# A Lyme disease mRNA vaccine targeting Borrelia burgdorferi OspA induces strong immune responses and prevents transmission in mice

Djamel Tahir, Virginie Geolier, Hugo Bruant, Anne Le Fleche-Mateos, Adeline Mallet, Marie Varloud, Céline Civat, Yves Girerd-Chambaz, Sandrine Montano, Corinne Pion, et al.

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## To cite this version:

Djamel Tahir, Virginie Geolier, Hugo Bruant, Anne Le Fleche-Mateos, Adeline Mallet, et al. A Lyme disease mRNA vaccine targeting Borrelia burgdorferi OspA induces strong immune responses and prevents transmission in mice. Molecular Therapy - Nucleic Acids, 2025, 36 (2), pp.102514. 10.1016/j.omtn.2025.102514. pasteur-05068089

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# A Lyme disease mRNA vaccine targeting *Borrelia burgdorferi* OspA induces strong immune responses and prevents transmission in mice

Djamel Tahir,<fnref n="1" />,² Virginie Geolier,<fnref n="1" /> Hugo Bruant,<fnref n="1" /> Anne Le Flèche-Matéos,<fnref n="1" /> Adeline Mallet,² Marie Varloud,³ Céline Civat,⁴ Yves Girerd-Chambaz,⁴ Sandrine Montano,⁴ Corinne Pion,⁴ Elisabeth Ferquel,<fnref n="1" /> Vincent Pavot,⁴,<fnref n="5" /> and Valérie Choumet<fnref n="1" />,<fnref n="5" />

Lyme borreliosis (LB), caused by *Borrelia burgdorferi* sensu lato, is one of the most common tick-borne diseases in the northern hemisphere. Given its increasing global incidence, LB remains a major public health concern and the development of an effective vaccine is recognized as a key component of the overall disease prevention strategy. Here, we present results obtained with newly developed lipid nanoparticle-encapsulated mRNA vaccine candidates encoding the outer surface protein A (OspA) of *B. burgdorferi* sensu stricto (Bbss) serotype 1 (mRNA-OspA) with or without a secretion signal (SS) or a transmembrane domain. We evaluated the immunogenicity and protective efficacy of the mRNA-OspA vaccine candidates in a tick-fed mouse challenge model compared with an adjuvanted OspA protein subunit vaccine and the licensed canine vaccine Recombitek Lyme. At the doses tested, the mRNA-OspA vaccines induced significantly higher OspA-specific immunoglobulin G titers than the protein-based vaccines, as well as functional antibodies measured by serum bactericidal assay against Bbss strain B31. Complete protection against transmission was observed in the group immunized with the mRNA-OspA without SS. Overall, these data demonstrate that an mRNA-OspA vaccine can be effective against LB infection and could be used in the future for the prevention of Lyme disease.

## INTRODUCTION

Lyme disease or Lyme borreliosis (LB), is a tick-borne bacterial disease that is one of the most common vector-borne diseases in the northern hemisphere, particularly in North America and Europe, but also in temperate regions of Asia.<fnref n="1" /> LB is a multisystemic zoonotic infection caused by the *Borrelia burgdorferi* sensu lato (Bbsl) complex, which belongs to an ancient phylum of bacteria called spirochetes. *Borrelia* spp. are the only members of this phylum that are transmitted to vertebrate hosts by an arthropod vector.² Among the numerous genospecies of the complex, at least 20 have been identified. Four species are known to be responsible for most LB cases in humans. These four species can be further subdivided into seven

different outer surface protein A (OspA) serotypes (ST1-ST7) as described by Wilske et al.³ in 1993. In the United States, LB is caused by *B. burgdorferi* sensu stricto (Bbss) serotype 1, in contrast with Europe, where four *Borrelia* species cause most LB cases: Bbss (ST1), *B. afzelii* (ST2), *B. garinii* (ST3, ST5, ST6, and ST7) and *B. bavariensis* (ST4).³ In addition, *B. spielmanii*, *B. lusitaniae*, *B. bissettii*, and *B. valaisiana* have also been isolated from patients diagnosed with LB in Europe, demonstrating that the disease can also be caused by less common *Borrelia* species.⁴,<fnref n="5" />

The spirochetes responsible for LB are transmitted by hard ticks of the *Ixodes* genus. In Europe, the primary vector tick species for Bbsl is *I. ricinus*, commonly known as the sheep or castor bean tick, followed by *I. persulcatus* ticks.⁶,⁷ Among the North American *Ixodes* species, the blacklegged tick, *I. scapularis*, is the most implicated vector for Bbss. However, other tick species (e.g., *I. pacificus*, *I. affinis*, *I. angustus*, *I. hexagonus*, *I. minor*, *I. muris*, *I. ovatus*, and *I. spinipalpis*) have demonstrated competence as vectors for one or more *Borrelia* species.⁷

In humans, the disease is characterized by early (non-disseminated and disseminated) and late phases and by a wide range of clinical manifestations that can affect the skin, central and peripheral nervous system, joints, heart, and eyes.⁸ However, the most observed symptom is a characteristic skin lesion, known as erythema migrans, that develops at the site of infected tick bite.⁶ It is worth noting that

Received 16 September 2024; accepted 7 March 2025;
https://doi.org/10.1016/j.omttn.2025.102514.

**Correspondence:** Djamel Tahir, Institut Pasteur, Environnement et Risques Infectieux, Université Paris Cité, 75015 Paris, France.

**E-mail:** djamel.tahir@yahoo.fr

**Correspondence:** Vincent Pavot, Sanofi Vaccines R&D, Campus Mérieux, 69280 Marcy l'Etoile, France.

**E-mail:** vincent.pavot@sanofi.com

**Correspondence:** Valérie Choumet, Institut Pasteur, Environnement et Risques Infectieux, Université Paris Cité, 75015 Paris, France.

**E-mail:** valerie.choumet@pasteur.fr

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<footnote n="1">¹Institut Pasteur, Environnement et Risques Infectieux, Université Paris Cité, 75015 Paris, France; ²Institut Pasteur, Ultrastructural Bio-Imaging Core Facility, 75015 Paris, France; ³Ceva Santé Animale, 10 Avenue de la Ballastière, 33500 Libourne, France; ⁴Sanofi Vaccines R&D, Campus Mérieux, 69280 Marcy l'Etoile, France</footnote>

<footnote n="5">⁵These authors contributed equally</footnote>
specific clinical presentations are often associated with particular *Borrelia* species. For example, acrodermatitis chronica atrophicans, a chronic skin disease observed in Europe, is associated with *B. afzelii* infection, neurological disorders are associated with *B. garinii* infection, and arthritis in North America is primarily attributed to Bbss infection.⁶,⁹

Each year, approximately 476,000 Americans and 200,000 Europeans are diagnosed and treated for LB, with associated costs reaching up to $3 billion annually in the United States alone.¹⁰ In 2019, a total of 34,945 confirmed and probable cases of LB were reported to the CDC in the United States, marking a 4% increase compared with 2018.¹¹ Globally, the high incidence of LB underscores its significant socioeconomic burden, particularly in endemic regions. For instance, Lyme disease is estimated to cost the United States healthcare system between $712 million and $1.3 billion annually.¹⁰⁻¹²

Additionally, the impact of climate change on the distribution of *Ixodes* spp. ticks, the primary vectors of *B. burgdorferi*, and their reservoir hosts cannot be overlooked. Shifting environmental conditions are likely contributing to the global spread of LB.¹³

Taken together, these factors emphasize the urgent need for the development and implementation of effective vaccines as a strategy to mitigate the substantial health and economic impacts of Lyme disease in North America and Europe.

In the development of vaccines against human and canine LB, the primary focus has been on a subunit vaccines.¹⁴⁻¹⁷ Among the potential candidates, OspA, a major outer membrane lipoprotein of Bbsl, has emerged as a promising target, especially in the context of subunit vaccines.¹⁸ It was shown that OspA exhibits relatively low variability in its sequence and expression levels across strains, compared with other outer surface proteins of *B. burgdoferi*.¹⁰,¹⁸ OspA is highly expressed by *B. burgdorferi* and anchors the bacteria in the midgut of unfed ticks. During blood feeding, OspA is rapidly downregulated, allowing the bacteria to migrate to the salivary glands and then to be transmitted to the host. OspA can also be expressed on the surface of the spirochetes grown in artificial media but is generally not expressed during infection of mammalian hosts.¹⁹,²⁰ Therefore, one vaccine strategy is to induce circulating anti-OspA antibodies (Abs) that are then ingested with the blood meal when the tick feeds on vaccinated hosts for several hours/day.¹⁷ Anti-OspA Abs will then bind and kill the bacteria in the tick midgut before transmission can occur.¹⁹⁻²¹

Although there is currently no human LB vaccine on the market, two monovalent OspA ST1 vaccines—ImuLyme from Pasteur Mérieux-Connaught²² and LYMErix from SmithKline Beecham¹⁸,²³—have been evaluated in clinical efficacy trials, and the former was licensed for human use in the United States between 1998 and 2002. Retrospective analysis of clinical efficacy trials has identified a protective level of anti-OspA immunoglobulin G (IgG) titer equiv-

alent to 1,400 ELISA units.²⁴,²⁵ This concentration has been shown to protect against Lyme disease for at least the following season. Clinical trials with the other OspA-based vaccine candidate ImuLYME (Pasteur Mérieux-Connaught) also showed a correlation between anti-OspA IgG titers and protection. In fact, OspA vaccine recipients who contracted Lyme disease had significantly lower serum bactericidal activity and anti-OspA ELISA titers after three immunizations than OspA vaccine recipients who did not contract Lyme disease.²⁶

More recently, a hexavalent protein subunit vaccine VLA15 for LB was developed by Valneva Austria GmbH. This vaccine candidate consists of the C-terminal fragments of six different OspA serotypes (ST1–ST6), joined together in pairs to create three fusion proteins.²⁷ In 2023, VLA15 vaccine reached the phase 3 of clinical trials (Clinical Trials registration numbers NCT03769194, NCT03970733, and NCT05477524).²⁷,²⁸

It is worth noting that numerous OspA-based LB vaccines are commercially available for the immunization of dogs against LB.¹⁴,¹⁷ To our knowledge, only one recently published article reported the development of an mRNA-based vaccine against Bbss.²⁹ This emerging approach for prevention and treatment offers numerous advantages over conventional methods, including safety, efficacy, adaptability, rapid vaccine production, and cost-effectiveness.³⁰

In this study, we (1) developed four mRNA-lipid nanoparticle (LNP) vaccine candidates encoding the OspA of Bbss and (2) evaluated their immunogenicity and efficacy using a tick-fed mouse model. The different mRNA constructs encoded the native OspA sequence, OspA with an hemagglutinin secretion signal (HA-SS), a deglycosylated OspA or a transmembrane (TMB) OspA (as described in patent applications WO2023111262 and WO2023214082). The HA-SS and the TMB were added to secrete OspA or to express it at the cell surface to modulate immunogenicity. The deglycosylated sequence was used to avoid post-translational N-glycosylations that could negatively impact OspA conformation and/or mask critical epitopes. The immunogenicity and protective efficacy of the four mRNA constructs were evaluated in a tick-fed murine challenge model and compared with two adjuvanted subunit vaccines (OspA ferritin ST1 and a combination of OspA ferritin ST1 and OspA ferritin ST2)¹⁶ and to the licensed dog vaccine Recombitek Lyme.

