Leptospiral LigA-C mRNA-based immunization protects against Leptospira interrogans Serovar Australis infection

Leptospiral LigA-C mRNA-based immunization protects against Leptospira interrogans Serovar Australis infection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Leptospiral LigA-C mRNA-based immunization protects against Leptospira interrogans Serovar Australis infection Perumal Saranya, Panneerselvam Selvambika, Dianne Langford, Kalimuthusamy Natarajaseenivasan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6665688/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Leptospirosis, caused by the pathogenic Leptospira species, is one of the most prevalent zoonotic infections worldwide. Developing effective diagnostics and vaccines remains a critical challenge in combating this disease. Traditional bacterin-based vaccines have notable limitations, including short-lived immunity and serovar specificity. This study evaluated the leptospiral immunoglobulin-like protein A (LigA-C) as a vaccine candidate, focusing on its ability to elicit protective immune responses against leptospirosis. Using a hamster model, we demonstrated the protective efficacy of LigA-C mRNA immunization and explored the underlying immune mechanisms.LigA-C/mRNA immunization induced a robust antibody response, with significant increases observed following booster doses. Cytokine profiling revealed that elevated levels of IL-4, TNF-α, and IFN-γ were positively correlated with survival in immunized hamsters, while IL-10 levels were inversely correlated with protection. Immunized groups achieved 100% survival and exhibited minimal histopathological lesions in the kidneys and lungs. In contrast, control animals succumbed to infection within 11–15 days post-challenge with Leptospira interrogans serovar Australis strain Ballico.These findings indicate that LigA-C mRNA immunization confers protection through both humoral and cellular immune responses. The high survival rate and reduced pathology highlight the potential of LigA-C mRNA as a promising vaccine candidate for the prevention of leptospirosis. Biological sciences/Immunology Biological sciences/Microbiology Health sciences/Diseases Leptospirosis Leptospira interrogans LigA subunit vaccine mRNA vaccine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Vaccination is one of the most effective medical strategies for preventing and controlling infectious diseases 1 . The need for novel vaccine approaches that ensure safe, long-lasting, and cross-protective immunity has become increasingly critical 2-5 . In this context, subunit vaccines have gained significant attention and demonstrated efficacy against several infectious agents, including Leptospira species 6 . Identifying conserved protective antigens expressed by multiple pathogenic Leptospira serovars and utilizing them in recombinant DNA or protein-based vaccine formulations could offer a safe, effective, and cross-protective solution against leptospirosis 7 . In recent years, mRNA vaccines have emerged as a groundbreaking platform with broad applicability, ranging from prophylactic interventions to therapeutic treatments, and from personalized medicine to global health solutions 8 . Their ability to stimulate both humoral and cellular immune responses makes mRNA vaccines a promising technology. Moreover, Leptospiral outer membrane proteins (OMPs) play a central role in pathogenesis and are key markers for distinguishing pathogenic from non-pathogenic strains. Among these, the immunoglobulin-like proteins LigA and LigB are surface-exposed adhesins expressed in vivo, which interact with the host extracellular matrix and homeostatic proteins. LigA, in particular, has been extensively studied for its diagnostic and vaccine potential, with its immunogenic epitopes showing promise in experimental models 9 . The present study aimed to evaluate whether LigA-C, delivered via mRNA or protein-based immunization, could induce protective immunity in hamsters challenged with a virulent strain of Leptospira interrogans investigated the potential of LigA-C-specific mRNA to activate a robust immune response in a hamster model. We evaluated the expression levels of pro-inflammatory cytokines (TNF-α, interferon-γ [IFN-γ]), the anti-inflammatory cytokine interleukin-10 (IL-10), and interleukin-4 (IL-4), a cytokine involved in antibody production. Overall, the study assessed the immunogenicity and protective efficacy of LigA-C-specific mRNA as a vaccine candidate against leptospiral infection. Results Cloning, expression, purification and Western blot analysis of recombinant protein The L. interrogans serovar Australis strain BallicoligA-C terminus gene, cloned into the pET15b expression vector, was confirmed by colony PCR and restriction endonuclease analysis (REA) (Fig. 1A). The recombinant LigA-C was expressed upon induction with 1.0 mM IPTG, and its molecular mass (58 kDa) was verified by SDS-PAGE (Fig. 1B). Immunoblot analysis showed that polyclonal rabbit antibodies specific to recombinant LigA-C recognized a 58 kDa protein band (Fig. 1C). mRNA transcript analysis The in vitro transcribed mRNA and the amplification of a 1530 bp product of the ligA-C gene from cDNA were visualized on a 1.2% 1X TAE agarose gel (Fig. 1D), stained with EtBr, and documented using a gel documentation system (Bio-Rad, USA). To confirm the specificity of the primers, the mRNA transcript was subjected to PCR, resulting in a significant CT value (Fig. 1E). The results indicate that the transcript targeting the ligA-C gene was specific for detecting pathogenic leptospires. No amplification was observed in the non-template controls throughout the experiments. Preparation of Antigens for Immunization For mRNA-based immunization, the linearized pDNA was in vitro transcribed and