The continuous emergence of SARS-CoV-2 variants of concern (VOCs) necessitates the development of safe and effective broad-spectrum vaccines to mitigate severe COVID-19 outcomes. Existing vaccines, while effective against severe disease, show waning protection against symptomatic infection over time and reduced efficacy against emerging VOCs like Beta, Delta, Gamma, and Omicron variants and their sublineages. These variants exhibit varying degrees of resistance to vaccine-induced immunity. This research investigates a novel vaccine strategy focusing on a Gamma variant-adapted RBD vaccine to address these challenges. The rationale behind this approach stems from the observation that variant-adapted vaccines can elicit broader neutralizing antibody responses and potentially overcome the limitations of vaccines designed against earlier strains. This study aims to evaluate the immunogenicity, breadth of neutralization, and protective efficacy of a Gamma-adapted RBD vaccine in preclinical models, providing crucial data for its potential development as a broad-spectrum vaccine against SARS-CoV-2.

Numerous studies have highlighted the waning efficacy of initial COVID-19 vaccines against emerging VOCs. Research on variant-specific vaccines, particularly those targeting Beta and Omicron variants, demonstrates the potential for improved cross-reactivity and broader protection. Studies have shown that variant-adapted vaccines can induce higher neutralizing antibody titers against multiple VOCs and that heterologous boosting with variant-specific vaccines can enhance immune responses compared to homologous boosting. The use of adjuvants, such as Alum, has also been extensively explored to enhance vaccine immunogenicity and efficacy.

Two RBD-dimer antigens were designed, one from the ancestral Wuhan-Hu-1 strain and another from the Gamma variant, incorporating mutations K417T, E484K, and N501Y. These antigens were expressed in CHO-S cells, purified, and adjuvanted with aluminum hydroxide (Alum). BALB/c mice were immunized with a two-dose regimen. Antibody responses were assessed by ELISA, measuring IgG titers against Ancestral, Gamma, and Omicron BA.4/5 RBD antigens. Neutralizing antibody activity was evaluated using a virus neutralization assay against Ancestral and Gamma SARS-CoV-2 variants. Specific B cell and plasmablast populations were analyzed by flow cytometry. T cell responses (CD4+ and CD8+) were assessed by intracellular cytokine staining (IFN-γ, TNF-α, IL-2) and multiplex cytokine analysis of splenocyte supernatants. In protection studies, K18-hACE2 transgenic mice were immunized and challenged intranasally with Omicron BA.5. Viral RNA loads were quantified by RT-qPCR, and lung pathology was assessed histopathologically. Heterologous boosting experiments were conducted using mice primed with different vaccine platforms (BNT162b2, BBIBP-CorV, ChAdOx1-S) followed by a booster dose of the Gamma RBD vaccine. Immunoinformatics tools were used to predict linear B cell epitopes. Statistical analysis was performed using appropriate methods, such as one-way ANOVA, Kruskal-Wallis test, Mann-Whitney U test, and log-rank test.

The Gamma-adapted RBD vaccine demonstrated superior immunogenicity compared to the ancestral RBD vaccine. It induced significantly higher levels of anti-Gamma and anti-Ancestral RBD IgG antibodies. The Gamma vaccine also elicited broader neutralizing antibody activity against Ancestral and Gamma variants. Analysis of B cell populations revealed a higher proportion of specific B cells and plasmablasts in mice immunized with the Gamma vaccine. The Gamma vaccine also induced a significant increase in the frequencies of Gamma RBD-specific IFN-γ+, TNF-α+, and IL-2-producing CD4+ T cells, along with mixed Th1/Th2 cytokine responses. Importantly, the Gamma RBD vaccine conferred significant protection against both ancestral and Omicron BA.5 SARS-CoV-2 challenge in K18-hACE2 transgenic mice, showing reduced viral RNA loads and less severe lung pathology compared to control groups. Heterologous boosting with the Gamma RBD vaccine significantly enhanced neutralizing antibody titers and cross-reactivity against Omicron BA.1 and BA.5 compared to homologous boosting with other vaccine platforms. Immunoinformatics analysis revealed that a conserved epitope containing the N501Y substitution in the Gamma RBD increased antigenicity and potentially contributed to its enhanced immunogenicity. The study further demonstrated the ability of Gamma RBD-adapted vaccine to induce long-lasting antibody response with neutralizing activity against different VOCs for up to 253 days after immunization.

The results demonstrate the superiority of the Gamma-adapted RBD vaccine over the ancestral RBD vaccine in terms of immunogenicity, breadth of neutralizing antibody response, and protective efficacy in a preclinical mouse model. The enhanced immunogenicity likely stems from the presence of key mutations (K417T, E484K, N501Y) in the Gamma RBD, which may create more immunogenic epitopes and improved binding to the ACE2 receptor. The vaccine's ability to induce both robust humoral and cellular immune responses is particularly encouraging. This is consistent with the notion that both antibody responses and T-cell responses are required for durable protection against SARS-CoV-2. The success of the Gamma RBD vaccine as a heterologous booster further underscores its potential as a universal booster vaccine. These findings strongly support the advancement of this Gamma-adapted RBD vaccine candidate for clinical development. The broad cross-reactivity against various VOCs makes it a promising approach to address the challenges posed by the continuously evolving virus.

This study demonstrates the significant potential of a Gamma-adapted subunit RBD vaccine as a highly immunogenic and broad-spectrum vaccine candidate against SARS-CoV-2. The vaccine's superior performance compared to ancestral RBD vaccines, its efficacy as a heterologous booster, and its ability to protect mice from infection warrant further investigation and clinical trials. Future research should focus on evaluating its efficacy and safety in human clinical trials, exploring different adjuvant formulations to further enhance its immunogenicity, and investigating potential improvements to the vaccine design.

The study was conducted in preclinical mouse models, and the results may not fully translate to human responses. The use of transgenic K18-hACE2 mice for challenge studies, while providing a relevant model, might not perfectly recapitulate human disease pathogenesis. Although the study included heterologous boosting, it did not cover all available vaccine platforms, and additional studies might be necessary to evaluate compatibility with other vaccination regimens.