The mRNA construct was designed based on structural architecture of the Moderna mRNA-1273 which has been shown to be highly effective in ensuring stability, protein expression and immunogenicity. Specifically, we incorporated the 5’ untranslated region (UTR), the 3’ UTR, and the poly(A) tail in the mRNA sequence for the TP0954 antigen. Plasmids corresponding to the full sequence of TP0954 was amplified using E. coli Stabl3 (ThermoFisher Scientific, Waltham, MA, USA). Following plasmid extraction, linearized templates were generated via digestion with the BspQI restriction enzyme and purified using DNA magnetic beads (Vazyme, Nanjing, China). Subsequently, in vitro transcription (IVT) was performed to synthesize messenger RNA (mRNA), using T7 RNA polymerase (Vazyme, Nanjing, China), CleanCap (Syngenbio, Nanjing, China), and deoxyribonucleoside triphosphates (dNTPs)-where uridine triphosphate (UTP) was substituted with m¹ψ-5′ triphosphate (Syngenbio, Nanjing, China). After completion of the IVT reaction, the synthesized mRNA was purified using RNA magnetic beads (Vazyme, Nanjing, China). An ultraviolet spectrophotometer quantified mRNA concentration and formaldehyde-denaturing agarose gel electrophoresis assessed mRNA integrity and purity.

HEK293T cells were cultured in DMEM supplemented with 10% fetal bovine serum and 100 U/mL penicillin–streptomycin at 37°C in a humidified 5% CO₂ atmosphere. Cells were transfected with 2 µg mRNA using Lipofectamine 3000 and cultured for 48 h. Cells were then harvested, washed with PBS, and lysed in RIPA buffer supplemented with 1 mM PMSF. Total proteins were separated on 4-20% Bis-Tris gradient gels and transferred to PVDF membranes. After blocking, membranes were incubated with primary antibodies overnight at 4°C, followed by HRP-conjugated secondary antibodies. Protein bands were visualized using chemiluminescence and imaged with a ChemiDoc MP Imaging System.

The prepared mRNA solutions were encapsulated in lipid nanoparticles (LNPs). mRNA solutions were formulated at a concentration of 200 μg/mL in 25 mM sodium acetate buffer (pH 5.5). The LNP formulation comprised four components: cationic lipid, phosphatidylcholine, cholesterol, and polyethylene glycol (PEG)-lipid, at a molar ratio of 50:10:38.5:1.5, which were dissolved in anhydrous ethanol. mRNA and LNP components were mixed at a 3:1 flow ratio using a microfluidic device to form an mRNA-LNP mixture. Subsequently, ethanol was removed and replaced with 25 mM Tris-HCl buffer (pH 7.5) using a 100 kDa ultrafiltration centrifugal filter, and the solution was concentrated to produce the mRNA-LNP vaccine. Encapsulation efficiency was determined using the Quant-iT™ RiboGreen RNA Kit (ThermoFisher Scientific, Waltham, MA, USA), while particle size and polydispersity index were measured with a Nanoparticle Size and Zeta Potential Analyzer (Malvern Panalytical, Malvern, UK).

Female BALB/c mice (6–8 weeks old, specific-pathogen-free) were used in this study, supplied by GemPharmatech Co., Ltd. (Nanjing, China). Four groups were established by random allocation, with 5 mice per group. Mice were intramuscularly injected with TP0954 mRNA vaccine at different doses (1 μg, 5 μg, 10 μg) into the hind limb. For the LNP control group, mice received an equivalent volume of LNPs alone. Primary immunization was administered on day 0, with a booster dose given on day 14. Blood samples were collected via cardiac puncture on day 14 and 28. On day 28, following anesthesia, spleens were harvested. This animal study was approved by the Ethics Committee of the Hospital of Dermatology, Chinese Academy of Medical Sciences.