purified using the ethanol precipitation method. For the subunit vaccine, the LigA-C protein was overexpressed in E. coli , achieving the expected size of 58 kDa. The recombinant protein was purified with a good yield and used for immunization. The heat-killed vaccine was prepared from L. interrogans serovar Australis strain Ballico, and sterility testing confirmed the absence of leptospiral growth. The antigen formulation for the first booster, administered on day 0, was prepared with 100 μg of protein in Alhydrogel (Sigma, St. Louis, MO). A second booster of 100 μg protein was given on day 14 through intramuscular injection.The mRNA concentration wasdeterminedby testing the LD 50 , and was found to be 100 μg. Antibody response in immunized hamsters The specific antibody response in immunized hamsters was assessed using an IgG-ELISA at three different time points: 0, 14, and 28 days post-immunization. The results, shown in (Fig. 2A), demonstrated that all immunization strategies induced significant antibody levels. Hamsters immunized with mRNA showed higher IgG levels than the control groups at 14 days post-inoculation (P<0.0001), with these levels continuing to rise at 28 days post-inoculation, similar to the recombinant subunit immunization. No significant antibody levels were detected in the negative control groups (PBS or Alhydrogel). However, no significant difference (p>0.05) was observed in antibody production between the protein subunit and mRNA vaccine groups. Cytokine expression profile The induction of both Th1 and Th2 type cytokines was assessed on day 28 using total RNA isolated from the blood samples of vaccinated hamsters. The abundance of these cytokines was measured through qRT-PCR analysis (Fig. 2B,C,D,E). The results revealed that the expression levels of the anti-inflammatory cytokine IL-10 in all vaccinated groups were inversely correlated with survival. In contrast, the expression of the Th2 cytokine IL-4 and the inflammatory cytokines TNF-α and IFN-γ in the mRNA/rLigA-C vaccinated groups was increased and positively correlated with hamster survival following the challenge. Groups that received the mRNA/heat-killed leptospires showed significant differences (P<0.0001) in cytokine levels compared to the control groups. However, immunization with the recombinant protein subunit vaccine elicited a cytokine response that was significantly less pronounced (P<0.05). Prophylactic effects of the antigenic candidates The protective efficacy of the immunizations was assessed up to 28 days post-challenge, based on survival rates and histopathological findings in vital organs. The survival data (Fig. 3) demonstrated that animals immunized with mRNA/rLigA-C, followed by leptospiral challenge, were significantly protected, with a survival rate of 100% (P<0.01). All hamsters in the positive control group, which were administered killed whole leptospires, also showed 100% protection (P<0.01). In contrast, animals that received PBS + Alhydrogel succumbed to the infection, with median survival times of 11 and 14.5 days, confirming the virulence of the challenge strain. Histopathological examination of the lungs of deceased animals revealed alveolar hemorrhages, edema, capillary congestion, and leukocyte infiltration (Fig. 4). In the kidneys, the predominant lesions observed included interstitial nephritis, renal tubular failure, and necrosis (Fig. 5). From the hamster models, all vaccines showed 100% protection, but the various types of lesions formation in lungs and kidneys showed variation in organ protection against the Leptospira from one vaccine group to another group. These findings suggest that leptospiral mRNA immunization can induce sterilizing immunity against leptospirosis. Discussion Vaccination remains the most cost-effective and reliable strategy to prevent infectious diseases. However, developing an efficient vaccine against leptospirosis that offers cross-protection against various pathogenic serovars continues to be a significant challenge 10,11 .Current bacterin vaccines have notable limitations, including side effects, short-lived immunity, and serovar-specific protection, underscoring the need for innovative approaches 12–15 . Subunit recombinant vaccines, composed of purified antigens, represent a promising alternative. Nevertheless, these vaccines are inherently weakly immunogenic due to the absence of pathogen-associated molecular patterns and require adjuvants to enhance immune activation 16 .In this study, we evaluated mRNA and protein subunit vaccines using rLigA-C as the antigen and Alhydrogel as the adjuvant. mRNA vaccines offer several advantages, including efficient antigen delivery, stimulation of both humoral and cellular immunity, cost-effective mass production, and the elimination of purification steps 17 . Subunit vaccines are inherently safe and predominantly elicit humoral immunity, although their efficacy may be affected by antigen misfolding during production under denaturing conditions 18 . Leptospira outer membrane proteins (OMPs), such as the well-characterized Lig proteins, are key adhesins that mediate attachment to the host extracellular matrix (ECM). Immunization with full-length Lig proteins or their conserved C-terminal domains has previously shown significant protection in animal models 19–22 . The protection is largely attributed to anti-Lig antibodies, which block bacterial adhesion to the ECM, preventing invasion and subsequent infection. Consistent with these findings, the rLigA-C antigen elicited a robust antibody response against both the recombinant protein and the native protein in leptospiral whole-cell lysates (WCL). Our results demonstrated that rLigA-C vaccination, with a 100µg dose selected based on prior studies, provided protective immunity in hamsters 