A single subcutaneous or intranasal immunization with adenovirus-based SARS-CoV-2 vaccine induces robust humoral and cellular immune responses in mice

Abstract

Optimal vaccines are needed for sustained suppression of SARS-CoV-2 and other novel coronaviruses. Here, we developed a recombinant type 5 adenovirus vector encoding the gene for the SARS-CoV-2 S1 subunit antigen (Ad5.SARS-CoV-2-S1) for COVID-19 immunization and evaluated its immunogenicity in mice. A single immunization with Ad5.SARS-CoV-2-S1 via S.C. injection or I.N delivery induced robust antibody and cellular immune responses. Vaccination elicited significant S1-specific IgG, IgG1, and IgG2a endpoint titers as early as 2 weeks, and the induced antibodies were long lasting. I.N. and S.C. administration of Ad5.SARS-CoV-2-S1 produced S1-specific GC B cells in cervical and axillary LNs, respectively. Moreover, I.N. and S.C. immunization evoked significantly greater antigen-specific T-cell responses compared to unimmunized control groups with indications that S.C. injection was more effective than I.N. delivery in eliciting cellular immune responses. Mice vaccinated by either route demonstrated significantly increased virus-specific neutralization antibodies on weeks 8 and 12 compared to control groups, as well as BM antibody forming cells (AFC), indicative of long-term immunity. Thus, this Ad5-vectored SARS-CoV-2 vaccine candidate showed promising immunogenicity following delivery to mice by S.C. and I.N. routes of administration, supporting the further development of Ad-based vaccines against COVID-19 and other infectious diseases for sustainable global immunization programs.

Keywords: Adenovirus; COVID-19; Infectious diseases; Recombinant DNA vaccines; SARS-CoV-2.

Figure 1

Adenoviral‐vectored SARS‐CoV‐2‐S1 vaccine. (A) A shuttle vector carrying the codon‐optimized SARS‐CoV‐2‐S1 gene encoding N‐terminal 1–661 was designed as shown in the diagram. The vector was used to generate recombinant type 5 replication‐deficient adenoviruses (Ad5) by homologous recombination with the adenoviral genomic DNA. ITR, inverted terminal repeat; RBD, receptor binding domain. (B) Detection of the SARS‐CoV‐2‐S1 protein by western blot with the supernatant of A549 cells infected with Ad5.SARS‐CoV‐2.S1 (Ad5.S1) (10 MOI) using antispike protein of SARS‐CoV rabbit polyclonal antibody (lane 2). Mock (AdΨ5)‐infected cells were treated the same and used as a negative control (lane 1). As a positive control, 100 ng of recombinant SARS‐CoV‐2‐S1 (Sino biological, 1–685 amino acids with ten histidine tag) was loaded (lane 3). The supernatants were resolved on SDS‐10% polyacrylamide gel after being boiled in 2% SDS sample buffer with β‐ME. The images of original Western blots used for preparation of Fig. 1B are shown in Supporting information Fig. S5.

Figure 2

Antigen‐specific antibody responses in mice immunized with adenoviral vectored SARS‐CoV‐2‐S1 vaccine. BALB/c mice were immunized S.C. or I.N. with 1.5 × 10¹⁰ vp of Ad5.SARS‐CoV‐2‐S1 (Ad5.S1) or AdΨ5, while mice were immunized subcutaneously with PBS as a negative control. On weeks 0, 2, 4, 6, 8, 12, and 24 after vaccination, the sera from mice were collected, diluted, and SARS‐CoV‐2‐S1‐specific antibodies were quantified by ELISA to determine the (A) IgG (weeks 0, 2, 4, 6, 8, 12, and 24), (B) IgG1 (weeks 0, 2, 4, 6, and 24), and (C) IgG2a (weeks 0, 2, 4, 6, and 24) endpoint titers. Horizontal lines indicate geometric mean antibody titers. Significance was determined by Kruskal–Wallis test, followed by Dunn's multiple comparisons (*p < 0.05). Representative data are from one of two independent experiments (n = 5 mice per group for each experiment).

