A Zika virus vaccine expressing premembrane-envelope-NS1 polyprotein

Abstract

Current efforts to develop Zika virus (ZIKV) subunit vaccines have been focused on pre-membrane (prM) and envelope (E) proteins, but the role of NS1 in ZIKV-specific immune response and protection is poorly understood. Here, we develop an attenuated recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing ZIKV prM-E-NS1 as a polyprotein. This vectored vaccine candidate is attenuated in mice, where a single immunization induces ZIKV-specific antibody and T cell immune responses that provide protection against ZIKV challenge. Co-expression of prM, E, and NS1 induces significantly higher levels of Th2 and Th17 cytokine responses than prM-E. In addition, NS1 alone is capable of conferring partial protection against ZIKV infection in mice even though it does not induce neutralizing antibodies. These results demonstrate that attenuated rVSV co-expressing prM, E, and NS1 is a promising vaccine candidate for protection against ZIKV infection and highlights an important role for NS1 in ZIKV-specific cellular immune responses.

Fig. 1

Recombinant rVSV expressing ZIKV antigens are immunogenic in mice. a Expression of ZIKV E truncations by VSV vector. BSRT7 cells were infected with each recombinant virus expressing ZIKV antigen at an MOI of 3.0. At 16 h post-infection, cells were lysed in 500 μl of lysis buffer, and 10 μl of lysate was analyzed by SDS-PAGE and were blotted with anti-ZIKV E protein monoclonal antibody. b Expression of full-length ZIKV E protein by VSV vector. BSRT7 cells were infected with the indicated recombinant virus expressing ZIKV antigen at an MOI of 3.0. Cell lysates were harvested at 16 h post-infection, and analyzed by western blot. c Expression of NS1 protein by VSV vector. Same cell lysates from b were subjected to western blot analysis using anti-ZIKV NS1 antibody. d Comparison of the expression of ZIKV E truncations with or without anchor C signal peptide by VSV vector. BSRT7 cells were infected with each recombinant virus at an MOI of 3.0. Cell lysates were harvested at 16 h post-infection, and analyzed by western blot. e Kinetics of ZIKV E protein expression by the VSV vector. Top panel: BSRT7 cells were infected with each recombinant virus at an MOI of 3.0. Cytoplasmic extracts were harvested at the indicated time points. Equal amounts of total cytoplasmic lysate were analyzed by SDS-PAGE, followed by western blot analysis. Bottom panel: Equal amounts of total cytoplasmic lysate were blotted with anti-β-actin antibody. f Electron microscopy analysis of ZIKV virus-like particles (VLPs). ZIKV VLPs were negatively stained with 1% ammonium molybdate and visualized by a transmission electron microscope. rVSV-prM-E crude indicates a mixture of ZIKV VLPs and VSV virions from supernatant harvested from BSRT7 cells infected by rVSV-prM-E. ZIKV VLPs were further purified from rVSV-prM-E or rVSV-prM-E-NS1-infected cells. No VLPs were found in rVSV-E. ZIKV Cambodian strain was grown in Vero cells, purified, and used as a control. The yellow arrow indicates a VSV particle; white arrows indicate ZIKV VLPs; and yellow stars indicate ZIKV virions. g Kinetics of ZIKV-specific ELISA antibody induced by rVSV expressing ZIKV antigens. Groups of five female BALB/c mice were inoculated intranasally with a single dose (10⁶ PFU) of rVSV or rVSV expressing ZIKV antigens. For DNA vaccine, mice were immunized intramuscularly with 50 µg of pCI-prM-E, and boosted with same dose two weeks later. Serum samples were collected weekly and analyzed by ELISA for ZIKV-specific serum IgG Ab. Data are expressed as the geometric mean titers (GMT) of five mice ± standard deviation. Western blots shown are the representatives of three independent experiments

