mRNA vaccine targeting an outer surface protein provides protection against multiple Lyme disease strains

Infection status of the mice was determined by culturing spirochetes from explants of the mouse skin and ears. The presence of motile spirochetes was detected in the samples using dark-field microscopy. All mice injected with the empty LNP control were positive for B. burgdorferi infection in both the skin and ears (Fig. 1B), demonstrating robust infection via the needle challenge. In comparison, all skin and ear samples collected from mice vaccinated with OspC-A mRNA were negative for the presence of spirochetes. Furthermore, qPCR was performed to quantify the bacterial burden across a wider array of tissues, including the ear, bladder, lymph nodes, tibiotarsal joint, and heart. Significant B. burgdorferi DNA loads were detected in all of these tissues in the control mice, whereas the bacterial DNA was below the limit of detection in all tissues of the OspC-A mRNA vaccinated mice (Fig. 1C). Taken together, these results suggest that the candidate OspC-A mRNA vaccine completely protects against infection and dissemination of B. burgdorferi strains carrying OspC type A in mice one month after boost vaccination.

Dissemination of B. burgdorferi to the lymph nodes causes extensive extrafollicular proliferation of B cells⁵⁷. This accumulation of B cells leads to swelling of the lymph nodes systemically and especially close to the site of infection^58,59. Accordingly, we observed severe enlargement of the axillary lymph nodes in all control mice, whereas all OspC-A mRNA vaccinated mice had lymph nodes of a similar size to uninfected mice. In addition, white blood cell (WBC) counts from axillary lymph nodes of control mice were approximately fourfold higher than the WBC counts from the OspC-A mRNA group (Fig. 2C, D). Inflammation of the axillary lymph nodes in the control mice was characterized by marked follicular and medullary cord plasma cell hyperplasia and mild paracortical hyperplasia (Fig. 2E, F). Conversely, hyperplasia was not detected in any OspC-A mRNA vaccinated mice. In summary, these data demonstrate that the candidate OspC-A mRNA vaccine completely protects mice against carditis and lymphadenopathy associated with B. burgdorferi infection.

Enzyme-linked immunosorbent assay (ELISA) was used to measure anti-OspC antibody titers in the serum of mice collected at 4 weeks post-boost. Vaccination with OspC-A mRNA produced significant anti-OspC antibody titers relative to the control (Fig. 3B). OspC-A mRNA vaccination induced both IgG1 and IgG2a subtypes, with an IgG2a:IgG1 ratio greater than 1 (average = 5 ± 7.8), indicating a potential Th1-biased immune response (Fig. 3C, D).

Complement-mediated killing and phagocytosis are two of the principal mechanisms of B. burgdorferi clearance by the immune system^60–63. Antibody binding to B. burgdorferi surface proteins can mark the spirochetes for phagocytosis in a process referred to as opsonization or antibody-dependent cellular phagocytosis (ADCP). To evaluate whether the antibodies generated by vaccination with OspC-A mRNA were capable of inducing ADCP, fluorescently labeled B. burgdorferi were incubated with a RAW 267.4 mouse macrophage cell line in the presence of vaccinated serum. Since OspC is not highly expressed by B. burgdorferi in vitro, the B31-A3 strain used in this assay was engineered to constitutively express OspC (B. burgdorferi-OspC)^64,65. Serum from mice vaccinated with OspC-A mRNA induced significantly higher levels of phagocytosis than serum from the control group, demonstrated by a greater percentage of fluorescent macrophages and a higher median fluorescence intensity (Fig. 3E). Additionally, incubation of B. burgdorferi-OspC with serum from mice vaccinated with OspC-A mRNA in the presence of guinea pig serum, resulted in a twofold decrease in live spirochetes after 24 h, compared to the control vaccine (Fig. 3F). Overall, these data suggest that the antibody response elicited by the candidate vaccine is functional and capable of inducing pertinent effector functions.

