Development of an mRNA-lipid nanoparticle vaccine against Lyme disease

Abstract

Lyme disease is the most common vector-borne infectious disease in the United States, in part because a vaccine against it is not currently available for humans. We propose utilizing the lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) platform to generate a Lyme disease vaccine like the successful clinical vaccines against SARS-CoV-2. Of the antigens expressed by Borrelia burgdorferi, the causative agent of Lyme disease, outer surface protein A (OspA) is the most promising candidate for vaccine development. We have designed and synthesized an OspA-encoding mRNA-LNP vaccine and compared its immunogenicity and protective efficacy to an alum-adjuvanted OspA protein subunit vaccine. OspA mRNA-LNP induced superior humoral and cell-mediated immune responses in mice after a single immunization. These potent immune responses resulted in protection against bacterial infection. Our study demonstrates that highly efficient mRNA vaccines can be developed against bacterial targets.

Keywords: Borrelia burgdorferi; Lyme disease; OspA; antibody; antigen; bacteria; lipid nanoparticle; mRNA; nucleoside-modification; spirochete; vaccine.

Graphical abstract

Figure 1

Nucleoside-modified OspA mRNA-LNP vaccination induces antigen-specific T cell responses in mice (A) Mice were vaccinated intramuscularly with a single dose of 3 μg of OspA mRNA-LNP or 3 μg of Luc mRNA-LNP or 1 μg of rOspA + alum. Splenocytes were stimulated with an OspA overlapping peptide pool 12 days after immunization, and cytokine production by CD4⁺ and CD8⁺ T cells was analyzed by flow cytometry. Percentages of OspA-specific (B) CD4⁺ and (C) CD8⁺ T cells producing IFN-γ, IL-2, and TNF-α and frequencies of combinations of cytokines produced by (D) CD4⁺ and (E) CD8⁺ T cells are shown. Values from OspA-immunized mice are compared to values from animals immunized with Luc mRNA-LNP or rOspA + alum (B, C, D, and E). Each symbol represents one animal, and data represent mean ± SEM (n = 9–10 mice per group). Data from two independent experiments are shown. Statistical analysis: one-way ANOVA with Bonferroni correction, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 2

Nucleoside-modified OspA mRNA-LNP yields Tfh cell and antigen-specific GC B cell responses in mice Mice were vaccinated intramuscularly with OspA or Luc mRNA-LNP or rOspA + alum as described in Figure 1. Tfh and GC B cell responses in inguinal and popliteal lymph nodes were analyzed at day 12 post immunization. (A and B) Tfh cell (B220⁻CD4⁺CD62L⁻PD-1⁺CXCR5⁺) representative gating strategy (A) and frequencies and absolute numbers (B). (C and D) Antigen-specific GC B cell (CD19⁺CD3⁻FAS⁺GL7⁺OspA-AF488⁺/OspA-AF647⁺) representative gating strategy (C) and frequencies and absolute numbers, including non-specific GC B cells (D). Each symbol represents one animal, and data represent mean ± SEM (n = 10 mice per group). Data from two independent experiments are shown. Statistical analysis: one-way ANOVA with Bonferroni correction, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 3

Nucleoside-modified OspA mRNA-LNP vaccination elicits potent antigen-specific MBC and LLPC responses in mice Mice were vaccinated intramuscularly with OspA or Luc mRNA-LNP or rOspA + alum as described in Figure 1. MBCs and LLPCs in spleen and bone marrow, respectively, were analyzed 8 weeks post immunization. (A and B) Antigen-specific MBC (IgD⁻Dump[CD4, CD8a, Ter-119, F4/80]⁻CD19⁺B220⁺CD38⁺GL7⁻OspA-AF488⁺/OspA-AF647⁺) representative gating strategy (A) frequency and absolute numbers (B). (C and D) Antigen-specific LLPC (IgD⁻Dump[CD4, CD8a, Ter-119, F4/80]^-B220⁻CD138⁺ OspA-AF488⁺/OspA-AF647⁺) representative gating strategy (C) and frequency (D). (E) Quantification of bone marrow OspA-specific IgG1, IgG2a, and IgG2b antigen secreting cells (ASCs). In (B), (D), and (E), each symbol represents one animal, and data represent mean ± SEM (n = 10–15 mice per group except for naive, in which n = 3–6). Data from two to three independent experiments are shown. Statistical analysis: one-way ANOVA with Bonferroni correction, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 4

Nucleoside-modified OspA mRNA-LNP vaccination generates long-term antibody responses in mice (A) Mice were vaccinated intramuscularly with a single dose of 3 μg of OspA or Luc mRNA-LNP or 1 μg of rOspA + alum (represented by upward arrow) and bled retro-orbitally at 2 weeks (represented by blood droplet). Mice were bled again at 4 weeks and boosted with the same vaccine doses and then subsequently bled up to 24 weeks. (B) OspA-specific IgG endpoint titers from OspA mRNA-LNP-immunized mice are compared to values from animals immunized with 3 μg Luc or 1 μg rOspA + alum (and boosted with same dose at 4 weeks). Each symbol represents one animal, and data represent mean ± SEM (n = 9–10 mice per group). The horizontal dotted line represents the limit of detection. Titers below the limit of detection are reported as half of the limit of detection. Data from two independent experiments are shown. Statistical analysis: one-way ANOVA with Bonferroni correction of log transformed data, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 5

Nucleoside-modified OspA mRNA-LNP vaccination protects mice from infection with Borrelia burgdorferi (A) Mice were vaccinated with a single dose of OspA or Luc mRNA-LNP or rOspA + alum (represented by upward arrow) as described in Figure 1. 28 days after immunization, mice were challenged subcutaneously (SQ) with 10⁵Borrelia burgdorferi (strain N40) and then sacrificed 25 days later. Bladder, heart, joint (knee), and skin (ear) tissues were harvested for detection of B. burgdorferi infection. (B) qPCR results showing Borrelia-specific gene (flaB) normalized to mouse β-actin in bladder, heart, joint, and skin. Each symbol represents one animal, and data represent mean ± SEM (n = 9–10 mice per group). (C) Binding of sera samples 25 days post-infection to C₆ protein by ELISA. (D) Numbers of protected mice by treatment group. Data from two independent experiments are shown. Statistical analysis: one-way ANOVA with Bonferroni correction, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.