Incorporation of SARS-CoV-2 spike NTD to RBD protein vaccine improves immunity against viral variants

Abstract

Emerging SARS-CoV-2 variants pose a threat to human health worldwide. SARS-CoV-2 receptor binding domain (RBD)-based vaccines are suitable candidates for booster vaccines, eliciting a focused antibody response enriched for virus neutralizing activity. Although RBD proteins are manufactured easily, and have excellent stability and safety properties, they are poorly immunogenic compared to the full-length spike protein. We have overcome this limitation by engineering a subunit vaccine composed of an RBD tandem dimer fused to the N-terminal domain (NTD) of the spike protein. We found that inclusion of the NTD (1) improved the magnitude and breadth of the T cell and anti-RBD response, and (2) enhanced T follicular helper cell and memory B cell generation, antibody potency, and cross-reactive neutralization activity against multiple SARS-CoV-2 variants, including B.1.1.529 (Omicron BA.1). In summary, our uniquely engineered RBD-NTD-subunit protein vaccine provides a promising booster vaccination strategy capable of protecting against known SARS-CoV-2 variants of concern.

Keywords: Immunology; Virology.

Graphical abstract

Figure 1

Development of novel SARS-CoV-2 subunit vaccine (A) Schematic drawing of the spike protein of SARS-CoV-2. S, signal peptide; NTD, N-terminal domain; RBD, receptor binding domain; TM transmembrane domain. Numbers refers to the amino acid position in the spike protein. (B) Cartoon of VAANZ-W_RRN construct showing the position of the tags, the antigenic domains, and the cleavage points. L, leader sequence; F, FLAG tag; Fc, human Fc domain of IgG1; scissors are the location of the HRV-3C protease cleavage sites. (C) Summary of the RBD proteins produced. Virus variant, mutations present, length in amino acids and theoretical mass (kDa) are reported.

Figure 2

Protection following immunization with VAANZ-Δ_RRN is equivalent to protection from previous infection (A) Schematic of immunization and SARS-CoV-2 challenge protocol. K18-hACE2 transgenic mice were either unvaccinated, immunized with VAANZ-Δ_RRN, or challenged with sublethal dose of ancestral SARS-CoV-2. All mice groups received 1 × 10⁴ TCID₅₀ of SARS-CoV-2 on Day 35 and monitored for weight loss (B) and survival (C) for 16 days post infection. Viral titers from lung and nasal turbinates at day 3 post infection (D). Symbols and error bars indicate mean from groups of 10 as shown in C. p values were calculated by a mixed-effect analysis (B) or Mantel-Cox Log-Rank test (C), each group were compared to unvaccinated controls. (E) Anti-RBD IgG in sera over time from C57BL/6 mice immunized with VAANZ-Δ_RRN/AddaVax or 6 μg of inactivated SARS-CoV-2 virus/AddaVax. Symbols indicate geometric mean ± SD from groups of n = 10. Mice were immunized with 2 doses of PBS or 50 μg of specified protein vaccine and AddaVax, spaced 3 weeks apart and assessed 1 week after the second dose. All data are pooled from two independent experiments. p values in D-E were calculated by one-way ANOVA with Tukey’s multiple comparison. ∗∗, p < 0.01; ∗∗∗∗, p < 0.0001; ns, not significant.

Figure 3

VAANZ-Δ _RRN induces stronger immune responses than Δ_RBD-RBD vaccine Responses to VAANZ-Δ_RRN were compared to Δ_RBD-RBD. (A and B) RBD-specific and S1-specific IFNγ producing cells and their ratio (B) is shown. (C) Plots gated on CD4⁺CD44⁺TCRb⁺ cells show T_FH CD4 T cells expressing PD-1 and BCL6. Percent among CD4⁺ cells (D) and number (E) of T_FH cells. Anti-RBD IgG (F) and S1-binding B cells (G–J) were measured. (G) S1-specific tetramer on IgD⁻B220⁺ cells. S1⁺B cells as total number (H) number within subset (I and J) Percent among B cell subset. Data are pooled from 2-3 independent experiments, symbols indicate individual mice from groups of n = 5–10. Bars indicate mean (except in B and H) or geometric mean (B, H). p values were calculated by one-way ANOVA with Tukey’s multiple comparison, except in A, I-J, which were calculated by two-way ANOVA ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗∗, p < 0.0001.

Figure 4

Enhanced breadth of antibody neutralization generated by VAANZ-Δ_RRN compared to Δ_RBD-RBD (A and B) Mice were immunized as in Figure 3 legend. Box and whisker graph comparing pseudotyped lentivirus neutralization antibody titers against SARS-CoV-2 variants from (A) human sera of individuals immunized with BNT162b2 or (B) serum from mice immunized with VAANZ-Δ_RRN. Blue line shows geometric mean ± SD ID₅₀ of human BNT162b immunized serum, pink line shows geometric mean ± SD ID₅₀ of mouse serum immunized with VAANZ-W_RRN, both against ancestral variant. Mid line shows mean, boxes show upper and lower quartile, whiskers show range. (C) Antibody dissociation assay was performed to calculate affinity index of anti-RBD IgG from VAANZ-Δ_RRN and Δ_RBD-RBD immunized mice. (D) Inhibition of interaction between RBD (from Omicron BA.1 and Beta variants) and hACE2 by sera from immunized mice. (E) Pseudotyped lentivirus neutralization antibody titers (ID₅₀) against Omicron BA.1 variant. (F) NTD targeting antibodies were measured as a ratio of S1-ACE2 and RBD-ACE2 SVNT antibody titers (IC₅₀). (G–I) K18-hACE2 transgenic mice were either unvaccinated, immunized with VAANZ-Δ_RRN or challenged with sublethal dose of ancestral SARS-CoV-2. All mice groups received 1 × 10⁴ TCID₅₀ of Beta variant SARS-CoV-2 on Day 35 and monitored for weight loss (G) and survival (H) for 16 days post infection. Viral titers from lung digests and nasal turbinates at day 3 after infection (I). Boxplots in A-B show median, upper and low quartile range and outliers. Bars in C–F indicate geometric means from groups n = 5–10 mice pooled from 2 independent experiments. p values were calculated by one-way ANOVA with Tukey’s multiple comparison ∗∗, p < 0.01; ∗∗∗∗, p < 0.0001; ns, not significant.