All immunized mice developed IgG Abs against OspA after the first injection. One week after the booster dose, high levels of anti-Bbss Abs were detected, with mRNA-based vaccines eliciting significantly higher titers compared with adjuvanted OspA subunit vaccines. Both vaccine types induced bactericidal Abs, as shown by a serum bactericidal activity assay (SBA). Vaccine efficacy against LB infection ranged from 50% to 60% for protein-based vaccines and 55% to 100% for mRNA-LNP based vaccines, with the non-secreted mRNA formulation achieving 100% protection. These findings suggest that mRNA-OspA vaccines effectively induce robust IgG titers and could prevent Lyme disease transmission.
A

<table><thead><tr><th></th><th>Intracellular</th><th>Supernatant</th><th>Transmembrane</th></tr></thead><tbody><tr><td>mRNA-OspA ST1 Native</td><td>++++</td><td>-</td><td>-</td></tr><tr><td>mRNA-HA-SS-OspA ST1 Native</td><td>-</td><td>++++</td><td>-</td></tr><tr><td>mRNA-HA-SS-OspA ST1 Gly(-)</td><td>-</td><td>++++</td><td>-</td></tr><tr><td>mRNA-TMB-OspA ST1 Native</td><td>+</td><td>-</td><td>+++</td></tr><tr><td>mRNA-TMB-OspA ST1 Gly(-)</td><td>++</td><td>+</td><td>+++</td></tr></tbody></table>

<figure><img src="image_4.png" /><figcaption>**Figure 1.** *In vitro* expression of mRNA-OspA in HEK293T</figcaption></figure>

(A) Western blots of mRNA-OspA ST1 native, mRNA-HA-SS-OspA ST1 native, mRNA-HA-SS-OspA ST1 Gly(-), mRNA-TMB-OspA ST1 native, and mRNA-TMB-OspA ST1 Gly(-) in intracellular fraction, cell supernatant and crude extracts (transmembrane). Cell's supernatants, crude extracts and intracellular compartments were collected after 48 or 72 h. A negative control (buffer) and a positive control (recombinant OspA) were also used. Blotted proteins (duplicates) were detected with polyclonal rabbit antibodies that recognize OspA. Antigenicity of secreted OspA ST1 antigen delivered by mRNA in HEK293 cells was also assessed. Transfected cell supernatants were used to perform sandwich ELISAs with functional monoclonal antibodies (B) LA-2, (C) 857-2, and (D) 221-7. (E) Depict localization of mAbs target epitopes on OspA ST1.

RESULTS

**Characterization of mRNA-OspA vaccine candidates**

The *in vitro* expression of mRNA-OspA was evaluated following mRNA transfection into HEK cells. As illustrated in Figures 1 and S1, protein expression varied across cellular compartments depending on the mRNA construct. Figure 1A demonstrates that the native mRNA-OspA construct (without the HA-SS or TMB domain) resulted in predominantly intracellular OspA expression, with no detectable secretion into the supernatant. In contrast, the mRNA-OspA construct containing the HA-SS (mRNA-HA-SS-OspA ST1) successfully directed OspA expression into the cell supernatants, as expected. Variability in band sizes observed for the native OspA construct reflects different levels of N-glycosylation. Meanwhile, the mutated OspA construct lacking glycosylation motifs (Gly(-)) expressed a single band corresponding with a uniform OspA size, as anticipated due to the absence of glycosylation.

Figures 1A and S1 highlight the effect of incorporating a TMB domain into the OspA construct. In this case, OspA expression was confined to the cell membrane (crude extract), consistent with its membrane-associated localization. Once again, the Gly(-) mutant exhibited a single band representing uniform OspA size, while the native form showed multiple bands due to varying levels of glycosylation.

As shown in Figure 1, the antigenicity of OspA ST1 antigen delivered by mRNA in HEK293T cells was effective. Transfected cell supernatants were used to perform sandwich ELISAs with functional mAbs LA-2, 857-2, and 221-7 (Figures 1B, 1C, and 1D, respectively). The mAb LA-2 targets the C-terminus of OspA ST1 (Figure 1E) only and has been shown to correlate with protection in clinical studies.³¹,³² MAbs 221-7 and 857-2 (Figure 1E) were selected as lead candidates based on borreliacidal activity and protection in mice against tick-mediated transmission of *B. burgdorferi*, as shown in Wang et al.³³
<figure><img src="image_5.png" /><figcaption>**Figure 2. Schematic representation of the study procedures and timeline**</figcaption></figure>

One hundred C3H/HeNRj mice were allocated into 10 groups and intramuscularly immunized with each vaccine preparation on days 0 and 21. Two weeks later (35 days p.i.), mice were infested with *Bbss*-infected *Ixodes ricinus* nymphs. HA-SS, HA secretion signal; Gly(-), deglycosylated; TMB, HA transmembrane domain.

Secreted OspA ST1 (mRNA-HA-SS-OspA ST1) produced by mRNA was correctly recognized by the 3 functional mAbs, showing that the antigen was in the correct conformation after *in vitro* expression in human cells (HEK293T) and that natural glycosylation did not mask critical epitopes (Figures 1B–1D). Mutation of N-glycosylation sites inhibited the binding of the mAb LA-2, possibly due to the induction of a wrong conformation in the C-terminal epitope. This was not observed with 221-7 and 857-2 antibodies.

## Clinical monitoring of mice after immunizations and challenge

No specific local or systemic adverse events were noted within the following hours or days after the receipt of vaccine doses. Four mice were euthanized throughout the trial after reaching humane end points.

## Immunogenicity of mRNA-OspA vaccine candidates in mice

To assess the immunogenicity of mRNA-OspA ST1 candidates, C3H/HeNRj mice were immunized with the four mRNA-OspA vaccine candidates, AF03-adjuvanted ST1 and ST2 OspA-ferritin nanoparticles or Recombitek Lyme, a non-adjuvanted canine vaccine containing full-length lipidated recombinant OspA from the same serotype (Figure 2). Empty LNPs, empty ferritin (+AF03) and PBS were used as negative controls. Ab responses to these immunogens were determined by ELISA to recombinant OspA.

The OspA-specific IgG titer responses of mice immunized with the four mRNA-OspA candidates, the two OspA-ferritin and the Recombitek Lyme are shown in Figures 3A and S2. Except for the negative control groups (PBS, empty LNPs, and empty ferritin), IgG titers significantly increased after the first immunization (day 0) and

reached a peak on day 35 (Figure S2) for all immunized groups (plateau between day 35 and day 45). At each time point, the homogeneity of anti-OspA IgG titers was assessed, and no statistically significant differences were found in OspA-specific IgG titers among mice within the same vaccine group. In addition, the level of Abs obtained from individuals belonging to the same vaccine group followed globally a similar trend to those observed in pooled sera (mRNA-OspA = mRNA-HA-SS-OspA = mRNA-HA-SS-OspA-gly(-) = mRNA-TMB-OspA > OspA-ferritin ST1 = OspA-ferritin ST1+ OspA ferritin ST2 > Recombitek). Anti-OspA IgG titers for all vaccinated groups remained at high levels until 6 weeks post immunization (p.i.), which was followed by a continuous decrease over time (up to 70 to 92 days p.i.) (Figures 3 and S2). Finally, whatever the vaccine candidate, IgG levels were reduced in all immunized groups 8 weeks p.i. (day 45 versus days 70; *p* values varied between 0.0002 and 0.0004) (Figures 3 and S2). As expected, no immune response was detected against OspA in the control groups (PBS, empty LNPs, and empty ferritin) (Figures 3A and B).

Mice immunized with the mRNA-LNP vaccine candidates generated a humoral immune response approximately 5–10 times greater (GMT > $10^6$ ELISA units) than in mice immunized with protein-based OspA vaccine (GMTs between $10^5$ and $5 \times 10^5$ ELISA units), as shown on day 45 p.i. (Figure 3C). No significant differences were detected in Ab titers between the four mRNA-based vaccines. Similarly, there was no significant difference in Ab titers between the three protein-based vaccine groups (Figure 3).

Mice immunized with all tested vaccines displayed a broad Ab response with detectable levels of IgG1, IgG2a, IgG2b, and IgG2c.
<caption>Figure 3. Humoral immune responses to protein- and mRNA-based vaccines</caption>

(A) Protein-based vaccines (B) mRNA-based vaccine candidates. OspA-specific IgG ELISA titers were measured following IM vaccination of C3H/HeNRj mice (n = 10 mice per group) on day 0 and day 21 with mRNA vaccines coding for OspA ST1 with or without secretion signal (HA-SS) or transmembrane domain (TMB). Bars represent geometric mean titers (GMTs) ± 95% confidence interval (10 or 9 mice in each group on days 0, 21, 28, 45, and 70, and 5 or 4 mice in each group on day 92). Horizontal dotted lines represent the limit of detection (LOD) of the assay. (C) Humoral immune responses to mRNA-OspA vaccine candidates compared with protein-based vaccines during plateau phase reached between day 35 and day 45. Individual mice are shown. OspA-specific IgG ELISA titers measured on day 45 p.i. Bars represent geometric mean titers ± 95% confidence interval. \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001.

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<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>Figure 4. Antibody titers of different IgG subclasses measured on day 45 post-immunization.</figcaption></figure>

Titers are presented in individual value with the geometric mean indicated for all groups (n = 6 mice in each group). (A) IgG1 and (B) IgG2a. \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001.