23 and indicated that a 100 µgbooster dose of mRNA has an acceptable safety profile 24 .Previous research has highlighted the central role of humoral immunity in protection against leptospirosis 25 . The protective efficacy observed here suggests that LigA domains involved in ECM attachment and complement regulation are sufficient to induce immunity, warranting further exploration of these mechanisms. This study corroborates earlier findings on LigA's immune protective properties 2,9,19,22,23,26–28 . Hamsters were chosen as the experimental model due to their suitability for leptospirosis immunization studies, given that mice are not ideal for this purpose 23 . Both rLigA-C and mRNA vaccines induced significant protective immunity in the hamster model, with comparable antibody titers between the two approaches. While variability in immunogenicity was observed across experiments.The overall differences in antibody responses and survival rates between vaccinated and control groups were statistically significant, underscoring the efficacy of these immunization platforms.The LigA-C mRNA vaccine induced a significant antibody response after booster doses, suggesting its potential to generate protective immunity against leptospirosis. Additionally, it activated both humoral and cell-mediated immunity (CMI), as evidenced by increased Th1 (IFN-γ) and Th2 (IL-4) cytokine levels. While pro-inflammatory cytokines such as TNF-α were not significantly elevated compared to controls, the vaccines did induce significant IL-10 activation, supporting a balanced immune response. These findings,aligned with previous studies,show that excessive TNF-α levels are associated with severe leptospirosis 29 . The histopathological analysis revealed that rLigA-C and mRNA immunizations provided substantial protection, as immunized animals displayed minimal lung lesions and absence of severe kidney damage, in contrast to severe pathology observed in control animals. While the heat-killed vaccine also elicited some protection, its immune response was insufficient to completely eliminate bacterial colonization. Notably, mRNA and rLigA-C vaccines achieved 100% survival in vaccinated hamsters, with no detectable lesions in vital organs, further supporting their superior efficacy. Alhydrogel, an adjuvant commonly used in animal immunization and approved for human use 30 played a crucial role in enhancing immune responses in this study. The significant reduction in histopathological damage and the robust immune responses observed highlight the potential of LigA-C mRNA vaccines as a viable strategy for preventing leptospirosis. Although current adjuvants and delivery systems could further optimize these vaccines, LigA demonstrates a distinct advantage over other OMPs by inducing substantial protection in both recombinant protein and mRNA vaccine formats. In conclusion, this study demonstrates that rLigA-C-based protein and LigA-C-mRNA vaccines elicit robust antibody responses and provide sterilizing immunity against lethal leptospiral infections in a hamster model. The conserved region of LigA-C effectively activates humoral and cell-mediated immunity, suggesting its potential for developing a universal leptospirosis vaccine for humans and animals. Materials and methods Leptospiral bacterins The leptospiral bacterins (heat-killed antigens) used for immunization were prepared following previously described protocols 31 . In brief, Leptospira interrogans serovar Australis strain Ballico was subjected to centrifugation at 12,000 × g, washed four times with phosphate-buffered saline (PBS), and then heat-inactivated by incubation at 100°C for 2 hours. The inactivated cells were washed four additional times with PBS and resuspended in PBS to achieve a final concentration of approximately 10⁸ leptospires per 0.1 ml. Sterility was confirmed by the absence of bacterial growth when cultured on EMJH medium and blood agar at both 30°C and 37°C. Cloning and expression of LigA-C The L. interrogans serovar Australis strain BallicoligA-C terminus gene was cloned into the pET15b vector as previously described 9 and provided by the Medical Microbiology Laboratory, Department of Microbiology, Bharathidasan University, Tiruchirappalli-24. Recombinant clones were validated through colony PCR and restriction endonuclease analysis (REA). The LigA-C recombinant protein was overexpressed and purified using the Escherichia coli expression system,following established protocols 9 .Recombinant plasmids were transformed into E. coli BL21 (DE3) cells (Novagen, Madison, WI), and expression of the His₆-tagged LigA-C protein was induced by adding 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to LB medium (pH 7.5) once the cultures reached an optical density (OD₆₀₀) of 0.6. Cells were harvested 2 to 3 hours post-induction. The recombinant His₆-tagged protein was purified using immobilized metal affinity chromatography (IMAC) with a Ni²⁺ resin column (Bio-Rad, USA) under denaturing conditions in a buffer containing 8 M urea, as per the manufacturer’s protocol. The protein was then extensively dialyzed, and its purity was confirmed by SDS-PAGE analysis. SDS-PAGE and Immunoblotting SDS-PAGE was conducted using 10% polyacrylamide gels and a discontinuous buffer system as previously described 32 . Affinity-purified proteins were mixed with 2× SDS-PAGE sample buffer (125 mM Tris-HCl, 4% SDS, 2% glycerol, 1% β-mercaptoethanol, 0.5% bromophenol blue), boiled for 5 minutes, and loaded onto the gels. Electrophoresis was performed in a Mini-PROTEAN tetra cell apparatus (Bio-Rad, USA) using Tris-glycine running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3) at 100 V for 2 hours.The separated proteins were transferred onto nitrocellulose membranes (Bio-Rad, USA) using transfer buffer (20 