HEK293T cells (~80% confluent) in T-850 flasks with 50 mL of culture media were co-transfected with a plasmid for expression of the VEEV structural proteins (capsid and envelope proteins), and a second plasmid for the transcription of the saRNA SARS-CoV-2 Spike RBD using FectoPRO. The culture media were changed 24 h after transfection with FBS-free DMEM, and culture supernatants were harvested at 72 h after transfection and filtered through a 0.45-µm filter. Next, the VEEV replicon particles were purified from the filtered culture supernatant using Cellufine sulfate resins. Briefly, the Cellufine sulfate resins were washed with water and 300 mM sodium chloride in 10 mM sodium phosphate buffer, and then mixed with culture supernatant (adjusted to 300 mM with 5 M sodium chloride), followed by incubation at room temperature with orbital shaking for 1 h for particle binding. The resins were spun down at 1699 × g for 2 min, and subsequently washed once with 300 mM sodium chloride in 10 mM sodium phosphate buffer. VEEV replicon particles were eluted with 450 mM sodium chloride in 10 mM sodium phosphate buffer and concentrated with Amicon Ultra-15 centrifugal filter unit-100K. For titration, Vero cells were infected with the concentrated replicon particles and infectious unit (IU) per mL were calculated from the percentage of VEEV-positive cells by Flow Cytometry.

Several vaccines have been widely used to counteract the global pandemic caused by SARS-CoV-2. However, due to the rapid emergence of SARS-CoV-2 variants of concern (VOCs), further development of vaccines that confer broad and longer-lasting protection against emerging VOCs are needed. Here, we report the immunological 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 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.