Immunogenic and efficacious SARS-CoV-2 vaccine based on resistin-trimerized spike antigen SmT1 and SLA archaeosome adjuvant

Abstract

The huge worldwide demand for vaccines targeting SARS-CoV-2 has necessitated the continued development of novel improved formulations capable of reducing the burden of the COVID-19 pandemic. Herein, we evaluated novel protein subunit vaccine formulations containing a resistin-trimerized spike antigen, SmT1. When combined with sulfated lactosyl archaeol (SLA) archaeosome adjuvant, formulations induced robust antigen-specific humoral and cellular immune responses in mice. Antibodies had strong neutralizing activity, preventing viral spike binding and viral infection. In addition, the formulations were highly efficacious in a hamster challenge model reducing viral load and body weight loss even after a single vaccination. The antigen-specific antibodies generated by our vaccine formulations had stronger neutralizing activity than human convalescent plasma, neutralizing the spike proteins of the B.1.1.7 and B.1.351 variants of concern. As such, our SmT1 antigen along with SLA archaeosome adjuvant comprise a promising platform for the development of efficacious protein subunit vaccine formulations for SARS-CoV-2.

Figure 1

Levels and specificity of antibodies induced by SmT1-based vaccine formulations. C57BL/6 mice (n = 8/group) were immunized i.m. with SmT1 (2 or 10 µg) with or without adjuvant on Days 0 and 21. Serum was collected on Day 28 and analyzed by ELISA to determine the antibody titers (A). Grouped data is presented as geometric mean + 95% confidence interval. Statistical significance of differences for the adjuvanted groups vs. the antigen alone control group is shown: ****p < 0.0001 by one-way ANOVA followed by Tukey's multiple comparisons test. Serum (n = 5–6 per group) was also analyzed by peptide microarray to identify specific epitopes recognized by the vaccine-induced IgG antibodies (B). The median of the mean fluorescent intensity values obtained with each 15mer peptide are represented.

Figure 2

Comparison of immunogenicity of various spike-derived antigen formats delivered with SLA adjuvant. C57BL/6 mice (n = 10/group) were immunized i.m. with 2 µg of SmT1, spike NTD, RBD, S1 or S2 alone or with SLA archaeosomes on Days 0 and 21. Serum was collected on Days 28 and analyzed by ELISA to determine the antibody titers (A). Grouped data is presented as geometric mean + 95% confidence interval. Serum from Day 28 was also analyzed at a final serum dilution of 1:75 by surrogate neutralization assay (B). Grouped data is presented as mean + standard error of mean. Splenocytes were harvested on Day 28 and analyzed by IFN-γ ELISpot when stimulated by spike peptide pools (C). Values obtained with media alone were subtracted from those measured in the presence of the peptides. Grouped data is presented as mean + standard error of mean (SEM). Statistical significance of differences for the adjuvanted subdomain groups vs. SmT1 + SLA group is shown: *p < 0.05, **p < 0.01 and ****p < 0.0001 by one-way ANOVA followed by Tukey's multiple comparisons test.

Figure 3

Antigen dose response of SmT1-SLA adjuvanted vaccine formulations. C57BL/6 mice (n = 10/group) were immunized i.m. with 3 µg of SmT1 alone or 0.01–3 µg of antigen with SLA archaeosomes on Days 0 and 21. Serum was collected on Days 20 (A) and 28 (B) and analyzed by ELISA to determine the antibody titers. Grouped data is presented as geometric mean + 95% confidence interval. Serum from Day 28 was also analyzed at a final serum dilution of 1:75 by surrogate neutralization assay (C). Grouped data is presented as mean + standard error of mean. Splenocytes were harvested on Day 28 and analyzed by IFN-γ ELISpot when stimulated by spike peptide pools (D). Values obtained with media alone were subtracted from those measured in the presence of the peptides. Grouped data is presented as mean + standard error of mean (SEM). Statistical significance of differences for the adjuvanted groups vs. the antigen alone control group is shown: ***p < 0.001 and ****p < 0.0001 by one-way ANOVA followed by Tukey's multiple comparisons test.