Male New Zealand White (NZW) rabbits (8–13 weeks old, weighing 2.5–3.0 kg) were used in this study. The rabbits were randomly assigned to three groups with three animals per group. Animals in the TP0954 mRNA vaccine immunized group received an intramuscular injection of 5 μg dose of TP0954 mRNA vaccine into the hind limb. Animals in the recombinant TP0954 protein immunized group received an intramuscular injection of 100 μg dose of purified TP0954 protein mixed with an equal volume of TiterMax Gold Adjuvant. The 100 µg dose of protein was chosen based on precedent in rabbit models of syphilis vaccination to ensure sufficient antigen exposure and reproducible immune responses (18). The LNP group was administered an equivalent volume of LNPs. Primary immunization was performed on day 0, with a booster immunization given on day 14. Blood samples were collected from the ear veins on days 14 and 28.

Two weeks following the booster immunization, dorsal fur of all rabbits was shaved, and the exposed skin was disinfected with 75% ethanol. Each rabbit was intradermally challenged at 8 distinct sites: at each site, 0.1 mL of a suspension containing 1×10⁶ freshly isolated T. pallidum Nichols strain per mL (prepared in 0.9% saline) was administered. T. pallidum Nichols strain was propagated in healthy male NZW rabbits via intratesticular inoculation and harvested as previously described by Lukehart et al. (19) The development of lesions at the challenge sites was recorded and photographed daily. Additionally, the diameter of each lesion was measured daily. Blood samples used for serological test were collected from the ear veins on day 0, 7, 14, 21 post challenge. 21 days post T. pallidum challenge, rabbits from each group were euthanized, the lesions and organs were harvested for subsequent experiments.

To monitor short-term systemic responses and safety signals after immunization, consistent with international guidance on nonclinical evaluation of vaccines. Rectal temperature and body weight of mice were measured for 7 days after primary immunization. Blood sample was collected in each group at 4 weeks following the first immunization. Serum was then separated after centrifugation. Serum biochemical indicators were automatically measured using an automatic biochemistry analyzer (Chemray 800, China). 2 weeks post the final immunization, all mice were euthanized, the organs were harvested, fixed, embedded in paraffin, and sectioned for histopathological analysis. Following conventional hematoxylin and eosin (H&E) staining, the tissue sections were examined under a light microscope.

To evaluate the specific antibody response, blood sample was collected in each group at 2 and 4 weeks following the primary immunization. The blood was then centrifuged in order to separate the serum. The specific antibody levels were determined by indirect ELISA. The 96-well plates were coated with 1 ug/mL purified recombinant TP0954 protein. Excess antigen was washed off with PBST. Then 200 mL of blocking buffer (1g BSA dissolved in 50mL PBS) was added and incubated at 37 °C for 2 hours. Then the wells were washed three times with PBST. Rabbit serum was two-fold serially diluted starting from a dilution of 1:100. After 1 hour of incubation at 37 °C, the wells were washed three times with PBST again. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:10,000) or horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:10,000) was subsequently added to the wells and incubated at 37 °C for 1 hour. The plates were washed three times with wash buffer, and 100 mL of TMB peroxidase substrate was added per well and incubated at 37 °C for 15 minutes. Then 100 mL stop solution was added to stop the color reaction. The absorbance at 450 nm (A450 value) was detected with a microplate reader ((Infinite 200 Pro, Switzerland)). The endpoint titer was considered to be the last serum dilution with readings higher than the 2.1-fold of the negative controls.

Two weeks post the final immunization, mice were euthanized, and their spleens were harvested for preparation of splenic cell suspensions. 21 days post T. pallidum challenge, rabbits were all euthanized and spleens were harvested. Each spleen was homogenized through a 200-mesh cell strainer to obtain a splenic cell suspension. The suspension was centrifuged at 2000 rpm for 10 minutes. Following centrifugation, red blood cell (RBC) lysate was added to lyse RBCs, and the mixture was incubated for 5 minutes. Phosphate-buffered saline (PBS) was then added to terminate the lysis reaction. After another centrifugation step (2000 rpm, 5 minutes), PBS was added to wash the splenic cell suspension. A third centrifugation (2000 rpm, 5 minutes) was performed, and the resulting splenocytes were resuspended in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) for subsequent detection assays.

