Safety and immunogenicity of an optimized self-replicating RNA platform for low dose or single dose vaccine applications: a randomized, open label Phase I study in healthy volunteers

Abstract

Self-replicating RNA (srRNA) technology, in comparison to mRNA vaccines, has shown dose-sparing by approximately 10-fold and more durable immune responses. However, no improvements are observed in the adverse events profile. Here, we develop an srRNA vaccine platform with optimized non-coding regions and demonstrate immunogenicity and safety in preclinical and clinical development. Optimized srRNA vaccines generate protective immunity (according to the WHO defined thresholds) at doses up to 1,000,000-fold lower than mRNA in female mouse models of influenza and rabies. Clinically, safety and immunogenicity of RBI-4000, an srRNA vector encoding the rabies glycoprotein, was evaluated in a Phase I study (NCT06048770). RBI-4000 was able to elicit de novo protective immunity in the majority of healthy participants when administered at a dose of 0.1, 1, or 10 microgram (71%, 94%, 100%, respectively) in a prime-boost schedule. Similarly, we observe immunity above the WHO benchmark of protection following a single administration in most participants at both 1 and 10 microgram doses. There are no serious adverse events reported across all cohorts. These data establish the high therapeutic index of optimized srRNA vectors, demonstrating feasibility of both low dose and single dose approaches for vaccine applications.

Fig. 1. Optimized srRNA vaccines against influenza elicit protective immune responses at ultra-low doses in mice.

A Humoral responses as measured by ELISA for anti-hemagglutinin H5 total IgG antibody titers and H1 endpoint IgG antibody titers, 14 days following one or two doses of srRNA/LNP vaccines (n = 5 mice per bar). B Neutralizing antibodies were measured by Hemagglutination Inhibition (HI) assay against H5 and H1, using A/Vietnam/1203/04 and A/California/07/09, respectively, 14 days following one or two doses of srRNA/LNP vaccines (n = 5 mice per bar). Line refers to WHO Correlate of Protection (CoP) threshold. C Splenic T cell responses against H5 and H1 HA, assayed by ELISpot, 14 days following two vaccinations. Symbols represent individual animals (n = 5 mice per bar). Bars represent geometric mean and error bars show 95% CI. Statistical significance was determined by using Mann–Whitney tests vs Saline (black lines) or Kruskal–Wallis test vs 10 ng dose group (blue lines), where *p = 0.0476 (HI assay) and *p = 0.0159 (ELISpot), **p = 0.0079, ***p < 0.0005, ****p < 0.0001.

Fig. 2. RBI-4000 elicits protective immune responses and protects from viral challenge in mice.

Serum-neutralizing antibody titers, assessed by rapid fluorescence foci inhibition test (RFFIT), at indicated time points following one (A) or two vaccinations (n = 10 mice per group per time point) (B) with RBI-4000 (n = 5 mice per every time point after day 49). C Splenic T cell responses against rabies virus glycoprotein (RABV-G), assayed by ELISpot, 14 days following two vaccinations with RBI-4000. Symbols represent individual animals. D Probability of survival in a model of rabies virus lethal challenge in previously vaccinated with one (prime) or two doses (P-B) of RBI-4000 (n = 10 mice per group). Bars represent geometric mean and error bars show 95% CI. Statistical significance was determined by using a Kruskal–Wallis test for bar graphs or by two-way ANOVA with multiple comparisons for line graphs and Kaplan–Meier for survival, where *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001. Exact p values are listed in the Source Data file.

Fig. 3. Clinical trial design.

Enrollment and randomization of participants on RBI-4000-101 Phase I clinical trial.

Fig. 4. Clinical safety of RBI-4000.

A Local solicited and B Systemic solicited adverse reactions following 1 and 2 doses of RBI-4000 and RabAvert. Cohort 3 only received a single dose of RBI-4000. Maximum severity of each symptom is plotted. Reactogenicity was described as grade 1 (blue), grade 2 (green), or grade 3 (yellow).

Fig. 5. RBI-4000 elicits RVNA response in all dose cohorts in humans.

A Serum of trial participants was assayed for RVNA titers by RFFIT, after a single (Prime) or two (Boost) administrations of RBI-4000. Percentage of participants within each cohort with an RVNA titer above the LLOD of the assay at any time during the study is plotted in gray bars. Percentage of participants within each cohort with a RVNA titer above the WHO-defined indirect immune measure of protection threshold (RVNA ≥ 0.5 IU/mL) at any time during the study is plotted in blue bars (n = 17 Cohort 1, n = 18 Cohort 2–4 and n = 12 Cohort 5). B Longitudinal analysis of serum RVNA titers as measured by RFFIT in each cohort is plotted. Dashed line represents the WHO-defined indirect protection threshold (RVNA ≥ 0.5 IU/mL). Symbols represent individual titers. Box represents 25–75th percentile, line represents median and whiskers represent min and max values (n = 17 Cohort 1, n = 18 Cohort 2–4 and n = 12 Cohort 5). C RVNA titers following immunization with RBI-4000 at Day 29 (Prime only) and Day 71 (Prime-Boost) are shown for each dose level. Peak RVNA titers for RabAvert are shown for Day 15. Dashed line represents the WHO-defined indirect protection threshold (RVNA ≥ 0.5 IU/mL). Symbols represent individual titers (n = 17 Cohort 1, n = 18 Cohort 2–4 and n = 12 Cohort 5). Box represents 25–75th percentile, line represents median and whiskers represent the min and max values. Statistical significance was determined by using a Kruskal–Wallis analysis, where *p < 0.0402, **p < 0.0041, ****p < 0.0001.