Figure 3

Antigen‐specific humoral responses in mice immunized with Ad5.SARS‐CoV‐2‐S1. Formation of GC reactions and Ig isotype switching in draining LNs. BALB/c mice were vaccinated S.C. or I.N. with 1.5 × 10¹⁰ vp of Ad5.SARS‐CoV‐2‐S1 (Ad5.S1) or AdΨ5, while mice were immunized S.C. with PBS as a negative control. Cervical and axillary LNs were harvested 14 days after I.N. and S.C. vaccination and single‐cell suspensions of LNs were stained and analyzed by flow cytometry to determine the frequencies of S1‐specific GC‐B cells and their IgM, IgG1, and IgG2a isotype distribution. (A) S1‐specific GC B cells in cervical LNs. (B) IgM, IgG1, and IgG2a isotype distribution of S1‐specific B cells in cervical LNs. (C) S1‐specific GC B cells in axillary LNs. (D) IgM, IgG1, and IgG2a isotype distribution of S1‐specific B cells in axillary LNs. As outlined in the gating strategy presented in Supporting information Fig. S2, %GC of S1⁺ B cells in A and C were calculated as %CD95⁺ CD38^– of live CD19⁺ S1⁺, and frequencies of isotype‐specific GC B‐cell subsets in B and D were calculated as %Isotype⁺ of live CD19⁺ S1⁺ CD95⁺ CD83^– cells. Results are mean ± SD. Groups were compared by one‐way Welch's ANOVA, followed by Dunnett's T3 multiple comparisons, and significant differences are indicated by *p < 0.05. Data are from two independent experiments (n = 5 mice per group for each experiment) that are indicated by circles or triangles.

Figure 4

Antigen‐specific cellular responses in mice immunized with Ad5.SARS‐CoV‐2‐S1. BALB/c mice were immunized S.C. or I.N. with 1.5 × 10¹⁰ vp of Ad5.SARS‐CoV‐2‐S1 (Ad5.S1) or AdΨ5, or subcutaneously with PBS as a negative control. Twelve days after vaccination, splenocytes were isolated and stimulated with SARS‐CoV‐2 S1 PepTivator, followed by intracellular cytokine staining (ICS) and flow cytometry to identify SARS‐CoV‐2 S1‐specific T cells (see Supporting information Fig. S4 for complete gating strategy). Frequencies of SARS‐CoV‐2 S1‐specific (A) CD8⁺ IFN‐γ⁺ and (B) CD4⁺ IFN‐γ⁺ T cells, presented after subtracting background responses detected in corresponding unstimulated splenocyte samples. Results are mean ± SD. Groups were compared by one‐way Welch's ANOVA, followed by Dunnett's T3 multiple comparisons, and significant differences are indicated by *p < 0.05. Data are from two independent experiments (n = 5 mice per group for each experiment) that are indicated by circles or triangles.

Figure 5

Neutralizing antibody responses in mice immunized with Ad5.SARS‐CoV‐2 S1. BALB/c mice were immunized S.C. or I.N. with 1.5 × 10¹⁰ vp of Ad5.SARS‐CoV‐2‐S1 (Ad5.S1) or AdΨ5, while mice were immunized subcutaneously with PBS as a negative control. Neutralizing antibodies in serum of mice 8 or 12 weeks after immunization were measured using a microneutralization assay (NT₉₀) with SARS‐CoV‐2. Serum titers that resulted in a 90% reduction in cytopathic effect compared to the virus control were reported. Horizontal lines represent geometric mean neutralizing antibody titers. Groups were compared by Kruskal–Wallis test at each time point, followed by Dunn's multiple comparisons. Significant differences relative to the PBS control are indicated by *p < 0.05. The minimal titer tested was 5, and undetectable titers (those with NT₉₀ serum titers < 5) were assigned a value of 2.5. Data are from a single experiment (n = 5 mice per group).