Mouse and rabbit splenic lymphocytes collected as described above were seeded in 6-well culture plates at a density of 1×10⁷ cells per mL. Each well was treated with 10 μg/mL recombinant TP0954 protein, with PBS serving as the blank control; the plates were then incubated at 37 °C for 24 hours. After incubation, cell culture supernatants were harvested to determine the secretion levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-2 (IL-2) from mouse and rabbit splenocytes. Assays were performed according to the manufacturer’s instructions for the respective ELISA kits (Elabscience, China; Ruisaiqi Biotechnology, China). The absorbance at 450 nm was measured using a microplate reader (Infinite 200 Pro, Switzerland).

DNAs were extracted from tissues of T. pallidum-infected rabbits by using a TIANamp Genomic DNA Kit (Tiangen, Beijing, China) following the manufacturer’s protocol. PCR amplification was performed by using a LightCycle 96 apparatus (Roche, Basel, Switzerland). Quantitative analysis of T. pallidum gDNA and rabbit gDNA was achieved by using primers targeting T. pallidum DNA Polymerase I (polA) gene and rabbit collagenase-1 precursor (MMP-1) gene, respectively. The sequences of the primers are given as followings: polA Sense: 5’-TACGGTGCAAGTGCTCAGAC-3’, Antisense: 5’-CAGGCACATTGTCGGAGGAA-3’; MMP-1 Sense: 5’-TTGCTTCTTCACACCAGAATGCTGT-3’, Antisense: 5’-GCGTGATCAGGCACTATGTAGCAAT-3’. The primers for quantitative real-time PCR (qPCR) amplification were provided by Genscript Biotech (Nanjing, China). As directed by the manufacturer, qPCRamplifications were performed in 20 ul Taq Pro Universal SYBR QPCR Master Mix (Vazyme, Nanjing, China) reaction mixture. Standard curves were generated for polA and MMP-1 by using 10-fold dilutions in series from 10⁷ to 10¹ reproductions of T. pallidum gDNA and 2-fold dilutions in series of rabbit gDNA from 200 to 1.56 ng/ul. The following conditions were used for the PCR of polA and MMP-1: 95 °C for 5 min, 40 cycles of 95 °C for 10 sec, 60 °C for 30 sec. Following is an analysis of melt-curves: 95 °C for 10 sec, 65 °C for 60 sec, and 97 °C for 1 sec.

On day 0, 7, 14, 21 post T. pallidum challenge, blood was collected from every rabbit and serum was separated. T. pallidum particle agglutination assay (TPPA) and rapid plasma reagin test (RPR) were then conducted according to the instruction of TPPA Detection Kit (FUJIREBIO INC, Tokyo, Japan) and RPR Diagnostic Kit (KEHUA BIO-ENGINEERING, Shanghai, China) respectively.

On day 21 post-challenge, skin lesions from each rabbit were excised, and a 6-mm punch biopsy was used to obtain standardized tissue subsamples from each lesion. These cutaneous lesion specimens were fixed, embedded in paraffin, and sectioned for histopathological analysis. Following conventional hematoxylin and eosin (H&E) staining, the tissue sections were examined under a light microscope to observe and assess the degree of inflammatory cell infiltration.

Statistical analysis was performed with GraphPad Prism 8.0 software. Data were presented as Means ± SDs. The following tests were used to assess significance: two-tailed unpaired Student’s t-test between two comparisons; one-way ANOVA test and Chi-square Test of Independence for multiple comparisons. A significance level of * p < 0.05, ** p < 0.01, and *** p < 0.001 is considered to indicate significant differences in the statistical results.

The full sequence of TP0954 protein was selected as the antigenic targets for mRNA synthesis. The mRNA was constructed by integrating the coding sequence with 5’ cap, 5’UTR, 3’UTR and a poly(A) tail (Figure 1). The sequence of TP0954 mRNA was provided in Supplementary Table 1. Then, it was cloned into pUC57 plasmid. Following plasmid amplification and extraction, linearized template was generated via digestion with the BspQI restriction enzyme (Figure 1). High purity mRNA for vaccine purpose was obtained using in vitro transcription from linearized DNA (Figure 1). Western blot analysis confirmed the expression of a 54 kDa fragment, indicating successful translation and expression of TP0954 protein (Figure 1). TP0954 mRNA was encapsulated in LNPs, with an average size of 96.86 nm (Figure 1), and polymer dispersity index (PDI) value of LNP showed good stability of the LNP dispersion system (Figure 1). The encapsulation efficiency of mRNA-LNP vaccine was higher than 90% (Figure 1). Transmission Electron Microscope (TEM) showed spherical nanoparticles, and no obvious particle aggregation was observed (Figure 1).