Our results suggest that IgG2a is the primary subclass expressed in C3H/HeN mice. We therefore chose to show only IgG1 and IgG2a responses (Figure 4). Interestingly, as observed for the total IgG titers, mice immunized with OspA mRNA-LNP had higher IgG1 and IgG2a titers compared with those vaccinated with the protein-based vaccines. A statistically significant difference was observed between the two groups of vaccines for IgG1, whereas only a trend was noted for IgG2a. While no difference was detected in IgG2a isotype titers measured between the different formulations (mRNA- or protein-based vaccines), there was one significant difference between mRNA-OspA-native and mRNA-HA-SS-OspA-gly(-) for the IgG1 isotype.

Overall, the subclass response for IgG1 was higher than that for the IgG2a in the OspA-ferritin ST1 + OspA-ferritin ST2, Recombi-tek, mRNA-OspA, and mRNA-HA-SS-OspA treatment groups. There was no such difference in the other treatment groups (Figure S3).

## Serum bactericidal assay

All pooled sera from immunized mice exhibited high borreliacidal activity in presence of complement (Figure 5A). OspA-ferritin ST1 + OspA-ferritin ST2 vaccine group had the lowest SBA titer with 4,096, while all other groups exhibited SBA titer greater than 16,384 (Table S1). Among mRNA vaccines, mRNA-OspA-ST1-native tends to exhibit superior efficacy, in inducing borreliacidal activity (Figure 5). At all tested serum dilutions, the borreliacidal activity of mRNA-OspA-ST1-native was significantly superior to that of OspA-ferritin ST1 (*p* < 0.02) and OspA-ferritin ST1 + OspA-ferritin ST2 (*p* < 0.0006) (Figure 5A). There was no significant difference in killing activity between the protein-based vaccine groups (OspA-

ferritin ST1, OspA-ferritin ST1 + OspA-ferritin ST2 and Recombi-tek). The survivability of *B. burgdorferi* was confirmed in the culture mixtures by dark-field microscope (DFM) as described in Figure 5B and Table S1 illustrated the survival rates of spirochetes after 48 h of incubation with various dilutions of mouse sera.

## Protective effect of the vaccine candidates against Bbss

Mice were infested by *Borrelia*-infected ticks on D35 p.i. No statistical differences were found in the number of fully engorged ticks collected from mice in the different groups. *B. burgdorferi* DNA was early detected in ear biopsy samples from week 3 after tick infestation. For the negative control groups, the cumulative *ante-mortem*-positive biopsies were 100% (9/9), 80% (8/10), and 88.8% (8/9) for empty ferritin, empty LNPs, and PBS, respectively (Table 1). Results obtained using xenodiagnosis showed almost similar results for these three groups (Table 1). *Post-mortem* screening (using culture and PCR) showed dissemination and persistence of spirochetes in almost all tested organs of the three control groups demonstrating an infection rate of 100%.

In groups immunized with OspA vaccine candidates, regardless of the vaccine platform, the number of mice found positive was statistically lower (*p* ≤ 0.028) compared with the control groups. Interestingly, no Bbss DNA or live spirochetes (tissue culture) were detected in mice from the mRNA-OspA-native group during both, *ante-* and *post-mortem* screenings demonstrating full protection against transmission. We observed a significant difference (*p* < 0.0001) in the rate of infected mice between vaccinated versus control non-vaccinated groups. The infection rates were 0%, 40%, 33.3%, and 44.4% for mRNA-OspA native, mRNA-HA-SS-OspA, mRNA-HA-SS-OspA-gly(-), and mRNA-TMB-OspA, respectively (Table 1). The infection
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>Figure 5. Functional antibody responses determined by serum bactericidal assay</figcaption></figure>

(A) Viable numbers of *B. burgdorferi* B31 (log bacterial count) after 48 h of incubation with different serum pools collected on day 45. The x axis represents the serial 2-fold dilutions (from 1:8 to 1:16,384) of each serum pool. A mean of $2 \times 10^8$ spirochetes were counted in the negative control wells. The red dashed horizontal line represents the bactericidal activity for which the sera dilution is able to kill 50% ($1 \times 10^5$) of spirochetes present in the well. Spirochetes survivability was counted by DFM. (B) Survival of *B. burgdorferi* ss strain B31 GCB in the presence of guinea pig complement and sera collected from mice immunized with protein- or mRNA-based vaccines coding for OspA ST1 with or without secretion signal (HA-SS) or transmembrane domain (TMB). The viability of spirochetes (in green) was assessed after 48 h of incubation, using fluorescent microscope (Leica Microsystems). Scale bar, 200 µm. Gly(-), deglycosylated.

rate was 50%, 40%, and 40% for OspA-ferritin ST1, OspA-ferritin ST1 + OspA-ferritin ST2, and Recombitek, respectively (Table 1).

The overall vaccine efficacy for each vaccine candidate ranged from 50% to 60% in mice immunized with the protein-based vaccines and from 55% to 100% (Figure S4) in mice immunized with mRNA-LNP based vaccines, with a significantly greater efficacy

($p < 0.02$) of 100% with the mRNA-OspA-native formulation compared with the other vaccine candidates. No significant difference was observed between the other vaccine candidates (Figure S4).

DISCUSSION

Despite remaining susceptible to antibiotic treatments (e.g., doxycycline, amoxicillin, cefuroxime, cefotaxime, etc.), LB remains a
<table><caption>Table 1. Vaccine efficacy following tick-fed mouse challenge with Bbss</caption><thead><tr><th rowspan="2">Vaccine candidate</th><th rowspan="2">Total mice included</th><th colspan="2">Ante mortem</th><th colspan="2">Post mortem</th><th rowspan="2">Cumulative number of infected mice (%)</th></tr><tr><th>PCR ear biopsies (pos/tested)</th><th>Xenodiagnosis (pos/tested)</th><th>Culture: Heart, meninges, bladder (pos/tested)</th><th>PCR: Heart, (pos/tested)</th></tr></thead><tbody><tr><td>OspA-ferritin ST1</td><td>10</td><td>5/10</td><td>1/10</td><td>2/5</td><td>3/10</td><td>5/10 (50)</td></tr><tr><td>OspA-ferritin ST1+ OspA-ferritin ST2</td><td>10</td><td>3/10</td><td>1/10</td><td>3/5</td><td>2/10</td><td>4/10 (40)</td></tr><tr><td>mRNA-OspA</td><td>10</td><td>0/10</td><td>0/10</td><td>0/5</td><td>0/10</td><td>0/10 (0)</td></tr><tr><td>mRNA-HA-SS-OspA</td><td>10</td><td>1/10</td><td>1/10</td><td>2/5</td><td>3/10</td><td>4/10 (40)</td></tr><tr><td>mRNA-HA-SS-OspA-gly(-)</td><td>9</td><td>1/9</td><td>2/9</td><td>1/3</td><td>3/9</td><td>3/9 (33.3)</td></tr><tr><td>mRNA-TMB-OspA</td><td>9</td><td>1/9</td><td>3/9</td><td>2/5</td><td>3/9</td><td>4/9 (44.4)</td></tr><tr><td>Recombitek</td><td>10</td><td>4/10</td><td>2/10</td><td>3/5</td><td>2/10</td><td>4/10 (40)</td></tr><tr><td>Empty ferritin</td><td>9</td><td>9/9</td><td>9/9</td><td>4/5</td><td>4/9</td><td>9/9 (100)</td></tr><tr><td>Empty LNPs</td><td>10</td><td>8/10</td><td>9/10</td><td>6/6</td><td>7/10</td><td>10/10 (100)</td></tr><tr><td>PBS</td><td>9</td><td>8/9</td><td>9/9</td><td>8/9</td><td>7/9</td><td>9/9 (100)</td></tr></tbody></table>

Mice were challenged at Day 35 p.i. (2 weeks post-booster). Numbers of infected mice per vaccinated group are shown using different screening techniques.

prevalent disease in the northern hemisphere.⁶ Recent studies estimate that approximately 476,000 people in the United States and 200,000 people in Western Europe are diagnosed with the disease each year.³⁴,³⁵ Lyme disease is transmitted by the bite of *Ixodes* ticks, which carry the spirochete bacteria Bbsl. After a tick bite, while some strains of spirochetes do not cause systemic infection, others can spread efficiently from the bite site through the skin and into the bloodstream, eventually reaching distant tissues and organs. This dissemination can lead to severe systemic diseases such as neuroborreliosis, arthritis and carditis. Some patients, for reasons not yet understood, continue to experience persistent symptoms known as post-treatment Lyme disease syndrome, even after undergoing both oral and intravenous antibiotic treatments.³⁶

The mRNA technology is mainly used to develop vaccines against viral targets, because bacterial proteins encoded by mRNA in eukaryotic cells can undergo post-translational modifications, such as glycosylations, that can affect their conformation and immunogenicity. In the present study, we investigated the immunogenicity and efficacy of mRNA vaccine candidates encoding Bbss OspA serotype 1 in mice. Because *B. burgdorferi* is an extracellular pathogen and humoral immunity has been shown to be protective against this organism, vaccine studies have focused on identifying borreliant antigens that are (1) surface exposed, (2) conserved among different strains and genospecies of *Borrelia* spirochetes, and (3) produced during tick transmission and mammalian infection.¹⁵ Any outer surface protein that fulfills these three basic requirements (such as OspA) is considered an excellent candidate for vaccine studies. However, until recently, most studies have focused on OspA in the context of protein-based vaccines.

