Plant-expressed Zika virus envelope protein elicited protective immunity against the Zika virus in immunocompetent mice

Abstract

Zika virus infection causes multiple clinical issues, including Guillain-Barré syndrome and neonatal malformation. Vaccination is considered as the only strategy for the prevention of ZIKV-induced clinical issues. This study developed a plant-based recombinant vaccine that transiently expressed the ZIKV envelope protein (ZikaEnv:aghFc) in Nicotiana benthamiana and evaluated the protective immunity afforded by it in immunocompetent mice. ZikaEnv:aghFc induced both humoral and cellular immunity at a low dose (1-5 μg). This immune-inducing potential was enhanced further when adjuvanted CIA09A. In addition, antigen-specific antibodies and neutralizing antibodies were vertically transferred from immunized females to their progeny and afforded both protective immunity to ZIKV and cross-protection to Dengue virus infection. These results suggest that our plant-based ZIKV vaccine provides a safe and efficient protective strategy with a competitive edge.

Figure 1

Expression and purification of Zika Env:hFc and Zika Env:aghFc. (A) Schematic representation of Zika Env:hFc and Zika Env:aghFc. NB, new chaperone-binding protein (signal peptide); hFc, constant fragment of human immunoglobulin G; HDEL, histidine–aspartate–glutamate–leucine peptide (endoplasmic-reticulum-retention signal); L, peptide linker (GGGGSGGGGS); aghFc, aglycosylated hFc; DEL, aspartate–glutamate–leucine peptide (endoplasmic-reticulum-retention signal with the last lysine from aghFc). (B) Expression and solubility of recombinant Zika Env:hFc and Zika Env:aghFc in Nicotiana benthamiana. Each fraction was subjected to Western blot analysis using an HRP-conjugated anti-human IgG antibody. T, plant total extract; S, soluble fraction; P, pellet fraction. (C) Purification of Zika Env:hFc and (D) Zika Env:aghFc by Protein A affinity chromatography. The total plant extract and flow-through were subject to Western blotting (left panels), and eluents were stained with Coomassie Brilliant Blue after polyacrylamide gel electrophoresis (right panels). T, total plant extract; FT, flow-through; E, eluents. Bovine serum albumin (BSA) was used as a standard protein for quantitation. Original blots are presented in Supplementary Fig. 1.

Figure 2

Antibody responses elicited by ZikaEnv:hFc and ZikaEnv:aghFc in the presence of each adjuvant or an adjuvant combination. (A) Experimental strategies. C57BL/6 mice (n = 5) were immunized with ZikaEnv:hFc and ZikaEnv:aghFc with each adjuvant or with an adjuvant combination, or with PBS at weeks 0, 2, and 6. The adjuvants used are indicated in all figures. To measure the humoral immune response, blood samples were collected at weeks 0, 1, 4, and 7. (B, C) An indirect enzyme-linked immunosorbent assay (ELISA) was performed using ZikaEnv:hFc and ZikaEnv:aghFc as coating antigens. Serum samples were diluted at 1:100 and OD 450 values were measured using indirect ELISA. The data are presented as the mean ± SD of five mice per group. Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001. (D) Virus neutralization titers against ZIKV, as determined using the plaque reduction neutralization test (PRNT) Mouse sera utilized for PRNT measurement were taken 7 weeks post immunization with each adjuvant, an adjuvant combination, or PBS.

Figure 3

Cellular immune responses elicited by ZikaEnv:hFc and ZikaEnv:aghFc in the presence of each adjuvant or an adjuvant combination. C57BL/6 mice (n = 5) were immunized with ZikaEnv:hFc and ZikaEnv:aghFc with each adjuvant or with an adjuvant combination, or PBS at weeks 0, 2, and 6. The adjuvants used are indicated in all figures. Mice were euthanized at 7 days after the last immunization. To measure the cellular immune response, splenocytes were isolated from five mice per group. (A, B) Frequencies of IFN-γ- and TNF-α-expressing CD4 + and CD8 + T cells, as determined by flow cytometry. (C–F) Splenocytes were stimulated with the same dose of (1 μg/mL) of ZikaEnv:hFc, ZikaEnv:aghFc, or PBS. The supernatants were harvested after 48 h of incubation and used to measure the concentrations of IFN-γ, TNF-α, IL-4, and IL-12 using ELISA. The data are presented as the mean ± SD of five mice per group. Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Humoral immune responses elicited according to the amount of Zika Env:hFc and ZikaEnv:aghFc. (A) Experimental strategies. C57BL/6 mice (n = 5) were immunized with ZikaEnv:hFc, ZikaEnv:aghFc, or PBS at weeks 0, 2, and 6. To measure the humoral immune response, blood samples were collected at weeks 0, 1, 4, and 7. (B, C) An indirect enzyme-linked immunosorbent assay (ELISA) was performed using ZikaEnv:hFc and ZikaEnv:aghFc as coating antigens. Serum samples were diluted at 1:100 and OD 450 values were measured using indirect ELISA. The data are presented as the mean ± SD of five mice per group. Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001. (D) Virus neutralization titers against ZIKV, as determined using the plaque reduction neutralization test (PRNT). Mouse sera utilized for PRNT measurement were taken 7 weeks post immunization with ZikaEnv:hFc, ZikaEnv:aghFc, or PBS.

