A single-dose F1-based mRNA-LNP vaccine provides protection against the lethal plague bacterium

Abstract

Messenger RNA (mRNA) lipid nanoparticle (LNP) vaccines have emerged as an effective vaccination strategy. Although currently applied toward viral pathogens, data concerning the platform's effectiveness against bacterial pathogens are limited. Here, we developed an effective mRNA-LNP vaccine against a lethal bacterial pathogen by optimizing mRNA payload guanine and cytosine content and antigen design. We designed a nucleoside-modified mRNA-LNP vaccine based on the bacterial F1 capsule antigen, a major protective component of Yersinia pestis, the etiological agent of plague. Plague is a rapidly deteriorating contagious disease that has killed millions of people during the history of humankind. Now, the disease is treated effectively with antibiotics; however, in the case of a multiple-antibiotic-resistant strain outbreak, alternative countermeasures are required. Our mRNA-LNP vaccine elicited humoral and cellular immunological responses in C57BL/6 mice and conferred rapid, full protection against lethal Y. pestis infection after a single dose. These data open avenues for urgently needed effective antibacterial vaccines.

Fig. 1.. Construct design, physicochemical characterization of mRNA-LNPs formulations used throughout the study, and immunological responses elicited by SP-cp-caf1 mRNA-LNPs.

(A) Schematic representation of mRNA constructs used throughout the study. Elements encoded are the following: mammalian signal peptide sequence (SP), circular permutated (cp), caf1 sequence (caf1), high guanine and cytosine (GC) content (GC), and human Fc sequence (hFc). Poly(A), polyadenylate. (B) LNP formulation used throughout the study. DSPC, distearoyl-sn-glycero-3-phosphocholine; PEG-DMG, dimyristoyl-rac-glycero-3-methoxypolyethylene glycol. (C) Chemical structure of the proprietary ionizable lipid—lipid 14. (D) Table summarizing LNP physicochemical aspects. (E) A representative cryo–electron microscopy (cryo-EM) image of LNP-encapsulated SP-cp-caf1 mRNA. Scale bar, 100 nm.

Fig. 2.. Increased GC content leads to development of anti-F1 humoral response and partially protects mice from a lethal Y. pestis challenge in the bubonic plague model.

(A) Western blot analysis of F1 expression in samples collected from transfected HeLa cells at different hours posttransfection (hpt). (B) Schematic diagram of immunization, sample collection, and challenge. (C) F1-specific cellular response determined by enzyme-linked immunosorbent spot (ELISpot). (D and E) Anti-F1 IgG titers determined by enzyme-linked immunosorbent assay (ELISA). (F) Survival curve of C57BL/6 mice vaccinated with three doses of SP-cp-caf1 (GC), rF1 + alum, or luciferase. Statistical analysis was performed using a one-way analysis of variance (ANOVA) followed by post hoc Newman-Keuls test (ELISpot data), a two-way ANOVA with Tukey’s multiple comparisons test (*P < 0.05, **P < 0.01, and ***P < 0.001,) (ELISA data), or log-rank (Mantel-Cox) test (for survival plot). ns, not significant.

Fig. 3.. SP-cp-caf1-hFc and ΔSP-cp-caf1 mRNA LNPs elicit robust cellular and humoral immune responses and protect mice against a lethal Y. pestis challenge in the bubonic plague model.

(A) Western blot analysis of F1 expression in HeLa cells. (B) Schematic diagram of immunization and sample collection. (C) F1-specific cellular response determined by ELISpot. (D) Anti-F1 IgG titers determined by ELISA. (E) Survival curve of C57BL/6 mice vaccinated with three doses of ΔSP-cp-caf1, SP-cp-caf1-hFc, rF1 + alum, luciferase, or alum alone. (F) Survival curve of C57BL/6 mice vaccinated with a single dose of ΔSP-cp-caf1, SP-cp-caf1-hFc, or luciferase. Statistical analysis was performed using a one-way ANOVA followed by post hoc Newman-Keuls test, two-way ANOVA with Tukey’s multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001) (for immune responses) or log-rank (Mantel-Cox) test (****P < 0.0001) (for survival plot).