OspC-ACIKN vaccination induced significant serum IgG titers at 3 weeks after the boost against all included OspC types relative to the control (Fig. 6B). Following bacterial challenge, infection status was determined by culture of the skin surrounding the bacterial injection site for 28 days, and live spirochetes were identified by dark-field microscopy. 90–100% of control mice were positive for motile spirochetes in the skin across all B. burgdorferi strains (Fig. 6C). In comparison, only 10% of the mice vaccinated with OspC-ACIKN mRNA were positive for motile spirochetes with the type A strain, 20% were positive for the type K strain, and no mice vaccinated with OspC-ACIKN were positive for the type I strain. Interestingly, OspC-ACIKN vaccination only reduced infection with type C and type N strains by 10% compared to the control. These results were further confirmed by quantification of the B. burgdorferi DNA burden in the ear, bladder, tibiotarsal joint, and heart, which found that vaccination with OspC-ACIKN significantly reduced the tissue spirochete loads of the type A, I, and K strains but not the type C and N strains (Fig. 6D and Fig. S2).

We previously demonstrated that infection with each of the five B. burgdorferi strains causes inflammation of the heart base and hyperplasia of the axillary lymph nodes in mice at 2 weeks post-infection⁶⁶. Here, we observed that the infection status, as determined by qPCR and explant culture, was associated with the development of carditis and lymphadenopathy. All control mice developed mild-to-marked Lyme carditis following infection with each strain (Fig. 6E). Additionally, OspC-ACIKN vaccinated mice with breakthrough infection of any strain developed minimal-to-marked carditis, with the exception of one mouse in the group challenged with the type C strain, which was previously shown to cause the lowest levels of carditis⁶⁶. Overall, vaccination with OspC-ACIKN significantly reduced the development of Lyme carditis from infection with the type A and type I strains, but not types C, K, or N.

Similarly, control mice had severe lymph node enlargement and consistently developed moderate-to-severe lymph node hyperplasia following infection with all strains (Fig. 6F, G). Additionally, OspC-ACIKN vaccinated mice with breakthrough infection developed mild-to-severe lymph node hyperplasia. In comparison, OspC-ACIKN vaccinated mice that were protected from infection had significantly reduced lymph node cellularity and hyperplasia. Vaccination with OspC-ACIKN therefore reduced lymph node pathology resulting from infection with B. burgdorferi strains type A, I, and K, but not C and N. Overall, the OspC-ACIKN vaccine afforded significant protection against pathologies associated with infection of three of the five B. burgdorferi strains.

Given that OspC-ACIKN vaccination of the mice provided minimal protection against B. burgdorferi strains with OspC types C and N, we performed a follow-up study to evaluate whether increasing the vaccine dose from 1 to 3 µg of mRNA would better prevent infection. In this study, mice were vaccinated with 3 µg of mRNA of each of the five OspC types or the luciferase mRNA as a control. Mice were bled for serum collection and then challenged 4 weeks post-boost with B. burgdorferi strain OspC type C, as this strain resulted in the highest rates of infection in mice vaccinated with 1 µg of OspC-ACIKN (Fig. 6C). Finally, the mice were euthanized on day 70 for tissue collection.

Infection with the OspC type C strain did not cause detectable carditis in half of the control mice (Fig. 7D). We have previously reported that the OspC type C strain causes the least carditis of the five strains evaluated in this study⁶⁶. Consequently, vaccination with OspC-ACIKN did not significantly reduce inflammation in the heart compared to the control mice. In contrast, following the type C challenge, control mice developed significant hyperplasia of the axillary lymph nodes, as demonstrated by the substantially elevated white blood cell counts (Fig. 7E) and histopathological analysis (Fig. 7F). Mice vaccinated with 3 µg of OspC-ACIKN had significantly lower white blood cell counts and less hyperplasia of the axillary lymph nodes compared to the control group. Taken together, these results demonstrate that increasing the vaccination dose of mRNA can lead to improved protection against infection and pathology with the type C strain.

