Study of the Effects of Several SARS-CoV-2 Structural Proteins on Antiviral Immunity

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike (S) protein is a critical viral antigenic protein that enables the production of neutralizing antibodies, while other structural proteins, including the membrane (M), nucleocapsid (N) and envelope (E) proteins, have unclear roles in antiviral immunity. In this study, S1, S2, M, N and E proteins were expressed in 16HBE cells to explore the characteristics of the resultant innate immune response. Furthermore, peripheral blood mononuclear cells (PBMCs) from mice immunized with two doses of inactivated SARS-CoV-2 vaccine or two doses of mRNA vaccine were isolated and stimulated by these five proteins to evaluate the corresponding specific T-cell immune response. In addition, the levels of humoral immunity induced by two-dose inactivated vaccine priming followed by mRNA vaccine boosting, two homologous inactivated vaccine doses and two homologous mRNA vaccine doses in immunized mice were compared. Our results suggested that viral structural proteins can activate the innate immune response and elicit a specific T-cell response in mice immunized with the inactivated vaccine. However, the existence of the specific T-cell response against M, N and E is seemingly insufficient to improve the level of humoral immunity.

Keywords: SARS-CoV-2; humoral immunity; innate immune; specific T-cell response; structural protein.

Figure 1

Schematic depicting the vaccination protocol. Group I-I (n = 15) was vaccinated with two doses of inactivated SARS-CoV-2 vaccine (28-day interval), and groups I-I-m (7 d), I-I-m (14 d) and I-I-m (21 d) (n = 15 per group) were, respectively, vaccinated with the third dose of mRNA booster on days 7, 14 and 21 after the second dose. Group m (n = 10) was vaccinated with a dose of mRNA vaccine, and groups m-m (3 d), m-m (7 d), m-m (14 d) and m-m (21 d) (n = 10 per group) were vaccinated with the second dose of mRNA booster on days 3, 7, 14 and 21. Control mice were injected with phosphate-buffered saline (PBS) alone. Spleens of immunized animals were collected on the 3rd, 7th, 14th, 21st and 28th days after the last immunization for an ELISpot assay. Serum samples were collected on the 3rd and 28th days for ELISA and neutralizing antibody assays (n = 2–3 per group).

Figure 2

Innate immune response elicited by various SARS-CoV-2 structural proteins in 16HBE cells. (a) Western blot analysis to examine the expression of recombinant plasmids. 16HBE cells were transiently transfected with five recombinant plasmids and cell lysate was harvested 48 h later. (b,c) Expression of innate/inflammatory cytokines in 16HBE cells after transfection at different times. The relative expression levels of innate/inflammatory cytokines in 16HBE cells were normalized to their levels in the blank control group (transfection blank pcDNA3.1(+)) using the comparative Ct (ΔΔCt) method. Scheirer–Ray–Hare test was conducted. The data are shown as the mean ± SD based on data from two independent experiments. *, S1 vs. control. #, S2 vs. control. ▲, M vs. control. ●, N vs. control. ▼, E vs. control. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001, ^▲ p < 0.05, ^▲▲ p < 0.01, ^▲▲▲ p < 0.001, ^● p < 0.05, ^●● p < 0.01, ^●●●● p < 0.0001, ^▼ p < 0.05, ^▼▼ p < 0.01, ^▼▼▼ p < 0.001, ^▼▼▼▼ p < 0.0001.

Figure 3

Specific T-cell responses in mice immunized with inactivated SARS-CoV-2 vaccine (n = 15), analyzed by ELISpot assay. (a–c) The ELISpot responses show IFN-γ-, IL-4- and IL-17-secreting T cells among splenic lymphocytes after stimulation with the antigenic proteins S1, S2, M, N and E. The black dotted line represents the control group. Scheirer–Ray–Hare test was conducted. Bars represent the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control.

Figure 4

Specific T-cell responses in mice immunized with SARS-CoV-2 mRNA vaccine analyzed by ELISpot assay. Groups m-m (3 d), m-m (7 d), m-m (14 d) and m-m (21 d) (n = 10 per group) received booster immunization on days 3, 7, 14 and 21 after the first mRNA vaccine dose, respectively. The black dotted line represents the control group. (a) The results of the ELISpot assay specific for IFN-γ and IL-4 suggested specific T-cell responses against S1 and S2. (b) The results of the ELISpot assay specific for IFN-γ and IL-4 suggested specific T-cell responses against M, N and E. Scheirer–Ray–Hare test was conducted. Bars represent the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control.

Figure 5

Antibody responses elicited by two doses of an mRNA vaccine or two doses of an inactivated vaccine followed by an mRNA vaccine boost. (a) The specific IgG antibody levels against Wuhan-Hu-1 strain S1 and S2 were detected by ELISA at 3 days and 28 days post-boost immunization. Scheirer–Ray–Hare test was conducted. (b) The specific IgG antibody levels against Omicron strain (B.1.1.529) S1 were detected by ELISA at 3 days and 28 days post-boost immunization. Scheirer–Ray–Hare test was conducted. (c) Pseudovirus-neutralizing antibody assay against the Omicron strain (B.1.1.529) and virus-neutralizing antibody assay against the Wuhan-Hu-1 strain. One-way ANOVA (nonparametric or mixed) was conducted. Bars represent the mean ± SD. Group I-I (n = 15) was vaccinated with two doses of inactivated SARS-CoV-2 vaccine (28-day interval), and groups I-I-m (7 d), I-I-m (14 d) and I-I-m (21 d) (n = 15 per group) were, respectively, vaccinated with the third dose of mRNA booster on days 7, 14 and 21 after the second dose. Group m (n = 10) was vaccinated with a dose of mRNA vaccine, and groups m-m (3 d), m-m (7 d), m-m (14 d) and m-m (21 d) (n = 10 per group) were vaccinated with the second dose of mRNA booster on days 3, 7, 14 and 21. Bars represent the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, no significance.