Our mRNA vaccine candidates have been designed and produced encoding the OspA antigen from Bbss ST1 as a native protein, secreted (by adding a HA-SS) or expressed as a transmembrane protein by adding a TMB domain (as described in patent applications

WO2023111262 and WO2023214082). These mRNA vaccine candidates were compared with the protein-based vaccine candidate OspA-ferritin¹⁶ and the licensed canine vaccine Recombitek Lyme.³⁷

*In vitro* characterization of the mRNA-OspA vaccine candidates showed a strong expression in HEK293T cells, resulting in efficient expression of the OspA antigen. When an HA-SS was added, we observed secretion of OspA and efficient transmembrane expression when the HA-TMB domain was added. In addition, antigenicity data showed that the secreted OspA efficiently bound to three conformational monoclonal Abs (LA-2, 857-2, and 221-7), except for the deglycosylated OspA version, which was unable to bind the LA-2 mAb, indicating that the amino acid mutations affected the C-terminal structure of the LA-2 epitope, but not the central and N-terminal regions targeted by the mAbs 857-2 and 221-7, respectively.³³

We then evaluated the immunogenicity and efficacy of our mRNA-OspA vaccine candidates in a tick-fed mouse challenge model. The strength of our experimental design lies in the inclusion of tick-infected mice under laboratory conditions with Bbss B31 and the implementation of multiple screening techniques. These techniques include PCR analysis of ear biopsies taken at multiple time points, xenodiagnosis for *ante-mortem* screening, and PCR testing of specific organs coupled with tissue culture for *post-mortem* screening, all of which contribute to the determination of the infectious status of each mouse. The combination of all these techniques increases sensitivity and minimizes the occurrence of false negatives. In addition, we used the highly invasive Bbss strain B31 (invasive group A), so that non-detection of spirochetes in the organs of immunized mice would not be due to the non-invasive nature of the strain used. Furthermore, the selection of C3H/HeN mice as model in this study is well justified, as this strain is widely recognized as a standard and reliable choice for the study of *B. burgdorferi* and LB in the laboratory. Notably, C3H/HeN mice exhibit a high susceptibility to *B. burgdorferi* infection, leading to significant bacterial loads
in various tissues.³⁸⁻⁴¹ This characteristic makes them an excellent model for investigating bacterial dissemination and persistence within the host.³⁹

In this study, we showed that mRNA-OspA demonstrated superior immunogenicity and protective efficacy compared with adjuvanted OspA-ferritin and Recombitek Lyme vaccines when administered twice, 3 weeks apart.

No significant difference in OspA-specific Ab titers was observed in mice immunized with the different mRNA-based vaccine preparations: native or modified forms. However, a statistically significant difference was observed in the Ab titers measured in the groups of mice immunized with protein-based vaccines. For example, mice immunized with OspA-ferritin ST1 or OspA-ferritin ST1 + OspA-ferritin ST2 produced higher IgG titers than mice immunized with Recombitek Lyme. This finding is consistent with a previous study reporting that the OspA-ferritin nanoparticle vaccine elicited a superior Ab response compared with the full-length lipidated recombinant OspA vaccine.¹⁶

Although the level of Abs increased after the booster doses in all vaccine groups, this level tended to decrease with time, especially after 70 days p.i. It has been reported that a non-lipidated recombinant OspA failed to induce a durable Ab response against *B. burgdorferi* in mice.⁴² The same observation has been reported in dogs vaccinated with different available vaccines (lipidated or not) against *B. burgdorferi* infection. In fact, it has been observed that dogs vaccinated according to the standard vaccination schedule (1 dose/year) showed a drastic decrease in Ab levels after 4 months, which is probably insufficient to prevent *Borrelia* infection.⁴³ The administration of an additional booster vaccination on day 180 prevented the decrease in Ab levels and established sufficient immunity to prevent infection.⁴³ In contrast, recently published data showed that a single immunization with OspA mRNA-LNP generated durable levels of antigen-specific Abs in mice that persisted for at least 5 months after the initial immunization.²⁹

We also evaluated the functionality of the anti-OspA Ab responses using the SBA assay. Bacterial killing activity was observed on sera from each of the seven vaccination groups. Sera from mice vaccinated with OspA mRNA-LNP provided greater bactericidal activity at high dilutions compared with those from mice immunized with recombinant OspA vaccines. In addition, sera from mice immunized with mRNA-OspA native generated a significantly higher bactericidal activity compared with other animals immunized with mRNA-HA-SS-OspA, mRNA-HA-SS-OspA-gly(-) or mRNA-TMB-OspA.

In this study, all OspA vaccine candidates (mRNA and protein-based vaccines) prevented *B. burgdorferi* transmission from infected ticks to immunized mice with different levels of efficacy (from 50% to 100%). Interestingly, mice immunized with mRNA-OspA native and exposed to Bbss-infected ticks were 100% protected (10/10) throughout the follow-up period. In their study, Pine et al.²⁹ reported that a single immunization with OspA mRNA-LNP successfully protected all mice

from infection when animals were challenged by subcutaneous injection of *B. burgdorferi*. However, others have shown that transmission of *B. burgdorferi* by injection is significantly less efficient than by tick bite.⁴⁴,⁴⁵ This is likely since many bioactive molecules in tick saliva play an important role in enhancing the transmission of *B. burgdorferi* from the tick to the host.⁴⁶

OspA-based LB vaccines should inhibit the transmission of spirochetes from the ticks to the host after a blood meal containing anti-OspA Abs. This blocking effect is highly dependent on the concentration of anti-OspA Abs present in the host's bloodstream and introduced into the tick's gut at the beginning of a blood meal. However, we cannot rule out the possibility that a certain number of spirochetes may evade the bactericidal effect and consequently migrate from the tick midgut, spread into the hemolymph, and reach the tick's salivary glands before being transmitted to the host. In this situation, infection can still occur if the number of *Borrelia* injected into the host by the ticks exceeds the infectious dose. Some studies have reported that relatively low numbers of *Borrelia* are sufficient to establish infection in mice, typically ranging from a few hundred to a few thousand spirochetes.⁴⁷,⁴⁸ It is noteworthy that, in our study, each mouse was challenged with 12 Bbss-infected nymphs, which is twice as many as in the study by Kamp et al.¹⁶ In their study, Sajid et al.⁴⁹ reported that immunization with an mRNA vaccine encoding 19 salivary proteins of *I. scapularis* can induce acquired resistance to *I. scapularis* and prevent *B. burgdorferi* infection in guinea pigs when they are later exposed to a single *B. burgdorferi*-infected tick. However, when immunized guinea pigs were challenged with three nymphs, *B. burgdorferi* infection occurred in 60% (3/5).⁵⁰ This could be explained by the fact that the infectious dose is too high to be counteracted by the vaccination in this animal model. This is a strong reminder that the level of naturally occurring infestation (i.e., the number of tick bites) should be carefully considered when designing preclinical and clinical efficacy studies of LB vaccines. Indeed, while dogs can occasionally be simultaneously infested by numerous ticks, this is much less common in humans with the rate of simultaneous occurrence of two (or more) tick bites being 1.2% (23/1,891).⁵¹

It is noteworthy that the same vaccine candidate (adjuvanted OspA-ferritin nanoparticles) was previously evaluated by Kamp et al.¹⁶ using *I. scapularis* infected with Bbss strain N40 (ST1). The authors demonstrated an efficacy of 100%, while in our study 50% efficacy was obtained using *I. ricinus* (European tick species) and Bbss strain B31 (U.S. isolated strain). The same observation was made for the commercial canine vaccine (Recombitek Lyme) used here as a positive control, which showed lower protective efficacy in mice (60%) compared with that reported in dogs (100%).⁵² Therefore, the choice of tick species and *Borrelia* strain/serotypes is another critical factor to consider in the preclinical evaluation of LB vaccines.

To better characterize the humoral immune response of the different vaccines, we measured OspA-specific IgG subclasses (IgG1 and IgG2a) in individual sera collected at day 45 p.i. Results showed that mice immunized with any of the four mRNA-OspA LNP vaccine
candidates tended to have higher IgG2a titers than mice immunized with OspA-ferritin or Recombitek Lyme. In mice, IgG2a are associated with a CD4 Th1 profile and efficiently bind complement enhancing borreliacidal effects in vitro.⁵³⁻⁵⁵ However, in previous experiments with mice hyperimmunized with OspA, complement was not required to kill spirochetes within nymphs or to block transmission from nymphs to the vaccinated host.⁵³ In our study, mRNA-OspA native and mRNA-HA-SS-OspA were found to elicit the most efficient in vitro bactericidal activity, but only mice immunized with the mRNA-OspA native were 100% protected.

In further experiments, it would be interesting to assess the viability of spirochetes in the midgut and salivary glands of adult ticks (emerged from nymphs engorged on immunized mice). This would help us to determine whether the vaccines completely block the migration of spirochetes from the midgut to the salivary glands during tick feeding on immunized mice.

As shown in previous studies, Ab-mediated immunity to OspC can prevent the dissemination of *B. burgdorferi* in the host during early infection.⁵⁶,⁵⁷ In this scenario, the combination of both OspA and OspC in vaccine development is expected to facilitate the induction of humoral immune responses that synergistically target spirochetes in both ticks and mammals. This combination has been shown to be effective in dogs, with VanguardrLyme being the only chimeric recombinant canine Lyme vaccine that helps to provide broad-spectrum coverage against OspA, found in the tick, and seven types of OspC found in the dog.⁵⁸ The vaccine has demonstrated broad efficacy in protecting dogs against infection and preventing the onset of clinical manifestations of LB.⁵⁹

Despite the prevalence of LB, no licensed human vaccine is currently available. In this study, we demonstrate that a novel mRNA-OspA vaccine has the potential to induce protective immunity against tick-borne *B. burgdorferi* infection. The immunogenicity and protective efficacy of the mRNA-OspA vaccine exceeded that of the adjuvanted and non-adjuvanted recombinant OspA proteins including the canine Lyme vaccine Recombitek. Although primarily used for viral infections, these results suggest that the mRNA-LNP vaccine platform may also be effective in preventing bacterial infections. With further preclinical and clinical development, the mRNA-OspA-LNP vaccine could become a viable strategy for the prevention of LB and could be combined with mRNA encoding OspC antigens to cover the circulating serotypes in endemic areas.

# MATERIALS AND METHODS

## Protein and mRNA vaccine designs and production

The sequences for OspA ST1 and ST2 were that of *B. burgdorferi* strain B31 (GenBank: WP_010890378.1) and *Borrelia afzelii* strain PKO (GenBank: WP_011703777.1), respectively. The ferritin sequence was that of *Helicobacter pylori* (GenBank: WQU12245.1). OspA-ferritin fusions were designed through a short flexible linker (GS) followed by a short sequence (ESQVRSQF) from bull frog ferritin added to the amino terminus of the *H. pylori* ferritin to

help with radial projection of the antigen from the core. A few additional point mutations were introduced in the ferritin part to enhance recombinant expression for various reasons (glycosylation, cysteine removal, deamination site, etc.).

The OspA ST1 sequence was mutated according to Gross et al. to eliminate a putative human LFA-1 epitope.⁶⁰ All sequences were synthesized (Genscript) and subcloned into proper vectors for recombinant expression in *E. coli* (ferritin fusions) or mRNA synthesis to produce mRNAs.