Fig. 2

ZIKV antigen expression and antibody response by MTase-defective rVSV (mtdVSV) vector. a Single-step growth curve of mtdVSVs. Confluent BSRT7 cells were infected with individual viruses at an MOI of 3.0. After 1 h of incubation, the inoculum was removed, the cells were washed with DMEM, and fresh medium (containing 2% fetal bovine serum) was added, followed by incubation at 37 °C. Samples of supernatant were harvested at the indicated intervals over a 48-h time period, and the viral titer was determined by plaque assay. Data are the GMT of three independent experiments ± standard deviation. b Expression of ZIKV antigens by the mtdVSV in cell lysates. BSRT7 cells were infected with each recombinant virus at an MOI of 3.0. At 24 h post-infection, cell lysates were harvested and analyzed by western blot using antibody against ZIKV E or NS1 protein. c Kinetics of ZIKV E protein expression in cell lysates. Top panel: BSRT7 cells were infected with each recombinant virus at an MOI of 3.0. At 12, 24, and 36 h post-infection, cell lysates were harvested and analyzed by western blot using antibody against ZIKV E protein. Bottom panel: Equal amounts of total cytoplasmic lysate were blotted with anti-β-actin antibody. d Kinetics of ZIKV E protein release into cell culture supernatants. Cell culture supernatants were harvested from virus-infected cells at the indicated time points, and 10 µl of supernatant was analyzed by western blot using E-specific antibody. e ZIKV NS1 protein released into the cell culture supernatant. Cell culture supernatants were harvested from virus-infected cells at 36 h post-infection, and 10 µl of supernatant was analyzed by western blot using ZIKV serum antibody. f Dynamics of mouse body weight after inoculation with mtdVSV. Five six-week-old female BALB/c mice in each group were intranasally inoculated with DMEM or 10⁶ PFU of rVSV or mtdVSV expressing ZIKV antigens. The body weight for each mouse was evaluated at indicated time points. The average body weights of five mice were shown. All mice in rVSV group were dead and euthanized at day 7. g Kinetics of ZIKV specific antibody induced by mtdVSV expressing ZIKV antigen. Serum samples were collected weekly and analyzed by ELISA for ZIKV-specific serum IgG Ab. The titers are expressed as the GMT of five mice ± standard deviation. h ZIKV specific neutralizing antibody titer at week 5 post-inoculation. i ZIKV NS1-specific antibody detected by ELISA at week 5 post-inoculation. The western blot gels presented are a representative of three independent experiments. Mouse body weights are mean of five mice ± standard deviation

Fig. 3

MTase-defective rVSV (mtdVSV)-based vaccine induces ZIKV-specific T helper cell responses. Six-week-old BALB/c mice were immunized with each vaccine candidate (5 mice per group). Mice were euthanized at day 35 post-immunization, the spleen was isolated from each mouse, homogenized, a cell suspension prepared, split into three wells (triplicate per mouse) and cultured in 96-well microtiter plates in the presence of 20 µg/ml of ZIKV E protein for 5 days. a Proliferation of CD4+ T cells. The frequencies of ZIKV-specific Th1 (IFN-γ⁺CD4⁺ and TNF-α⁺CD4⁺) (b), Th2 cells (IL-4⁺CD4⁺, IL-5⁺CD4⁺) (c), Th17 (IL-17A⁺ CD4⁺) (d), and Tfh (IL-21⁺ CD4⁺) (e) cells were determined by flow cytometry after intracellular staining with the corresponding anti-cytokine. Data were expressed as mean % positive cells (the mean of 15 samples: 3 wells × 5 mice) ± standard deviation. Asterisk indicates that the group was statistically different with unstimulated and DMEM groups. P-value in from left to right for each panel by Student’s t-test: a ****P = 3.55 × 10⁻⁹, ****P = 4.10 × 10⁻⁶, ****P = 4.21 × 10⁻⁷. b **P = 0.00676, **P = 0.00394, ****P = 7.58 × 10⁻⁶, ****P = 3.32 × 10⁻⁵. c *P = 0.0243, **P = 0.00180, *P = 0.0304, *P = 0.0149, ***P = 0.000409, ****P = 7.72 × 10⁻⁶, *P = 0.0102. d **P = 0.00749, ****P = 2.52 × 10⁻⁶, ***P = 0.000907. e ***P = 0.000313, ***P = 0.000162

