Fig 1.
Characteristics of BCVax delta strain spike protein.
(A) Linear diagram of the delta S protein sequence. The mutation sites of delta strain including T19R, G142D, Δ156–157, R158G, L452R, T478K, D614G, P681R, and D950N were as indicated. (B) SDS-PAGE analysis with Instant Blue staining of delta-S protein under de-naturing conditions. (C) SEC-UV analysis of delta S protein after His-tag removal and purification. (D) Human ACE2 binding activity evaluation of delta S protein using ForteBio Octet analyzer. Data shown in colored lines representing different concentrations of S protein. KD values were calculated using a 1:1 global fit model (Octet). %HMWS: percentage of high molecular weight species.
Fig 2.
Characterization of AB801-ISCOM with dynamic light scattering (DLS) and transmission electron microscopy (TEM)s.
(A) Dynamic light scattering (DLS) analysis of AB801-ISCOM. Duplicate analysis was performed to demonstrate the consistency. (B) Transmission electron microscopy (TEM) Image of AB801-ISCOM. (C) The negative stain EM structural analysis of BCVax, consisting of delta S protein and AB801-ISCOM. The EM image showed delta S protein in trimer structure with homogenous distribution. The size of delta S protein and AB801-ISCOM is about 20 nm and 40 nm, respectively. The upper right image shows magnified delta S protein trimer. The lower right image shows magnified AB801-ISCOM in cage like structure.
Fig 3.
Evaluation of the immunogenicity of BCVax in BALB/c mice.
(A) Treatment and sampling schedule. The delta S protein (10 μg) combined with or without adjuvant candidates of aluminum hydroxide plus CpG 1018 (50 μg plus 10 μg, triangle), AB801 (5 or 10 μg, square), or AB801-ISCOM (5 or 10 μg, circle, grey bars) were immunized to BALB/c mice on day 0 and day 14. (B) anti-S protein IgG titer of wild type, delta, and omicron strains in immunized mouse serum collected on day 28. (C) Neutralizing antibody titers of sera of immunized mice against SARS-CoV-2 pseudoviruses D614G, B.1.1.7, 507Y.V2, P.1, B.1.617.2, BA.1, BA.2, and BA.4/5 variants. Bars indicate the geographic mean titer (GMT), and the error bars represent 95% confidence intervals. Data were analyzed using one-way ANOVA. (D) pNT50 value of AB801-ISCOM groups against D614G, B.1.1.7, 507Y.V2, P.1, B.1.617.2, BA.1, BA.2, and BA.4/BA.5 variants. (*p <0.05).
Fig 4.
T cell responses in BCVax immunized BALB/c mice.
The delta S protein (10 μg) combined with or without adjuvant candidates of aluminum hydroxide plus CpG 1018 (50 μg plus 10 μg, triangle), AB801 (5 or 10 μg, square), or AB801-ISCOM (5 or 10 μg, circle, grey bars) were immunized to BALB/c mice on day 0 and day 14, splenocytes were harvested on day 28 for analysis. (A) Flow cytometry analysis of CD4 T cell populations of CD4+IFN-γ+ and CD4+IL-4+ cells. (B) CD8 T cell populations of CD8+IFN-γ+ and CD8+GranzymeB+ cells were evaluated using immunized mice splenocytes. (C) The number of IL-2, IFN-γ, and IL-4 secreting cells from immunized splenocytes per 2.5E+5 cells were analyzed by ELISPOT assay. (D) The ratio of IFN-γ/IL-4 of ELISPOT results. Bars represent mean ± SD. Statistically significant differences were indicated (*p < 0.05).
Fig 5.
Evaluation of the immunogenicity of BCVax candidates in BALB/c mice with booster injection.
Groups of mice were immunized with 10 μg of delta S protein combined with different dosage of AB801-ISCOM (7.5, 5, or 2 μg) on day 0 and day 14 (white bars). Another group of mice were received first two injections of 10 μg delta S protein with 7.5 μg AB801-ISCOM on day 0 and day 14, and a booster of same dosage on day 56 (gray bars). (A) The treatment schedule and sampling time. (B) anti-S protein IgG titers of delta, omicron BA.2 and BA.5 strains in immunized mouse serum collected on day 84. (C) The time course of serum IgG response. (D) pseudovirus neutralizing activity of sera collected at day 84 of immunized mice against SARS-CoV-2 pseudoviruses B.1.617.2, BA.2, and BA.4/BA.5 variants. Bars indicate the GMT, and the error bars represent 95% confidence intervals. Data were analyzed using one-way ANOVA. (E) CD8 T cell populations of CD8+IFN-γ+ and CD8+GranzymeB+ cells were evaluated using immunized mice splenocytes harvested on day 84. Bars represent mean ± SD. Statistically significant differences were indicated (*p < 0.05).