Unmodified rabies mRNA vaccine elicits high cross-neutralizing antibody titers and diverse B cell memory responses

Abstract

Licensed rabies virus vaccines based on whole inactivated virus are effective in humans. However, there is a lack of detailed investigations of the elicited immune response, and whether responses can be improved using novel vaccine platforms. Here we show that two doses of a lipid nanoparticle-formulated unmodified mRNA vaccine encoding the rabies virus glycoprotein (RABV-G) induces higher levels of RABV-G specific plasmablasts and T cells in blood, and plasma cells in the bone marrow compared to two doses of Rabipur in non-human primates. The mRNA vaccine also generates higher RABV-G binding and neutralizing antibody titers than Rabipur, while the degree of somatic hypermutation and clonal diversity of the response are similar for the two vaccines. The higher overall antibody titers induced by the mRNA vaccine translates into improved cross-neutralization of related lyssavirus strains, suggesting that this platform has potential for the development of a broadly protective vaccine against these viruses.

Fig. 1. Induction of Type I IFN and intermediate monocytes after mRNA vaccination.

a Outline of study design with respect to group and vaccine assignment, group composition, vaccine dose/route of administration, number of vaccine doses, time interval between each dose and sampling timeline. Red, green and blue will be the color coding for data referring to groups 1, 2 and 3 respectively throughout the manuscript. Full black circles and empty white circles across the study timeline indicate blood draws and bone marrow aspirates respectively. b Dot plot of the detectable plasma cytokine and chemokine levels showing significant differences between the groups measured by a 30-plex assay. The values shown refer to the levels measured at day 0 (prior to prime immunization), day 1 (peak response) and day 14 (return to steady-state levels). Direct comparisons are shown for each cytokine between combined groups 1 and 2 (as at this time of the study, they both received one dose of the mRNA vaccine and are therefore equivalent) and group 3. n = 18 biologically independent animals. Statistical differences were assessed at day 1 comparing groups using Mann–Whitney U test. Cytokine concentrations undetectable or below lower limit of quantitation (LLOQ) are shown as LLOQ value. Dotted lines indicate LLOQ. Representative flow cytometry plots (c) and longitudinal data (d) showing the differentiation and temporary fluctuation of intermediate monocytes. Data are shown for day 0 (prior to prime immunization), day 1 (peak innate response) and day 14 (return to steady-state levels). Complete gating strategy for the identification of monocytes is shown in Supplementary fig. 11b. Statistical differences in (d) were assessed at day 1 using Mann–Whitney U test. n = 18 biologically independent animals. All statistical tests comparing the study groups were two-tailed tests. All error bars indicate mean ± SD.

Fig. 2. Higher level humoral and cellular responses with mRNA vaccination.

a Neutralizing antibody titers are shown across the whole study timeline. Arrows indicate immunizations. Statistical significance was calculated for peak antibody responses (week 6) and at study end (week 50) using Kruskal–Wallis test as per Supplementary Fig. 4a. Connecting lines indicate group means. The dashed line across refers to the RVNA titer of 0.5 IU/mL which is suggested by the WHO as the VNT threshold. n = 18 biologically independent animals. b Total IgG titers expressed as the half-maximal effective concentration (EC₅₀) calculated with a sample dilution series measured by ELISA are shown across the whole study timeline. Arrows indicate immunizations. Connecting lines indicate group means. n = 18 biologically independent animals. Statistical significance was calculated for peak antibody responses (week 6) and at study end (week 50) using Kruskal–Wallis test as per Supplementary Fig. 4b. c Antigen-specific plasmablasts are measured by B cell ELISpot prior to boost (baseline) and four days after the boost. n = 18 biologically independent animals. Statistical differences were assessed at day 4 using Mann–Whitney U test. Error bars indicate mean ± SD. d Longitudinal data of antigen-specific antibody-secreting plasma cells in the bone marrow enumerated by B cell ELISpot at the different study timepoints. n = 18 biologically independent animals. Statistical significance was calculated for peak antibody responses (week 6) and at study end (week 50) using Kruskal–Wallis test. Representative flow cytometry plots (e) and longitudinal data (f) showing the generation and expansion of antigen-specific memory B cells. Data are shown for week-1 (prior to prime immunization), 2 weeks after prime immunization (week 2), 2 weeks after boost (week 6 from study start) and from a later time point (week 18) from which single cells have also been sorted. n = 18 biologically independent animals. Statistical significance was calculated at weeks 2, 6, and 18 using Kruskal–Wallis test. Error bars indicate mean ± SD. Representative flow cytometry plots (g) and longitudinal data (h) showing the generation of antigen-specific Th1 cells identified by their ability to produce intracellular IL-2 and IFN-γ upon stimulation with a pool of overlapping peptides covering the whole RABV-G protein. Data are shown for unstimulated and stimulated cells collected prior to immunization (week 0), 2 weeks after prime (week 2) and 2 weeks after boost (week 6 from study start) immunizations. n = 18 biologically independent animals. Statistical significance was calculated at week 6 using Kruskal–Wallis test. All statistical tests comparing the study groups were two-tailed tests. Error bars indicate mean ± SEM.