The B. burgdorferi strains used in this paper were characterized in detail in a previous publication, including B31-A3 (OspC type A), JD1 Clone SK143 (OspC type C), Bb16-54 (OspC type I), 297 Ah130 (OspC type K), and Bb16-126 (OspC type N)⁶⁶. Strain B31-A3, and strain B31-A3 engineered to constitutively express OspC (B31-A3-OspC), were kindly provided by Dr. Patricia Rosa (National Institute of Allergy and Infectious Disease, MD, USA)^65,84. Strain JD1 and strain 297 Ah130 were kindly provided by Dr. George Chaconas (University of Calgary, AB, Canada). Strain Bb16-54 and strain Bb16-126 were obtained from the B. burgdorferi isolate collection of the Public Health Agency of Canada. Maintenance of the B. burgdorferi strains was performed as previously described^66,85,86. In brief, B burgdorferi was cultured at 35 °C and 1.5% CO₂ in BSK-H media containing 6% rabbit serum and supplemented with 5 µg/mL Amphotericin B, 100 µg/mL Phosphomycin, and 50 µg/mL Rifampicin. Cultures were grown to mid-log phase and were not subcultured to prevent plasmid loss. Cell counts were determined using a Petroff-Hausser counting chamber under dark-field microscopy.

Female C3H/HeN mice were purchased from Charles River (Senneville, Quebec, Canada). Mice arrived at 6 weeks old and were allowed to acclimatize for 1 week before experimental procedures began. All animal procedures in this study were reviewed and approved by the Health Canada Animal Care Committee and were conducted under protocols #2020-011, #2023-003, and #2024-003 according to institutional guidelines.

mRNA encoding firefly luciferase, ospC type A of B. burgdorferi strain B31 (GenBank accession #AAC66329.1↗), ospC type C of strain JD1 (GenBank accession #ADQ31294.1↗), ospC type I of strain Bb16-54 (GenBank accession #PHOQ01000005.1↗), OspC type K of strain 297 (GenBank accession #NC_018983.1↗), or ospC type N of strain Bb16-126 (GenBank accession #PHQD00000000.1↗) were commercially synthesized by TriLink Biotechnologies (San Diego, CA, USA) with a CleanCap®AG Cap 1 structure and a 120A polyadenylated tail. mRNA was DNAse and phosphatase-treated and purified by a silica membrane. OspC mRNA contained a human tyrosinase signal peptide (MLLAVLYCLLWSFQTSAGHFPRA; GenBank accession #AH003020↗) at the N-terminus. All mRNA was fully substituted with N1-methyl-pseudo-uridine and was codon optimized for expression in mice.

LNPs were produced using a NanoAssemblr BT™ or Ignite™ instrument (Precision Nanosystems, Inc., Vancouver, BC) as described previously, encapsulating mRNA instead of plasmid DNA⁸⁵. The percent molar ratio was 50:10:38.5:1.5, respectively, of lipids heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102) (MedKoo Biosciences, Morrisville, NC, USA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Avanti Polar Lipids, Inc., Alabaster, AL, USA), ovine cholesterol (Millipore Sigma, Burlington, ON, CA), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000, Millipore Sigma). Lipids in ethanol were mixed at a ratio of 3:1 with aqueous 50 mM sodium citrate, pH 4, containing the mRNA at an N/P ratio of 6. For empty LNP controls, the aqueous phase contained 50 mM sodium citrate only.

LNP size was characterized by nanoparticle tracking analysis (NTA) using a NanoSight 300 instrument (Malvern Panalytical, Westborough, MA, USA) as described previously⁸⁵. Average particle size ranged from 68 to 83 nm. The efficiency of nucleic acid encapsulation was determined using Triton X-100 (Millipore Sigma) and SYBR™ Gold dye (Thermo Fisher, Ottawa, ON, CA) as described previously⁸⁵. In brief, LNPs were disrupted with 1% Triton X-100 in a 96-well plate, and nucleic acids were quantified by the addition of 1X SYBR™ Gold dye and measured using a Synergy MX plate reader (Ex/Em: 495/537 nm) (BioTek, Winooski, Vermont, USA). Disrupted LNPs were measured alongside undisrupted LNPs and a nucleic acid standard curve. The encapsulation efficiency was determined by subtracting the unencapsulated mRNA from the total amount of mRNA in a sample and then dividing by the total amount of mRNA. The encapsulation efficiencies ranged from 88 to 98%.