To preliminarily evaluate the in vivo toxicity of mRNA vaccine, the body temperature and body weight of immunized mice were monitored for 7 days after the primary immunization. There were no significant differences in body temperature and body weight after vaccination between immunized mice and LNP immunized mice (Figure 2). This observation indicated that mRNA vaccine may have good safety. Based on blood biochemical indicators, we further evaluated the functions of the heart, liver, and kidneys. Among them, creatine kinase (CK) and lactate dehydrogenase (LDH) are indicators of cardiac function; Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are indicators of liver function; Creatinine (CREA) and uric acid (UA) are indicators of renal function. As shown in (Figure 2), there were no significant differences in cardiac function, liver function, and renal function between mRNA vaccine immunized mice and LNP immunized mice, and all data were within the normal range. Compared with the control group, there were no obvious pathological changes in heart, liver, spleen, lung and kidney in immunized mice (Figure 2). Therefore, our mRNA vaccine showed good safety.

To determine the immune response induced by TP0954 mRNA vaccine, groups of BALB/c mice (n = 5) were immunized intramuscularly twice with 1, 5 or 10 µg mRNA vaccine with empty LNP immunization as control. The interval between every injection was two weeks. The blood samples were collected at week 2 and week 4 (Figure 3). The antigen-specific antibodies were detected by indirect ELISA. As shown in Figure 3, 1 µg dose of TP0954 mRNA vaccine did not induce significant antigen-specific humoral immune responses. After intramuscular injection with 5 µg dose of TP0954 mRNA vaccine, the serum IgG titers significantly increased after initial immunization, and were further elevated after the second immunization. Moreover, immunization with 5 µg dose of TP0954 mRNA vaccine could induce almost the same level of IgG titer as 10 µg induced. Therefore, 5µg dose of mRNA vaccine might be the optimal dose to elicit antigen-specific humoral immune responses. IFN-γ, TNF-α and IL-2 are critical Th1-related cytokines, which is essential for early clearance of T. pallidum in lesions. Therefore, we also examined the levels of IFN-γ, TNF-α and IL-2 produced by mouse splenic cells by ELISA (Figure 3). The increased levels of IFN-γTNF-α and IL-2 induced by 5 and 10 µg dose of TP0954 mRNA vaccine were higher than those of 1 µg dose. Therefore, 5 µg dose of TP0954 mRNA vaccine was sufficient to elicit Th1-related immune responses in BALB/c mice.

Based on the results of mRNA vaccine elicited antigen-specific humoral and T-cell immune responses in BALB/c mice, 5 µg dose of TP0954 mRNA vaccine was used for further immunization in rabbits. To determine whether TP0954 mRNA vaccine was able to induce specific antibody response in rabbits, blood sample was collected in each group at 2 and 4 weeks following the first immunization (Figure 4). Then we performed serial two-fold dilutions of serum to detect specific antibody titers. As shown in Figure 4, TP0954 mRNA vaccine induced higher titers of specific antibody starting at week 2 compared with LNP-immunized rabbits, but lower titers compared with TP0954 protein vaccine immunized rabbits. Moreover, antibody levels in animals immunized TP0954 mRNA and protein vaccine after the second immunization were similarly high and were higher compared with primary immunization. These results indicated that TP0954 mRNA vaccine was able to induce strong antigen-specific humoral response. 21 days post T. pallidum challenge, rabbits were all euthanized and spleens were harvested. With stimulation with 10 μg/mL recombinant TP0954 protein for 24 hours, after incubation, cell culture supernatants were harvested to determine the secretion levels of IFN-γ, TNF-α and IL-2. Compared to that of rabbits immunized with LNP, the production of IFN-γ, TNF-α and IL-2 was significantly increased in the rabbits immunized with TP0954 protein vaccine. And TP0954 mRNA vaccine induced significantly higher level of IFN-γ, TNF-α and IL-2 compared to TP0954 protein vaccine, indicating that TP0954 mRNA vaccine could induce stronger Th1- related immune responses in NZW rabbits (Figure 4).

