Rabies, a lethal viral zoonosis, causes approximately 60,000 human deaths annually, predominantly in Africa and Asia. The rabies virus (RABV) glycoprotein (G) is the primary target of protective immunity, making it a crucial target for vaccine development. Current rabies vaccines, including inactivated, live attenuated, and recombinant subunit vaccines, have limitations such as requiring multiple doses (inactivated), potential for reversion to virulence (live attenuated), or limited protection (recombinant). mRNA vaccines offer a promising alternative due to their rapid production, scalability, and flexibility in immunogen design. They also avoid genomic integration, enhancing safety. Previous studies have demonstrated the protective efficacy of RABV-G-expressing mRNA vaccines in mice and dogs, but further evaluation in more clinically relevant animal models like non-human primates is needed to assess safety and immunogenicity. This study builds upon previous findings demonstrating the strong immune response induced by LVRNA001 in mice and dogs, further evaluating its safety and efficacy in dogs, mice, and cynomolgus macaques.

The literature extensively covers various rabies vaccine approaches. Inactivated vaccines, while safe, necessitate multiple doses, imposing a significant economic burden, especially in developing nations. Live-attenuated vaccines carry the risk of virulence reversion. Recombinant vaccines, including those based on vaccinia virus or modified vaccinia virus Ankara (MVA), have shown limited protective efficacy. The rise of mRNA vaccine technology offers a faster, more cost-effective, and safer alternative, boasting flexibility in immunogen design and avoiding genomic integration. Previous studies have shown promising results with mRNA rabies vaccines in mice and dogs, inducing strong protective immune responses. However, the lack of data using non-human primate models for comprehensive safety and immunogenicity evaluations remains a gap.

The study employed multiple animal models: dogs, mice, and cynomolgus macaques. In dogs, pre-exposure prophylaxis involved two immunizations with LVRNA001 (10 µg or 50 µg) at 0 and 7 days, followed by challenge with a lethal dose of RABV on day 35. Post-exposure prophylaxis involved infection followed by LVRNA001 immunization (10 or 50 µg at 0 and 7 days). Control groups received an inactivated vaccine or PBS. Survival rates and neutralizing antibody titers were assessed. In mice, various vaccination schedules (single dose, 0/7, 0/14 days) were tested with LVRNA001 (1.67 µg or 5 µg) and an inactivated vaccine. Pre- and post-exposure efficacy against various RABV strains (China I-VII clades) was evaluated. Acute toxicity studies were performed in mice, while chronic toxicity studies were carried out in cynomolgus macaques with repeated doses of LVRNA001 (50 µg or 150 µg) over 5 weeks, followed by a 4-week convalescence period. Parameters such as body weight, temperature, hematological and biochemical markers, lymphocyte subsets, and histopathology were assessed. Reproductive toxicity studies in rats assessed the effects of LVRNA001 on fertility, maternal performance, and postnatal outcomes during embryofetal development and littering phases. Neutralizing antibody titers were measured using fluorescent antibody virus neutralization tests (FAVN) and RFFIT; IFN-γ secreting cells were analyzed by ELISpot assays; lymphocyte subsets were analyzed by flow cytometry; and histopathological examinations were conducted. Statistical analysis involved one-way or two-way ANOVA with Tukey's post-hoc test.

LVRNA001 provided complete protection against lethal RABV challenge in dogs in both pre- and post-exposure scenarios. In mice, LVRNA001 demonstrated 100% survival rates in pre-exposure prophylaxis against various RABV strains, significantly outperforming the inactivated vaccine. Post-exposure prophylaxis in mice showed higher survival rates with LVRNA001 (88.57% with 5 µg, 0/14 schedule) compared to the inactivated vaccine (52.86%). In cynomolgus macaques, repeated administration of LVRNA001 (50 µg or 150 µg) caused no significant changes in body weight, temperature, hematological or biochemical markers, or lymphocyte subsets. Histopathological examination revealed only mild, transient inflammation at the injection site. LVRNA001 induced high neutralizing antibody titers (>20,000 IU/mL) and a Th1-biased immune response. Reproductive toxicity studies in rats showed no significant effects of LVRNA001 on fertility, maternal performance, reproductive processes, or postnatal outcomes.

The study confirmed the protective efficacy of LVRNA001 against lethal RABV infection in dogs and mice, demonstrating its broad-spectrum efficacy across different RABV strains. The lack of significant adverse effects in chronic and reproductive toxicity studies underscores its safety profile. The robust immunogenicity observed in cynomolgus macaques, characterized by high neutralizing antibody titers and a Th1-biased immune response, aligns with the known correlates of protection against rabies. While differences in antibody production kinetics were observed between mice and dogs, the overall protection conferred by LVRNA001 in both species was significant. The observed transient increases in inflammatory markers (FIB, CRP) in macaques receiving LNPs likely reflect a normal inflammatory response to injection, resolving during the convalescence period. This study demonstrates that LVRNA001 offers a potentially superior alternative to existing rabies vaccines.

LVRNA001 demonstrates significant promise as a safe and effective rabies vaccine candidate. Its efficacy in both pre- and post-exposure scenarios in multiple animal models, coupled with the absence of significant toxicity, positions it favorably for further development. Future research should focus on clinical trials to confirm these preclinical findings in humans and to optimize dosage and immunization schedules for maximal efficacy and safety.

While the study utilized multiple animal models, the results may not perfectly translate to human responses. Further investigation in larger animal studies and clinical trials is needed to assess the long-term safety and efficacy of LVRNA001 in humans. The specific mechanisms underlying the species-specific differences in antibody production kinetics observed between mice and dogs warrant further investigation.