Development of CpG-adjuvanted stable prefusion SARS-CoV-2 spike antigen as a subunit vaccine against COVID-19

Abstract

The COVID-19 pandemic is a worldwide health emergency which calls for an unprecedented race for vaccines and treatment. In developing a COVID-19 vaccine, we applied technology previously used for MERS-CoV to produce a prefusion-stabilized SARS-CoV-2 spike protein, S-2P. To enhance immunogenicity and mitigate the potential vaccine-induced immunopathology, CpG 1018, a Th1-biasing synthetic toll-like receptor 9 (TLR9) agonist was selected as an adjuvant candidate. S-2P in combination with CpG 1018 and aluminum hydroxide (alum) was found to be the most potent immunogen and induced high titer of neutralizing antibodies in sera of immunized mice against pseudotyped lentivirus reporter or live wild-type SARS-CoV-2. In addition, the antibodies elicited were able to cross-neutralize pseudovirus containing the spike protein of the D614G variant, indicating the potential for broad spectrum protection. A marked Th1 dominant response was noted from cytokines secreted by splenocytes of mice immunized with CpG 1018 and alum. No vaccine-related serious adverse effects were found in the dose-ranging study in rats administered single- or two-dose regimens of S-2P combined with CpG 1018 alone or CpG 1018 with alum. These data support continued development of CHO-derived S-2P formulated with CpG 1018 and alum as a candidate vaccine to prevent COVID-19 disease.

Figure 1

Induction of neutralizing antibodies by CpG 1018 and aluminum hydroxide-adjuvanted SARS-CoV-2 S-2P 2 weeks post-second injection. BALB/c mice (N = 6 per group) were immunized with 2 dose levels of CHO cell-expressed SARS-CoV-2 S-2P adjuvanted with CpG 1018, aluminum hydroxide or a combination of both 3 weeks apart and the antisera were harvested at 2 weeks after the second injection. The antisera were subjected to neutralization assay with pseudovirus expressing SARS-CoV-2 spike protein to determine the ID₅₀ (left) and ID₉₀ (right) titers of neutralization antibodies.

Figure 2

Total anti-S IgG titers in mice immunized with S-2P with adjuvants. Sera from BALB/c mice in Fig. 1 (N = 6 per group) immunized with 0, 1 or 5 μg of S-2P with CpG 1018, aluminum hydroxide or combination of both were quantified for the total amount of anti-S IgG with ELISA.

Figure 3

Neutralization of wild-type SARS-CoV-2 virus by antibodies induced by SARS-CoV-2 S-2P adjuvanted with CpG 1018 and aluminum hydroxide. The antisera were collected as described in Fig. 2 (N = 6 per group) and subjected to a neutralization assay with wild-type SARS-CoV-2 to determine neutralization antibody titers.

Figure 4

Inhibition of pseudoviruses carrying D614D (wild-type) or D614G (variant) versions of the spike protein by mice immunized with S-2P with CpG 1018 and aluminum hydroxide. The antisera of BALB/c mice immunized with 1 or 5 μg of S-2P with 10 μg CpG 1018 and 50 μg aluminum hydroxide as in Fig. 1 (N = 5 per group due to assay capacity) were collected. Neutralization assays were performed with pseudoviruses with either D616D or D614G spike proteins.

Figure 5

Correlations between SARS-CoV-2 pseudovirus ID_90, wild-type SARS-CoV-2 ID₅₀, and total anti-S IgG titers in mice. Values of ID₅₀/ID₉₀/IgG titers above lower detection limit (> 40) (N = 29) were tabulated and correlations were calculated with Spearman’s rank correlation coefficient for wild-type SARS-CoV-2 ID₅₀ vs pseudovirus ID₉₀ (left), wild-type SARS-CoV-2 ID₅₀ vs total anti-S IgG titer (middle), and pseudovirus ID₉₀ vs total anti-S IgG titer (right).

Figure 6

Th1-dependent cytokine production induced by SARS-CoV-2 S-2P adjuvanted with CpG 1018, aluminum hydroxide, or CpG 1018 with aluminum hydroxide in mice. The antisera of BALB/c mice (N = 6 per group) were collected at 2 weeks after the second injection, the splenocytes were harvested and incubated with S-2P protein (2 μg), concanavalin A (0.1 μg; data not shown) for positive control, or complete RPMI 1640 medium only for negative control. After 20 h incubation, the levels of IFN-γ (left) and IL-2 (right) were analyzed by ELISA.

Figure 7

Th2-dependent cytokine production induced by SARS-CoV-2 S-2P adjuvanted with CpG 1018, aluminum hydroxide, or CpG 1018/aluminum hydroxide in mice. The antisera of BALB/c mice (N = 6 per group) were collected at 2 weeks after the second injection, the splenocytes were harvested and stimulated as in Fig. 6. After 20 h incubation, the levels of IL-4 (left), IL-5 (middle), and IL-6 (right) released from the splenocytes were analyzed. For detection of cytokines, the culture supernatant was harvested to analyze the levels of cytokines by ELISA.

Figure 8

IFN-γ/IL-4, IFN-γ/IL-5, and IFN-γ/IL-6 ratios. IFN-γ, IL-4, IL-5, and IL-6 values from the cytokine assays (N = 6 per group) were used to calculate ratios. Ratio values greater than 1 indicate Th1 bias whereas ratio less than 1 indicate Th2 bias responses.