Protection from COVID-19 disease in hamsters vaccinated with subunit SARS-CoV-2 S1 mucosal vaccines adjuvanted with different adjuvants

Abstract

Introduction: Adjuvant plays an important role in directing the immune responses induced by vaccines. In previous studies, we have shown that a mucosal SARS-CoV-2 S1 subunit vaccine adjuvanted with a combination of CpG, Poly I:C and IL-15 (named CP15) induced effective mucosal and systemic immunity and conferred nearly sterile protection against SARS-CoV-2 viral replication in macaque models.

Methods: In this study, we used a hamster model, which mimics the human scenario and reliably exhibits severe SARS-CoV-2 disease similar to hospitalized patients, to investigate the protection efficacy of the vaccines against COVID-19 disease. We compared the weight loss, viral loads (VLs), and clinical observation scores of three different vaccine regimens. All three regimens consisted of priming/boosting with S1 subunit vaccines, but adjuvanted with alum and/or CP15 administrated by either intramuscular (IM) or intranasal (IN) routes: Group 1 was adjuvanted with alum/alum administrated IM/IM; Group 2 was alum-IM/CP15-IN; and Group 3 was CP15-IM/CP15-IN.

Results: After challenge with SARS-CoV-2 WA strain, we found that the alum/CP15 group showed best protection against weight loss, while the CP15 group demonstrated best reduction of oral SARS-CoV-2 VLs, suggesting that the protection profiles were different. Sex differences for VL and clinical scores were observed. Humoral immunity was induced but not correlated with protection. Moreover, S1-specific binding antibody titers against beta, omicron BA.1, and BA.2 variants showed 2.6-, 4.9- and 2.8- fold reduction, respectively, compared to the Wuhan strain.

Discussion: Overall, the data suggested that adjuvants in subunit vaccines determine the protection profiles after SARS-CoV-2 infection and that nasal/oral mucosal immunization can protect against systemic COVID-19 disease.

Keywords: COVID-19; SARS-CoV-2; hamster model; intranasal vaccine; mucosal adjuvant.

Figure 1

The Alum/CP15 group, which had been vaccinated with S1 protein adjuvanted with alum/CP15, showed significant protection against weight loss after challenge with SARS-Cov-2 WA strain in Syrian golden hamsters. (A). Schematic diagram of vaccination/challenge schedules, and vaccination groups. (B, C). Kinetics of body weight loss (B) and the area under curve of body weight loss (C) in hamsters after challenge with SARS-CoV-2 WA strain. N=5 for each group, and each dot in (C) represents one animal. Two-way ANOVA and Kruskal-Wallis tests with Dunn’s multiple comparison corrections were used to compare between the vaccinated groups and the control group. Mean ± SEM are shown.

Figure 2

The CP15 group showed significant oral swab viral load reduction after challenge with SARS-Cov-2 WA strain in Syrian golden hamsters. After challenge with SARS-CoV-2 WA strain, oral swabs from each hamster were collected at different time points and viral loads were measured as TCID50. (A, B). Oral swab viral loads of each hamster from different groups (A) and the summary (means) of each group (B) are shown. (C) The area under the curve of oral swab viral loads from each group are shown. N=5 for each group, and each symbol represents one animal in (A). The dots denote female, while the triangles denote male in (C). Two-way ANOVA and Kruskal-Wallis tests with Dunn’s multiple comparison corrections were used to compare between the vaccinated groups and the control group. Dashed line shows the detection limit. Mean ± SEM are shown.

Figure 3

Sex difference in viral load reduction after challenge with SARS-CoV-2 WA strain in Syrian golden hamsters. (A, B). Kinetics of viral load reduction in vaccinated and naïve female (A) and male (B) hamsters. (C, D). Comparisons of area under curve of viral load reduction between female and male hamsters after SARS-CoV-2 WA strain infection. Each symbol represents one animal in (C, D). Two-way ANOVA analyses were used to compare the vaccinated groups and control group. Mann-Whitney test was used to compare the females and males. Dashed lines show the detection limit. Mean ± SEM are shown.

Figure 4

Clinical scores of the hamsters after SARS-CoV-2 Washington strain challenge. (A–C). After SARS-CoV-2 Washington strain infection, animals were monitored, and clinical scores were given each day. The clinical score was based on the observation whether ruffled fur and/or hunched back was present in the animals: milder ruffled fur =1; ruffled fur= 2, and hunched back=1, and if none of them was present the animal will be given a score of zero. The clinical score was calculated based the sum of ruffled fur and hunched back scores. Kinetics of clinical score changes in all the animals (A), females (B), and males (C) of difference groups. (D, E). Comparisons of area under curve of clinical score changes in females and males. N=5 for each group. The triangles denote males, and the dots denote females in (D). Each dot represents one animal in (E). Two-way ANOVA and Mann-Whitney analysis were used to compare between the vaccinated groups and the control group. Mean ± SEM are shown.

Figure 5

Vaccine-induced humoral immunity did not correlate with protection after challenge with SARS-CoV-2 WA strain in Syrian golden hamsters. (A, B). PRNT titers (A) and S1-specific binding antibody titers (B) against SARS-CoV-2 WA/Wuhan strain in the serum of the vaccinated and naïve animals (4 weeks after the second vaccination). (C) S1-specific binding antibody correlated with PRNT titers. (D, E). S1-specific binding antibody titers did not correlate with weight loss (D) or viral load reduction (E) of the vaccinated animals. Spearman analyses were used for the correlations. (F). S1-specific binding antibody titers against S1 of SARS-CoV-2 wild type (Wuhan) strain, beta, Omicron, and Omicron A.2 variants. Each symbol represents one animal. Non-parametric one-way ANOVA analyses with Dunn’s multiple comparison correction was used to compare the titers against wild type and the variants. Dashed line shows the titers of the naïve animals. Means ± SEM are shown.

Figure 6

Binding antibody titers against S1 variants, which were lower than those of vaccinated with beta variant S1, did not correlate with weight loss or viral loads (VL) in the hamsters after SARS-CoV-2 Washington strain challenge. (A). To compare the humoral immune responses, we measured the serum binding antibody titers against Wuhan (Wild type, WT) strain from both this cohort, as well as another cohort of hamsters, which were vaccinated with the same regimen as group1&2 of this cohort except the S1 protein was substituted by S1 protein from the beta variant. Serum binding antibody titers against S1 (WT) were lower in this cohort, which were vaccinated with S1 from WT, than those of animals vaccinated with the beta variant S1. (B, C). Binding antibody titers against S1 variants (4 weeks after the second vaccination) did not correlate with weight loss (B) or VL (C). Spearman analyses were used for the correlations. .