Replication-competent vesicular stomatitis virus vaccine vector protects against SARS-CoV-2-mediated pathogenesis

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of human infections and hundreds of thousands of deaths. Accordingly, an effective vaccine is of critical importance in mitigating coronavirus induced disease 2019 (COVID-19) and curtailing the pandemic. We developed a replication-competent vesicular stomatitis virus (VSV)-based vaccine by introducing a modified form of the SARS-CoV-2 spike gene in place of the native glycoprotein gene (VSV-eGFP-SARS-CoV-2). Immunization of mice with VSV-eGFP-SARS-CoV-2 elicits high titers of antibodies that neutralize SARS-CoV-2 infection and target the receptor binding domain that engages human angiotensin converting enzyme-2 (ACE2). Upon challenge with a human isolate of SARS-CoV-2, mice expressing human ACE2 and immunized with VSV-eGFP-SARS-CoV-2 show profoundly reduced viral infection and inflammation in the lung indicating protection against pneumonia. Finally, passive transfer of sera from VSV-eGFP-SARS-CoV-2-immunized animals protects naïve mice from SARS-CoV-2 challenge. These data support development of VSV-eGFP-SARS-CoV-2 as an attenuated, replication-competent vaccine against SARS-CoV-2.

Figure 1.. Immunogenicity of VSV-eGFP-SARS-CoV-2.

A. Scheme of vaccination and SARS-CoV-2 challenge. B-D. Four-week-old female BALB/c mice were immunized with VSV-eGFP or VSV-eGFP-SARS-CoV-2. Some of the immunized mice were boosted with their respective vaccines four weeks after primary vaccination. IgG responses in the sera of vaccinated mice were evaluated three weeks after priming or boosting by ELISA for binding to SARS-CoV-2 S (B) or RBD (C) or two weeks after priming or boosting by focus reduction neutralization test (FRNT) (D) (n = 15 per group; one-way ANOVA with Dunnett’s post-test: **** P < 0.0001).

Figure 2.. VSV-eGFP-SARS-CoV-2 protects mice against SARS-CoV-2 infection.

Three weeks after priming or boosting with VSV-eGFP or VSV-eGFP-SARS-CoV-2, immunized animals were treated with anti-Ifnar1 mAb and one day later, animals were transduced with 2.5 × 10⁸ PFU of AdV-hACE2 by intranasal administration. Five days later, animals were challenged with 3 × 10⁵ PFU of SARS-CoV-2 via intranasal administration. A-E. At 4 or 8 dpi tissues were harvested and viral burden was determined in the lung (A-B), spleen (C), heart (D), and nasal washes (E) by plaque (A) or RT-qPCR (B-E) assay (n = 7–8 mice per group; Kruskal-Wallis test with Dunn’s post-test (A-E): ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). Dotted lines indicate the limit of detection. F. SARS-CoV-2 RNA in situ hybridization of lungs of mice vaccinated with VSV-eGFP or VSV-eGFP-SARS-CoV-2 and challenged with SARS-CoV-2 at 4 dpi. Images show low- (left; scale bars, 100 μm), medium- (middle; scale bars, 100 μm), and high-power magnifications (right; scale bars, 10 μm; representative images from n = 3 per group).

Figure 3.. VSV-eGFP-SARS-CoV-2 protects mice from SARS-CoV-2 lung inflammation

A. Lungs of VSV-eGFP or VSV-eGFP-SARS-CoV-2 immunized mice were evaluated at 4 dpi for cytokine and chemokine expression by RT-qPCR assay. Data are shown as fold-change in gene expression compared to fully naive, age-matched animals after normalization to Gapdh (n = 7–8 per group, Kruskal-Wallis test with Dunn’s post-test: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). B. Hematoxylin and eosin staining of lung sections from immunized mice at 8 dpi with SARS-CoV-2 (3 × 10⁵ PFU). Images show low- (left; scale bars, 250 μm), medium- (middle; scale bars, 50 μm), and high-power magnifications (right; scale bars, 25 μm; representative images from n = 3 mice per group).

Figure 4.. Vaccine-induced sera limits SARS-CoV-2 infection.

A. Passive transfer of immune sera and SARS-CoV-2 challenge scheme. Ten-week-old female BALB/c mice were treated with anti-Ifnar1 mAb and one day later, animals were transduced with 2.5 × 10⁸ PFU of AdV-hACE2 by intranasal administration. Four days later, animals were administered 100 μL of pooled immune sera collected from VSV-eGFP or VSV-eGFP-SARS-CoV-2 vaccinated mice after one or two immunizations. One day later, animals were challenged with 3 × 10⁵ PFU of SARS-CoV-2 via intranasal administration. B-F. At 4 dpi tissues were harvested and viral burden was determined in the lung (B-C), spleen (D), heart (E), and nasal washes (F) by plaque (B) or RT-qPCR (C-F) assays (n = 7 mice per group; Kruskal-Wallis test with Dunn’s post-test (B-F): * P < 0.05, ** P < 0.01, **** P < 0.0001). Dotted lines indicate the limit of detection. G. Lungs of mice treated with immune sera were evaluated at 4 dpi for cytokine expression by RT-qPCR assay. Data are shown as fold-change in gene expression compared to naive, age-matched animals after normalization to Gapdh (n = 7 per group, Kruskal-Wallis test with Dunn’s post-test: * P < 0.05, ** P < 0.01).