Receptor-binding domain recombinant protein on alum-CpG induces broad protection against SARS-CoV-2 variants of concern

Abstract

We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. This formulation is equivalent to the Corbevax^TM vaccine that recently received emergency use authorization by the Drugs Controller General ofIndia. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum + CpG. We also evaluated mice immunized with RBD/alum + CpG and boosted with RBD/alum. Mice were immunized twice intramuscularly at a 21-day interval. Compared to two doses of the /alum formulation, the RBD/alum + CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 and (Delta) variants. Neutralizing antibody titers against the B.1.1.529 (BA.1, Omicron) variant exceeded those in human convalescent plasma after Wuhan infection but were lower than against the other variants. Interestingly, the second dose did not benefit from the addition of CpG, possibly allowing dose-sparing of the adjuvant in the future. The data reported here reinforces that the RBD/alum + CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2, including variants of concern.

Keywords: COVID-19; Corbevax; Subunit vaccine; TLR9 adjuvant; Virus neutralization.

Fig. 1

Humoral and cellular immune response in immunized mice. A) Total IgG titers at day 35, measured by ELISA. B) IgG2a/IgG1 titer ratios. Geometric mean and 95% confidence intervals are shown. C) Heatmap of secreted cytokine levels measured in the supernatants from splenocytes re-stimulated with the SARS-CoV-2 RBD protein. Median values were calculated within each treatment group for each cytokine and plotted. D) IC50 values measured by a neutralization assay using a lentiviral SARS-CoV-2 Wuhan pseudovirus. Mann-Whitney tests were performed to evaluate for statistical significance between different groups. p > 0.12(ns), p < 0.033 (*), p < 0.001 (***).

Fig. 2

Comparison of RBD219-N1C1-specific immune responses after one dose with CpG and a boost without CpG versus two vaccine doses with CpG. Geometric mean and 95% confidence intervals are shown. A) Total IgG titers measured from mouse sera against RBD protein, B) IgG2a/IgG1 titer ratios from mouse sera against RBD protein, C) Heatmap of secreted cytokines measured in supernatants from splenocytes re-stimulated with RBD protein. D) IC50 values measured by a neutralization assay using a lentiviral Wuhan SARS-CoV-2 pseudovirus. Mann-Whitney tests were performed to evaluate for statistical significance between different groups. p > 0.12(ns), p < 0.033 (*). The dotted lines indicate the limit of quantification.

Fig. 3

Neutralizing activity by sera from vaccinated mice against Alpha and Beta variants of the SARS-COV-2 pseudovirus A) Neutralization of various pseudovirus mimicking different variants of concern: Wuhan SARS-CoV-2 (blue), B.1.1.7 (Alpha) SARS-CoV-2 variant (red), B.1.351 (Beta) SARS-CoV-2 variant (green). The bar graph represents the geometric means of technical replicates (n = 6, except for the group vaccinated with 7ug RBD/Alum n = 2). The dotted line indicates the limit of quantification. B) Live virus neutralization (PRNT assay): Original SARS-CoV-2 (blue), B.1.351 (Beta) SARS-CoV-2 variant (green). The dotted line indicates the limit of quantification. The bar graph represents the geometric means of technical replicates (n = 2). C) Correlation graph, comparing IC50s of the live virus and pseudovirus neutralization.

Fig. 4

Neutralizing activity by sera from vaccinated mice against Delta and Omicron variants of the SARS-CoV-2 pseudovirus. Neutralization of various pseudovirus mimicking different variants of concern: Wuhan SARS-CoV-2 (left panel), B.1.617.2 (Delta) SARS-CoV-2 variant (middle panel), B.1.351 (Omicron) SARS-CoV-2 variant (right panel). The human NIBSC convalescent plasma was included as a positive control, serum from mice vaccinated with alum + CpG was included as a negative control. Data points are averages of technical replicates (n = 2). The dotted line indicates the limit of quantification p < 0.0021 (**), p < 0.0001 (****).