Expression of the OspC proteins was confirmed by mass spectrometry following transfection of HEK293T cells in vitro with OspC-ACIKN mRNA encapsulated in LNPs (Fig. S3). HEK293T cells were incubated with 500 ng of OspC-ACIKN mRNA encapsulated in LNPs at 37 °C and 5% CO₂ After 24 h, the cells were lysed in Radio-Immunoprecipitation Assay buffer and then pulse sonicated. The samples were processed with a single pot solid phase-enhanced sample preparation (SP3) strategy, consisting of reduction with 10 mM DTT followed by alkylation with 20 mM IAA. Protein was bound to SeraMag Magnetic carboxylate beads in the presence of 80% ACN, then impurities were removed with three washes of 80% ACN. Trypsin was added at a ratio of 1:25 enzyme: protein and incubated overnight at 37 °C.

Tryptic peptides were analyzed using an Orbitrap Astral Mass Spectrometer coupled with a Vanquish Neo UHPLC System (Thermo Fisher Scientific Inc.). For each injection, 1 ul (500 ng peptides) were analyzed by loading onto a PepMap Neo C18 trap and desalting with 0.1% formic acid in water (solvent A) before separating on a 75 μm I.D. × 150 mm fused silica analytical column packed in-house with 3-μm ReproSil-Pur C18 beads (100 Å; Dr. Maisch GmbH, Ammerbuch, Germany). Chromatographic separation was achieved at a flow rate of 500 nl/min. The gradient was set as 5–45% buffer B in 28.5 min, the spray voltage was set to 2.6 kV, and the temperature of the heated capillary was 300 °C. Scan range was from 380 to 980 m/z for both MS and DIA. The DIA window was set to 2 Da, with HCD energy of 25%. The mass resolution was 240,000 for ms1. A real-time internal calibration by the lock mass was used. All data were recorded with Xcalibur software (Thermo Fisher Scientific, San Jose, CA).

The software package DiaNN (v2.1.0) was used to analyze the data with a library-free search against the reviewed Uniprot Human database (downloaded 20,241,120, entries) containing the sequences of the five OspC types added. The resulting protein and peptide results were filtered to view only OspC. Relative abundance was manually calculated by the Top3 method by averaging the three most abundant precursors, ignoring shared peptides. The relationship between MS signal response and protein concentration is constant within a CV of less than ±10%.

For the monovalent protection study, 8-week-old female C3H/HeN mice were randomly assigned to groups and injected intramuscularly in the hind leg with LNPs containing 1 µg of mRNA encoding OspC-A (OspC-A mRNA, n = 5) or an empty LNP negative control containing a matched amount of lipid (Control, n = 5) (Fig. 1A). For the polyvalent protection study, 8-week-old female C3H/HeN mice were randomly assigned to groups and injected intramuscularly with LNPs containing 3 µg of each mRNA encoding the five OspC types (OspC-ACIKN, n = 10) or 3 µg of mRNA encoding firefly luciferase (Control, n = 10) (Fig. 6A). The total injection volume was 50 µL divided between the two hind legs. Mice received an identical boost injection 4 weeks later and were bled 3 weeks after the boost injection for the collection of serum. Bacterial challenge was performed 4 weeks after the boost injection by subcutaneous needle injection between the shoulder blades.

For the challenge, spirochetes were grown to mid-log phase, centrifuged at 5000×g for 20 min, and resuspended in 100 µL of incomplete BSK-H media without rabbit serum. For the monovalent challenge, mice were injected with 10⁶ spirochetes of strain B31-A3. For the OspC-ACIKN vaccine study challenge, mice were injected with 10⁶ spirochetes of strain B31-A3 (OspC type A), Bb16-54 (OspC type I), or 297 Ah130 (OspC type K), or 10⁴ spirochetes of strain JD1 (OspC type C) or Bb16-126 (OspC type N). Challenge doses were determined based on our previous study characterizing strain infectivity⁶⁶. An additional group of five mice per study were injected with phosphate-buffered saline (PBS) instead of LNPs at day 0 and 28 and incomplete BSK-H media without bacteria instead of challenge at day 56 to serve as naive controls for histopathological analysis. Fourteen days after the challenge (d70), mice were euthanized by cardiac puncture exsanguination and cervical dislocation under isoflurane anesthesia for sample collection.

