Immunogenicity and protective efficacy of SARS-CoV-2 recombinant S-protein vaccine S-268019-b in cynomolgus monkeys

Abstract

The vaccine S-268019-b is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)-protein vaccine consisting of full-length recombinant SARS-CoV-2 S-protein (S-910823) as antigen, mixed with the squalene-based adjuvant A-910823. The current study evaluated the immunogenicity of S-268019-b using various doses of S-910823 and its vaccine efficacy against SARS-CoV-2 challenge in cynomolgus monkeys. The different doses of S-910823 combined with A-910823 were intramuscularly administered twice at a 3-week interval. Two weeks after the second dosing, dose-dependent humoral immune responses were observed with neutralizing antibody titers being comparable to that of human convalescent plasma. Pseudoviruses harboring S proteins from Beta and Gamma SARS-CoV-2 variants displayed approximately 3- to 4-fold reduced sensitivity to neutralizing antibodies induced after two vaccine doses compared with that against ancestral viruses, whereas neutralizing antibody titers were reduced >14-fold against the Omicron variant. Cellular immunity was also induced with a relative Th1 polarized response. No adverse clinical signs or weight loss associated with the vaccine were observed, suggesting safety of the vaccine in cynomolgus monkeys. Immunization with 10 µg of S-910823 with A-910823 demonstrated protective efficacy against SARS-CoV-2 challenge according to genomic and subgenomic viral RNA transcript levels in nasopharyngeal, throat, and rectal swab specimens. Pathological analysis revealed no detectable vaccine-dependent enhancement of disease in the lungs of challenged vaccinated monkeys. The current findings provide fundamental information regarding vaccine doses for human trials and support the development of S-268019-b as a safe and effective vaccine for controlling the current pandemic, as well as general protection against SARS-CoV-2 moving forward.

Keywords: Coronavirus disease 2019; Cynomolgus monkeys; Recombinant protein vaccine; S-268019-b; Severe acute respiratory syndrome coronavirus 2.

Fig. 1

Immunogenicity of vaccine S-268019-b. (A) Schematic overview of the vaccination schedule and experimental groups. Cynomolgus monkeys (n = 4/group) were vaccinated on day 1 (D1) and again 3 weeks later on day 22 (D22) with vaccine S-268019-b containing various doses of SARS-CoV-2 recombinant S-protein S-910823 as antigen and adjuvant A-910823. Vaccinations were performed intramuscularly in a total volume of 500 µL. The animals were monitored for weight and clinical signs, and blood was collected as indicated for analysis of antibody content, hematology, and peripheral blood cell types. (B and C) Anti-SARS-CoV-2 antibody levels in cynomolgus monkeys immunized with vaccine S-268019-b. The animals were vaccinated on day 1 and again 3 weeks later on day 22 with vaccine S-268019-b containing various doses of SARS-CoV-2 recombinant S-protein S-910823 as antigen and adjuvant A-910823. Sera were collected on day −1 (white bar), day 21 (gray bar), and day 36 (black bar) and analyzed by ELISA for (B) anti-SARS-CoV-2 S-protein antibody and (C) the receptor binding domain (RBD). Open circles represent titers of individual animals. Each bar represents geometric mean titer (GMT) with error bars indicating 95% confident interval. (D and E) Neutralizing antibody levels in cynomolgus monkeys immunized with vaccine S-268019-b. (D) The animals were vaccinated on day 1 and again 3 weeks later on day 22 with vaccine S-268019-b containing various doses of SARS-CoV-2 recombinant S-protein S-910823 as antigen and adjuvant A-910823. Sera were collected on day −1 (white bar), day 21 (gray bar), and day 36 (black bar) and analyzed for the ability to neutralize SARS-CoV-2 WK-521. World Health Organization (WHO) reference plasma 20/150 of pooled human convalescent plasma (1,473 international unit/mL) was used as a positive control.

Fig. 2

Neutralizing antibody levels in cynomolgus monkeys immunized with vaccine S-268019-b against SARS-CoV-2 variants. Day 36 sera collected from cynomolgus monkeys vaccinated on day 1 and day 22 with S-268019-b containing 10 μg of antigen S-268019 and adjuvant A-910823 were analyzed. The 50% pseudovirus-neutralization titers (pVNT50) against SARS-CoV-2 variant pseudotyped lentiviruses (A) and neutralizing antibody titers against live SARS-CoV-2 (B) are shown. Open circles represent titers of individual animals. Each bar represents the geometric mean titer (GMT) with error bars indicating 95% confident interval.

