Immunogenicity of Multiple Doses of pDNA Vaccines against SARS-CoV-2

Abstract

Since its identification in Wuhan, China, in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has resulted in 46 million cases and more than one million deaths worldwide, as of 30 October 2020. Limited data exist on the magnitude and durability of antibodies generated by natural infection with SARS-CoV-2 and whether they can provide long-lasting immunity from reinfection. Vaccination has proven the most effective measure for controlling and preventing pandemics and, thus, development of a vaccine against COVID-19 is a top priority. However, the doses required to induce effective, long-lasting antibody responses against SARS-CoV-2 remain undetermined. Here, we present the development of SARS-CoV-2 vaccine candidates encoding the viral spike (S) gene, generated using plasmid (p)DNA technology, and we demonstrate the eliciting of S-specific antibodies in mice after three and four doses. The magnitude of binding and neutralizing antibody responses with three doses of synthetic, codon-optimized, full-length S (S.opt.FL) vaccine is comparable to that generated after four doses, suggesting that three doses are sufficient to elicit robust immune responses. Conversely, four doses of S1.opt pDNA vaccine, containing the S globular head, are required to elicit high levels of neutralizing antibodies. Furthermore, the S.opt.FL pDNA vaccine induces the highest serum levels of interferon (IFN)-γ, a marker for activation of cellular immune responses. Overall, our data show that three doses of S.FL pDNA vaccine elicit potent neutralizing antibody responses, with preclinical data that support the immunogenicity of these COVID-19 vaccine candidates and provide justification for further translational studies.

Keywords: COVID-19; SARS-CoV-2; antibody; coronavirus; immunity; pDNA; vaccine; virus.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Schematic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein. (A) The primary structure of S with its domains: signal peptide (SP), receptor binding domain (RBS), fusion peptide (FP), heptad repeat (HR), transmembrane (TM), and cytoplasmic tail (CT). (B) Side and top view of the three-dimensional structure of the trimeric spike protein in the perfusion confirmation. Image created from the structure with Protein Data Bank (PDB) identifier VXX6.

Figure 2

Optimizations of the full-length SARS-CoV-2 spike gene. (A) Distribution of codon usage frequency of the spike gene. Codon Adaptation Index (CAI) = 0.94. (B) Codon distribution percentage computed as codon quality group. (C) GC content adjustment with average equal to 55.69. (D) Restriction analysis for the S.opt.FL and S1.opt using single-cut digestion with BamHI and double-cut digestion with BamHI and NheI.

Figure 3

Schematic of the C57BL/6 mice immunization with SARS-CoV-2 vaccines. (A) Immunization groups and doses for the plasmid (p)DNA vaccines. All immunizations were received intramuscularly with 100 ug per dose, except the phosphate-buffered saline (PBS) control group. (B) The bleeding and immunization regime for the C57BL/6 mice.

Figure 4

Serum endpoint immunoglobulin G (IgG) ELISA titers against autologous full-length spike (S) protein. (A). Total IgG S antibodies were measured in mice sera 2 weeks after the third immunization. Serum starting concentration was 1:50. (B) Total IgG S antibodies were measured in mice sera 2 weeks after the fourth immunization. The highest dilution that gave an optical density (OD)450 twofold higher than that of the prebleed sera (week 0) was designated as the antibody endpoint titer. Antibody titers were expressed as mean endpoint titers ± standard error of the mean (SEM) for each vaccine group with an individual scatter dot plot (n = 6). Data were compared by one-way ANOVA followed by Tukey’s multiple comparison test. ns: no significant difference. The asterisks refer to the level of significance: **** p < 0.0001; ns: no significant difference.

Figure 5

Box-and-whisker plot of surrogate virus neutralization test (sVNT). (A) Titer of anti-receptor-binding domain (RBD) IgG antibodies from serially diluted mice vaccinated sera taken 2 weeks after the third immunization. (B) Titer of anti-RBD IgG antibodies from serially diluted mice vaccinated sera taken 2 weeks after the fourth immunization. Cutoff titer was calculated as the serum highest dilution showing a cutoff value >20%. Data were analyzed with one-way ANOVA with Tukey’s multiple comparison test. The asterisks refer to the level of significance: * p < 0.033; ns: no significant difference.

Figure 6

In vivo IFN-γ responses following C57BL/6 mice vaccinations. Comparing serum IFN-γ levels in each of vaccine constructs (S.opt.FL, S1.opt, and S.opt.FL+S1.opt) 2 weeks following second immunization in each vaccine construct using pooled mice sera from each group. Endpoint concentration was determined by titers expressed (mean ± SD). Data were analyzed with one-way ANOVA with Tukey’s multiple comparison test. The asterisks refer to the level of significance: *** p < 0.0002; ns: no significant difference.