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  Title	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
  ID	5712820
  External ID	7447705526
  Author	Unknown
  Published Date	Jan. 7, 2025, 11:20 a.m.
  Summary	Next-generation srRNA vectors result in protective immune responses at ultra-low doses in mice The prototypical first-generation srRNA vaccines are mostly derived from the same alphavirus, Venezuelan
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  URL	https://www.nature.com/articles/s41467-025-55843-9
  Sentiment	Negative: 0.01486    Neutral: 0.23632    Positive: 0.74883
  Relevance	0.00373

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  Content ID	3450601
  Key	dbc14eb2-b7ff-4c50-87b8-e46e6afeab8a
  Content	Next-generation srRNA vectors result in protective immune responses at ultra-low doses in mice The prototypical first-generation srRNA vaccines are mostly derived from the same alphavirus, Venezuelan Equine Encephalitis Virus (VEEV) strain TC-83, and have shown to be able to elicit equal immunogenicity to mRNA but at much lower doses27,28,29. This increase in the effective dose is suggested to be due to higher and more durable expression of the encoded transgene (or antigen)28,29,30 Although this version of the VEEV-based vector has been clinically validated and advanced in a recently approved commercial vaccine31, no intentional design improvements have been made to the technology in over three decades that confer enhanced protein expression or vaccine immunogenicity32,33,34,35. We hypothesized that bioactivity could be optimized by more closely mimicking the natural alphavirus genome. Optimizations to the vector are summarized in Supplementary Fig 1 and ref. 25. First, we strictly designed the DNA vector to template a minimum number of ribonucleotides at the 5′ and 3′ ends of the srRNA that are not naturally present in alphaviruses25. Minimal cloning adapters (~30 nt) up and downstream of the gene(s) of interest on the DNA template allow for rapid, modular transgene replacement and ensure optimal expression. Additionally, while previous reports established the canon of a minimal ~30 residue polyA tail being sufficient for srRNAs36,37, we increased the templated polyA tail to better match the alphavirus length (~68 residues)38 and found a significant enhancement in dose-dependent bioactivity in vitro25. These modifications also led to a notable increase in activity in vivo, evidenced by enhanced protein expression as assessed in mice administered srRNA encoding a reporter transgene for firefly luciferase (Fluc) compared to the parental sequence (Supplementary Fig. 2). Longitudinal assessment of bioluminescence, as a result of active Fluc expression, over 40 days demonstrated that improvements made to the VEEV sequence resulted in a higher peak and greater durability of protein expression when compared to the original vector (labeled as prototypical VEEV TC-83). To further assess if increased protein expression from this optimized vector led to better bioactivity of vaccines, we generated constructs encoding the influenza hemagglutinin (HA) antigen from two distinct pandemic strains, H5N1 and H1N1, in this improved backbone, and evaluated these in mouse immunogenicity studies at ultra-low doses ranging from 1 pg to 10 ng (Fig. 1). A single dose of this srRNA vaccine resulted in seropositivity, as measured by the presence of HA-specific antibodies, in all dose groups for H5, and down to 10 pg for H1 strains (Fig. 1A, Dose 1). A second (boost) dose of vaccine enhanced titers of HA-specific IgG for both H5 and H1 in a dose-dependent manner (Fig. 1A, Dose 2). A WHO-defined immune correlate of protection exists for influenza and is based on the titer of HA-neutralizing antibodies as measured in a standardized hemagglutination inhibition (HI) assay39,40,41. An HI titer of 40 (serum diluted 1 to 40) has also been established as protective in viral challenge studies in mice42. We observed HI titers above this established threshold for protection in animals immunized with vaccine doses as low as 1 pg for the H5N1 strain after two administrations (Fig. 1B). HI titers for H1N1 were observed in animals that received 10 ng down to 10 pg of srRNA vaccine, showing the robustness of the technology for two separate influenza strains. Importantly, a single administration of vaccine led to partial neutralization for H5N1 and H1N1 at higher doses (Fig. 1B). 