Fig. 4

MTase-defective rVSV (mtdVSV)-based vaccine protects BALB/c mice from viremia. Mice were intranasally inoculated with DMEM or a single dose (10⁶ PFU) of each recombinant virus. For DNA vaccine, mice were vaccinated intramuscularly with 50 µg of pCI-prM-E, and were boosted with the same dose two weeks later. At week 5, mice were intraperitoneally administered 1.8 mg of anti-IFNAR1 blocking antibody and 24 h later challenged intravenously with 10⁶ PFU of ZIKV Cambodian strain. a Dynamic of viremia in unimmunized mice after challenge with ZIKV. After challenge, blood samples were collected at the indicated time until day 24 and the presence of ZIKV RNA was quantitated by real-time RT-PCR and calculated to PFU equivalent RNA/ml. Data were expressed as GMT of 10 mice ± standard deviation. b Quantification of VSV RNA in BALB/c mice. Brains were harvested at the termination of the study. The VSV RNA was measured by real-time RT-PCR using primers annealing to the VSV L gene. Data were expressed together with the GMT of 10 mice (black bars). P value (by Student’s t-test) from top to bottom: ****P = 4.25 × 10⁻⁷, ***P = 0.000710, ***P = 0.000371. c Kinetics of ZIKV E-specific antibody induced by mtdVSV expressing ZIKV antigen. Serum samples were collected weekly and analyzed by ELISA for ZIKV-specific serum IgG Ab. Data are expressed as the GMT of five mice ± standard deviation. d Kinetics of ZIKV NS1-specific antibody induced by mtdVSV expressing ZIKV antigen. e mtdVSV-based vaccine protects BALB/c mice from viremia at day 3 post-challenge. The level of viremia was measured by real-time RT-PCR at day 3 post-challenge. P value (by Student’s t-test) from top to bottom: ****P = 1.02 × 10⁻⁵, ****P = 6.06 × 10⁻⁵, **P = 0.00345, ***P = 0.00310. f mtdVSV-based vaccine protects BALB/c mice from viremia at day 7 post-challenge. The level of viremia was measured by real-time RT-PCR at day 7 post-challenge. P value (by Student’s t-test) from top to bottom: ***P = 3.89 × 10⁻⁵, *P = 0.0201. Significance was calculated using t-test. N.S. indicates not significant

Fig. 5

MTase-defective rVSV (mtdVSV)-based vaccine induces high levels of ZIKV-specific antibody in A129 mice. A129 mice were immunized intramuscularly with pCI, pCI-prM-E, or pCI-NS1 at a dose of 50 µg DNA per mouse, and were boosted with the same plasmid at the same dose two weeks later. For VSV-based vaccines, mice were administered intramuscularly using a single dose (1 × 10⁵ PFU). The body weight for each mouse was evaluated at indicated time points (a). The average body weights of five mice were shown. At day 7, two out of five mice in rVSV-prM-E-NS1 group were dead and the other three terminated at day 10. After immunization, blood samples were collected at weeks 1 and 3. ZIKV E-specific antibody was measured by ELISA at weeks 1 (b) and 3 (c) post-immunization. ZIKV-specific neutralizing Ab was measured at weeks 1 (d) and 3 (e) post-immunization. ZIKV NS1-specific Ab was measured by ELISA at weeks 1 (f) and 3 (g) post-immunization. ELISA titers shown are GMT of 5 mice ± standard deviation. At the termination of this experiment, brains were collected and the presence of the VSV RNA was quantified by real-time RT-PCR using primers annealing to the VSV L gene (h). Antibody and viral load data are expressed as the GMT of five mice (black bars). Exact P value (by Student’s t-test) in each panel: b ****P = 1.36 × 10⁻⁶; d ****P = 5.44 × 10⁻⁵; g ****P = 6.70 × 10⁻⁵; h ****P = 4.32 × 10⁻⁶, N.S., not significant

