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.
Introduction
Lyme disease, caused by various species and strains of the bacteria Borrelia burgdorferi sensu lato, is the most common vector-borne illness in the United States. Its prevalence and geographic distribution have increased significantly since it was labeled a nationally notifiable condition in 1991. The Centers for Disease Control and Prevention report an estimated 476,000 cases of Lyme disease in the United States annually. Early infection manifests itself in a skin rash known as erythema migrans. Influenza-like symptoms often follow, but symptoms respond rapidly to antibiotics. Lyme disease can be difficult to diagnose, which may lead to delayed treatment and subsequently more severe complications. Carditis, arthritis, and neurological issues are characteristic of late stages of infection. Lyme disease can impact patients for the remainder of their lives, and, to make matters worse, contracting one strain does not equate with immunity against heterologous strains. Thus, developing a broadly protective prophylactic vaccine is crucial to preventing new and repeated cases of Lyme disease.
Lyme disease is transmitted through the bite of an Ixodes tick carrying the spirochete Borrelia burgdorferi. Infection begins with tick saliva contaminating the site of the bite; it spreads by activating host proteases that digest extracellular matrix components and employing mechanisms to evade immune response in the affected individual. B. burgdorferi expresses numerous proteins that are potential targets for vaccination. One such antigen is outer surface protein A (OspA), an abundantly expressed surface lipoprotein that anchors the bacterium to the tick midgut. It is rapidly downregulated upon feeding; therefore, early targeting is essential. There are many diverse strains of B. burgdorferi in the United States known to infect humans. OspA is a desirable vaccine target compared to other antigens because it is widely conserved among these strains. In 1998, GlaxoSmithKline (GSK) released phase III clinical trial results for LYMErix, an alum-adjuvanted recombinant OspA protein vaccine. Within a year, rates of Lyme disease had decreased by 76% among individuals who were vaccinated. However, a seminal paper was published the same year LYMErix was put on the market that revealed that OspA contained an epitope that was homologous to a peptide in hLFA-1, postulating that this cross-reactivity could lead to the development of treatment-resistant Lyme arthritis.
Although OspA vaccination was later proven to not cause this autoimmune response, LYMErix was removed from the market in 2002, just 4 years after its release. Since then, baited OspA-based vaccines have been developed that successfully block transmission of B. burgdorferi to ticks from their reservoir hosts, mainly Peromyscus leucopus (the white-footed mouse). Additionally, OspA-containing Lyme disease vaccines are commercially available for the immunization of dogs. Finally, the most promising candidate for a human Lyme disease vaccine (VLA15), which has begun phase III efficacy studies (NCT05477524), is OspA-based.
There has been no FDA-approved vaccine for Lyme disease since the demise of LYMErix, and cases continue to rise, underscoring the need for a preventative approach. An ideal vaccine is safe and efficacious, inducing broad protection in a variety of immunological backgrounds. Many preclinical Lyme disease vaccine candidates are inadequate as they elicit only strain-specific immunity after administration of multiple vaccine doses.
Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA-based vaccines demonstrated their safety and potency against various infectious diseases in preclinical studies as well as in human trials, and significantly contributed to the mitigation of the devastating effects of the coronavirus disease 2019 (COVID-19) pandemic. The mRNA-LNP vaccine platform induced potent antibody and cellular immune responses in both animals and humans, highlighting the viability of this novel modality for vaccine delivery. Here, we demonstrate that, in mice, OspA-encoding nucleoside-modified mRNA-LNP displays superior immunogenicity to an alum-adjuvanted OspA protein subunit vaccine at doses of 3 μg and 1 μg, respectively. The robust, antigen-specific humoral and cellular responses corresponded to protection against the heterologous N40 strain of B. burgdorferi. Applying nucleoside-modified OspA mRNA-LNP to B. burgdorferi could potentially reduce the prevalence of Lyme disease in the United States.
Results
OspA mRNA-LNP vaccination yields robust innate immune cell infiltration and antigen-specific CD4+ and CD8+ T cell responses in mice
Prior to the immunization studies, protein production from the OspA mRNA was demonstrated in vitro (Figure S1). Neuro 2-a cells were transfected with OspA- and firefly luciferase (Luc, negative control)-encoding mRNAs, and OspA protein in cell lysates was detected by western blotting.
Next, Balb/c mice were immunized intramuscularly with a single dose of 3 μg mRNA-LNP encoding OspA or 1 μg alum-adjuvanted OspA protein (rOspA + alum, positive control). Frequencies of innate cells (neutrophils, dendritic cells [DCs], macrophages, and monocytes) at the site of injection were measured at days 1, 3, and 5 post immunization. Non-injected animals (day 0) were used as controls. In both vaccine groups, infiltration of neutrophils peaked at day 1 and DCs, macrophages, and monocytes at day 3, and all populations decreased by day 5 (Figures S2 and S3).
In a separate study, Balb/c mice were immunized intramuscularly with a single dose of 3 μg OspA or Luc mRNA-LNP or 1 μg rOspA + alum. OspA-specific CD4+ and CD8+ T cell responses were evaluated after 12 days by intracellular cytokine staining of splenocytes (Figure 1A). The OspA mRNA-LNP vaccine elicited significantly higher levels of antigen-specific CD4+ and CD8+ T cells expressing Th1-associated cytokines (interferon [IFN]-γ, interleukin [IL]-2, and tumor necrosis factor [TNF]-α) than either Luc mRNA-LNP or rOspA + alum (Figures S4, 1B, and 1C). The only subpopulation that was not significantly increased compared to other experimental groups was IL-2+ CD8+ T cells, which are not major producers of this cytokine. Both CD4+ and CD8+ T cell responses to immunization with OspA mRNA-LNP were polyfunctional (Figures 1D and 1E). Hence, these data show that a single dose of OspA mRNA-LNP produced antigen-specific cellular immune responses in the spleens of mice.