The following four mRNAs were produced as previously described⁶¹: (1) mRNA-OspA-ST1-native, (2) with the HA-SS (mRNA-HA-SS-OspA-ST1-native), (3) a deglycosylated OspA sequence (mRNA-HA-SS-OspA-ST1-glyco(-)), and (4) with an HA TMB domain for cell surface expression (mRNA-TMB-OspA-ST1-native). Briefly, mRNAs encoding the amino acid of OspA ST1 (Table S2) were synthesized by *in vitro* transcription using RNA polymerase with a plasmid DNA template encoding the desired gene using unmodified nucleotides. The resulting purified precursor mRNA was reacted further via enzymatic addition of a 5' cap structure (Cap 1) and a 3' poly(A) tail of approximately 200 nucleotides in length as determined by gel electrophoresis.

## LNP formulation for mRNA vaccines

Preparation of mRNA/LNP formulations was described previously.⁶² In brief, an ethanolic solution containing a mixture of lipids (ionizable lipid, phosphatidylethanolamine, cholesterol, and polyethylene glycol-lipid) at a fixed lipid-to-mRNA ratio was combined with an aqueous buffered solution of target mRNA at an acidic pH under controlled conditions, resulting in a suspension of uniform LNPs. The nanoparticle suspensions were then subjected to ultrafiltration and diafiltration into a suitable diluent system (10% trehalose), diluted to the desired final concentration, filtered, and stored frozen at -80°C until use. The final pH was neutral, the size between 100 and 130 nm (using Dynamic Light Scattering) and an encapsulation efficiency (EE) of 90%-93%. The quantification of total mRNA and incorporation efficiency (%EE) was performed using the Ribogreen assay.

## mRNA *in vitro* expression

HEK293T cells in suspension (5 mL at 2 × 10⁶ cells/mL; shaker 125 mL) were transfected with 5 µg naked mRNA at 1 µg/µL mixed with equal volume of TransIT-mRNA Reagent and mRNA Boost Reagent-TransIT-mRNA Transfection Kit Mirus (Ref MIR 2250) for 2-5 min. The solution was added to the cells and incubated at 37°C, 100 rpm, 8% CO₂ for 48-72 h.

For western blot analysis of mRNA-transfected cells, after transfection, cells and medium were collected and centrifuged (500×g) to collect supernatants. The cell pellets were lysed using Lysozyme (Ready-Lyse Lysozyme Solution- Lucigen ref R1804M), Benzonase (Sigma-ref E1014), and a protease inhibitor cocktail (Sigma-ref P8340) for 10 min at 20°C under 800 rpm. The cell pellet was then centrifuged (11,000×g) to collect supernatants and crude extracts.
Extracts from mRNA-transfected HEK293T cells were denatured (95°C) and analyzed by both polyacrylamide gel electrophoresis using 4%-12% Bis-Tris/MES gel (Invitrogen) and western blot. Transfer to a nitrocellulose membrane (Bio-Rad) was performed using a semi-dry transfer system (Trans-Blot Turbo Transfer System, Bio-Rad). Blotted proteins were detected with polyclonal (rabbit) Abs that recognize OspA (anti-OspA polyclonal/Rabbit; Abcam, ref ab10608; 1:2,000) and a secondary Ab (anti-rabbit IgG Goat Antibody DyLight 800; Rockland, ref. 611-145-002; 1:2,000). Blots were imaged with Odyssey Infrared Imager (LICORbio).

## Characterization of mRNA encoded OspA ST1 antigenicity by sandwich ELISA with functional monoclonal Abs

For characterization with the OspA ST1-specific functional monoclonal Ab (mAb) LA-2, capture mAb 857-2 (R&D Biotech, Internal Order)³³ was coated at 2.5 µg/mL in PBS on microtiter plates (Greiner Bio-one). Plates were incubated overnight at 4°C and then blocked with PBS-Tween 0.05%-milk 5% for 1 h at room temperature (RT). Transfection supernatants were serially diluted 2-fold in dilution buffer (PBS-Tween 0.05%-milk 1%) and incubated for 1.5 h at RT. After PBS-0.05% Tween washes, detection of proteins attached to the coating were performed by incubation with the mAb LA-2 at 1:1,000 (Absolute Antibody, Cat. Ab01070-3.0-BT) for 1.5 h at RT and then with a goat anti-mouse IgG horseradish peroxidase (HRP) (Jackson Laboratories). After washes, plates were developed using 3,3,5,5-tetramethylbenzidine (TMB; Tebu-bio, cat. TMB100-1000) for 30 min at RT in the dark and stopped with 1 normal (N) solution of HCl (VWR ProLabo). Optical densities (ODs) were read at 450 nm and 650 nm.

For characterization with the cross-specific anti-OspA functional mAbs 857-2 and 221-7,³³ the mAbs were coated at 5 µg/mL in PBS on microtiter plates (Greiner Bio-one, cat. 655061). Plates were incubated overnight at 4°C and then blocked with PBS-Tween 0.05%-milk 5% for 1 h at RT. Transfection supernatants were serially diluted 2-fold in dilution buffer (PBS-Tween 0.05%-milk 1%) and incubated for 1.5 h at RT. After PBS-0.05% Tween washes, detection of proteins attached to the coating were performed by incubation at RT of an anti-OspA mouse polyclonal serum (internal) for 1.5 h. Plates were washed and incubated with a goat anti-mouse IgG HRP (Jackson Laboratories, cat. 115-036-062) for 1.5 h at RT. After washing, plates were developed using TMB for 30 min in the dark at RT. The colorimetric reaction was stopped with 1 N HCl (VWR Prolabo, cat 30024290). ODs were read at 450 nm and 650 nm.

## Production of antigens used as protein benchmark comparators

mRNA-OspA vaccine candidates were compared with the benchmark Lyme dog non-adjuvanted vaccine Recombitek Lyme (Boehringer Ingelheim) at 1 µg/dose (50 µL) and OspA-ferritin (ST1 and ST1 + ST2) at 1.7 µg/dose + AF03 adjuvant (2.5% squalene-based adjuvant). OspA-ferritin ST1 and ST2 antigens were produced by Sanofi Breakthrough Lab according to the Material and method previously published.¹⁶

## Mice and ethical statement

Specific-pathogen-free C3H/HeNRj mice (females, 5 weeks-old) were purchased from Janvier Labs. Mice were housed under pathogen-free conditions and given free access to food and water. Animals were housed in approved animal biosafety level two facilities and all experiments were performed according to the standards of animal welfare and procedures approved by the Institutional Animal Care and Use Committee at Pasteur Institute and the French Ministry of Education, Research and Innovation (APAFIS#20705-2019052010239405). Furthermore, all animal experiments were conducted in strict compliance with European guidelines (EU directive on animal testing 2010/63/EU).

## Bacterial strains and culture conditions

The low-passage-number, infectious Bbss used in this experiment is a clone of type strain B31 (ATCC 35210; American Type Culture Collection). *Borrelia* cultures were maintained in complete Barbour-Stoenner-Kelly medium (BSK-H complete, Sigma Aldrich) at 33°C. The spirochete cell viability, motility and concentration were determined using a DFM by counting spirochetes in a Petroff Hausser counting chamber (Hausser Scientific).

## Ticks and Bbss-infected tick generation

All larvae of *I. ricinus* ticks used in this study were obtained from Insect Services laboratory (Insect Services GmbH, Berlin, Germany). Ticks were maintained at 18°C-20°C and relative humidity of 90%-95% until their utilization. C3H/HeNRj mice at 7 weeks of age were inoculated intradermally with 100 µL of BSK-H containing Bbss strain B31 (equivalent of $10^5$ *Borrelia/mouse*). The inoculation was done on the median line on the lower back region after being shaved. The infectious status of each mouse was determined by nested PCR (nPCR) using DNA extracted from ear biopsies performed 3 and 4 weeks after inoculation. All positive mice were then infested with pathogen-free *I. ricinus* ticks (~200 larvae/mouse) and allowed to feed to repletion. Fully engorged ticks were collected, washed two times with sterile water then dried before being incubated in optimal conditions (22°C and 90% ± 5% relative humidity) for their molting into nymphs. To determine the percentage of ticks infected with *B. burgdorferi*, DNA from 96 randomly selected ticks (nymphs) were extracted and tested by nPCR as described later in details. About 3000 infected ticks (nymphs) were generated and used for this experiment. The infection rate determined by nPCR was 70.8% (68/96).

## Mouse immunization experiment

One hundred pathogen-free C3H/HeNRj mice were included in this trial. On day -1, mice were randomly assigned to 10 treatment groups of 10 mice each (Table 2). Mice from each treatment group received two doses of recombinant OspA or mRNA-OspA vaccine candidates respectively at days 0 and 21 (Figure 2). The dose and composition of each vaccine candidate are detailed in Table 2. Mice were immunized via intramuscular injection of 50 µL of each vaccine preparation into the quadriceps muscle. All animals were monitored for local reactions or clinical signs p.i.
<table><caption>Table 2. Experimental animal vaccine composition/preparation, and administration vaccine dose to each mouse group</caption><thead><tr><th>Group name</th><th>Target antigen</th><th>Vaccine description</th><th>Final injected volume (concentration per mouse)</th></tr></thead><tbody><tr><td>OspA-ferritin ST1 + AF03</td><td>OspA of Bbss, serotype 1</td><td>non-lipidated OspA coupled to the ferritin nanoparticles ST1 in 2.5% squalene-based adjuvant (AF03)</td><td>50 µL (1.7 µg/dose)</td></tr><tr><td>OspA-ferritin ST1 + OspA-ferritin ST2 + AF03</td><td>OspA of Bbss (serotype 1) and B. afzelii (serotype 2)</td><td>non-lipidated OspA coupled to the ferritin nanoparticles serotype 1 (1.7 µg/dose) and serotype 2 (1.7 µg/dose) in 2.5% squalene-based adjuvant (AF03)</td><td>50 µL (1.7 µg of each antigen/dose)</td></tr><tr><td>mRNA-OspA-ST1-native</td><td>OspA of Bbss, serotype 1</td><td>mRNA-LNP: LNP-formulated, mRNA vaccine coding for OspA ST1. The administered dose was prepared in 10% trehalose</td><td>50 µL (10 µg/dose)</td></tr><tr><td>mRNA-HA-SS- OspA ST1-native</td><td>OspA of Bbss, serotype 1</td><td>mRNA-LNP coding for OspA ST1 with a SS from flu HA. The administered dose was prepared in 10% trehalose</td><td>50 µL (10 µg/dose)</td></tr><tr><td>mRNA-HA-SS- OspA ST1-glyco(-)</td><td>OspA of Bbss, serotype 1</td><td>mRNA-LNP coding for OspA ST1 with a -SS from HA and mutation of N-glycosylation sites. The administered dose was prepared in 10% trehalose</td><td>50 µL (10 µg/dose)</td></tr><tr><td>mRNA-TMB-OspA ST1-native</td><td>OspA of Bbss, serotype 1</td><td>mRNA-LNP coding for OspA ST1 with a SS and a TMB domain from HA. The administered dose was prepared in 10% trehalose</td><td>50 µL (10 µg/dose)</td></tr><tr><td>Recombitek Lyme (veterinary licensed vaccine)</td><td>OspA of Bbss, serotype 1</td><td>Recombitek Lyme (ST1), a lipidated OspA (0.1 mg/mL) non-adjuvanted vaccine, prepared in PBS</td><td>50 µL (1 µg/dose)</td></tr><tr><td>Empty ferritin + AF03 (negative control)</td><td>-</td><td>Empty ferritin nanoparticles in 2.5% squalene-based adjuvant (AF03)</td><td>50 µL (1.4 µg/dose)</td></tr><tr><td>Empty LNPs (negative control)</td><td>-</td><td>empty LNPs (mRNA free) prepared in 10% trehalose</td><td>50 µL</td></tr><tr><td>PBS (negative control)</td><td>-</td><td>phosphate-buffered saline solution</td><td>50 µL</td></tr></tbody></table>

