Cytomegalovirus-vectored COVID-19 vaccines elicit neutralizing antibodies against the SARS-CoV-2 Omicron variant (BA.2) in mice

Abstract

Cytomegalovirus (CMV) has been used as a novel viral vector for vaccine development and gene therapy. Coronavirus disease 2019 is an infectious disease caused by the SARS-CoV-2 virus, which is highly mutable and is still circulating globally. The study showed that the CMV viral vector caused transient systemic infection and induced robust transgene expression in vivo. CMV vectors expressing different SARS-CoV-2 proteins were immunogenic and could elicit neutralizing antibodies against a highly mutated Omicron variant (BA.2). The expression level of receptor-binding domain (RBD) protein was higher than that of full-length S protein using CMV as a vaccine vector, and CMV vector expression RBD protein elicited higher RBD-binding and neutralizing antibodies. Moreover, the study showed that CMV-vectored vaccines would not cause unexpected viral transmission, and pre-existing immunity might impair the immunogenicity of subsequent CMV-vectored vaccines. These works provide meaningful insights for the development of a CMV-based vector vaccine platform and the prevention and control strategies for SARS-CoV-2 infection.

Keywords: SARS-CoV-2; cytomegalovirus; immunogenicity evaluation; variant of concern; viral vector-based vaccine.

Fig 1

Construction of a luciferase-tagged MCMV and in vivo tracking of its dissemination. (A) Strategy for constructing MCMV-Luc. The luciferase expression cassette was inserted into the IE2 locus of MCMV via a modified CRISPR-Cas9 gene editing technology. (B) In vitro monitoring of virus growth in 3T3 cells with luciferase as a reporter. MCMV-Luc was used to infect 3T3 cells, and an in vitro luciferase assay was performed every day post-infection. MCMV-WT infection and mock infection were used as controls. (C) The bioluminescent signals were detected 4 days post-infection of mock, MCMV-WT, and MCMV-Luc using an IVIS Imaging System (Xenogen). (D) In vivo tracking of MCMV dissemination in mice. MCMV-Luc and/or MCMV-WT were used to infect mice (i.p.; 10⁷ TCID₅₀), and the bioluminescent signals were detected every 2 days using an in vivo luciferase assay.

Fig 2

Evaluation of the immunogenicity of CMV-vectored vaccine expressing SARS-CoV-2 key constituent proteins. (A) Strategy for constructing MCMV-RBD/N/S-full. The RBD/N/S-full protein expression cassettes were inserted into the IE2 locus of MCMV via a modified CRISPR-Cas9 gene editing technology. (B) Detection of target gene expression in MCMV-RBD/N/S-full infected cells using Western blot. MCMV-RBD/N/S-full infected and uninfected cells were lysed, and the proteins were separated by SDS-PAGE and transferred to PVDF membranes. The expression of RBD (lane 2) or S-full (lane 4) in the infected cell lysates was probed with a monoclonal mouse antibody specific to RBD, and N protein (lane 6) was probed with a monoclonal rabbit antibody. Uninfected cells (lanes 1, 3, and 5) were set as control, and Beta-actin was used as an endogenous reference. (C) Schematic diagram of immunization and sample collection. Three groups of mice (n = 4) were immunized with MCMV-RBD, MCMV-N, or MCMV-S-full at weeks 0, 4, and 8, and sera were collected at weeks 0, 2, 4, 6, 8, 10, and 12. (D) The SARS-CoV-2-specific IgG antibody titer was determined by indirect ELISA. The MCMV-RBD and MCMV-S-full immunized mice sera collected at different time points were serially diluted and tested using RBD-based indirect ELISA, and the MCMV-N immunized mice sera were tested using N-based indirect ELISA. (E) The endpoint dilution titer of MCMV-RBD/N/S-full immunized mice sera collected at week 12 was measured by indirect ELISA (cutoff: P/N > 2.5). (F) The NT₅₀ of MCMV-RBD and MCMV-S-full immunized mice sera collected at different time points were determined using luciferase-tagged pseudovirus expressing Spike protein of the Wuhan-Hu-1 strain. (G) The NT₅₀ of MCMV-RBD and MCMV-S-full immunized mice sera collected at different time points were determined using luciferase-tagged pseudovirus expressing SARS-CoV-2 Omicron variant (BA.2) Spike protein. (H) Comparison of the NT₅₀ of MCMV-RBD immunized mice sera collected at different time points against pseudovirus expressing Spike protein of the Wuhan-Hu-1 strain and the BA.2 variant. (I) Comparison of the NT₅₀ of MCMV-S-full immunized mice sera collected at different time points against pseudovirus expressing Spike protein of the Wuhan-Hu-1 strain and the BA.2 variant. Data are shown as mean ± SD. Significance was calculated using the two-tailed Student’s t-test for two groups. n.s., not significant; *P < 0.05; **P < 0.01; and ***P < 0.001.

Fig 3

Evaluation of unimmunized mice sera antibodies caged with CMV-vectored vaccine-immunized mice. (A) Measure of RBD-binding IgG antibodies in the MCMV-RBD immunized or unimmunized mice sera. (B) Measure of neutralizing antibodies specific to MCMV in the MCMV-RBD immunized or unimmunized mice sera. (C) Measure of RBD-binding IgG antibodies in the MCMV-S-full immunized or unimmunized mice sera. (D) Measure of neutralizing antibodies specific to MCMV in the MCMV-S-full immunized or unimmunized mice sera. (E) Measure of RBD-binding IgG antibodies in the MCMV-N immunized or unimmunized mice sera. (F) Measure of neutralizing antibodies specific to MCMV in the MCMV-N immunized or unimmunized mice sera.

Fig 4

The impacts of pre-existing immunity on the efficacy of CMV-vectored vaccines. (A) Schematic diagram of cross-immunization and sample collection. Four groups of mice with/without pre-existing immunity were cross-immunized with MCMV-RBD, MCMV-N, or MCMV-Zika-E-full, and sera were collected at weeks 0, 1, 2, and 3. (B) Evaluation of RBD- or N-binding IgG antibodies in the group I/II/III mice sera pre- or post-cross-immunization. RBD-binding IgG antibodies in groups I and III mice sera were detected by RBD-based indirect ELISA, and N-binding IgG antibodies in the group II mice sera were detected by N protein-based indirect ELISA. (C) Evaluation of neutralizing antibodies against SARS-CoV-2 Wuhan-Hu-1 strain in groups I and III mice sera pre- or post-cross-immunization. (D) Evaluation of neutralizing antibodies against SARS-CoV-2 Omicron variant (BA.2) in groups I and III mice sera pre- or post-cross-immunization. (E) Evaluation of neutralizing antibodies against MCMV viral vector in groups I, II, III, and IV mice sera pre- or post-cross-immunization. (F) Evaluation of SARS-CoV-2- or ZIKV-specific antibodies in groups I, II, III, and IV mice sera pre- or post-cross-immunization. N protein-binding antibodies in group I mice sera were detected by N protein-based indirect ELISA; RBD-binding antibodies in group II mice sera were detected by RBD-based indirect ELISA; ZIKV E protein-binding antibodies in groups III and IV mice sera were detected by E-DIII-based indirect ELISA.