Fig. 3. High quality neutralizing antibodies induced by both vaccines.

a Spearman correlation between neutralization (RVNA) and total binding expressed as half-maximal effective concentration (EC₅₀) of total IgG binding to RABV-G measured by ELISA. b Simulation of antibody decay over time down to the EC₅₀ of 80.9 corresponding to 0.5 IU/mL for each group based on antibody half-life estimates. Simulations out to ten years were performed, using the model fits for each animal. The time to binding assay target was calculated for each animal, and summaries per group are reported. The 95% CI is shown. c Antibody avidity by ELISA. The percentage of binding remaining after mild urea wash is shown for plasma collected 1 month after prime immunization (week 4), 1 month after boost (week 8 from study start) and at a later time point (week 18). n = 18 biologically independent animals. Statistical significance was calculated using Kruskal–Wallis test. Error bars indicate mean ± SD.

Fig. 4. Somatic hypermutation is similarly induced by mRNA and Rabipur and is not required for neutralization.

a Violin plots showing the mean level of SHM of antigen-specific Sanger sequences per animal across different groups and timepoints. Lines represent group mean. Statistical significance was calculated using Mann–Whitney test for non-paired data and Wilcoxon signed-rank test for paired data. b Maximum likelihood tree inferred by the multiple sequence alignment of VDJH amino acid antigen-specific sequences for all groups. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The scale bar indicates the distance of 0.2 substitutions per sequence position. This analysis involved 334 amino acid sequences. The reference rabies antibodies binding to Site I and to Site III are indicated with magenta and cyan bars respectively. Twelve sequences were selected for cloning monoclonal antibodies based on lowest, medium and highest SHM (named LowRab1-4, MedRab1-4 and HighRab1-4) as well as based on proximity with the reference antibodies when possible. c Top and side view of a trimeric RABV-G model with antigenic sites I and III highlighted in magenta and cyan respectively. PMDB ID: PM0079619. d Binding potency (EC₅₀) of the cloned mAbs is shown together with that of the reference rabies antibodies and negative control. Light gray, dark gray and black bars indicate the mAbs with the lowest, medium and highest SHM (LowRab, MedRab and HighRab) respectively. The magenta and cyan bars refer to the reference rabies antibodies binding to Site I and to Site III respectively. e Summary of competition between the cloned mAbs with the reference rabies mAbs. The color gradient from red to blue indicates high to no competition. f Spearman correlation between % of SHM and neutralization (IC₅₀) in the different cloned mAbs. The dashed line indicates the limit of detection (LOD) for neutralization which corresponds to the highest concentration of mAb used for the assay (5 μg/mL). All statistical tests comparing the study groups were two-tailed tests.

Fig. 5. mRNA induces higher levels of diverse and cross-neutralizing antibody compared to Rabipur.

a Heatmap showing IGHV allele usage for each vaccinated group per animal including both Sanger sequences and bulk HTS. Total IgG repertoires were subsampled to 100,000 sequences per individual, 1.2 million sequences are shown. The scale was adjusted for the total number of sequences for each group, the resulting value was log10 transformed. b Bar graph showing the proportion of different clusters of clonally related sequences in the merged datasets (defined as those with the same V and J allele, exact HCDR3 length match, at least one identical nucleotide junction, and 80% amino acid identity of HCDR3). c Sample coverage estimation based on clonotype counts for each group, where a value of 1 represents that the entire population was sampled. The continuous colored lines represent the mean, while the surrounding color for each line represents a 95% confidence interval. d Species richness estimation (Chao1) for clonotype counts for both groups per individual. Estimation was subsampled 100x to the lowest number of sequences, individual mean values were plotted. The boxplot centre measurement is the mean, the bounds of box represent the interquartile range (50% percentile), the whiskers represent 1.5 times upper or lower interquartile range. n = 10 biologically independent animals. Statistical significance was calculated using Mann–Whitney test, p-value = 0.48. e Summary of plasma competition between circulating antibodies with seven reference rabies mAbs, quantified as ug/mL site-competing antibody per plasma sample. Plasma competition was measured at week 8 (two weeks post boost). n = 18 biologically independent animals. Error bars indicate geometric mean ± geometric SD. f Serum neutralizing titers against several lyssavirus strains measured at week 47 post study start (42 weeks post boost). Data is shown as WHO International Units. 0.5 IU/mL was considered as the cutoff for positive neutralization and is displayed as a dotted line. n = 18 biologically independent animals. Statistical significance was calculated using Mann–Whitney test. All statistical tests comparing the study groups were two-tailed tests. Error bars indicate geometric mean ± geometric SD.