saRNA vaccine expressing membrane-anchored RBD elicits broad and durable immunity against SARS-CoV-2 variants of concern

The global COVID-19 pandemic, caused by SARS-CoV-2, has resulted in millions of cases and deaths, placing a significant strain on global health systems and economies. The rapid evolution of SARS-CoV-2, giving rise to numerous variants of concern (VOCs) with varying degrees of transmissibility and immune escape, necessitates the development of vaccines that provide broad and long-lasting protection. While several COVID-19 vaccines have been successfully deployed, concerns remain regarding the relatively short duration of their immune responses and their effectiveness against emerging VOCs. This research focuses on addressing these challenges by developing a novel vaccine platform based on self-amplifying RNA (saRNA) technology. SaRNA vaccines, unlike mRNA vaccines, utilize an alphavirus replication mechanism to amplify the expression of the target antigen, potentially leading to a stronger and more sustained immune response. This study investigates the immunogenicity and protective efficacy of a saRNA vaccine encoding a membrane-anchored receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. The membrane-anchored design aims to mimic the natural presentation of the RBD on the viral surface, potentially enhancing the immune response. The study's objective is to determine if this saRNA vaccine can induce broad and durable immunity against multiple SARS-CoV-2 VOCs, offering a potential solution to the ongoing challenge of variant-specific immunity.

Existing literature highlights the limitations of currently approved COVID-19 vaccines. Studies demonstrate a decline in vaccine effectiveness against emerging variants, necessitating the development of more robust and broadly protective vaccines. Several studies have explored the use of self-amplifying RNA (saRNA) as a vaccine platform, showing its potential to induce stronger and more long-lasting immune responses compared to mRNA vaccines. Previous research has also indicated that focusing the immune response on the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which is crucial for viral entry into host cells, can lead to effective neutralizing antibody responses. The membrane-anchored presentation of the RBD has been suggested as a strategy to enhance immunogenicity. The rationale for this study builds upon these prior findings, aiming to combine the advantages of the saRNA platform and RBD-focused immunogen design for enhanced vaccine efficacy against multiple SARS-CoV-2 variants.

This study employed a self-amplifying RNA (saRNA) platform to express a membrane-anchored receptor-binding domain (RBD-TM) of the SARS-CoV-2 spike protein. The RBD-TM construct was designed with an N-terminal signal sequence and a C-terminal transmembrane domain (TMD) from influenza hemagglutinin protein to facilitate efficient membrane anchoring and multivalent display, potentially enhancing immunogenicity. The saRNA was formulated into lipid nanoparticles (LNPs) for efficient delivery. Immunogenicity studies were conducted in mice, hamsters, and non-human primates (NHPs). Mice were immunized with different doses of saRNA RBD-TM LNPs and compared to a subunit vaccine. Antibody titers and neutralizing activity against different SARS-CoV-2 variants (including Wuhan-Hu-1, Alpha, Beta, Gamma, Delta, and Omicron) were measured using ELISA and neutralization assays. Splenocytes were analyzed to assess T-cell responses (IFN-γ, TNF-α, IL-2 production). Hamsters were immunized with saRNA RBD-TM LNPs encoding either Wuhan-Hu-1 or Gamma variant RBDs. They were challenged with Wuhan-Hu-1 or Gamma viruses, and weight loss was monitored to assess protective efficacy. In NHPs, immunogenicity and protective efficacy were evaluated using similar approaches. Longitudinal studies were conducted to assess the durability of immune responses, following antibody titers and neutralizing activity over time (up to 12 months). Flow cytometry was used to analyze B and T cell responses in blood and lymph nodes. Various assays (ELISA, neutralization assays, flow cytometry, etc.) were employed to comprehensively evaluate the immune response generated by the saRNA RBD-TM vaccine. Statistical analysis included one-way ANOVA, Mann–Whitney test, Wilcoxon matched-pairs signed-rank test, and Spearman’s rank test, with p<0.05 considered statistically significant. Ethical considerations were addressed with appropriate animal care and use protocols.

The saRNA RBD-TM vaccine induced significantly higher IgG antibody titers against various SARS-CoV-2 RBD variants compared to a subunit vaccine in mice. These antibodies demonstrated significant neutralizing activity against multiple VOCs. Both saRNA RBD-TM and saRNA secreted RBD induced stronger CD4 T cell responses compared to an mRNA-based S2P vaccine, displaying cross-reactivity with multiple VOCs. The saRNA RBD-TM vaccine also elicited robust RBD-specific CD8+ T cell responses. In hamsters, both saRNA RBD(Wuhan-Hu-1)-TM and saRNA RBD(Gamma)-TM protected against weight loss following challenge with either Wuhan-Hu-1 or Gamma viruses, indicating vaccine efficacy. Importantly, in NHPs, RBD-specific antibodies against VOCs persisted for at least 12 months post-immunization with saRNA RBD-TM LNPs. The Gamma RBD-based vaccine elicited higher antibody titers and demonstrated more durable neutralizing activity compared to the Wuhan-Hu-1 RBD vaccine in NHPs. Analysis of blood and lymph node samples revealed strong B and T cell responses, including germinal center B cells and CD4+ T cells, correlating with high antibody titers and durable immunity. Half-life calculations indicated a prolonged antibody persistence in NHPs immunized with saRNA RBD(Gamma)-TM.

This study demonstrates the potential of a saRNA vaccine expressing a membrane-anchored RBD to elicit broad and durable immunity against multiple SARS-CoV-2 variants. The superior immunogenicity and prolonged antibody persistence observed in NHPs, particularly with the Gamma RBD-based vaccine, is particularly significant. The robust T cell responses further contribute to the comprehensive immune protection. The findings suggest that the saRNA platform, combined with the strategic design of the RBD-TM construct, effectively enhances the magnitude, breadth, and longevity of the immune response. This strategy offers an advantage over current vaccines, addressing concerns regarding waning immunity and variant escape. The results support further development and investigation of this vaccine candidate for improved COVID-19 prevention.

This study demonstrates the efficacy of a novel saRNA vaccine expressing a membrane-anchored RBD-TM in inducing broad and durable immunity against SARS-CoV-2 VOCs. The prolonged antibody persistence and strong cellular immune responses observed in NHPs suggest this vaccine platform holds significant promise as a potential solution for long-term protection against COVID-19, overcoming limitations of existing vaccines. Future studies should focus on clinical trials to evaluate safety and efficacy in humans and explore further optimization of the vaccine design for even broader protection and improved durability.

While the study demonstrates promising preclinical results, the findings need to be validated in human clinical trials. The animal models used may not perfectly recapitulate the human immune response. The long-term durability of the immune response beyond 12 months needs to be further investigated. The study did not include all existing SARS-CoV-2 variants; therefore, broader testing against future emerging variants would strengthen the conclusions.