mM Tris, 190 mM glycine, 20% methanol, pH 8.3). Membranes were then blocked with 4% non-fat dry milk in Tris-buffered saline (20 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5). Hyperimmune rabbit sera were used as the primary antibody, followed by incubation with a secondary antibody (anti-rabbit IgG conjugated with horseradish peroxidase [HRP], Sigma, St. Louis, MO). Detection was carried out using the West Pico Signal chemiluminescence kit (Thermo Fisher Scientific, USA), and the results were documented using the Fusion Solo STM imaging system (VilberLourmat, Paris, France). In vitro transcription of mRNA and purification The plasmid DNA (pDNA) was linearized using restriction endonuclease digestion (REA) and used as a template for in vitro transcription. The mRNA was synthesized in a 50 µL reaction mixture containing 5× transcription buffer, 2 mM rNTP mix, 40 U RNase H, 30 U T7 RNA polymerase, 0.5 µg linearized pDNA template, and DEPC-treated Milli-Q water. The reaction was incubated at 37°C for 5 hours. Following transcription, the pDNA template was digested with 2 U DNase I at 37°C for 30 minutes, and the reaction was terminated by adding 0.5 M EDTA (pH 8.0) and heating at 65°C for 10 minutes.The synthesized mRNA was purified using the ethanol precipitation method with 3 M sodium acetate (pH 5.2) as described previously 33 . The quality and quantity of the purified mRNA was checked by agarose gel electrophoresis and mRNA quantification using a Biophotometer (Eppendorf, Hamburg, Germany) 34 .The purified mRNA was stored at − 80°C until further use. Conventional PCR and Real-time qPCR The specificity and quantity of the gene were assessed using conventional PCR and qRT-PCR with the LigA-C terminus forward primer (5’-CCGCTCGAGACAGAGCAAGTCACCTGGA-3’) and reverse primer (5’-CGCGGATCCTATGGCTCCGTTTTAATAGAGGC-3’).For conventional PCR, the 50 µL reaction mixture consisted of 1× Taq buffer, 0.2 mM dNTPs, 10 pM of each primer, 1.25 U of Taq DNA polymerase, 40 ng of cDNA as a template, and Milli-Q water to make up the final volume.Quantification of mRNA was performed using LigA-C gene-based real-time PCR on the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Immunization and challenge experiments Golden Syrian hamsters, aged 5–6 weeks, were divided into six groups and each group contains 5–6 animals (n = 5–6) and immunized intramuscularly. One group received 100 µg of naked mRNA dissolved in ringer’s buffer solution, while another group received 100 µg of recombinant LigA-C (rLigA-C) protein formulated with Alhydrogel. A negative control group was immunized with an emulsion of Alhydrogel and PBS, and a positive control group was immunized with 10⁸ heat-killed whole leptospires. Immunizations were administered at 14-day intervals.Twenty-eight days after the initial immunization, all hamsters were challenged intraperitoneally with 5× the median lethal dose (LD₅₀) of L. interrogans serovar Australis strain Ballico (1.3 × 10³ leptospires), following the protocol described in previous studies 35 . The animals were monitored daily for survival, and those surviving were euthanized on day 28 post-challenge.Blood samples were collected from the jugular vein on day 0 (pre-immune), day 14 (post-first dose), and day 28 (post-second dose). Serum samples were stored at − 80°C for subsequent analysis.The study protocols were approved by the Institutional Ethics Committee of Bharathidasan University (BDU/IAEC/2011/29). Humoral immune response ELISA was conducted to evaluate the humoral immune response. Polystyrene 96-well microtiter plates (Nunc; Thermo Scientific, USA) were coated overnight at 4°C with 0.2 µg/well of recombinant LigA (rLigA) antigen in carbonate coating buffer (pH 9.6). The plates were then washed three times with PBS containing 0.05% Tween 20 (PBST) and blocked with 4% non-fat milk in PBST.Hamster serum samples, diluted 1:100, were added to the wells and incubated at 37°C for 1 hour, followed by three washes with PBST. Goat anti-hamster IgG conjugated to peroxidase (Sigma-Aldrich, St. Louis, MO) was diluted 1:4000, added to the wells, and incubated at 37°C for 1 hour. The plates were then washed five times with PBST.The immune reaction was visualized using o-phenylenediamine dihydrochloride (Sigma-Aldrich, St. Louis, MO) as the substrate. The reaction was terminated by adding 0.1 M sulfuric acid (Merck, Germany), and the optical density (OD) was measured at 490 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Sera samples were assayed in triplicate, and mean values were calculated. RNA isolation, cDNA synthesis, and qRT-PCR Total RNA was extracted from the blood samples of immunized hamsters using the RNeasy Mini Kit (Qiagen, USA) following the manufacturer's protocol. Complementary DNA (cDNA) synthesis was performed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) as per the manufacturer’s instructions.Quantitative PCR (qPCR) was conducted with SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA) in a 20 µl reaction volume containing 50 ng of cDNA, 10 µl of Master Mix, and 0.2 µM of each primer. The qPCR cycling conditions included an initial denaturation step at 95°C for 3 minutes, followed by 39 amplification cycles (95°C for 5 seconds, 58°C or 60°C for 30 seconds, and a variable extension time at 72°C).Melting curve analysis was performed immediately after amplification at a linear temperature transition rate of 0.1°C/s from 70°C to 95°C, with continuous fluorescence detection. Cytokine gene expression was quantified using the relative CT (ΔΔCT) method. Briefly, the fold change of each target gene was normalized to the CT value of the GAPDH housekeeping gene (ΔCT) and compared to a calibrator sample, represented by the same normalized gene in the PBS or Alhydrogel-immunized hamster group (ΔΔCT). The