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. Full size image An additional strength of srRNA technology is its ability to elicit cellular immunity30,43,44,45. Robust HA-specific T cell responses were detected in all mice immunized with H5 at all doses of the vaccine, down to the lowest tested dose of 1 pg (Fig. 1C). H1-specific T cell responses were detectable across the tested dose range of 10 ng to 10 pg. Lack of a dose response for the higher end of the dose range suggests saturation was achieved at 1 ng for H1 and 100 pg for H5 (Fig. 1C). Additional analysis to assess the quality of the T cell responses using intracellular cytokine staining was performed. Both H5 and H1-encoding srRNA vaccines generated polyfunctional HA-specific CD8 and CD4 T cell responses, as defined by the concurrent production of several effector cytokines by the same T cell. The HA responses included CD8 T cells having a majority double- (IFNγ, TNF) or triple-cytokine producing (IFNγ, IL-2, TNF) effector phenotype, and also CD4 T cells with a majority triple-cytokine producing population (Supplementary Fig. 3). To assess if the enhanced bioactivity of new srRNA vectors translated to clinical improvements, we developed RBI-4000, a next-generation srRNA vaccine encoding RABV-G. Rabies virus was selected as a model pathogen due to an immune naive participant population, as opposed to influenza, where much of the population has been previously exposed to the virus. In addition, similarly to influenza, a WHO indirect measure of protection exists for rabies virus and is defined by the presence of serum rabies virus neutralizing antibodies (RVNA) at or above 0.5 IU/mL, thus allowing clear and objective interpretation of vaccine-induced immune responses46. Preclinical testing of RBI-4000 in mouse immunogenicity studies demonstrated that a single immunization could lead to protective RVNA titers down to a 1.5 ng dose level, the lowest dose tested (Fig. 2A). Durability of RVNA titers was established in animals that received either a single 1.5 mcg dose of RBI-4000 or two doses ranging from 1.5 ng to 1.5 mcg. Compared to an approved commercial vaccine, RabAvert, administered at 1/10th of the human dose (the maximum dose feasible in mice), RBI-4000 generated RVNA responses that were higher in magnitude and that exhibited a slower or no decay rate over a greater than 6-month period above the 0.5 IU/mL protective threshold (Fig. 2B). These sustained neutralizing antibody responses were accompanied by strong total T cell responses to RABV-G in all animals that received RBI-4000 (Fig. 2C). Polyfunctionality in the RABV-G-specific CD8 and CD4 T cells was confirmed in animals that received RBI-4000, as demonstrated by a double or triple-cytokine-producing effector phenotype (Supplementary Fig. 4). 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. Full size image We further confirmed that RBI-4000 could confer protective immunity in a mouse model of lethal rabies virus challenge. Mice received RBI-4000 prophylactically as a single 1 mcg administration or two administrations across a dose range of 0.01, 0.1, or 1 mcg. RabAvert provided a positive control in the study. 100% of mice that received RBI-4000 across all dose cohorts were protected from lethal rabies virus challenge, as with RabAvert (Fig. 2D). Interestingly, 30% (3/10) animals that received a srRNA construct encoding luciferase as a control (labeled as “Irrelevant”) were also protected. This non-specific effect is thought to be due to the inflammatory profile of LNP-formulated srRNA drug product that is known to trigger innate immune mediators which can lead to an anti-viral type I immune response47,48. Collectively, these data demonstrated that RBI-4000 was able to elicit robust immune responses, inclusive of polyfunctional effector T cells and RVNA titers that attained the WHO-defined metric against rabies infection. Clinical evaluation of optimized srRNA platform in humans Based on the preclinical data, we evaluated RBI-4000 in a clinical study to assess the application of the benefits of this new technology. RBI-4000-101 is a multicenter, open-label, randomized, active control Phase I clinical trial (NCT06048770) to assess the safety and immunogenicity of RBI-4000. The study enrolled 89 healthy volunteers, aged 18–45 years with no prior exposure or vaccination to rabies (Fig. 3, Demographics in Table 1). The study included four cohorts of ascending doses of RBI-4000 and an active comparator of the commercially available rabies vaccine, RabAvert. Participants in Cohorts 1 (0.1 mcg), 2 (1 mcg), and 4 (10 mcg) received a prime-boost dosing schedule of RBI-4000, 8 weeks apart, while Cohort 3 participants received a single dose of 10 mcg. The 8-week interval for RBI-4000 dosing was selected as it is optimal for generation of immune responses, specifically for srRNA-based vaccines9. RabAvert was administered as per the CDC guidelines, i.e., two doses 1 week apart, on Days 1 and 849. Fig. 3: Clinical trial design. Enrollment and randomization of participants on RBI-4000-101 Phase I clinical trial. Full size image Table 1 Demographic characteristics of the participants of RBI-4000-101 clinical trial Full size table Immunogenicity and safety data through 85 days post initial vaccination is included in this report. Long-term durability of RBI-4000 immunogenicity is continuing to be assessed. Clinical safety showed minimal reactogenicity at all doses tested Safety analysis includes all participants who received any dose of RBI-4000 or RabAvert. Vaccination was generally well tolerated with no anaphylactic reactions. There were no serious adverse events, no dose-limiting adverse events, and a maximally tolerated dose of RBI-4000 was not reached. Adverse