Figure 4

Humoral immune response with SmT1-based vaccine formulations adjuvanted with SLA, CpG and Poly(I:C) alone or in combination. C57BL/6 mice (n = 10/group) were immunized i.m. with 1 µg of SmT1 alone or with SLA, CpG and Poly(I:C) alone or in combination on Days 0 and 21. Serum was collected on Days 20 (A) and 28 (B) and analyzed by ELISA to determine the antibody titers. Grouped data is presented as geometric mean + 95% confidence interval. Serum from Day 28 was also analyzed at a final serum dilution of 1:75 by cell-based surrogate neutralization assay (C). Serial dilutions of serum were also tested in a SARS-CoV-2 plaque reduction neutralization titer assay (D) or plate-based spike-ACE2 neutralization assay (E). Grouped data is presented as mean + standard error of mean. Statistical significance of differences between the adjuvanted groups vs. the unadjuvanted control group is shown: ***p < 0.001 and ****p < 0.0001 by one-way ANOVA followed by Tukey's multiple comparisons test.

Figure 5

Cellular immune response with SmT1-based vaccine formulations adjuvanted with SLA, CpG and Poly(I:C) alone or in combination. C57BL/6 mice were immunized i.m. with 1 µg of SmT1 alone or with SLA, CpG and Poly(I:C) alone or in combination on Days 0 and 21. Splenocytes were harvested on day 28 and analyzed by IFN-γ⁺ ELISpot (n = 10/group) when stimulated by spike peptide pools or media alone (A). Similarly, intracellular cytokine staining (ICCS; n = 5/group) was conducted on the splenocytes to measure levels of IFN-γ⁺ CD4⁺ or IFN-γ⁺ CD8⁺ T cells when stimulated by a spike peptide pools or media alone (B). Grouped data is presented as mean + standard error of mean. The frequency of cells expressing IFN-γ, TNF-α, and IL-2 alone or in combination as assessed by ICCS is displayed (median per group) with the total number of cytokine-positive cells per million CD4⁺ T cells indicated below the pie chart (C). Values obtained with media alone were subtracted from those measured in the presence of the peptide pool. Statistical significance of differences between the adjuvanted groups vs. the antigen alone control group is shown: **p < 0.01, ***p < 0.001 and ****p < 0.0001 by one-way ANOVA followed by Tukey's multiple comparisons test.

Figure 6

Efficacy of SmT1-based vaccine formulations adjuvanted with SLA, CpG or SLA + CpG in hamster challenge model. Syrian Golden hamsters (n = 6/group) were immunized i.m. with 3 µg of SmT1 alone or with SLA and alone or in combination on Days 0 ± 21. As negative controls, hamsters received either vehicle or adjuvant alone. On Day 35, hamsters were challenged intranasally with 1 × 10⁵ PFU of SARS-CoV-2 (A). On Day 5, lungs were collected and viral load measured by plaque assay (B). Grouped data is presented as mean + standard error of mean. Statistical significance of differences for the groups vs. the vehicle control group is shown: **p < 0.01, ***p < 0.001 and ****p < 0.0001 by two-way (A) or one-way (B) ANOVA followed by Tukey's multiple comparisons test.

Figure 7

Humoral immune response with SmT1-based vaccine formulations in hamsters. Syrian Golden hamsters (n = 6/group) were immunized i.m. with 3 µg of SmT1 alone or with SLA, CpG or SLA + CpG on Days 0 ± 21. Serum was collected on Days 21 (A) and 34 (B) and analyzed by ELISA to determine the antibody titers. Grouped data is presented as geometric mean + 95% confidence interval. Serum from Day 34 was also analyzed at a final serum dilution of 1:25 (C) and 1:75 (D) by cell-based surrogate neutralization assay. Grouped data is presented as mean + standard error of mean. Statistical significance of differences for the adjuvanted groups vs. the antigen alone control group is shown: **p < 0.01, ***p < 0.001 and ****p < 0.0001 by one-way ANOVA followed by Tukey's multiple comparisons test.