## OspA-specific IgG titer determination by ELISA

A total of seven blood samples (about 300 µL) were collected from the submandibular vein on days 0, 21, 28, 35, 45, 70, and 92 p.i. (Figure 2) to determine seroconversion and measure the kinetics of anti-Bbss IgG titers (anti-OspA). Briefly, 96-well ELISA plates (CAT#655 061, Greiner Bio-One) were coated with 1 µg/mL Bbss antigen (OspA His-tag) and incubated overnight at +4°C then blocked in 5% powdered milk for 1 h. Serum was individually (or pooled from 10 mice per group) diluted in PBS-Tween 0.05% with 1% milk, and serial dilutions were applied to the plate and incubated for 1.5 h at 37°C. After washing three times with PBS with 0.05% Tween (Tween 20), plates were then incubated with HRP-conjugated goat anti-mouse IgG Ab (1:5,000; diluted in PBS-Tween 0.05% with 1% powdered milk PBS) for 1.5 h at 37°C. Finally, TMB was used as substrate for 30 min in the dark at RT. The colorimetric reaction was stopped with 1N HCl (Riel-de-Haen). OD was measured at 450 nm by a Tristar 5 multimode microplates Reader (Berthold Technologies). Specific OspA IgG titers were quantified through an internal pool of positive sera reference. The titer of this reference was previously calculated as the reciprocal dilution to obtain an OD of 1. OD values were converted into Ab titers expressed in arbitrary units.

The determination of Bbss-specific Abs (IgG subclasses) was performed by ELISA using anti-isotypes IgG1 (1071-05), IgG2a (A90-107P), IgG2b (1093-05), and IgG2c (A90-136P; Southern Biotech) following the same described protocol.

## Mouse challenge with Bbss-infected ticks

On day 35, each mouse was infested with 12 Bbss-infected *I. ricinus* nymphs. After 5–6 days of tick attachment, fully engorged nymphs were collected and counted. In this trial, it was determined that if the count of engorged ticks (fed to repletion) reached at least five specimens, the respective mouse was categorized as correctly infested. However, in case where the number of engorged nymphs was less than five specimens, the mouse was immediately re-infested with 6 ± 2 ticks.

During the infestation phase, mice were housed in single-use individually ventilated mouse cages (Innovive). Once an animal was infested, any further handling was done inside isolators (Figure S5) designed to handle animals infected with pathogens from level 2 (or more) as required by internal rules of the Institut Pasteur.

## Determination of infectious status of mice

All mice were assayed for infection by different *ante-* and *post-mortem* screening tests. Mice were considered positive (infected) when any techniques demonstrated the presence of *B. burgdorferi* in the live animal, or when *B. burgdorferi* DNA was detected either in ticks (i.e., xenodiagnoses) or in biopsy/tissue of the sacrificed animal.

### Ante-mortem screening

#### Ear biopsies

Ear punch biopsies were collected once a week from day 0 to day 105 (Figure 2). Total DNA from biopsy samples was extracted using the DNeasy blood and tissue kit protocol from QIAGEN according to
the manufacturer's recommendations (QIAGEN) and then analyzed for *Borrelia* DNA as described below.

## Xenodiagnosis

Around 60 larval ticks (pathogen-free *I. ricinus*) were used in this assay. Ticks were placed on each mouse on day 75 p.i. (corresponding with day 40 post-infestation) (Figure 2). Larvae were allowed to feed to repletion, collected, and then kept in optimal conditions till their molting Ticks (n = 10/mouse) were selected, and DNA was extracted individually for PCR testing against *Borrelia*.

## Post-mortem screening

### Tissue culture for Bbss isolation

One-half of the mice were euthanized 70 days after exposure to Bbss-infected ticks, which corresponds to day 105 p.i. The other one-half were euthanized 105 days after tick exposure, corresponding with day 140 p.i. (Figure 2). Bladder, heart, and meninge tissues were aseptically sampled at necropsy for culture and/or PCR testing. Except for meninges, which were immediately placed into culture tubes, bladder and heart specimens were cut into pieces, ground, and homogenized in sterile Petri dish. The blended samples were divided into two equal parts. The first one-half was cultured in tubes containing BSK-H growth medium supplemented with 50 µg/mL rifampicin, 100 µg/mL Fosfomycin, and 5 µg/mL amphotericin. Tubes were incubated at 34°C and the cultures were examined weekly over a 6-week period to detect the presence of *B. burgdorferi* by darkfield microscopy. The second one-half of the blended samples was placed into tubes and kept at -80°C for further molecular biology analyses.

### Tissue samples screening by nPCR

Total genomic DNA was extracted from each blended samples (bladder and heart) using the QIAamp Tissue Kit (QIAGEN). nPCR amplifying a 250 base pairs (bp) fragment was performed using two pairs of primers as described previously.⁴⁵ Negative controls were processed with DNA-free water and DNA from non-infected tissues, and the positive control was genomic DNA of *B. afzelii*.

### Serum bactericidal assay

SBAs against Bbss were performed according to a protocol developed in house (protocol S1) based on literature on SBA and *Borrelia*.²⁶,³³ Here, Bbss B31 culture was done as described above, and the culture was adjusted with fresh BSK-H medium to obtain a final concentration of 10⁷ cells/mL. sera sampled from mice on D45 p.i. were used in this assay. Serum samples were pooled and homogenized. First, pooled serum samples were sterilized by passing through 0.2 µm-pore syringe filters (Sigma-Aldrich). The filtered sera are pre-diluted 1:4 in BSK-H medium, then a 2-fold serial dilution is performed. Serum samples were then heat-inactivated at 56°C for 30 min to remove endogenous complement activity. We added 50 µL of sterilized guinea pig complement sera (S1639-5ML; Sigma Aldrich) pre-diluted to 20% with sterile purified water to 100 µL of each serum dilution. A total of 50 µL BSK-H medium containing 10⁷ spirochetes/mL were added into and mixed with above-prepared serum samples. There-

fore, the final concentration of Bbss in each microtube (v = 200 µL) was 2.5 × 10⁶ spirochetes/mL. The SBA assay was also performed with BSK-H medium containing bacteria and complement only (no serum) as a negative control. All microtubes were incubated at 33°C for 48 h.

Viable numbers of Bbss were determined on each serum pool dilution (12 consecutive dilutions) using a DF microscope by counting spirochetes in a Petroff Hauser counting chamber. The serum bactericidal Ab titer was defined as the reciprocal of the highest serum dilution that produced 50% or greater killing compared with the killing observed for the negative control groups (empty ferritin, empty LNPs and PBS).

### Statistical analysis

Data generated during the study were entered into a Microsoft Excel (MS Excel 2016) database and analyzed using Prism version 10 software package (GraphPad, San Diego, CA, USA). First, the Shapiro-Wilk test was performed to verify the normality of the distribution. One-way ANOVA followed by Tukey's correction tests for multiple comparisons were used to test for statistical significance among immunized groups and the controls. Differences were considered significant if the *p* value was ≤0.05 (*), ≤0.01 (**), ≤0.001 (***), or ≤0.0001 (****).

The effectiveness of each vaccine candidate was calculated using the following formula:

$$ \text{Vaccine effectiveness (\%)} = 100 \times \frac{\text{NVAR-VAR}}{\text{NVAR}}, \text{ where NVAR and VAR are the attack rate in control non-vaccinated and vaccinated groups, respectively.} $$

## DATA AND CODE AVAILABILITY

All data are available within the article and its supplemental information file or from the authors upon request.

## ACKNOWLEDGMENTS

The authors thank Malaika Mohammad, Louise Laserre, Maewenn Tellier, and Olivia Meffre from Institut Pasteur for their technical assistance. We also extend our gratitude to Professor George Chaconas from the University of Calgary, Canada, for providing the Bbss strain GCB used in the SBA assessment. Last, we acknowledge Jean-Sébastien Bolduc from Sanofi for his critical review of this manuscript. The project was funded by Sanofi (Reference: S-RD20008-02A -CCSANOFI 2020 CONTRAT APPLIC CHOUMET) and the Institut Pasteur (Reference: 24502E Projet principal).

## AUTHOR CONTRIBUTIONS

mRNA and subunit vaccine candidate designs, production, characterization, and initial immunogenicity studies in mice for pre-selection of the mRNA vaccine candidates (as described in patent applications WO2023111262 and WO2023214082): V.P., Y.G.C, C.C., and C.P. Conceptualization of the immuno-challenge mouse study, V.C. and V.P.; methodology, V.C., D.T., C.C., S.M., E.F., and V.P.; validation, V.C., D.T., E.F., and V.P.; formal analysis, V.C., D.T., H.B., V.G., C.C., S.M., E.F., and V.P.; resources, V.C. and V.P.; data curation, V.C., D.T., S.M., E.F., and V.P.; writing—original draft preparation, D.T., E.F., and V.C.; writing—review and editing, D.T., E.F., V.C., and V.P.; funding acquisition, V.P. and V.C.

