Fig. 3. S_MG vaccination elicits stronger humoral and cellular immune responses than S_FG in BALB/c mice.

(A) Structural models of S_FG and S_MG protein vaccine are shown (according to Fig. 2C). Blue: glycans; gray: protein. S_FG was expressed by HEK293E without further modification. S_MG was obtained by enzymatic digestion to truncate all N-glycans of S_HM expressed by HEK293S GnTI¯ to single GlcNAc, whereas O-glycans were unmodified. (B) Immunization schedule using proteins as in (A) as immunogens in BALB/c mice (n = 5 in each experiment). S_FG (blue), S_{HM (}yellow), S_MG (red), and control (gray). Alum, aluminum hydroxide. (C) Anti–S protein IgG titers of serum samples were analyzed by ELISA. (D) Neutralization titers of serum samples were measured using pseudovirus with WT S protein. (E to G) IgG subtype analysis of sera, including IgG1 (E), IgG2a (F), and the IgG2a:IgG1 ratio (G). (H to K) The percentage of Tfh in activated nonregulatory CD4 T cells (H) and the percentages of IFN-γ (I)–, IL-4 (J)–, and IL-21 (K)–expressing Tfh cells (CD4⁺CD19⁻CD44^hiFoxp3⁻PD-1⁺CXCR5⁺) in the lymph nodes (LNs) of BALB/c mice by flow cytometry. (L) The percentage of granzyme B−producing CD8⁺ T cells (CD3⁺ B220⁻CD8⁺ CD49b⁻) in the LN of BALB/c mice analyzed by flow cytometry. (M) The ratio of S protein–specific B cells (CD3⁻CD19⁺S protein⁺) (percentage) normalized to fluorescence minus one (FMO) control staining (stained without S protein) (percentage) in the spleen is shown. (N) Kappa and lambda light chain usage is shown. (O and P) Heavy (O) and kappa (P) chain distribution of B cell repertoire analysis. Less than 5% usage is shown in white. (Q to S) Anti−S protein IgG titers (Q), pseudovirus neutralization titers (R), and authentic virus neutralization titers (S) are shown for serum isolated from BALB/c mice after three doses of indicated vaccines against SARS-CoV-2 WT (or D614G) and variants (number above each bar indicate fold of increase of S_MG compared to S_FG group). pNT₅₀ represents the reciprocal dilution achieving 50% neutralization. The dotted line in bar charts represents the lower limit of detection. Data are shown as means ± SEM and analyzed by two-sided Mann-Whitney U test to compare two experimental groups, except in (N), where five samples were pooled together and a chi-squared test was used. P values shown above each bar. *P < 0.05; **P < 0.01.