RelCoVax®, a two antigen subunit protein vaccine candidate against SARS-CoV-2 induces strong immune responses in mice

Abstract

The COVID-19 pandemic has spurred an unprecedented movement to develop safe and effective vaccines against the SARS-CoV-2 virus to immunize the global population. The first set of vaccine candidates that received emergency use authorization targeted the spike (S) glycoprotein of the SARS-CoV-2 virus that enables virus entry into cells via the receptor binding domain (RBD). Recently, multiple variants of SARS-CoV-2 have emerged with mutations in S protein and the ability to evade neutralizing antibodies in vaccinated individuals. We have developed a dual RBD and nucleocapsid (N) subunit protein vaccine candidate named RelCoVax® through heterologous expression in mammalian cells (RBD) and E. coli (N). The RelCoVax® formulation containing a combination of aluminum hydroxide (alum) and a synthetic CpG oligonucleotide as adjuvants elicited high antibody titers against RBD and N proteins in mice after a prime and boost dose regimen administered 2 weeks apart. The vaccine also stimulated cellular immune responses with a potential Th1 bias as evidenced by increased IFN-γ release by splenocytes from immunized mice upon antigen exposure particularly N protein. Finally, the serum of mice immunized with RelCoVax® demonstrated the ability to neutralize two different SARS-CoV-2 viral strains in vitro including the Delta strain that has become dominant in many regions of the world and can evade vaccine induced neutralizing antibodies. These results warrant further evaluation of RelCoVax® through advanced studies and contribute towards enhancing our understanding of multicomponent subunit vaccine candidates against SARS-CoV-2.

Keywords: Delta variant; Nucleocapsid; RBD; RelCoVax®; SARS-CoV-2; Subunit vaccine.

Fig. 1

Expression of RBD and N proteins. (A) Schematic representations of S and N proteins of SARS-CoV-2. Receptor binding domain or RBD of S protein as expressed extends from Arg 319 to Phe 541 and includes the receptor binding motif (RBM) . M is the transmembrane domain and IC is the intracellular domain of S protein. For N protein, full length protein is expressed. (B) Representative SDS-PAGE and western blot images for purified RBD and N proteins. L is the protein molecular weight ladder.

Fig. 2

Effect of adjuvants on immunogenicity of RBD and N proteins. (A) Dosing schedule for mice (N = 6 per group) used in this study. Each antigen is formulated with or without adjuvants in a total volume of 100 µL per dose. Two doses for each formulation were administered 14 days apart (day 1 and day 15) and serum samples were collected 13 days after each dose. (B) Anti-RBD IgG and (C) anti-N IgG end point titers for each mouse group. Group 1 to group 4 are the test groups. (+) and (-) signs indicates the presence and absence of each component in the formulation. The bars and the numbers indicate the geometric mean titer (GMT) for each group and error bars represent the 95% confidence interval (CI) for that group. The dashed lines indicate the limit of detection.

Fig. 3

Comparison of 1 µg and 10 µg antigen doses. (A) Dosing schedule for mice (N = 6 per group) to compare 1 µg and 10 µg doses of each antigen (no antigen is placebo group). All formulations including placebo contain 500 µg of alum and 200 µg of CpG oligonucleotide as adjuvants in a total volume of 100 µL per dose. Two doses for each formulation were administered 14 days apart (day 1 and day 15) and serum samples were collected 6 days and 13 days after each dose. (B) Anti-RBD IgG and (C) anti-N IgG end point titers for each mouse group. The bars and the numbers indicate the GMT for each group and error bars represent the 95% CI for that group. (+) indicates N = 5 since 1 animal in the 1 µg dose group died during blood collection on day 21 (no serum sample on day 28 for that mouse). The dashed lines indicate the limit of detection.

Fig. 4

Immunogenicity of two different formulations of RelCoVax®. (A) Dosing schedule for mice (N = 12 per group) receiving two different formulations (4 µg per dose and 10 µg per dose) of vaccine candidate with no antigen placebo group as control. Two doses for each formulation were administered 14 days apart (day 1 and day 15) and serum samples were collected on day 28 (13 days after dose 2). The 4 µg dose included 200 µg of alum and 80 µg of CpG oligonucleotide as adjuvants and 2 mg of 2-PE as a preservative while the 10 µg dose included 500 µg of alum and 200 µg of CpG oligonucleotide as adjuvants and 5 mg of 2-PE as a preservative. The placebo contained no antigen but included 500 µg of alum, 200 µg of CpG oligonucleotide and 5 mg of 2-PE per dose. Each mouse was administered a total volume of 200 µL per dose. (B) Anti-RBD IgG end point titers for each group and (C) anti-N IgG endpoint titers for each group. The bars and the numbers indicate the GMT for each group and error bars represent the 95% CI for that group. The dashed lines indicate the limit of detection. (D) SARS-CoV-2 neutralizing endpoint titers (PRNT₅₀) for 3 serum pools (4 mice per pool). The serum pools were prepared from the same serum samples tested in Fig. 4B and Fig. 4C. The bars and the numbers indicate the GMT for each group and error bars represent the 95% CI for that group. LLOD – lower limit of dilution, ULOD – upper limit of dilution.

Fig. 5

Assessment of cellular immune responses. (A) Dosing schedule for mice (N = 6 per group) receiving the vaccine formulation containing 10 µg of each antigen with 500 μg of alum and 200 μg of CpG oligonucleotide added as adjuvants and 5 mg of 2-PE added as preservative in a 200 µL dose volume. No antigen control (200 µL) was used as placebo. Two doses were administered 14 days apart (day 1 and day 15) and 3 mice from each group were sacrificed day 29 and day 30 respectively. Splenocytes were harvested after sacrifice for ELISpot assay. (B) Cellular immune responses as measured by IFN-γ ELISpot of splenocytes from immunized mice when stimulated with different antigens. The bars and the numbers indicate the mean number of IFN-γ spot forming cells (SFCs) per million splenocytes for each group and error bars indicate the standard error of mean (SEM).