Figure 5

Cellular immune responses elicited according to the amount of Zika Env:hFc and ZikaEnv:aghFc. C57BL/6 mice (n = 5) were immunized with ZikaEnv:hFc, ZikaEnv:aghFc, or PBS at weeks 0, 2, and 6. Mice were euthanized at 7 days after the last immunization. To measure the cellular immune response, splenocytes were isolated from five mice per group. (A, B) The frequencies of IFN-γ- and TNF-α-expressing CD4⁺ and CD8⁺ T cells are shown. (C–F) Splenocytes were stimulated with the same dose (1 μg/mL) of ZikaEnv:hFc, ZikaEnv:aghFc, or PBS. The supernatants were harvested after 48 h of incubation and were used to measure the concentrations of IFN-γ, TNF-α, IL-4, and IL-12 using ELISA. The data are presented as the mean ± SD of five mice per group. Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Humoral immune responses elicited by ZikaEnv:aghFc. (A) Experimental strategies. C57BL/6 mice (n = 5) were immunized with ZikaEnv:aghFc or PBS at weeks 0, 2, and 6. To measure the humoral immune response, blood samples were collected at weeks 0, 1, 4, and 7. (B) An indirect enzyme-linked immunosorbent assay (ELISA) was performed using ZikaEnv:aghFc as a coating antigen. Serum samples were diluted at 1:100 and OD 450 values were measured using indirect ELISA. The data are presented as the mean ± SD of five mice per group. Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001. (C) Virus neutralization titers against ZIKV, as determined using the plaque reduction neutralization test (PRNT). Mouse sera utilized for PRNT measurement were taken 7 weeks post immunization with ZikaEnv:aghFc or PBS.

Figure 7

Cellular immune responses elicited by ZikaEnv:aghFc. C57BL/6 mice (n = 5) were immunized with ZikaEnv:aghFc or PBS at weeks 0, 2, and 6. Mice were euthanized at 7 days after the last immunization. To measure the cellular immune response, splenocytes were isolated from five mice per group. (A) Frequencies of IFN-γ- and TNF-α-expressing CD4⁺ and CD8⁺ T cells. (B–E) Splenocytes were stimulated with the same dose (1 μg/ml) of ZikaEnv:aghFc or PBS. The supernatants were harvested after 48 h of incubation and used to measure the concentrations of IFN-γ, TNF-α, IL-4, and IL-12 using ELISA. The data are presented as the mean ± SD of five mice per group. Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8

Assessment of the protection against ZIKV challenge afforded by transfer of ZikaEnv:aghFc-specific Ab. (A) Experimental strategies. C57BL/6 mice (n = 5) were immunized with ZikaEnv:aghFc or PBS at weeks 0, 2, and 6. Immunized mice were bred as homozygous breeding pairs. (B–D) The survival rate, body weight, and clinical score of C57BL/6 neonatal mice (n = 5 per group) were monitored up to 3 weeks post-infection. Two-day-old neonatal mice were inoculated with 10⁶ TCID₅₀/mouse for the MR766 strain (A–C) or 10⁶ TCID₅₀/mouse for the PRVABC59 strain. (B) The survival rate is presented using Kaplan–Meier survival curves. (C) The body weight data are represented as the mean ± SD of five mice per group. (D) Mice were allocated a clinical score (range, 0–3) based on the most severe clinical sign observed, as follows: normal appearance (0); staggering walk, wide stance, or paralysis of the hind legs (1); 25% weight loss or labored breathing (2); and death (3). (E) Two-day-old neonatal mice were inoculated with 10⁶ TCID₅₀/mouse for the MR766 strain or 10⁶ TCID₅₀/mouse for the PRVABC59 strain. After 6 days for the MR766 ZIKV strain or after 12 days for the PRVABC59 ZIKV strain challenge, ZIKV RNA levels in the mouse brain were measured by qRT-PCR. The data are presented as the mean ± SD of five mice per group. (F) To measure ZikaEnv:aghFc-specific IgG antibodies, blood samples were collected on day 2. Serum samples were diluted at 1:100 and OD 450 values were measured using indirect ELISA. The data are presented as the mean ± SD of five mice per group. Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001. (G) Virus neutralization titers against ZIKV, as determined using the plaque reduction neutralization test (PRNT). To measure PRNT, blood samples were collected on day 2.

Figure 9

Assessment of the protection against DENV challenge afforded by the transfer of ZikaEnv:aghFc-specific Ab. C57BL/6 mice (n = 5) were immunized with ZikaEnv:aghFc or PBS at weeks 0, 2, and 6. Immunized mice were bred as homozygous breeding pairs. (A, B) The survival rate and clinical score of C57BL/6 neonatal mice (n = 5 per group) were monitored up to 3 weeks post-infection. Two-day-old neonatal mice were inoculated with DENV type 2 at 10⁶ TCID₅₀/mouse. (A) The survival rate is presented using Kaplan–Meier survival curves. (B) Mice were allocated a clinical score (range, 0–3) based on the most severe clinical sign observed, as follows: normal appearance (0); staggering walk, wide stance, or paralysis of the hind legs (1); 25% weight loss or labored breathing (2); and death (3). (C) After 12 days of DENV type 2 challenge, DENV RNA levels in the mouse brain were measured by qRT-PCR. (D) To measure ZikaEnv:aghFc-specific IgG antibodies, blood samples were collected on day 2. Serum samples were diluted at 1:100 and OD 450 values were measured using indirect ELISA. (E) Virus neutralization titers against ZIKV, as determined using the plaque reduction neutralization test (PRNT). To measure PRNT, blood samples were collected on day 2. The data are presented as the mean ± SD of five mice per group. Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001.