Human rotavirus (HRV) is still a leading cause of severe dehydrating gastroenteritis globally, particularly in infants and children. Previously, we demonstrated the immunogenicity of mRNA-based HRV vaccine candidates expressing the viral spike protein VP8* in rodent models. In the present study, we assessed the immunogenicity and protective efficacy of two mRNA-based HRV trivalent vaccine candidates, encoding VP8* of the genotypes P[8], P[6], or P[4], in the gnotobiotic (Gn) pig model of Wa (G1P[8]) HRV infection and diarrhea. Vaccines either encoded VP8* alone fused to the universal T-cell epitope P2 (P2-VP8*) or expressed P2-VP8* as a fusion protein with lumazine synthase (LS-P2-VP8*) to allow the formation and secretion of protein particles that present VP8* on their surface. Gn pigs were randomly assigned into groups and immunized three times with either P2-VP8* (30 µg) or LS-P2-VP8* (30 µg or 12 µg). A trivalent alum-adjuvanted P2-VP8* protein vaccine or an LNP-formulated irrelevant mRNA vaccine served as the positive and negative control, respectively. Upon challenge with virulent Wa HRV, a significantly shortened duration and decreased severity of diarrhea and significant protection from virus shedding was induced by both mRNA vaccine candidates compared to the negative control. Both LS-P2-VP8* doses induced significantly higher VP8*-specific IgG antibody titers in the serum after immunizations than the negative as well as the protein control. The P[8] VP8*-specific IgG antibody-secreting cells in the ileum, spleen, and blood seven days post-challenge, as well as VP8*-specific IFN-γ-producing T-cell numbers increased in all three mRNA-vaccinated pig groups compared to the negative control. Overall, there was a clear tendency towards improved responses in LS-P2-VP8* compared to the P2-VP8*mRNA vaccine. The demonstrated strong humoral immune responses, priming for effector T cells, and the significant reduction of viral shedding and duration of diarrhea in Gn pigs provide a promising proof of concept and may provide guidance for the further development of mRNA-based rotavirus vaccines.

Background: Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs), which are increasingly antibiotic resistant and frequently recur. Novel therapeutics are sought to treat and prevent recurrent UTIs (rUTIs), including vaccines. Key virulence factor FimH, which mediates bacterial adhesion to host cells and biofilm formation, is a promising target for a vaccine against UPEC. We assessed the immunogenicity of mRNA-based nanoparticle vaccines against UPEC containing FimH as the encoded antigen. Methods: Lipid nanoparticle (LNP)-formulated mRNA vaccines encoding FimH as a monomeric, pre-binding conformation protein (FimH _DG ), or a multimeric protein nanoparticle (PNP) through fusion to Helicobacter pylori ferritin (FimH _DG -Ferritin) were developed. Immunogenicity was assessed in vivo in female BALB/cAnNRj mice and female Wistar rats following three intramuscular (IM) injections of FimH _DG or FimH _DG –Ferritin mRNA vaccines, or comparator protein subunit vaccines. Antibody levels and functional response were measured in serum and urine by ELISA and bacterial adhesion inhibition (BAI) assays. T cell response was characterized by flow cytometry. Results: In both animal models, unmodified FimH _DG and FimH _DG -Ferritin mRNA vaccines induced higher functional serum antibody levels compared with the protein subunit vaccine control, at the tested dosages. FimH _DG -Ferritin resulted in greater binding antibody levels and higher splenic FimH-specific CD4 ⁺ and CD8 ⁺ T cell responses compared with monomeric FimH _DG in both models, resulting in its nomination as lead candidate vaccine design. Validation in rats demonstrated that N1mΨ nucleoside modification further enhanced FimH _DG -Ferritin immunogenicity compared with unmodified mRNA. Conclusions: The mRNA vaccine FimH _DG -Ferritin with N1mΨ-modified nucleosides is a promising candidate for further development as a vaccine against UPEC.

Human rotavirus (HRV) is still a leading cause of severe dehydrating gastroenteritis globally, particularly in infants and children. Previously, we demonstrated immunogenicity of mRNA-based HRV vaccine candidates expressing the viral spike protein VP8* in rodent models. In the present study, we assessed the immunogenicity and protective efficacy of two mRNA-based HRV trivalent vaccine candidates, encoding VP8* of the genotypes P[8], P[6] or P[4], in the gnotobiotic (Gn) pig model of Wa (G1P[8]) HRV infection and diarrhea. Vaccines either encoded VP8* alone fused to the universal T cell epitope P2 (P2-VP8*) or expressed P2-VP8* as a fusion protein with lumazine synthase (LS-P2-VP8*) to allow the formation and secretion of protein particles that present VP8* on their surface. Gn pigs were randomly assigned into groups and immunized three times with either P2-VP8* (30 µg) or LS-P2-VP8* (30 µg or 12 µg). A trivalent alum-adjuvanted P2-VP8* protein vaccine or an LNP-formulated irrelevant mRNA vaccine served as positive and negative control, respectively. Upon challenge with virulent Wa HRV, significantly shortened duration and decreased severity of diarrhea and significant protection from virus shedding was induced by both mRNA vaccine candidates compared to the negative control. Both LS-P2-VP8* doses induced significantly higher VP8*-specific IgG antibody titers in the serum after immunizations than the negative as well as the protein control. The P[8] VP8*-specific IgG anti-body-secreting cells in ileum, spleen and blood seven days post-challenge as well as VP8*-specific IFN--producing T cell numbers increased in all three mRNA-vaccinated pig groups compared to the negative control. Overall, there was a clear tendency towards improved responses in LS-P2-VP8* compared to P2-VP8*mRNA vaccine. The demonstrated strong humoral immune responses, priming for effector T cells, and significant reduction of viral shedding and duration of diarrhea in Gn pigs provide a promising proof of concept and may provide guidance for further development of mRNA-based rotavirus vaccines.

Background: Increasing antimicrobial resistance and the high health and economic burden of frequent, recurring urinary tract infections (rUTIs) drive the need for an efficacious UTI vaccine. Most UTIs are caused by uropathogenic E. coli (UPEC) expressing the virulence factor FimH, which mediates bladder colonisation. We have developed a lipid nanoparticle (LNP) mRNA vaccine, FimH _DG -Ferritin, as a UPEC vaccine candidate. Methods: FimH _DG -Ferritin mRNA constructs were introduced into HeLa cells through mRNA-transfection to assess the generation of immune-accessible protein nanoparticles (PNPs) in vitro by native polyacrylamide gel electrophoresis and negative stain electron microscopy. Following this, induction of humoral and cellular responses were evaluated in female BALB/c AnNRj mice and Wistar rats following either 2 or 3 intramuscular administrations of the FimH _DG -Ferritin mRNA vaccine. Finally, protective efficacy was assessed in a clinically relevant mouse model of rUTI. Results: In vitro PNP formation of the FimH _DG -Ferritin fusion protein with intact functional epitopes was confirmed. Immunogenicity was demonstrated in mice and rats; equivalent functional antibody levels were raised against homologous and heterologous UPEC strains. In a rUTI mouse model, FimH _DG -Ferritin mRNA vaccine administration induced the highest levels of functional antibodies among protein subunit comparators and significantly reduced bladder bacterial load versus negative controls. Conclusion: These preclinical data in rodents establish the FimH _DG -Ferritin mRNA vaccine as a protective candidate vaccine to prevent UTI caused by UPEC, supporting consideration for phase 1 clinical evaluation.