Two weeks after the booster immunization, each rabbit was challenged intradermally with 0.1 ml of 1×10⁶ freshly isolated T. pallidum Nichols strain per ml in 0.9% saline at each of 8 sites. During the subsequent 21 days, induration and ulceration were monitored daily for diameters. As shown in Figure 5, obvious indurations were present on rabbits by day 5, 3 and 3 in LNP group, TP0954 mRNA vaccine group and TP0954 protein vaccine group, respectively. Both rabbits immunized with TP0954 mRNA and protein vaccine showed delayed lesion development compared to the LNP group. On day 21, the average diameter of indurations in LNP group, TP0954 mRNA vaccine group, TP0954 protein vaccine group was 14.95 ± 2.72mm, 8.57 ± 2.27 mm, 11.24 ± 1.53 mm, respectively (Figure 5). Moreover, ulcerations presented on day 8 in LNP group, day 18 in TP0954 mRNA vaccine group and day 16 in TP0954 protein vaccine group (Figure 5). In addition, as shown in Figure 5, on day 21, the numbers of ulcerations were significantly less in TP0954 mRNA vaccine group (6/22) and TP0954 protein vaccine group (13/22) than LNP group (22/24). Similarly, the average size of ulcerations was also smaller in TP0954 mRNA vaccine group (4.25 ± 0.83mm) and TP0954 protein vaccine group (5.52 ± 1.45mm) than LNP group (8.77 ± 2.43mm). Lesions on the backs of the rabbits on 21 days post-intradermal challenge with T. pallidum were presented in Figure 5 (Pictures of high-definition, un-magnified and whole-field original image of the skin were presented in Supplementary Figure 1). All these results indicated that immunization with TP0954 mRNA vaccine could be able to significantly delay the development of both indurations and ulcerations, and showed better efficiency compared to TP0954 protein vaccine.

To evaluate the ability of mRNA vaccine immunization to prevent T. pallidum dissemination, all of the rabbits were killed at day 21 post-challenge. The total DNA was extracted from the cutaneous lesions at the initial infection sites as well as distal tissues or organs and qPCR was performed to determine T. pallidum DNA concentration to assess the treponemal burdens. As shown in Figure 6, all immunized rabbits showed decreased T. pallidum loads at cutaneous lesions. Moreover, The T. pallidum loads were significantly lower at cutaneous lesions in rabbits immunized with TP0954 mRNA vaccine compared with rabbits immunized with TP0954 protein vaccine. In addition, the T. pallidum burdens in the distal organs of the animals were also assessed (Figure 6). After immunization with TP0954 mRNA or protein vaccine, the burdens in the spleen, liver and kidney were also obviously decreased. And TP0954 mRNA vaccine immunization showed better efficiency in inhibiting T. pallidum dissemination compared with TP0954 protein vaccine. These results suggested that TP0954 mRNA vaccine prevented treponemal dissemination to distant organs, further confirming that TP0954 was a promising vaccine candidate.

On day 21 post-challenge, H&E stained sections from primary cutaneous lesion sites of rabbits were performed to evaluate the intensities of inflammatory infiltration and cell types. All lesions revealed various degrees of infiltrates of inflammatory cells. In the lesions of LNP immunized rabbits, neutrophils, lymphocytes, plasma cells and macrophages were all observed. And compared with rabbits immunized with LNP, rabbits immunized with TP0954 mRNA vaccine and TP0954 protein vaccine had increased levels of lymphocytes, plasma cells, macrophages (Figure 7).

As shown in Figure 8, the serum TPPA and RPR of all rabbits were negative on day 0 and 7 post challenge. The average titers of TP0954 mRNA and protein vaccine immunized rabbits were lower than those of LNP immunized rabbits on day 14 and 21. However, no statistical difference was observed probably due to the limited number of rabbits.