final values, representing the relative fold change between immunized and non-immunized hamsters, were calculated as the mean of triplicate measurements. Histopathology Surviving hamsters were euthanized on day 28 post-challenge. To euthanize the hamsters, the animals were exposed to inhalation anesthesia using an open drop jar method. Isoflurane (1–3%) was administered via the respiratory route at a dose of 0.05 ml/L; the volume in the chamber was vaporized by placing it on cotton at the bottom of the jar. The hamster was then placed inside a closed chamber for induction. The animal was closely monitored in the jar, focusing on its respiration rate. Once the animal lost the righting reflex and its breathing slowed, it was removed from the jar for further assessment process 36 . Kidney and lung tissues were harvested and fixed in 10% neutral buffered formalin (pH 7.0). The fixed tissues were then sectioned into 5–6 µm slices, stained with hematoxylin and eosin, and examined under a light microscope for histopathological analysis. Data analysis The Fisher exact test and log-rank test were employed to assess significant differences in mortality and survival, respectively, between the experimental groups. Cytokine and antibody levels were analyzed using the paired t-test and two-way ANOVA to compare group differences. A p-value of ≤ 0.05 was considered statistically significant in all analyses. Data were analyzed using GraphPad Prism 8.0 (GraphPad Software, Inc.). Declarations Acknowledgements This study was supported by the Indian Council of Medical Research (ICMR) grant IIRP-2023-6075/F1. 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Immunoprotective properties of recombinant LigA and LigB in a hamster model of acute leptospirosis. PloS one , 12 (7), e0180004. https://doi.org/10.1371/journal.pone.0180004 Faisal, S. M., Yan, W., Chen, C. S., Palaniappan, R. U., McDonough, S. P., & Chang, Y. F. (2008). Evaluation of protective immunity of Leptospira immunoglobulin like protein A (LigA) DNA vaccine against challenge in hamsters. Vaccine , 26 (2), 277-287. https://doi.org/10.1016/j.vaccine.2007.10.029 Cao, Y., Faisal, S. M., Yan, W., Chang, Y. C., McDonough, S. P., Zhang, N., ... & Chang, Y. F. (2011). Evaluation of novel fusion proteins derived from extracellular matrix binding domains of LigB as vaccine candidates against leptospirosis in a hamster model. Vaccine , 29 (43), 7379-7386. https://doi.org/10.1016/j.vaccine.2011.07.070 Coutinho, M. L., Choy, H. A., Kelley, M. M., Matsunaga, J., Babbitt, J. T., Lewis, M. S., ... & Haake, D. A. (2011). A LigA three-domain region protects hamsters from lethal infection by Leptospira interrogans. PLoS neglected tropical diseases , 5 (12), e1422. https://doi.org/10.1371/journal.pntd.0001422 Estavoyer, J. M., Racadot, E., Couetdic, G., Leroy, J., &Grosperrin, L. (1991). Tumor necrosis factor in patients with leptospirosis. Reviews of infectious diseases , 13 (6), 1245-1245. https://doi.org/10.1093/clinids/13.6.1245 Petrovsky, N., & Aguilar, J. C. (2004). Vaccine adjuvants: current state and future trends. Immunology and cell biology , 82 (5), 488-496. https://doi.org/10.1111/j.0818-9641.2004.01272.x Adler, B., & Faine, S. (1980). Immunogenicity of boiled compared with formalized leptospiral vaccines in rabbits, hamsters and humans. Epidemiology & Infection , 84 (1), 1-10. https://doi.org/10.1017/S0022172400026450 Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. nature , 227 (5259), 680-685. Green, M. R., & Sambrook, J. (2020). Precipitation of RNA with Ethanol. Cold Spring Harb Protoc , 101717 . DOI: 10.1101/pdb.prot101717 Den Roover, S., & Aerts, J. L. (2024). MRNACalc: An accurate RNA quantification tool in the era of modified nucleosides. Molecular Therapy-Nucleic Acids , 35 (2). DOI: 10.1016/j.omtn.2024.102226 Raja, V., Sobana, S., Mercy, C. S. A., Cotto, B., Bora, D. P., & Natarajaseenivasan, K. (2018). Heterologous DNA prime-protein boost immunization with RecA and FliD offers cross-clade protection against leptospiral infection. Scientific Reports , 8 (1), 6447. https://doi.org/10.1038/s41598-018-24674-8 Fox, James G., et al. “Inhalant Anesthetics.” Laboratory Animal Medicine, Third ed., Elsevier/Academic Press, 2015, pp. 1141–1142. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6665688","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":472108816,"identity":"79a25c84-3170-433b-b64a-ab05c39c7ef6","order_by":0,"name":"Perumal Saranya","email":"","orcid":"","institution":"Bharathidasan University","correspondingAuthor":false,"prefix":"","firstName":"Perumal","middleName":"","lastName":"Saranya","suffix":""},{"id":472108817,"identity":"fe8bb9a3-7b30-4818-a3b2-6f19f1855540","order_by":1,"name":"Panneerselvam Selvambika","email":"","orcid":"","institution":"Bharathidasan University","correspondingAuthor":false,"prefix":"","firstName":"Panneerselvam","middleName":"","lastName":"Selvambika","suffix":""},{"id":472108818,"identity":"3fc20834-3c4e-45fb-ad95-5c28fb68ff7a","order_by":2,"name":"Dianne Langford","email":"","orcid":"","institution":"Rowan University","correspondingAuthor":false,"prefix":"","firstName":"Dianne","middleName":"","lastName":"Langford","suffix":""},{"id":472108819,"identity":"03101eeb-d683-48e0-97e3-13b27c147681","order_by":3,"name":"Kalimuthusamy Natarajaseenivasan","email":"data:image/png;base64,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","orcid":"","institution":"Bharathidasan University","correspondingAuthor":true,"prefix":"","firstName":"Kalimuthusamy","middleName":"","lastName":"Natarajaseenivasan","suffix":""}],"badges":[],"createdAt":"2025-05-14 15:38:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6665688/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6665688/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84819946,"identity":"914e0f0b-998d-4686-9f00-ad1708da142e","added_by":"auto","created_at":"2025-06-17 