier number and the number of days postonset of symptoms (DPO). All sera were tested at a starting dilution of 1:75 and were serially diluted 3-fold in duplicate. A nonlinear regression best-fit curve was generated for each data set using GraphPad Prism 6. Error bars represent SEM.
FIG 5
FIG 5
Human sera can engage FcγRs when targeting NS1-transfected cells. (A to D) To examine the ability of human sera to activate NS1-specific Fc-mediated effector functions, HEK 293T cells were transfected with an NS1 (pCAGGS-NS1) expression plasmid. Each color represents an individual sample; the titles includes the patient identifier, and the keys include numbers of days postonset (DPO) of symptoms. A surrogate in vitro reporter assay for measuring Fc-FcγR interactions was performed as previously shown (Fig. 4) (32). All sera were tested at a starting dilution of 1:75 and were serially diluted 3-fold in duplicate. A nonlinear regression best-fit curve was generated for each data set using GraphPad Prism 6. Error bars represent SEM.
FIG 6
FIG 6
Cross-reactive envelope-specific antibodies do not elicit Fc-mediated responses. Sera from TBEV vaccinated patients were analyzed for binding to ZIKV proteins and the ability to elicit Fc-mediated effector functions. Sera were obtained through a screening of TBEV vaccinee samples as described previously in the work of Duehr et al. (49). ELISAs were performed on recombinant ZIKV envelope protein (A) or recombinant NS1 protein from PRVABC59 ZIKV (B). All sera were tested at a starting dilution of 1:40 and were serially diluted 4-fold in duplicate. “Acute ZIKV Infection” indicates serum from a patient with an acute infection confirmed by reverse transcription-PCR. (C) Vero cells were infected with PRVABC59 ZIKV, and a surrogate in vitro reporter assay was performed to measure Fc-FcγR interactions. All sera were tested at a starting dilution of 1:25 and were serially diluted 3-fold in duplicate.

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