Importantly, RBD-specific antibodies against VOCs are maintained for at least 12?months in NHPs. MKC3946 characteristics of a self-amplifying RNA (saRNA) vaccine expressing the SARS-CoV-2 Spike (S) receptor binding domain (RBD), which is membrane-anchored by fusing with an N-terminal signal sequence and a C-terminal transmembrane domain (RBD-TM). Immunization with saRNA RBD-TM delivered in lipid nanoparticles (LNP) efficiently induces T-cell and B-cell responses in non-human primates (NHPs). In MKC3946 addition, immunized hamsters and NHPs are protected against SARS-CoV-2 challenge. Importantly, RBD-specific antibodies against VOCs are maintained for at least 12?months in NHPs. These findings suggest that this saRNA platform expressing RBD-TM will be a useful vaccine candidate inducing durable immunity against emerging SARS-CoV-2 strains. Subject terms: Disease prevention, Viral infection, SARS-CoV-2, MKC3946 Vaccines Vaccines with broad and long-lasting protection against variants of concern are still limited. Here, the authors report a self-amplifying RNA (saRNA) vaccine expressing a membrane-anchored SARS-CoV-2 Spike RBD and show that it elicits broad, durable and protective immunity in small animal models and NHPs. Introduction SARS-CoV-2 has spread rapidly and globally with >607 million cases and 6.4 million deaths, causing a significant impact on global health and economies (WHO Coronavirus (COVID-19) Dashboard, https://covid19.who.int/). SARS-CoV-2 continues to spread with the emergence of new antigenic variants, and broadly protective vaccines that are capable of worldwide distribution are an urgent and high priority. There are currently several approved vaccines against coronavirus disease 20191 (COVID-19) that have contributed to reducing the impact of the pandemic. However, due to the relatively short duration of the immune responses induced by these vaccines, as well as the continued emergence of new VOCs2, further development of vaccine designs is required. In this study, we utilized a self-amplifying RNA (saRNA) MKC3946 platform technology to develop a COVID-19 vaccine. This single-cycle vector system utilizes an alphavirus RNA amplification system, the Venezuelan Equine Encephalitis Virus (VEEV)-based replicon expression vector3. This vector expresses the alphavirus nonstructural proteins (nsPs)1-4, which together replicate and transcribe the saRNA resulting in efficient expression of the gene(s) of interest. Due to this self-amplification process, the level and duration of expression of target antigens is higher and longer than that observed with mRNA vaccine platforms4. Therefore, prolonged presentation of the antigen to the immune system using this saRNA platform is expected. There are several preclinical reports using saRNA vectors to develop COVID-19 vaccine candidates5C10 expressing MKC3946 full length S protein, and these candidate vaccines induced high humoral and cellular responses as well as protection against COVID-19 challenge in animal models. It has been reported that over 90% of neutralizing antibodies from infected patients target the SARS-CoV-2 S Receptor Binding Domain (RBD)11. Given the correlation Rabbit Polyclonal to RAD51L1 between neutralizing antibody and protection, we designed the transgene inserted into the saRNA vector based on RBD sequences to focus the immune response on the highest concentration of neutralizing epitopes. It has been shown that efficient presentation of antigens to B cell receptors in multivalent arrays leads to a stronger signal induction12. To induce B cell responses in this manner, we fused the S protein signal sequence to the N terminus of the RBD, and the transmembrane-cytoplasmic tail domain (TM) from influenza hemagglutinin protein (HA) to the C terminus of the RBD13. This approach results in the display of the RBD sequence on the surface of transfected cells in a multivalent manner. In this study, we report the development of this RBD-based saRNA candidate vaccine, and the preclinical evaluation in mice, hamster and non-human primate models using lipid nanoparticle delivery systems. Results Designing an saRNA vaccine expressing a membrane-anchored, SARS-CoV-2 spike RBD We first designed candidate RBD vaccines and compared the expression characteristics of secreted RBD and membrane-anchored RBD (RBD-TM) constructs. The secreted RBD version was constructed using the SARS-CoV-2 signal sequence (amino acids.