Comparison of Wild Type DNA Sequence of Spike Protein from SARS-CoV-2 with Optimized Sequence on The Induction of Protective Responses Against SARS-Cov-2 Challenge in Mouse Model

Abstract

Genetic optimization of Nucleic Acid immunogens is important for potentially improving their immune potency. A COVID-19 DNA vaccine is in phase III clinical trial which is based on a promising highly developable technology platform. Here, we show optimization in mice generating a pGX-9501 DNA vaccine encoding full-length spike protein, which results in induction of potent humoral and cellular immune responses, including neutralizing antibodies, that block hACE2-RBD binding of live CoV2 virus in vitro. Optimization resulted in improved induction of cellular immunity by pGX-9501 as demonstrated by increased IFN-γ expression in both CD8+ and CD4 + T cells and this was associated with more robust antiviral CTL responses compared to unoptimized constructs. Vaccination with pGX-9501 induced subsequent protection against virus challenge in a rigorous hACE2 transgenic mouse model. Overall, pGX-9501 is a promising optimized COVID-19 DNA vaccine candidate inducing humoral and cellular immunity contributing to the vaccine's protective effects.

Keywords: COVID-19; DNA vaccine; SARS-CoV-2; optimizations; protective response; spike protein; wild-type sequence.

Figure 1.

Comparison of expression and antibody levels of optimized versus non-optimized spike sequences. 293 T cells were transfected by pGX-9501, pVAX1-S-WT, and pVAX1 for 48hrs and lysed for RT-PCR analysis (a) and Western blotting (b), respectively. (c) BALB/c mice were immunized with either construct at 25 μg dose by using the 3P EP by the IM route. Immune analysis was performed at 2 weeks in an S1Elisa assay.

Figure 2.

Effects of antibody production and functional assay. (a), The scheme of mice immunizations. (b), C57BL/6, or BALB/c mice (N = 6 per group) were either immunized with pVAX1 (blue circle) or vaccinated with pVAX1-S-WT (red square) and pGX-9501 (green triangle) intramuscularly, following by electroporation. Serum IgG binding titers (mean ± SEM) to SARS-CoV-2 pre-S1, S2, and RBD were measured on day 28. (c), Blocking abilities of RBD binding to the hACE2 with serum samples at serial dilutions on day 28. Data shown represent mean blocking efficiency (mean± SEM) for the five mice. Please add in the Single immunization group to the chart as well. Including bleeding.

Figure 3.

pGX-9501 protects against challenges with SARS-CoV-2 in BALB/c mice. Mice treated with the vaccine were challenged by SARS-CoV-2 (10⁵TCID50) in a volume of 100 μl 7 days after the second immunization (single dose group was challenged by virus 14 days after immunization). Five days after the challenge, Serum was collected for anti-s1 ELISA(a), and lung was harvested for measuring virus load by qRT-PCR (b). (c), Mice post vaccination were challenged by SARS-CoV-2 (10⁵TCID₅₀) in a volume of 100 μl 7 days after the second immunization (single dose group was challenged by virus 14 days after immunization). Serum was collected for ELISA to evaluate the Neutralizing antibody. (d), The histochemistry analysis of lung after H&E staining. €, Daily weight loss were monitored as shown.

Figure 4.

pGX-9501 promoted a biased CD8 T cell-based Th1-type cytokine phenotype and did not induce a TH2-associated phenotype. Single suspensions of splenocytes and lymphoid cells of lymph nodes harvested from C57BL/6 (a) or BALB/c (b) mice immunized were stimulated with 10 mg/mL SARS-CoV-2 peptide pools in vitro for 4 to 6 hours, and IFN-γ production from CD4⁺ T cells was analyzed by flow cytometry in both the C57BL/6 and BALB/c mice strains. Cytokine expression was studied using the SARS-CoV-2 peptide pool for immune stimulation.

Figure 5.

pGX-9501 induces effective specific cytotoxic lymphocyte(CTL) killing activity in vivo with enhanced IFN-g dominated cytokine expression in specific CD8 + T cells. Single suspension lymphocytes of spleens or lymph nodes from immunized C57BL/6 (a) and BALB/c (b) mice were stimulated with 10 mg/mL SARS-CoV-2 peptide pools in vitro for 4 to 6 hours. Levels of IFN-γ and TNF-α production in CD8 + T cells were measured by flow cytometry. C, Antigen-specific cytotoxic lymphocyte driven (CTL) killing ability was evaluated using an in vivo CTL assay. Target cells at 4*106/ml from naïve mice labeled with eFlour450 were incubated with 10 mg/mL SARS-CoV-2 peptide pools in vitro for 4–6 h before transferring into immunized mice by the intravenous route. The intensity of eFlour450 peptide labeled target cells were compared with the non-peptide labeled negative control cells after 5 hrs by flow cytometry to demonstrate in vivo killing. In vivo killing is only observed in the optimized pGX-9501 vaccinated animals.