All authors have read and agreed to the published version of the manuscript.
## DECLARATION OF INTERESTS

Authors have declared the following interests: V.P., C.C., Y.G.C., S.M., and C.P. are Sanofi employees and may own Sanofi shares.

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.omtn.2025.102514.

## REFERENCES

1. van den Wijngaard, C.C., Hofhuis, A., Simões, M., Rood, E., van Pelt, W., Zeller, H., and Van Bortel, W. (2017). Surveillance perspective on Lyme borreliosis across the European Union and European Economic Area. Euro Surveill. 22, 1-9. https://doi.org/10.2807/1560-7917.ES.2017.22.30569.

2. Brisson, D., Drecktrah, D., Eggers, C.H., and Samuels, D.S. (2012). Genetics of Borrelia burgdorferi. Annu. Rev. Genet. 46, 515-536. https://doi.org/10.1146/annurev-genet-011112-112140.

3. Wilske, B., Preac-Mursic, V., Göbel, U.B., Graf, B., Jauris, S., Soutschek, E., Schwab, E., and Zumstein, G. (1993). An OspA serotyping system for *Borrelia burgdorferi* based on reactivity with monoclonal antibodies and OspA sequence analysis. J. Clin. Microbiol. 31, 340-350. https://doi.org/10.1128/jcm.31.2.340-350.1993.

4. Stanek, G., and Reiter, M. (2011). The Expanding Lyme Borrelia Complex-Clinical Significance of Genomic Species? Clin Microbiol Infect 17, 487. https://doi.org/10.1111/j.1469-0691.2011.03492.x.

5. Collares-Pereira, M., Couceiro, S., Franca, I., Kurtenbach, K., Schäfer, S.M., Vitorino, L., Gonçalves, L., Baptista, S., Vieira, M.L., and Cunha, C. (2004). First Isolation of *Borrelia lusitaniae* from a Human Patient. J. Clin. Microbiol. 42, 1316-1318. https://doi.org/10.1128/JCM.42.3.1316-1318.2004.

6. Marques, A.R., Strle, F., and Wormser, G.P. (2021). Comparison of Lyme Disease in the United States and Europe. Emerg. Infect. Dis. 27, 2017-2024. https://doi.org/10.3201/eid2708.204763.

7. Eisen, L. (2020). Vector competence studies with hard ticks and Borrelia burgdorferi sensu lato spirochetes: A review. Ticks Tick. Borne. Dis. 11, 101359. https://doi.org/10.1016/j.ttbdis.2019.101359.

8. Strle, F., and Stanek, G. (2009). Clinical Manifestations and Diagnosis of Lyme Borreliosis. In *Lyme Borreliosis (KARGER)*, pp. 51-110. https://doi.org/10.1159/000213070.

9. Murray, T.S., and Shapiro, E.D. (2010). Lyme disease. Clin. Lab. Med. 30, 311-328. https://doi.org/10.1016/j.cll.2010.01.003.

10. Adrion, E.R., Aucott, J., Lemke, K.W., and Weiner, J.P. (2015). Health Care Costs, Utilization and Patterns of Care following Lyme Disease. PLoS One 10, e0116767. https://doi.org/10.1371/journal.pone.0116767.

11. Maksimyan, S., Syed, M.S., and Soti, V. (2021). Post-Treatment Lyme Disease Syndrome: Need for Diagnosis and Treatment. Cureus 13, e18703. https://doi.org/10.7759/cureus.18703.

12. DeLong, A., Hsu, M., and Kotsoris, H. (2019). Estimation of cumulative number of post-treatment Lyme disease cases in the US, 2016 and 2020. BMC Public Health 19, 352. https://doi.org/10.1186/s12889-019-6681-9.

13. Stone, B.L., Tourand, Y., and Brissette, C.A. (2017). Brave New Worlds: The Expanding Universe of Lyme Disease. Vector Borne Zoonotic Dis. 17, 619-629. https://doi.org/10.1089/vbz.2017.2127.

14. O'Bier, N.S., Hatke, A.L., Camire, A.C., and Marconi, R.T. (2021). Human and Veterinary Vaccines for Lyme Disease. Curr. Issues Mol. Biol. 42, 191-222. https://doi.org/10.21775/cimb.042.191.

15. Kenedy, M.R., Lenhart, T.R., and Akins, D.R. (2012). The role of *Borrelia burgdorferi* outer surface proteins. FEMS Immunol. Med. Microbiol. 66, 1-19. https://doi.org/10.1111/j.1574-695X.2012.00980.x.

16. Kamp, H.D., Swanson, K.A., Wei, R.R., Dhal, P.K., Dharanipragada, R., Kern, A., Sharma, B., Sima, R., Hajdusek, O., Hu, L.T., et al. (2020). Design of a broadly reactive Lyme disease vaccine. NPJ Vaccines 5, 33. https://doi.org/10.1038/s41541-020-0183-8.

17. Chen, W.-H., Strych, U., Bottazzi, M.E., and Lin, Y.-P. (2022). Past, present, and future of Lyme disease vaccines: antigen engineering approaches and mechanistic in-

sights. Expert Rev. Vaccines 21, 1405-1417. https://doi.org/10.1080/14760584.2022.2102484.

18. Dattwyler, R.J., and Gomes-Solecki, M. (2022). The year that shaped the outcome of the OspA vaccine for human Lyme disease. NPJ Vaccines 7, 10. https://doi.org/10.1038/s41541-022-00429-5.

19. Hovius, J.W.R., van Dam, A.P., and Fikrig, E. (2007). Tick-host-pathogen interactions in Lyme borreliosis. Trends Parasitol. 23, 434-438. https://doi.org/10.1016/j.pt.2007.07.001.

20. Hodzic, E., Tunev, S., Feng, S., Freet, K.J., and Barthold, S.W. (2005). Immunoglobulin-regulated expression of *Borrelia burgdorferi* outer surface protein A in vivo. Infect. Immun. 73, 3313-3321. https://doi.org/10.1128/IAI.73.6.3313-3321.2005.

21. Comstedt, P., Hanner, M., Schüler, W., Meinke, A., and Lundberg, U. (2014). Design and Development of a Novel Vaccine for Protection against Lyme Borreliosis. PLoS One 9, e113294. https://doi.org/10.1371/journal.pone.0113294.

22. Sigal, L.H., Zahradnik, J.M., Lavin, P., Patella, S.J., Bryant, G., Haselby, R., Hilton, E., Kunkel, M., Adler-Klein, D., Doherty, T., et al. (1998). A Vaccine Consisting of Recombinant *Borrelia burgdorferi* Outer-Surface Protein A to Prevent Lyme Disease. N. Engl. J. Med. 339, 216-222. https://doi.org/10.1056/NEJM199807233390402.

23. Steere, A.C., Sikand, V.K., Meurice, F., Parenti, D.L., Fikrig, E., Schoen, R.T., Nowakowski, J., Schmid, C.H., Laukamp, S., Buscarino, C., and Krause, D.S. (1998). Vaccination against Lyme Disease with Recombinant *Borrelia burgdorferi* Outer-Surface Lipoprotein A with Adjuvant. N. Engl. J. Med. 339, 209-215. https://doi.org/10.1056/NEJM199807233390401.

24. Sikand, V.K., Halsey, N., Krause, P.J., Sood, S.K., Geller, R., Van Hoecke, C., Buscarino, C., and Parenti, D.; Pediatric Lyme Vaccine Study Group (2001). Safety and Immunogenicity of a Recombinant *Borrelia burgdorferi* Outer Surface Protein A Vaccine Against Lyme Disease in Healthy Children and Adolescents: A Randomized Controlled Trial. Pediatrics 108, 123-128. https://doi.org/10.1542/peds.108.1.123.

25. Van Hoecke, C., Lebacq, E., Beran, J., and Parenti, D. (1999). Alternative Vaccination Schedules (0, 1, and 6 Months Versus 0, 1, and 12 Months) for a Recombinant OspA Lyme Disease Vaccine. Clin. Infect. Dis. 28, 1260-1264. https://doi.org/10.1086/514779.

26. Luke, C.J., Marshall, M.A., Zahradnik, J.M., Bybel, M., Menefee, B.E., and Barbour, A.G. (2000). Growth-Inhibiting Antibody Responses of Humans Vaccinated with Recombinant Outer Surface Protein A or Infected with *Borrelia burgdorferi* or Both. J. Infect. Dis. 181, 1062-1068. https://doi.org/10.1086/315359.

27. Comstedt, P., Schüler, W., Meinke, A., and Lundberg, U. (2017). The novel Lyme borreliosis vaccine VLA15 shows broad protection against *Borrelia* species expressing six different OspA serotypes. PLoS One 12, e0184357. https://doi.org/10.1371/journal.pone.0184357.

28. Bézay, N., Hochreiter, R., Kadlecek, V., Wressnigg, N., Larcher-Senn, J., Klingler, A., Dubischar, K., Eder-Lingelbach, S., Leroux-Roels, I., Leroux-Roels, G., and Bender, W. (2023). Safety and immunogenicity of a novel multivalent OspA-based vaccine candidate against Lyme borreliosis: a randomised, phase 1 study in healthy adults. Lancet Infect. Dis. 23, 1186-1196. https://doi.org/10.1016/S1473-3099(23)00210-4.

29. Pine, M., Arora, G., Hart, T.M., Bettini, E., Gaudette, B.T., Muramatsu, H., Tombácz, I., Kambayashi, T., Tam, Y.K., Brisson, D., et al. (2023). Development of an mRNA-lipid nanoparticle vaccine against Lyme disease. Mol. Ther. 31, 2702-2714. https://doi.org/10.1016/j.ymthe.2023.07.022.

30. Al Fayez, N., Nassar, M.S., Alshehri, A.A., Alnefaie, M.K., Almughem, F.A., Alshehri, B.Y., Alawad, A.O., and Tawfik, E.A. (2023). Recent Advancement in mRNA Vaccine Development and Applications. Pharmaceutics 15, 1972. https://doi.org/10.3390/pharmaceutics15071972.

31. Embers, M.E., and Narasimhan, S. (2013). Vaccination against Lyme disease: past, present, and future. Front. Cell. Infect. Microbiol. 3, 6. https://doi.org/10.3389/fcimb.2013.00006.

