A multivalent mRNA-LNP vaccine protects against Clostridioides difficile infection

Abstract

Clostridioides difficile infection (CDI) is an urgent public health threat with limited preventative options. In this work, we developed a messenger RNA (mRNA)-lipid nanoparticle (LNP) vaccine targeting C. difficile toxins and virulence factors. This multivalent vaccine elicited robust and long-lived systemic and mucosal antigen-specific humoral and cellular immune responses across animal models, independent of changes to the intestinal microbiota. Vaccination protected mice from lethal CDI in both primary and recurrent infection models, and inclusion of non-toxin cellular and spore antigens improved decolonization of toxigenic C. difficile from the gastrointestinal tract. Our studies demonstrate mRNA-LNP vaccine technology as a promising platform for the development of novel C. difficile therapeutics with potential for limiting acute disease and promoting bacterial decolonization.

Fig. 1.. mRNA-LNP vaccines elicit robust immune responses against C. difficile virulence factors.

(A) Maximum likelihood phylogenetic tree illustrating the amino acid sequence identity of TcdA, TcdB, and PPEP-1 between mRNA constructs and 137 C. difficile strains. (B-E) Mice were immunized intramuscularly (i.m.) once (C) or twice (B), (D), (E) with 1 or 5μg of bivalent mRNA-LNPs (TcdA/TcdB) (green), trivalent mRNA-LNPs (TcdA/TcdB/PPEP-1) (purple), or trivalent recombinant protein with alum (grey). Age-matched naïve mice served as unvaccinated controls. Two weeks after boost, TcdA (left), TcdB (middle), and PPEP-1 (right) specific antibodies in sera were measured by ELISA. Two weeks after prime, total number of T follicular helper (Tfh) cells in draining lymph nodes (dLN) (C) were measured by flow cytometry. (D, E) Two weeks after boost, total germinal center (GC) B cells (D) and antigen-specific B cells (E) were measured in spleen by flow cytometry. (F and G) Two weeks after immunization, mice were challenged intraperitoneally (i.p.) with 625ng recombinant TcdA (F) or 125ng recombinant TcdB (G) and monitored for survival. (B-G) n = 5–14 mice per group, 2 independent experiments. Data are represented as mean ± SEM with fold change (B), mean ± SD (C-E), or percent survival (F, G). Statistics by one-way ANOVA with Tukey’s multiple comparisons (C-E) or Log-rank (Mantel-Cox) test (F, G). Stats in (F) and (G) shown between 5μg mRNA-LNP vaccine against other conditions. *p <0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Fig. 2.. mRNA-LNP vaccines protect against lethal CDI and induce mucosal immunity.

(A) Schematic of experimental design. Mice were immunized i.m. with 1μg bivalent or trivalent mRNA-LNPs, treated with cefoperazone (abx) in their drinking water, and challenged with 10,000 spores of C. difficile (VPI 10463). (B - D) Mice were monitored for survival (B), weight loss (C), and clinical sickness (D) over the course of infection. (E) Ceca pathology scores on day 2 post-infection. (F and G) C. difficile colony-forming units (CFUs) (F) and C. difficile toxin titers (G) in stool over time. Dotted lines indicate assay limit of detection (LoD). (H and I) Mucosal IgA (H) and IgG (I) titers 2 weeks after boost or infection. For C.d only, mice received cefoperazone followed by 100,000 spores of CD196. Vaccine only received two 1μg trivalent mRNA-LNP immunizations. Vaccine + C.d, immunized then infected with VPI 10463. C.d + vaccine, infected with CD196 then immunized twice with 1μg trivalent mRNA-LNP. ND, not detectable. n = 4–5 mice per group, 2 independent experiments. Data are represented as mean ± SD (C-E), mean ± SEM (F-G), or box and whiskers (H-I). Statistics by Log-rank (Mantel-Cox) (B), two-way ANOVA with Tukey’s multiple comparisons (C-D), Mann-Whitney (F-G) or Kruskal-Wallis multiple comparisons test. *p <0.05, ** p<0.01, *** p<0.001, **** p<0.0001 vs unvaccinated.

Fig. 3.. mRNA-LNP vaccines provide long-term protection against CDI.

(A - B) Antigen-specific antibodies in sera (A) and feces (B) at 14 days and 40+ days after the last immunization with 1μg of bivalent or trivalent mRNA-LNPs. (C) Antigen-specific memory B cells in the spleen 40+ days post-immunization. (D to G) Mice were infected with 10,000 spores of C. difficile (VPI 10463) 40 days after last immunization. (D) Schematic of experimental design. (E) Survival, (F) weight loss, and (G) clinical sickness were monitored over course of infection. (H to K) Mice were infected with 10,000 VPI spores 2 weeks after the last immunization, and reinfected with 10,000 VPI spores 26 weeks after initial infection. (H) Schematic. (I) Survival, (J) weight loss, and (K) clinical scores. ND, not detectable. n = 4–10 mice per group. Data are represented as box and whiskers (A-B), mean ± SD (C, F-G, J-K), or percent survival (E, I). Statistics by Mann-Whitney (A-B), Log-rank (Mantel-Cox) (E, I), or two-way ANOVA with Tukey’s multiple comparisons test (F-G, J-K). *p <0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Fig. 4.. mRNA-LNP vaccine targeting vegetative and spore proteins protects against CDI and elicits antibodies in non-human primates.

(A) Amino acid sequence identity of CdeM from our mRNA construct compared to 137 C. difficile strains across 5 clades. (B) Antigen-specific antibodies in sera 14 days after the last immunization with 1μg of mRNA-LNPs. (C to I) Mice were immunized twice i.m. with 1μg of tetravalent (red), spore trivalent (TcdA/TcdB/CdeM) (blue), vegetative trivalent (TcdA/TcdB/PPEP-1) (purple), or CdeM monovalent (orange) mRNA-LNPs. Two weeks after last immunization, mice were treated with cefoperazone and infected with 10,000 VPI spores. (C) Survival curve. (D) Weight loss. (E) Clinical scores. (F to I) C. difficile CFUs (F, H) and toxin titers (G, I) in feces over time. (J) Antigen-specific IgG titers in a macaque at baseline (naïve), after prime (day 21), and boost (day 35) immunizations with 200μg of tetravalent mRNA-LNP. ND, not detectable. n = 5 mice/group, 2 independent experiments. Data represented as mean ± SD (A, D-E), percent survival (C), or mean ± SEM (B, F-I). Data in (J) show fold change compared to control macaque. Statistics by Log-rank (Mantel-Cox) (C), two-way ANOVA with Tukey’s multiple comparisons test (D, E), or Kruskal-Wallis multiple comparisons test (B, F-I). *p <0.05, ** p<0.01, *** p<0.001, **** p<0.0001.