Adenovirus Vaccine Containing Truncated SARS-CoV-2 Spike Protein S1 Subunit Leads to a Specific Immune Response in Mice

Abstract

The development of an efficient and safe coronavirus disease 2019 (COVID-19) vaccine is a crucial approach for managing the severe acute respiratory disease coronavirus 2 (SARS-CoV-2) pandemic in light of current conditions. In this study, we produced a shortened segment of the optimized SARS-CoV-2 spike gene (2043 bp, termed S1) that was able to encode a truncated S1 protein. The protein was tested to determine if it could elicit efficient immunization in mice against SARS-CoV-2. The presence of the S1 protein was confirmed with immunofluorescence and Western blotting. An adenovirus vaccine bearing the S1 gene fragment (Ad-S1) was administered intramuscularly to mice four times over 4 weeks. SARS-CoV-2 S1 protein humoral immunity was demonstrated in all immunized mice. The serum from immunized mice demonstrated excellent anti-infection activity in vitro. A robust humoral immune response against SARS-CoV-2 was observed in the mice after vaccination with Ad-S1, suggesting that the adenovirus vaccine may aid the development of vaccines against SARS-CoV-2 and other genetically distinct viruses.

Keywords: S1 gene; SARS-CoV-2; adenoviral vector; immunity; vaccines.

Figure 1

Construction of recombinant adenovirus vaccine and experimental strategy. (A) pAdeno-CMV-S1 is a recombinant adenovirus shuttle plasmid containing the target gene S1. pBHGloxΔE1,3Cre is the adenovirus skeleton plasmid. pBHGloxΔE1,3Cre and pAdeno-CMV-S1 were extracted with a QIAGEN plasmid extraction kit (Beijing North Yitao Trading Co., LTD., Beijing, China). One day before transfection, HEK293 cells were passaged in a 25 cm² cell culture flask. The culture medium was DMEM containing 5% FBS without antibiotics. On the second day, 60–80% of the cells were selected and the transfection solution was added to the cells. After 7 days of transfection, the cells were scraped off, centrifuged, the supernatant discarded, and the cells were resuscitated with PBS. The cells were freeze-thawed repeatedly at −80 °C and 37 °C. After centrifugation, the supernatant contained the primary virus solution. The recombinant adenovirus strain was purified, amplified, and obtained after further processing and designated Ad-S1. The vaccine was injected intramuscularly into the mice for follow-up studies. (B) The depicted time scale of immunization and blood withdrawal.

Figure 2

Identification of recombinant HAdV-C5–SARS-CoV-2 S1 protein and determination of adenovirus titer. (A) PCR detection of the recombinant plasmid with the target gene S1. (B) Western blot detection of SARS-CoV-2. Exponential-growth HEK293 cells were infected with SARS-CoV-2 for 48 h, then the cellular protein was extracted and separated by SDS-PAGE. SARS-CoV-2 expression was confirmed by Western blotting with goat anti-rabbit IgG. (C) Immunofluorescence microscopy of HEK-293 cells infected with Ad-S1 (green). Cells were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) to stain the nuclei. Scale bar 1000 μm. (D) Plaque assay was used for measurement. Micrographs depicting 10-5, 10-6, 10-7 and 10-8 dilution (from left to right).

Figure 3

Ad-S1 induced altered plasma levels of type 1/2 cytokines, type 1 interferons, and other cytokines. The results were detected by drawing blood from the mice 7 days after the first immunization. The data are presented as box and whisker scatterplots. Each circle represents a single individual. p-values were calculated using a two-sample t-test.

Figure 4

Ad-S1 induced high titers of antibodies (A) and neutralization activity (B,C) in mice. (A) ELISA of SARS-CoV-2-specific serum IgG from Ad-S1-immunized mice. (B) Analysis of pseudovirus activities of serum from the Ad-S1-immunized mice. (C) Analysis of neutralizing activities of serum from the Ad-S1-immunized mice.