Fig. 3

Cytokine production by peripheral blood monocytic cells (PBMCs) of cynomolgus monkeys immunized with vaccine S-268019-b. The animals were vaccinated on day 1 (D1) and again 3 weeks later on day 22 (D22) with vaccine S-268019-b containing various doses of SARS-CoV-2 recombinant S-protein S-910823 as antigen and adjuvant A-910823. PBMCs were collected at the indicated time points and analyzed using FluoroSpot assays for Th1 cytokine IFN-γ, Th2 cytokine IL-4, and Th2 cytokine IL-5. (A) Representative images of IFN-γ/IL-4/IL-5 FluoroSpot assay results for groups vaccinated with S-268019-b containing 10 µg S-910823 with or without A-910823. Individual images of assay results were captured for IFN-γ (top image), IL-4 (central image), and IL-5 (lower image). Results are shown as spot-forming cells (SFC) per 1 × 10⁶ cells for IFN-γ (B), IL-4 (C), and IL-5 (D) for the various vaccine antigen doses at day −1 (white bar), day 21 (gray bar), and day 36 (black bar). Open circles represent titers of individual animals. Each bar represents mean values with error bars indicating standard error of the mean (SEM). (E) Comparison of Th1 cytokine IFN-γ vs Th2 cytokine IL-4, and Th1 cytokine IFN-γ vs Th2 cytokine IL-5. Each colored circle represents an individual animal vaccinated with S-268019-b containing the indicated dose of antigen S-910823.

Fig. 4

Protective efficacy of S-268019-b immunization against SARS-CoV-2 infection in cynomolgus monkeys. (A) Schematic representation of the vaccination schedule and SARS-CoV-2 challenge experiments. Cynomolgus monkeys (n = 4/group) were vaccinated twice at 3 weeks interval as shown in Fig. 1A., followed by SARS-CoV-2 challenge 8–13 weeks later after the second vaccination. Blood and swab samles were collected on days 0, 1, 4, and 7 post infection and subjected to the indicated analyses. (B) Ratios of clinical scoring, body weight, and respiratory rate of immunized monkeys after challenge infection with SARS-CoV-2 relative to those prior to infection. Each symbol indicates the ratio of an individual monkey. Lines represent the mean clinical score ratio. ***p < 0.001 by Tukey’s multiple comparison test (S-910823+A-910823 vs S-910823 at 1 day post infection). (C) Neutralizing titers of sera collected from immunized monkeys. The serum samples were collected on day 0 just prior to infection, and on days 1, 4, and 7 post infection. Each point represent an individual neutralizing titers against SARS-CoV-2 and the bars represent the geometric mean titers. The error bars indicate a 95% confidence interval. The far-right open bar represents the neutralizing antibody titer of the WHO International Reference Panel High sample (20/150). The limit of detection was set as 10 for monkey sera. (D) Copy numbers of virus genomic and subgenomic RNA in swab samples collected from vaccinated monkeys challenged with a SARS-CoV-2 infection. Each symbol indicates the copy number of viral genomic RNA (upper panels) and subgenomic RNA (lower panels) of individual monkeys. Lines represent the mean copy numbers. No significant differences were found by the Tukey’s multiple comparison test.

Fig. 5

Histopathological evaluation of cynomolgus monkey lung tissue after infection with SARS-CoV-2. (A) Representative histopathological findings of the lungs of cynomolgus monkeys 7 days post-infection. Upper and middle rows are low- and high-magnification, respectively, of hematoxirin and eosin stained paraffin-embedded lung tissue sections. Bottom rows of each panel show high-magnification of representative immunohistochemistry results using an anti-SARS-CoV-2 nucleocapsid protein-specific antibody. Yellow arrows indicate eosinophils; black arrows indicate neutrophils. Al, alveoli, Br, bronchi, v, vessels. The bars represent 200 µm at low magnification and 20 µm at high magnification. (B) Histopathological scores of pulmonary tissues from immunized monkeys after SARS-CoV-2 challenge infection. Each point represents the histopathological score of an individual monkey. The bars represent mean scores of each experimental group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)