For the immunogenicity study, 8-week-old female C3H/HeN mice were randomly assigned to groups and injected intramuscularly with either 1 µg of mRNA encoding OspC-A encapsulated in an LNP (OspC-A mRNA, n = 10) or 1 µg of mRNA encoding firefly luciferase encapsulated in a LNP (Control, n = 10). Mice received an identical boost injection 4 weeks later and were euthanized 4 weeks after the boost for sample collection. Euthanasia was performed by cardiac puncture exsanguination and cervical dislocation under isoflurane anesthesia.

Live spirochetes can be cultured from mouse tissues to determine the mouse infection status and spirochete viability. The skin surrounding the bacterial injection site and the right ear were collected 2 weeks after challenge and cultured in complete BSK-H media for 28 days at 35 °C and 1.5% CO₂. Cultures were examined under dark-field microscopy across five fields of view and were considered positive if at least one motile spirochete was observed.

Two weeks after the bacterial challenge, the mice were euthanized, and the bladder, left ear, left tibiotarsal joint, inferior half of the heart, and left inguinal and medial iliac lymph nodes were collected and frozen in liquid nitrogen. DNA extraction from mouse tissues was performed using a DNAeasy kit (Qiagen, Toronto, ON, CA) according to the manufacturer’s instructions and qPCR analysis was performed as described previously, targeting the flaB gene of B. burgdorferi and standardized per 10⁶ mouse β-actin gene copies⁸⁵.

Quantification of lymphadenopathy was performed as described previously⁸⁶. Briefly, the left axillary lymph nodes were collected 2 weeks after bacterial challenge, pressed between two microscope slides, and filtered through a 70-µm cell strainer to create a single-cell suspension. White blood cell counts were determined using a Sysmex XT-2000iV hematology analyzer.

Histopathology analysis of the heart and axillary lymph nodes was performed as described previously^85,86. In brief, the superior half of the heart and the right axillary lymph nodes were collected 2 weeks after bacterial challenge into 10% neutral buffered formalin for routine histopathological processing and hematoxylin and eosin staining. Inflammation in the heart tissue and lymph nodes was graded on a scale from 0 to 5 based on International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice (INHAND) standards, where 0 was normal, 1 was minimal, 2 was mild, 3 was moderate, 4 was marked, and 5 was severe^87,88. Lymph nodes were graded individually 0–5 for follicular hyperplasia, paracortical hyperplasia, medullary cord plasma cell hyperplasia, and the combined pathology score represents the sum of these three grades.

OspC-specific antibody titers were quantified according to the method described previously^85,86. Briefly, recombinant OspC type A, C, I, K, or N were used to coat 96-well Nunc Maxisorp™ flat-bottom plates (Thermo Fisher) at a concentration of 1 µg/mL in 100 µL overnight at 4 °C. Plates were blocked with 1% (w/v) bovine serum albumin (IgG-Free, Protease-Free, Jackson ImmunoResearch, West Grove, PA) and incubated with serum collected 4 weeks after boost vaccination, serially diluted in PBS containing 0.05% Tween-20 (PBS-T) for 1 h at 37 °C. For total IgG ELISAs, the serum dilutions were twofold from 1:50 to 1:102,400. For IgG1 and IgG2a ELISAs, the serum dilutions were three-fold from 1:50 to 1:8,857,350. Following serum incubations and washing, HRP-conjugated goat anti-mouse IgG (Cytiva, Marlborough, MA), HRP-conjugated goat anti-mouse IgG1 (Jackson ImmunoResearch), or HRP-conjugated goat anti-mouse IgG2a (Jackson ImmunoResearch) was added to each plate at a 1:5000 dilution in PBS-T. Tetramethyl-benzidine (TMB) substrate (Cell Signaling Technology, Danvers, MA, USA) was added to each well, followed by 0.16 M sulfuric acid, and then the absorbance was read at 450 nm using a spectrophotometer. Endpoint titers were defined as the reciprocal of the highest dilution that resulted in an optical density (OD) greater than the average OD of all wells containing serum from control mice plus three times the standard deviation.