Fig. 6

NS1 alone provides partial protection against ZIKV challenge. a–c Experiment in A129 mice. A129 mice were immunized with a single dose (10⁵PFU) of mtdVSV-based vaccine or two doses (50 µg each, two weeks apart) of DNA vaccine. At week 4 post-immunization, mice were intraperitoneally challenged with 10⁵ PFU of ZIKV Cambodian strain. a Clinical score in A129 mice after ZIKV challenge. ZIKV-associated clinical signs were scored for each mouse. 1 = healthy; 2 = mild: ruffled fur but no neurological symptoms; 3 = moderate: ruffled fur, hindlimb weakness, and partial hindlimb paralysis; and 4 = severe: paralysis, moribund and early removal is required. b Body weight change in A129 mice after ZIKV challenge. After ZIKV challenge, body weight for each mouse was evaluated daily. The average body weights of five mice ± standard deviation were shown. c Body weight change of A129 mice in pCI-NS1 group after ZIKV challenge. Each line represents individual animal. One mouse had 10% of body weight loss and was recovered after ZIKV challenge. d Expression of ZIKV NS1 protein by rVSV-G1670A and pCI vectors. BSRT7 cells were infected by rVSV-G1670A-NS1 at an MOI of 3.0 or transfected with 4 µg of pCI-NS1, and cell lysates were harvested at day 3, and subjected to western blotting using ZIKV NS1 antibody. e-h Experiment in BALB/c mice. 4-week-old BLAB/c mice were immunized with two doses (50 µg each, two weeks apart) of pCI-NS1, or a single dose (10⁶ PFU) of mtdVSV-NS1 or mtdVSV-prM-E. At week 4 post-immunization, mice were administered 1.8 mg of anti-IFNAR1 blocking antibody and challenged intravenously with 10⁶ PFU of ZIKV Cambodian strain. e NS1-specific antibody response in BALB/c mice. P value (by Student’s t-test) from top to bottom: ****P = 3.95 × 10⁻⁵, ****P = 3.48 × 10⁻⁵, ****P = 1.51 × 10⁻⁵, **P = 0.00183. Data shown are the GMT of 5 mice ± standard deviation. f E-specific antibody responses in BALB/c mice. Data shown are the GMT of 5 mice ± standard deviation. g NS1 alone provided partial protection against viremia in BALB/c mice at day 3 post-challenge. The level of viremia was measured by real-time RT-PCR at day 3 post-challenge. P value (by Student’s t-test) from top to bottom: ****P = 4.15 × 10⁻⁶, ***P = 0.000161, ***P = 0.000767, ***P = 0.000187, *P = 0.0250. h NS1 alone provided protection against viremia in BALB/c mice at day 7 post-challenge. P value (by Student’s t-test) from top to bottom: **P = 0.00510, ***P = 0.000114, **P = 0.00277, *P = 0.0308. Data shown are the mean of 5 mice. N.S., not significant

Fig. 7

MTase-defective rVSV (mtdVSV)-based vaccine protects A129 mice from viremia and prevents ZIKV replication in vivo. After ZIKV challenge, the level of viremia in A129 mice was measured by real-time RT-PCR at days 3 (a) and 7 (b) post-challenge. At day 7 post-challenge, all mice were terminated, brain (c), lung (d), uterus (e), and spleen (f) tissues were harvested and analyzed for ZIKV RNA by real-time RT-PCR. Data shown are GMT of 5 mice. Data were analyzed using t-test and compared to the placebo DMEM group or the pCI group (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; N.S., not significant, Student’s t-test). g mtdVSV-based vaccine prevents ZIKV-induced encephalitis in A129 mice. Half of brain tissue from each mouse was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin-eosin (HE) for the examination of histological changes by light microscopy. Micrographs with 10× magnification (scale bar of 200 μm) are shown. P value (by Student’s t-test) from top to bottom in each panel: a ***P = 0.000392, ****P = 6.62 × 10⁻⁶, ****P = 1.80 × 10⁻⁵, *P = 0.0236; b ***P = 0.000236, ****P = 1.07 × 10⁻⁵, ****P = 4.67 × 10⁻⁵, *P = 0.0383; c ****P = 2.96 × 10⁻⁶, ****P = 1.98 × 10⁻⁵, ****P = 3.46 × 10⁻⁵, *P = 0.0476; d ****P = 9.80 × 10⁻⁹, ****P = 9.95 × 10⁻⁶, ****P = 2.07 × 10⁻⁶, **P = 0.00694; e ***P = 0.000905, ****P = 2.31 × 10⁻⁶, ****P = 1.95 × 10⁻⁹, **P = 0.00288; f ****P = 2.85 × 10⁻⁶, ****P = 4.46 × 10⁻⁶, ****P = 9.27 × 10⁻⁷, *P = 0.0478, N.S., not significant

Fig. 8

Validation the safety and immunogenicity of rVSV-prM-E-NS1 in A129 mice. A129 mice were immunized intramuscularly with 1 × 10⁵ PFU of rVSV-prM-E-NS1 or injected with 100 µl of saline. The body weight for each mouse was evaluated at the indicated time points (a). Data shown are mean of 5 mice ± standard deviation. After immunization, blood samples were collected at week 4. ZIKV E-specific antibody (b) and NS1-specific antibody (c) was measured by ELISA. ELISA titers are shown as GMT of 5 mice (black bars). At week 4 post-immunization, mice were challenged with ZIKV, and their body weight (d) and viremia (e) were monitored every 1 or 3 days until day 21. Data shown are mean (d) or GMT (e) of 5 mice ± standard deviation