32. Van Hoecke, C., Comberbach, M., De Grave, D., Desmons, P., Fu, D., Hauser, P., Lebacq, E., Lobet, Y., and Voet, P. (1996). Evaluation of the safety, reactogenicity and immunogenicity of three recombinant outer surface protein (OspA) lyme
vaccines in healthy adults. Vaccine **14**, 1620-1626. https://doi.org/10.1016/s0264-410x(96)00146-6.

33. Wang, Y., Kern, A., Boatright, N.K., Schiller, Z.A., Sadowski, A., Ejemel, M., Souders, C.A., Reimann, K.A., Hu, L., Thomas, W.D., and Klempner, M.S. (2016). Pre-exposure Prophylaxis With OspA-Specific Human Monoclonal Antibodies Protects Mice Against Tick Transmission of Lyme Disease Spirochetes. J. Infect. Dis. **214**, 205-211. https://doi.org/10.1093/infdis/jiw151.

34. Kugeler, K.J., Schwartz, A.M., Delorey, M.J., Mead, P.S., and Hinckley, A.F. (2021). Estimating the Frequency of Lyme Disease Diagnoses, United States, 2010-2018. Emerg. Infect. Dis. **27**, 616-619. https://doi.org/10.3201/eid2702.202731.

35. Sykes, R.A., and Makiello, P. (2017). An estimate of Lyme borreliosis incidence in Western Europe. J. Public Health **39**, 74-81. https://doi.org/10.1093/pubmed/fdw017.

36. Chung, M.K., Caboni, M., Strandwitz, P., D'Onofrio, A., Lewis, K., and Patel, C.J. (2023). Systematic comparisons between Lyme disease and post-treatment Lyme disease syndrome in the U.S. with administrative claims data. EBioMedicine **90**, 104524. https://doi.org/10.1016/j.ebiom.2023.104524.

37. Eschner, A.K., and Mugnai, K. (2015). Immunization with a recombinant subunit OspA vaccine markedly impacts the rate of newly acquired *Borrelia burgdorferi* infections in client-owned dogs living in a coastal community in Maine, USA. Parasit. Vectors **8**, 92. https://doi.org/10.1186/s13071-015-0676-x.

38. Barthold, S.W., Beck, D.S., Hansen, G.M., Terwilliger, G.A., and Moody, K.D. (1990). Lyme Borreliosis in Selected Strains and Ages of Laboratory Mice. J. Infect. Dis. **162**, 133-138. https://doi.org/10.1093/infdis/162.1.133.

39. Barthold, S.W., Persing, D.H., Armstrong, A.L., and Peeples, R.A. (1991). Kinetics of *Borrelia burgdorferi* dissemination and evolution of disease after intradermal inoculation of mice. Am. J. Pathol. **139**, 263-273.

40. Armstrong, A.L., Barthold, S.W., Persing, D.H., and Beck, D.S. (1992). Carditis in Lyme Disease Susceptible and Resistant Strains of Laboratory Mice Infected with *Borrelia Burgdorferi*. Am. J. Trop. Med. Hyg. **47**, 249-258. https://doi.org/10.4269/ajtmh.1992.47.249.

41. Barthold, S.W., de Souza, M.S., Janotka, J.L., Smith, A.L., and Persing, D.H. (1993). Chronic Lyme borreliosis in the laboratory mouse. Am. J. Pathol. **143**, 959-971.

42. Bockenstedt, L.K., Fikrig, E., Barthold, S.W., Kantor, F.S., and Flavell, R.A. (1993). Inability of truncated recombinant Osp A proteins to elicit protective immunity to *Borrelia burgdorferi* in mice. J. Immunol. **151**, 900-906.

43. Wilczek, C.K., Wenderlein, J., Hiereth, S., and Straubinger, R.K. (2022). A Retrospective Study with a Commercial Vaccine against Lyme Borreliosis in Dogs Using Two Different Vaccination Schedules: Characterization of the Humoral Immune Response. Vaccines **11**, 43. https://doi.org/10.3390/vaccines11010043.

44. Gern, L., Schaible, U.E., and Simon, M.M. (1993). Mode of Inoculation of the Lyme Disease Agent *Borrelia burgdorferi* Influences Infection and Immune Responses in Inbred Strains of Mice. J. Infect. Dis. **167**, 971-975. https://doi.org/10.1093/infdis/167.4.971.

45. Sertour, N., Cotté, V., Garnier, M., Malandrin, L., Ferquel, E., and Choumet, V. (2018). Infection Kinetics and Tropism of *Borrelia burgdorferi* sensu lato in Mouse After Natural (via Ticks) or Artificial (Needle) Infection Depends on the Bacterial Strain. Front. Microbiol. **9**, 1722. https://doi.org/10.3389/fmicb.2018.01722.

46. Jin, L., Jiang, B.-G., Yin, Y., Guo, J., Jiang, J.-F., Qi, X., Crispell, G., Karim, S., Cao, W.-C., and Lai, R. (2022). Interference with LTβR signaling by tick saliva facilitates transmission of Lyme disease spirochetes. Proc. Natl. Acad. Sci. USA **119**, e2208274119. https://doi.org/10.1073/pnas.2208274119.

47. Kasumba, I.N., Bestor, A., Tilly, K., and Rosa, P.A. (2016). Virulence of the Lyme disease spirochete before and after the tick bloodmeal: a quantitative assessment. Parasit. Vectors **9**, 129. https://doi.org/10.1186/s13071-016-1380-1.

48. Piesman, J. (1993). Dynamics of *Borrelia burgdorferi* Transmission by Nymphal *Ixodes dammini* Ticks. J. Infect. Dis. **167**, 1082-1085. https://doi.org/10.1093/infdis/167.5.1082.

49. Sajid, A., Matias, J., Arora, G., Kurokawa, C., DePonte, K., Tang, X., Lynn, G., Wu, M.-J., Pal, U., Strank, N.O., et al. (2021). mRNA vaccination induces tick resistance and prevents transmission of the Lyme disease agent. Sci. Transl. Med. **13**, eabj9827. https://doi.org/10.1126/scitranslmed.abj9827.

50. Fikrig, E. (2022). 16th International Conference on Lyme Borreliosis and other Tick-borne diseases (ICLB). In mRNA Vaccination Targeting Tick Antigens Induces Resistance to *Ixodes scapularis*, p. 41.

51. Perthame, E., Chartier, L., George, J.-C., Varloud, M., Ferquel, E., and Choumet, V. (2023). Case presentation and management of Lyme disease patients: a 9-year retrospective analysis in France. Front. Med. **10**, 1296486. https://doi.org/10.3389/fmed.2023.1296486.

52. Conlon, J.A., Mather, T.N., Tanner, P., Gallo, G., and Jacobson, R.H. (2000). Efficacy of a nonadjuvanted, outer surface protein A, recombinant vaccine in dogs after challenge by ticks naturally infected with *Borrelia burgdorferi*. Vet. Ther. **1**, 96-107.

53. Rathinavelu, S., Broadwater, A., and de Silva, A.M. (2003). Does Host Complement Kill *Borrelia burgdorferi* within Ticks? Infect. Immun. **71**, 822-829. https://doi.org/10.1128/IAI.71.2.822-829.2003.

54. Deenick, E.K., Hasbold, J., and Hodgkin, P.D. (2005). Decision criteria for resolving isotype switching conflicts by B cells. Eur. J. Immunol. **35**, 2949-2955. https://doi.org/10.1002/eji.200425719.

55. Collins, A.M. (2016). IgG subclass co-expression brings harmony to the quartet model of murine IgG function. Immunol. Cell Biol. **94**, 949-954. https://doi.org/10.1038/icb.2016.65.

56. Bockenstedt, L.K., Hodzic, E., Feng, S., Bourrel, K.W., de Silva, A., Montgomery, R.R., Fikrig, E., Radolf, J.D., and Barthold, S.W. (1997). *Borrelia burgdorferi* strain-specific Osp C-mediated immunity in mice. Infect. Immun. **65**, 4661-4667. https://doi.org/10.1128/iai.65.11.4661-4667.1997.

57. Izac, J.R., O'Bier, N.S., Oliver, L.D., Camire, A.C., Earnhart, C.G., LeBlanc Rhodes, D.V., Young, B.F., Parnham, S.R., Davies, C., and Marconi, R.T. (2020). Development and optimization of OspC chimerotope vaccinogens for Lyme disease. Vaccine **38**, 1915-1924. https://doi.org/10.1016/j.vaccine.2020.01.027.

58. Littman, M.P., Gerber, B., Goldstein, R.E., Labato, M.A., Lappin, M.R., and Moore, G.E. (2018). ACVIM consensus update on Lyme borreliosis in dogs and cats. J. Vet. Intern. Med. **32**, 887-903. https://doi.org/10.1111/jvim.15085.

59. Marconi, R.T., Garcia-Tapia, D., Hoevers, J., Honsberger, N., King, V.L., Ritter, D., Schwahn, D.J., Swearingin, L., Weber, A., Winkler, M.T.C., and Millership, J. (2020). VANGUARD®crLyme: A next generation Lyme disease vaccine that prevents *B. burgdorferi* infection in dogs. Vaccine X **6**, 100079. https://doi.org/10.1016/j.jvaccx.2020.100079.

60. Gross, D.M., Forsthuber, T., Tary-Lehmann, M., Etling, C., Ito, K., Nagy, Z.A., Field, J.A., Steere, A.C., and Huber, B.T. (1998). Identification of LFA-1 as a Candidate Autoantigen in Treatment-Resistant Lyme Arthritis. Science **281**, 703-706. https://doi.org/10.1126/science.281.5377.703.

61. Kalnin, K.V., Plitnik, T., Kishko, M., Zhang, J., Zhang, D., Beauvais, A., Anosova, N.G., Tibbitts, T., DiNapoli, J., Ulinski, G., et al. (2021). Immunogenicity and efficacy of mRNA COVID-19 vaccine MRT5500 in preclinical animal models. NPJ Vaccines **6**, 61. https://doi.org/10.1038/s41541-021-00324-5.

62. DeRosa, F., Smith, L., Shen, Y., Huang, Y., Pan, J., Xie, H., Yahalom, B., and Heartlein, M.W. (2019). Improved Efficacy in a Fabry Disease Model Using a Systemic mRNA Liver Depot System as Compared to Enzyme Replacement Therapy. Mol. Ther. **27**, 878-889. https://doi.org/10.1016/j.ymthe.2019.03.001.