ADCP activity was measured as previously described⁸⁶. In brief, mouse serum samples were collected 4 weeks after boost vaccination, diluted 1:10 in Dulbecco’s Modified Eagle Medium (DMEM) containing no serum or antibiotics, and heat-inactivated at 56 °C for 30 min. In a 96-well plate, 20 µL of diluted inactivated serum was incubated with B. burgdorferi-OspC stained with a FITC-conjugated anti-Borrelia polyclonal antibody (PA1-73005, Thermo Fisher) at a concentration of 3.75 × 10⁷ cells/mL for 15 min at 37 °C and 300 rpm to enable opsonization. Then, 10 µL of RAW 264.7 cells (ATCC TIB-71), a mouse macrophage cell line, at a concentration of 7.5 × 10⁶ cells/mL were added to each well before shaking for 15 min at 37 °C and 300 rpm and then incubating for 1 h at 37 °C without shaking. Phagocytosis was arrested by the addition of 100 µL of cold PBS, and ADCP activity was represented by the percentage of FITC + RAW 264.7 cells using a BD FACSymphony™ A1 flow cytometer.

Serum borreliacidal activity was measured as previously described⁸⁵. In brief, 4 µL of mouse serum samples collected 4 weeks after boost vaccination were incubated with 4 µL of mid-log phase B. burgdorferi-OspC culture diluted to 5 × 10⁷ cells/mL and 4 µL of guinea pig serum (GPS, Complement Technologies, Tyler, Texas, USA) for 24 hours at 35 °C and 1.5% CO₂. Motile cells were then quantified using a Petroff-Hausser counting chamber under dark-field microscopy, and the percentage of live spirochetes was determined by dividing the cell count by the average cell count of control samples without serum.

Four weeks after OspC-A mRNA or Control boost vaccination, the draining popliteal and inguinal lymph nodes were collected in RPMI media. The nodes were gently squished between two microscope slides and passed through a 70 µm cell strainer before centrifugation at 400×g for 5 min. Cells were resuspended in RPMI, and white blood cells were counted using a Sysmex XT-2000iV hematology analyzer.

The multiplex cytokine ELISA was performed using a ProcartaPlex 17-plex Immunoassay kit (EPX170-26087-901, Thermo Fisher) as described previously⁸⁶. In brief, 1 million lymph node cells in single-cell suspension from OspC-A vaccinated or control mice (n = 5 per group) were stimulated with a custom overlapping peptide pool of OspC type A (GenScript, Piscataway, NJ, USA) for 24 h at 37 °C before centrifugation at 400×g for 5 min. The supernatant was collected and frozen at −80 °C until the assay was performed according to the manufacturer’s instructions. A Luminex 200 system (Millipore Sigma) was used to read the plate, and MILLIPLEX Analyst software version 5.1 was used to determine the concentration of each cytokine.

