RNA vaccines have demonstrated efficacy against SARS-CoV-2 in humans, and the technology is being leveraged for rapid emergency response. In this report, we assessed immunogenicity and, for the first time, toxicity, biodistribution, and protective efficacy in preclinical models of a two-dose self amplifying messenger RNA (SAM) vaccine, encoding a prefusion-stabilized spike antigen of SARS-CoV-2 Wuhan-Hu-1 strain and delivered by lipid nanoparticles (LNPs). In mice, one immunization with the SAM vaccine elicited a robust spike-specific antibody response, which was further boosted by a second immunization, and effectively neutralized the matched SARS-CoV-2 Wuhan strain as well as B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta) variants. High frequencies of spike-specific germinal center B, Th0/Th1 CD4, and CD8 T cell responses were observed in mice. Local tolerance, potential systemic toxicity, and biodistribution of the vaccine were characterized in rats. In hamsters, the vaccine candidate was well-tolerated, markedly reduced viral load in the upper and lower airways, and protected animals against disease in a dose-dependent manner, with no evidence of disease enhancement following SARS-CoV-2 challenge. Therefore, the SARSCoV-2 SAM (LNP) vaccine candidate has a favorable safety profile, elicits robust protective immune responses against multiple SARS-CoV-2 variants, and has been advanced to phase 1 clinical evaluation (NCT04758962).

Vaccines containing mRNA with the capacity to self-amplify represent an alternative to the mRNA vaccines that came to prominence during the COVID-19 pandemic. To gain further insights on the safety profile of self-amplifying mRNA- (SAM-) vaccines, this preclinical toxicology study in rats evaluated the effect of (i) the type of delivery system (lipid nanoparticle [LNP] vs cationic nano-emulsion [CNE]); (ii) antigen-encoding sequence (rabies glycoprotein G vs SARS-CoV-2 Spike); and (iii) RNA amplification. Further analyses also evaluated gene expression in peripheral blood after vaccination, and the biodistribution of vaccine RNA. The SAM vaccines administered as two doses 2-weeks apart had acceptable safety profiles in rats, with respect to clinical signs, blood biochemistry, and macroscopic and microscopic pathology. A transient increase in ALT/AST ratio occurred only in female rats and in the absence of muscle and liver damage was dependent on RNA amplification and appeared related to the greater quantities of vaccine RNA in the muscle and livers of female rats vs male rats. The RNA and delivery-vehicle components, but not the nature of the antigen-coding sequence or the requirement for RNA amplification, affected aspects of the stimulation of innate-immune activity, which was consistent with the transient activation of type I and type II interferon signaling. The delivery vehicle, LNP, differed from CNE as vaccine RNA in CNE compositions appeared independently to stimulate innate-immune activity at 4 hours after vaccination. Our analysis supports further studies to assess whether these differences in innate-immune activity affect safety and efficacy of the SAM vaccine.

The novel self-amplifying mRNA (SAM) technology for vaccines consists of an engineered replication-deficient alphavirus genome encoding an RNA-dependent RNA polymerase and the gene of the target antigen. To validate the concept, the rabies glycoprotein G was chosen as antigen. The delivery system for this vaccine was a cationic nanoemulsion.To characterize the local tolerance, potential systemic toxicity and biodistribution of this vaccine, two nonclinical studies were performed. In the repeated dose toxicity study, the SAM vaccine was administered intramuscularly to rats on four occasions at two-week intervals followed by a four-week recovery period. SAM-related changes consisted of a transient increase in neutrophil count, alpha-2-macroglobulin and fibrinogen levels. Transient aspartate aminotransferase and alanine aminotransferase increases were also noted in females only. At necropsy, observations related to the elicited inflammatory reaction, such as enlargement of the draining lymph nodes were observed that were almost fully reversible by the end of the recovery period.In the biodistribution study, rats received a single intramuscular injection of SAM vaccine and then were followed until Day 60. Rabies RNA was found at the injection sites and in the draining lymph nodes one day after administration, then generally decreased in these tissues but remained detectable up to Day 60. Rabies RNA was also transiently found in blood, lungs, spleen and liver. No microscopic changes in the brain and spinal cord were recorded.In conclusion, these results showed that the rabies SAM vaccine was well-tolerated by the animals and supported the clinical development program.