Preclinical Immune Response and Safety Evaluation of the Protein Subunit Vaccine Nanocovax for COVID-19

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global health concern. The development of vaccines with high immunogenicity and safety is crucial for controlling the global COVID-19 pandemic and preventing further illness and fatalities. Here, we report the development of a SARS-CoV-2 vaccine candidate, Nanocovax, based on recombinant protein production of the extracellular (soluble) portion of the spike (S) protein of SARS-CoV-2. The results showed that Nanocovax induced high levels of S protein-specific IgG and neutralizing antibodies in three animal models: BALB/c mouse, Syrian hamster, and a non-human primate (Macaca leonina). In addition, a viral challenge study using the hamster model showed that Nanocovax protected the upper respiratory tract from SARS-CoV-2 infection. Nanocovax did not induce any adverse effects in mice (Mus musculus var. albino) and rats (Rattus norvegicus). These preclinical results indicate that Nanocovax is safe and effective.

Keywords: ACE2; CHO; COVID-19; RBD; SARS-CoV-2.

Figure 1

Recombinant SARS-CoV-2 spike (S) protein construct used for Nanocovax. Recombinant S protein (rSPP) construct containing native S protein sequence (aa 1–1213) followed by the arginine-9 tag. The S1/S2 furin cleavage sites (RRAR) and two amino acids (Kv) were mutated as noted.

Figure 2

High-yield production and characterization of recombinant SARS-CoV-2 spike (S) protein. Viable cell density (A). Viability of cells (B). Protein titer from day 7 to end of fed-batch culture (C). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of harvest sample (D). Western blotting of harvest sample (E). Purity of S protein (F). N- and C-terminal peptide sequencing and blasting to complete non-redundant database of protein sequence storage at the National Center for Biotechnology Information (NCBI) (G).

Figure 3

Immunogenicity of Nanocovax vaccine using different animal models. The BALB/c mice were intramuscularly injected twice 7 days apart. Blood was collected on day 14 to determine SARS-CoV-2 S protein-specific antibody IgG levels by ELISA (A). Syrian hamsters were intramuscularly injected twice 7 days apart. Blood was collected on day 28 and day 45 to determine SARS-CoV-2 S protein-specific antibody IgG levels by ELISA assay (B). Northern pig-tailed macaques were intramuscularly injected twice 7 days apart. Blood was collected on days 14, 28, and 45 to determine SARS-CoV-2 S protein-specific antibody IgG levels by ELISA assay (C). The data represent the mean ± SD, and p-values were determined by one-way ANOVA with the Newman–Keuls multiple comparison test and two-way ANOVA analysis with Bonferroni post-hoc tests (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant).

Figure 4

SARS-CoV-2-neutralizing antibodies. BALB/c mice were intramuscularly injected twice 7 days apart. Blood was collected on day 14 to determine SARS-CoV-2-neutralizing antibodies by in vitro surrogate virus neutralization assay (A). Syrian hamsters were intramuscularly injected twice 7 days apart. Blood was collected on days 28 and 45 to determine SARS-CoV-2-neutralizing antibodies by in vitro surrogate virus neutralization assay (B). Northern pig-tailed macaques were intramuscularly injected twice 7 days apart. Blood was collected on days 14, 28, and 45 to determine SARS-CoV-2-neutralizing antibodies by in vitro surrogate virus neutralization assay (C). The SARS-CoV-2 neutralizing antibodies in the BALB/c (D) and hamster (E) models were also measured by PRNT₅₀ assay. The data represent the mean ± SD, and p-values were determined by one-way ANOVA with the Newman–Keuls multiple comparison test and two-way ANOVA analysis with Bonferroni post-hoc tests (***p < 0.001; ns, not significant).

Figure 5

Challenge of Syrian hamsters with live SARS-CoV-2. Syrian hamsters were vaccinated twice 7 days apart. After vaccination, the hamsters were challenged with low and high levels of the SARS-CoV-2 virus on day 14. The weight gain in hamsters was monitored after challenge with high (A) and low levels of SARS-CoV-2 (B). SARS-CoV-2 load [envelope (E) gene] in lung samples 14 days post challenge was determined by real-time RT-PCR (C).

Figure 6

Repeat-dose toxicity study. The repeat-dose toxicity assay was performed on Rattus norvegicus by intramuscular injection once a week for 4 weeks. Pathological signals and weight changes were observed. Body weight gain (A). Relative organ weights (B). Indicators of hematological parameters (C). Biochemical parameters (D). Anatomical structure of the organs (E) and histological analysis of the liver, spleen, and kidneys (F) in repeat-dose test (magnification ×40). The data represent the mean ± SD, and p-values were determined by two-way ANOVA with Bonferroni post-hoc tests and one-way ANOVA with the Newman–Keuls multiple comparison test.