In a 96-well plate, 1 million mouse lymph node cells (n = 5 per group) in single-cell suspension from OspC-A vaccinated or control mice were stimulated individually with a custom 15-mer overlapping peptide pool of OspC type A containing 1 µg of each peptide (GenScript) in RPMI media for 18 h at 37 °C, 5% CO₂. Unstimulated lymph node cells were included as a negative control, and lymph node cells stimulated with phorbol 12-myristate-13-acetate (Sigma-Aldrich, St. Louis, MO, USA) at 20 ng/mL and ionomycin (Sigma-Aldrich) at 1 µg/mL (PMA+ionomycin) were included as a positive control. Following the incubation, lymph node cells were centrifuged at 500×g for 5 min, washed twice with FACS buffer, and resuspended in FACS buffer containing rat anti-mouse CD16/CD32 (Mouse Fc Block™, BD Biosciences, Mississauga, ON, CA). The plate was incubated for 15 min at 4 °C and then centrifuged at 500×g for 5 min. The lymph node cells were resuspended in antibody cocktail containing anti-CD3 Alexa Fluor® 700 (Clone 17A2, BD Biosciences), anti-CD4 BB700 (Clone RM4-5, BD Biosciences), anti-OX40 BV786 (Clone OX-86, BD Biosciences), anti-CD25 PE (Clone OX-39, BD Biosciences), and LIVE/DEAD™ Fixable Blue Dead Cell Stain (ThermoFisher) diluted in Brilliant Stain Buffer (BD Biosciences) and incubated for 30 min at 4 °C in the dark. Lymph node cells were washed twice with FACS buffer, fixed in Cytofix™ fixation buffer (BD Biosciences) for 15 min at room temperature, washed twice again and then resuspended in FACS buffer. Acquisition was performed using a BD FACSAria™ Fusion flow cytometer, and data analysis was performed using FlowJo v10 software (Treestar) according to the gating strategy depicted in Fig. S4.

The ELISpot assay was performed using a Mouse IFN-γ/IL-4 Double-Color ELISPOT kit according to the manufacturer’s instructions (ImmunoSpot by Cellular Technology Limited, Shaker Heights, Ohio, USA). For stimulation, 1 million lymph node cells in single-cell suspension from OspC-A vaccinated or control mice (n = 5 per group) were stimulated individually with a custom 15-mer overlapping peptide pool of OspC type A containing 1 µg of each peptide (GenScript). The plates were read using an ImmunoSpot® S6 Analyzer, and data analysis was performed using ImmunoSpot® v7.0 software.

Intracellular cytokine staining and flow cytometry analysis of antigen-specific T cells was performed as described previously using mouse lymph node cells instead of splenocytes⁸⁶. In brief, 1 million lymph node cells in single-cell suspension from OspC-A vaccinated or control mice (n = 5 per group) were stimulated individually with a custom 15-mer overlapping peptide pool of OspC type A containing 1 µg of each peptide (GenScript) for 5 h. Unstimulated and PMA+ionomycin-stimulated samples were included as negative and positive controls, respectively. GolgiPlug™ (Brefeldin A, BD Biosciences) was added to each well after the first hour. After the 5 h incubation, lymph node cells were washed with PBS, blocked with rat anti-mouse CD16/CD32 (Mouse Fc Block™, BD Biosciences), and surface stained for 30 min at 4 °C with anti-CD3 Alexa Fluor® 700 (Clone 17A2, BD Biosciences), anti-CD4 BB700 (Clone RM4-5, BD Biosciences), anti-CD8a FITC (Clone 53-6.7, BD Biosciences) and LIVE/DEAD™ Fixable Blue Dead Cell Stain (Thermo Fisher) diluted in Brilliant Stain Buffer (BD Biosciences). Lymph node cells were fixed and permeabilized before intracellular staining with anti-IFN-γ BV786 (Clone XMG1.2, BD Biosciences), anti-TNF BV650 (Clone MP6-XT22, BD Biosciences), and anti-IL-2 (Clone JES6-5H4, BD Biosciences) diluted in Brilliant Stain Buffer (BD Biosciences) for 30 min at 4 °C. Cells were resuspended in PBS and acquired immediately using a BD FACSAria™ Fusion flow cytometer. Data analysis was performed using FlowJo v10 software (Treestar) according to the gating strategy described previously⁸⁶.

Statistical significance of data or log10-transformed data were calculated using unpaired two-tailed t-tests or Fisher’s exact test at a significance value of α = 0.05. All statistical analyses were performed using GraphPad Prism 9 (Dotmatics Inc., Boston, MA, USA). ns not significant, *p value <0.05, **p value <0.01, ***p value <0.001, ****p value <0.0001.