events were limited to grades 1 and 2, with the exception of a single self-reported grade 3 fever event in a Cohort 2 participant (1 mcg dose, first administration only), which was not medically confirmed. Local reactogenic adverse events were more common than systemic, with pain or tenderness at the injection site being the most common symptoms. The most common systemic adverse event was headache. Myalgia and fatigue were also reported in over half of the participants at the 10 mcg dose. Overall, the boost dose (second administration) appeared better tolerated than the prime with fewer participants reporting adverse events, though more of the reported events were grade 2 after the boost versus grade 1 after the initial administration. Rates of adverse events are summarized in Fig. 4. There were no unsolicited related adverse events. 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). Full size image De novo protective immune responses were achieved at all doses clinically tested in humans Serial analysis of sera from immunized participants showed detectable RVNA titers in a dose-dependent manner following a single administration of RBI-4000 (Fig. 5A, gray bars). Furthermore, a single administration of RBI-4000 led to protective (RVNA ≥ 0.5 IU/mL) titers in the majority (67%) of participants that received a 1 mcg dose (Fig. 5A, Cohort 2, blue bars), while 89% and 94% of participants achieved RVNA titers at or above the protective threshold with a single dose of 10 mcg in Cohort 3 and 4, respectively. 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. Full size image As expected, a booster dose led to an increase in RVNA titers in Cohorts 1, 2, and 4. Two administrations of RBI-4000 at the lowest dose level tested of 0.1 mcg (Cohort 1), resulted in detectable RVNA in 95% of participants and over 70% of participants achieved the WHO-defined measure of indirect protection (RVNA 0.5 ≥ IU/mL). A dose-dependent increase in RVNA titers was observed with 100% of participants in the 1 mcg cohort (Cohort 2) with detectable RVNA titers after boost, with 94% achieving the WHO-established metric. Boosting at the highest dose level tested of 10 mcg (Cohort 4) resulted in 100% of participants achieving RVNA titers above 0.5 IU/mL, indicative of adequate responses to the rabies vaccine50. Longitudinal analysis of RVNA titers post administration of RBI-4000 showed a clear dose response with peak RVNA titers at Day 29 following the first, i.e., prime, administration (Fig. 5B, C). A dose-dependent enhancement of RVNA titers was observed in Cohorts 1, 2, and 4 that received a second or booster vaccination of RBI-4000, peaking 15 days following administration of RBI-4000. RVNA titers from participants who received RabAvert, an inactivated whole rabies virus vaccine, had differing kinetics from RBI-4000 (all dose levels), peaking at Day 15 following two administrations, proceeded with a rapid decay over the 85-day follow-up period (Fig. 5B). Assessing durability of the humoral immune responses is ongoing. Analysis of the RVNA responses at their peak (Day 29 for RBI-4000) showed a clear dose response (Fig. 5C), suggesting that although protective immune responses were achieved in the majority of participants in Cohort 2, 3, and 4, immune responses were not saturated. This, combined with the favorable safety profile, suggests that further dose escalation is possible and may yield even better immunity. After boosting, peak titers (Day 71 for RBI-4000) were higher across all levels tested and equivalent titers were observed for the 1 and 10 mcg dose cohorts (Fig. 5D, Cohorts 2 and 4). Importantly, peak titers of the RVNA response post-boost were not statistically different for Cohort 4 (Day 71) and RabAvert (Day 15). Also, the fold change of the boost was highest in those cohorts where the baseline RVNA level was lowest at Day 57, suggesting that the interval between prime and boost can be optimized to coincide with full contraction of the humoral immune response to achieve the highest boost of RVNA titers. This is in agreement with recent studies in both RNA and non-RNA-based vaccine platforms51,52,53,54. Three participants initially enrolled in the trial were subsequently identified as seropositive at baseline for RVNA. These participants were distributed one across each dose level and received a single administration of RBI-4000, prior to being replaced to complete the dose cohorts. They remained in the study for safety and RVNA evaluation, although excluded from the data presented in the main cohorts. Regardless of dose level of RBI-4000 received, each showed boosting of RVNA titers between 6 and 11-fold above their baseline levels, with little to no decay over time with RBI-4000 (Supplementary Fig. 5). Rabies-specific T cell responses were also assessed following each administration of RBI-4000 in a limited subset of participants based on sample availability at the time of this report. RBI-4000 was able to elicit antigen-specific T cells in a dose-dependent manner (Supplementary Fig. 6).
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