16:05:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":144965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eRestriction endonuclease analysis of \u003cem\u003eligA\u003c/em\u003e-C recombinant clone (Lane M –1 kb DNA ladder, 1 – pET15b plasmid, 2 – \u003cem\u003eligA\u003c/em\u003e-Cconstruct, 3 – Single digestion of pET15b, 4 – Single digestion of construct, 5 – Double digestion of construct with the released insert, 6 – \u003cem\u003eligA\u003c/em\u003e-C colony PCR) \u003cstrong\u003eB. \u003c/strong\u003eExpression and purification of LigA-C protein confirmation by SDS-PAGE(Lane M –Low range protein marker, 1 –Uninduced whole cell lysate, 2 –IPTG Induced whole cell lysate, 3 –Purified recombinant LigA-C protein) \u003cstrong\u003eC. \u003c/strong\u003eConfirmation of specificity of recombinant proteins by immunoblotting (1 –Uninduced whole cell lysate, 2 –IPTG Induced whole cell lysate, 3 –Purified recombinant LigA-C protein) \u003cstrong\u003eD. \u003c/strong\u003eConventional PCR(Lane M –1 Kb DNA ladder, 1 –IVT mRNA sample, 2 –Amplification of cDNA, 3 –Positive control LigA-C plasmid, 4 –Assay control) \u003cstrong\u003eE. \u003c/strong\u003eSpecificity of \u003cem\u003eligA\u003c/em\u003e-C qPCR for mRNA transcript.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6665688/v1/18744b12b0b3d3db7f5d3419.png"},{"id":84819135,"identity":"c459c7eb-7c93-45a7-ac1c-c9160c721507","added_by":"auto","created_at":"2025-06-17 15:57:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":83818,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eEvaluation of humoral immune response in control and immunized hamster groups by IgG ELISA. WCL or rLigA-C was used as antigen. Graphs represent the Mean ± SD of the OD of the sera obtained on day 0 (pre-vaccination), 14\u003csup\u003eth\u003c/sup\u003e (before booster) and 28\u003csup\u003eth\u003c/sup\u003e days (before challenge) \u003cstrong\u003eB-E. \u003c/strong\u003eEvaluation of cell mediated immune response in control and immunized hamster groups by qRT-PCR. (B) TNFα, (C) IL-10, (D) IL-4, (E) IFN-γ. The relative CT (ΔΔCT) method was used to quantify cytokine gene expression: CTs were normalized against the GAPDH gene CT (ΔCT) and then compared to the same normalized gene in the PBS or Alhydrogel immunized hamster group (calibrator). The control groups were set to 1. ns-no significance, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. P values were obtained through comparison with the negative control (PBS or Alhydrogel) using a Tukey’s multiple comparisons test by 2way ANOVA.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6665688/v1/a5718561df7cbcd346e9ef15.png"},{"id":84819136,"identity":"041ef19b-796f-49cf-a38a-8f858317716e","added_by":"auto","created_at":"2025-06-17 15:57:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":92685,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival graph of hamsters immunized with different vaccines after \u003cem\u003eLeptospira interrogans\u003c/em\u003estrain Ballico challenge with 10\u003csup\u003e8 \u003c/sup\u003eleptospires/ml. The Wilcoxon log-rank test was used to determine significant survival differences between immunised groups and the controls [PBS or Alhydrogel] (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6665688/v1/2d75479a2558aa413a20d395.png"},{"id":84819153,"identity":"5ba15d5a-1c2a-4296-aafd-0de63fb4ac65","added_by":"auto","created_at":"2025-06-17 15:57:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":740774,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathology of lung tissues stained with haematoxylin and eosin from hamsters that survived the lethal challenge. (A – normal hamsters, B,C– non-vaccinated control hamsters (PBS \u0026amp; Alhydrogel), D – heat killed leptospires, E – subunit vaccine, F – mRNA vaccine and challenged with \u003cem\u003eL. interrogans.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6665688/v1/9d09bd7acfdb8eaf79ef51f9.png"},{"id":84819141,"identity":"d45af891-342b-431f-b728-399d7d86530d","added_by":"auto","created_at":"2025-06-17 15:57:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":718165,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathology of kidney tissues stained with haematoxylin and eosin from hamsters that survived the lethal challenge. (A – normal hamsters, B,C– non-vaccinated control hamsters (PBS \u0026amp; Alhydrogel), D – heat killed leptospires, E – subunit vaccine, F – mRNA vaccine and challenged with \u003cem\u003eL. interrogans.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6665688/v1/78c0f532c94c4d6cf6fc0f9c.png"},{"id":92846755,"identity":"88fd3add-2985-4068-81a9-964001a4b3f6","added_by":"auto","created_at":"2025-10-06 09:39:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2419779,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6665688/v1/ac686cd6-40d9-48f2-ab42-124fc172151a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Leptospiral LigA-C mRNA-based immunization protects against Leptospira interrogans Serovar Australis infection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eVaccination is one of the most effective medical strategies for preventing and controlling infectious diseases\u003csup\u003e1\u003c/sup\u003e. The need for novel vaccine approaches that ensure safe, long-lasting, and cross-protective immunity has become increasingly critical\u003csup\u003e2-5\u003c/sup\u003e. In this context, subunit vaccines have gained significant attention and demonstrated efficacy against several infectious agents, including \u003cem\u003eLeptospira\u003c/em\u003e species\u003csup\u003e6\u003c/sup\u003e. Identifying conserved protective antigens expressed by multiple pathogenic \u003cem\u003eLeptospira\u003c/em\u003e serovars and utilizing them in recombinant DNA or protein-based vaccine formulations could offer a safe, effective, and cross-protective solution against leptospirosis\u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn recent years, mRNA vaccines have emerged as a groundbreaking platform with broad applicability, ranging from prophylactic interventions to therapeutic treatments, and from personalized medicine to global health solutions\u003csup\u003e8\u003c/sup\u003e. Their ability to stimulate both humoral and cellular immune responses makes mRNA vaccines a promising technology. Moreover, Leptospiral outer membrane proteins (OMPs) play a central role in pathogenesis and are key markers for distinguishing pathogenic from non-pathogenic strains. Among these, the immunoglobulin-like proteins LigA and LigB are surface-exposed adhesins expressed in vivo, which interact with the host extracellular matrix and homeostatic proteins. LigA, in particular, has been extensively studied for its diagnostic and vaccine potential, with its immunogenic epitopes showing promise in experimental models\u003csup\u003e9\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe present study aimed to evaluate whether LigA-C, delivered via mRNA or protein-based immunization, could induce protective immunity in hamsters challenged with a virulent strain of \u003cem\u003eLeptospira interrogans\u003c/em\u003e investigated the potential of LigA-C-specific mRNA to activate a robust immune response in a hamster model. We evaluated the expression levels of pro-inflammatory cytokines (TNF-α, interferon-γ [IFN-γ]), the anti-inflammatory cytokine interleukin-10 (IL-10), and interleukin-4 (IL-4), a cytokine involved in antibody production. Overall, the study assessed the immunogenicity and protective efficacy of LigA-C-specific mRNA as a vaccine candidate against leptospiral infection.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCloning, expression, purification and Western blot analysis of recombinant protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eL. interrogans\u003c/em\u003e serovar Australis strain BallicoligA-C terminus gene, cloned into the pET15b expression vector, was confirmed by colony PCR and restriction endonuclease analysis (REA) (Fig. 1A). The recombinant LigA-C was expressed upon induction with 1.0 mM IPTG, and its molecular mass (58 kDa) was verified by SDS-PAGE (Fig. 1B). Immunoblot analysis showed that polyclonal rabbit antibodies specific to recombinant LigA-C recognized a 58 kDa protein band (Fig. 1C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emRNA transcript analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe in vitro transcribed mRNA and the amplification of a 1530 bp product of the ligA-C gene from cDNA were visualized on a 1.2% 1X TAE agarose gel (Fig. 1D), stained with EtBr, and documented using a gel documentation system (Bio-Rad, USA). To confirm the specificity of the primers, the mRNA transcript was subjected to PCR, resulting in a significant CT value (Fig. 1E). The results indicate that the transcript targeting the ligA-C gene was specific for detecting pathogenic leptospires. No amplification was observed in the non-template controls throughout the experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of Antigens for Immunization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor mRNA-based immunization, the linearized pDNA was in vitro transcribed and purified using the ethanol precipitation method. For the subunit vaccine, the LigA-C protein was overexpressed in \u003cem\u003eE. coli\u003c/em\u003e, achieving the expected size of 58 kDa. The recombinant protein was purified with a good yield and used for immunization. The heat-killed vaccine was prepared from \u003cem\u003eL. interrogans\u003c/em\u003e serovar Australis strain Ballico, and sterility testing confirmed the absence of leptospiral growth. The antigen formulation for the first booster, administered on day 0, was prepared with 100 \u0026mu;g of protein in Alhydrogel (Sigma, St. Louis, MO). A second booster of 100 \u0026mu;g protein was given on day 14 through intramuscular injection.The mRNA concentration wasdeterminedby testing the LD\u003csub\u003e50\u003c/sub\u003e, and was found to be 100 \u0026mu;g.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibody response in immunized hamsters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe specific antibody response in immunized hamsters was assessed using an IgG-ELISA at three different time points: 0, 14, and 28 days post-immunization. The results, shown in (Fig. 2A), demonstrated that all immunization strategies induced significant antibody levels. Hamsters immunized with mRNA showed higher IgG levels than the control groups at 14 days post-inoculation (P\u0026lt;0.0001), with these levels continuing to rise at 28 days post-inoculation, similar to the recombinant subunit immunization. No significant antibody levels were detected in the negative control groups (PBS or Alhydrogel). However, no significant difference (p\u0026gt;0.05) was observed in antibody production between the protein subunit and mRNA vaccine groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytokine expression profile\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe induction of both Th1 and Th2 type cytokines was assessed on day 28 using total RNA isolated from the blood samples of vaccinated hamsters. The abundance of these cytokines was measured through qRT-PCR analysis (Fig. 2B,C,D,E). The results revealed that the expression levels of the anti-inflammatory cytokine IL-10 in all vaccinated groups were inversely correlated with survival. In contrast, the expression of the Th2 cytokine IL-4 and the inflammatory cytokines TNF-\u0026alpha; and