Immunogenicity of a vaccinia virus-based severe acute respiratory syndrome coronavirus 2 vaccine candidate

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines provide essential tools for the control of the COVID-19 pandemic. A number of technologies have been employed to develop SARS-CoV-2 vaccines, including the inactivated SARS-CoV-2 particles, mRNA to express viral spike protein, recombinant spike proteins, and viral vectors. Here, we report the use of the vaccinia virus Tiantan strain as a vector to express the SARS-CoV-2 spike protein. When it was used to inoculate mice, robust SARS-CoV-2 spike protein-specific antibody response and T-cell response were detected. Sera from the vaccinated mice showed strong neutralizing activity against the ancestral Wuhan SARS-CoV-2, the variants of concern (VOCs) B.1.351, B.1.617.2, and the emerging B.1.1.529 (omicron). This finding supports the possibility of developing a new type of SARS-CoV-2 vaccine using the vaccinia virus vector.

Keywords: COVID-19; SARS-CoV-2; spike protein; vaccine; vaccinia virus.

Figure 1

Construction of VACV recombinants expressing SARS-CoV-2 spike protein or RBD. (A) Schematic representation of VACV dTK-S and dTK-RBD. The SARS-CoV-2 S (with last 19aa deleted, dTK-S) and RBD sequences (319aa–529aa, dTK-RBD) were inserted into thymidine kinase (TK) locus of VACV Tiantan strain under the control of the early/late promoter p7.5 by homologous recombination. (B) Schematic representation of primers used for plaque purification and PCR verification. (C) PCR analysis of recombinant viruses confirmed the substitution of spike or RBD for the TK region. VACV, vaccinia virus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; RBD, receptor-binding domain; dTK-S, vaccinia virus-expressing spike protein; dTK-RBD, vaccinia virus-expressing receptor-binding domain; TK, thymidine kinase.

Figure 2

Expression of RBD and spike protein in cells infected by the recombinant VACV. (A) Vero cells were infected with parent VACV dTK, dTK-S, or dTK-RBD (MOI 0.01) for 24h. Cell lysates were analyzed by Western blotting using S2 or RBD-specific antibodies. (B) Vero cells were infected with VACV dTK, dTK-S, or dTK-RBD (MOI 0.05) for 24h. Cells were permeabilized and stained with anti-RBD antibody, followed by Alexa Fluor 488-conjugated donkey anti-rabbit antibody incubation. Percentages of FITC-positive cells were shown. (C) Immunofluorescence staining of HeLa cells infected with VACV dTK, dTK-S, or dTK-RBD (MOI 0.05) for 24h. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, or stained directly with anti-RBD antibody. Nuclei were stained by DAPI. (D) Immunofluorescence staining of syncytia formed by HeLa cells expressing human ACE2 (HeLa-ACE2). HeLa-ACE2 cells were infected with VACV dTK, dTK-S, or dTK-RBD (MOI 0.05) for 24 h, fixed, permeabilized, and stained with anti-RBD antibody. Nuclei were stained by DAPI. RBD, receptor-binding domain; VACV, vaccinia virus; dTK-S, vaccinia virus-expressing spike protein; dTK-RBD, vaccinia virus-expressing receptor-binding domain; MOI, multiplicity of infection; FITC, fluorescein isothiocyanate.

Figure 3

dTK-S VACV elicits effective humoral and cellular immunity in mice. (A) Immunization scheme. BALB/c mice aged 6–8 week were intramuscularly immunized with 1 × 10⁵ or 1 × 10⁶ PFU of control dTK, dTK-S, or dTK-RBD at week 0 and week 3. Sera were collected 2 weeks after each vaccination for further quantification of antibody responses. (B–D) Humoral responses in sera of immunized mice were evaluated. S1-binding antibody (B) and RBD-binding antibody (C) were analyzed by ELISA. The IgG2a/IgG1 ratio of S1-binding antibody in dTK-S immunized mice was detected by ELISA using HRP-conjugated isotype-specific antibodies (D). (E) T-cell response induced by dTK-S. CD8 and CD4 T cells expressing IFNγ, TNFα, IL-4, and IL-10 in the spleen of dTK- or dTK-S-immunized mice (high dose) were evaluated by intracellular cytokine staining, after stimulation with S peptides. Dotted lines indicate the lower limit of detection (LLOD). Animals with a response at or below the LLOD were put on LLOD. Statistical differences as determined by Student’s t-tests are indicated by asterisks; *p < 0.05, **p < 0.01; ***p < 0.001. 1st, sera collected 2 weeks after prime; 2nd, sera collected 2 weeks after boost; L, low dose (10⁵ PFU); H, high dose (10⁶ PFU); N, dTK immunization; S, dTK-S immunization; RBD, dTK-RBD immunization. dTK-S, vaccinia virus-expressing spike protein; VACV, vaccinia virus; PFU, plaque-forming unit; RBD, receptor-binding domain; HRP, horseradish peroxidase.

Figure 4

Neutralizing antibody responses in mice vaccinated by dTK-S. (A) Pseudovirus neutralization titers were determined for sera collected 2 weeks after dTK-S boost immunization. A 50% reduction relative to the uninfected control wells was shown as pseudovirus NT50. (B) Neutralization activities of the sera from the second dose of dTK-S vaccination against rVSV-eGFP-SARS-CoV-2. (C) VLP neutralization activities of sera from the second dose of dTK-S vaccination. (D) Neutralization activities of the sera from the second dose of dTK-S vaccination against SARS-CoV-2. (E) Neutralization activities of sera from the second dose of dTK-S vaccination for pseudoviruses carrying spike proteins of the SARS-CoV-2 Wuhan strain (WH-S) B.1.351 or B.1.617.2. (F) Neutralization activities of sera from the immunized mice for pseudoviruses carrying spike protein of the SARS-CoV-2 Wuhan strain (WH-S), B.1.351, or carrying single mutations K417N, E484K, N501Y, or D614G. (G) Neutralization activities of sera from the second dose of dTK-S vaccination for pseudoviruses carrying spike proteins of the SARS-CoV-2 Wuhan strain (WH-S) or B.1.1.529. Dotted lines indicate the lower limit of detection (LLOD). Animals with a response at or below the LLOD were put on LLOD. Statistical differences as determined by Student’s t-tests are indicated by asterisks; *p < 0.05, **p < 0.01; ***p < 0.001. dTK-S, vaccinia virus-expressing spike protein; VLP, virus-like particle.