IFN-\u0026gamma; in the mRNA/rLigA-C vaccinated groups was increased and positively correlated with hamster survival following the challenge. Groups that received the mRNA/heat-killed leptospires showed significant differences (P\u0026lt;0.0001) in cytokine levels compared to the control groups. However, immunization with the recombinant protein subunit vaccine elicited a cytokine response that was significantly less pronounced (P\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProphylactic effects of the antigenic candidates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protective efficacy of the immunizations was assessed up to 28 days post-challenge, based on survival rates and histopathological findings in vital organs. The survival data (Fig. 3) demonstrated that animals immunized with mRNA/rLigA-C, followed by leptospiral challenge, were significantly protected, with a survival rate of 100% (P\u0026lt;0.01). All hamsters in the positive control group, which were administered killed whole leptospires, also showed 100% protection (P\u0026lt;0.01). In contrast, animals that received PBS + Alhydrogel succumbed to the infection, with median survival times of 11 and 14.5 days, confirming the virulence of the challenge strain. Histopathological examination of the lungs of deceased animals revealed alveolar hemorrhages, edema, capillary congestion, and leukocyte infiltration (Fig. 4). In the kidneys, the predominant lesions observed included interstitial nephritis, renal tubular failure, and necrosis (Fig. 5). From the hamster models, all vaccines showed 100% protection, but the various types of lesions formation in lungs and kidneys showed variation in organ protection against the \u003cem\u003eLeptospira\u0026nbsp;\u003c/em\u003efrom one vaccine group to another group. These findings suggest that leptospiral mRNA immunization can induce sterilizing immunity against leptospirosis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eVaccination remains the most cost-effective and reliable strategy to prevent infectious diseases. However, developing an efficient vaccine against leptospirosis that offers cross-protection against various pathogenic serovars continues to be a significant challenge\u003csup\u003e10,11\u003c/sup\u003e.Current bacterin vaccines have notable limitations, including side effects, short-lived immunity, and serovar-specific protection, underscoring the need for innovative approaches\u003csup\u003e12\u0026ndash;15\u003c/sup\u003e. Subunit recombinant vaccines, composed of purified antigens, represent a promising alternative. Nevertheless, these vaccines are inherently weakly immunogenic due to the absence of pathogen-associated molecular patterns and require adjuvants to enhance immune activation\u003csup\u003e16\u003c/sup\u003e.In this study, we evaluated mRNA and protein subunit vaccines using rLigA-C as the antigen and Alhydrogel as the adjuvant. mRNA vaccines offer several advantages, including efficient antigen delivery, stimulation of both humoral and cellular immunity, cost-effective mass production, and the elimination of purification steps\u003csup\u003e17\u003c/sup\u003e. Subunit vaccines are inherently safe and predominantly elicit humoral immunity, although their efficacy may be affected by antigen misfolding during production under denaturing conditions\u003csup\u003e18\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLeptospira\u003c/em\u003e outer membrane proteins (OMPs), such as the well-characterized Lig proteins, are key adhesins that mediate attachment to the host extracellular matrix (ECM). Immunization with full-length Lig proteins or their conserved C-terminal domains has previously shown significant protection in animal models\u003csup\u003e19\u0026ndash;22\u003c/sup\u003e. The protection is largely attributed to anti-Lig antibodies, which block bacterial adhesion to the ECM, preventing invasion and subsequent infection. Consistent with these findings, the rLigA-C antigen elicited a robust antibody response against both the recombinant protein and the native protein in leptospiral whole-cell lysates (WCL).\u003c/p\u003e \u003cp\u003eOur results demonstrated that rLigA-C vaccination, with a 100\u0026micro;g dose selected based on prior studies, provided protective immunity in hamsters\u003csup\u003e23\u003c/sup\u003e and indicated that a 100 \u0026micro;gbooster dose of mRNA has an acceptable safety profile\u003csup\u003e24\u003c/sup\u003e.Previous research has highlighted the central role of humoral immunity in protection against leptospirosis\u003csup\u003e25\u003c/sup\u003e. The protective efficacy observed here suggests that LigA domains involved in ECM attachment and complement regulation are sufficient to induce immunity, warranting further exploration of these mechanisms. This study corroborates earlier findings on LigA's immune protective properties\u003csup\u003e2,9,19,22,23,26\u0026ndash;28\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHamsters were chosen as the experimental model due to their suitability for leptospirosis immunization studies, given that mice are not ideal for this purpose\u003csup\u003e23\u003c/sup\u003e. Both rLigA-C and mRNA vaccines induced significant protective immunity in the hamster model, with comparable antibody titers between the two approaches. While variability in immunogenicity was observed across experiments.The overall differences in antibody responses and survival rates between vaccinated and control groups were statistically significant, underscoring the efficacy of these immunization platforms.The LigA-C mRNA vaccine induced a significant antibody response after booster doses, suggesting its potential to generate protective immunity against leptospirosis. Additionally, it activated both humoral and cell-mediated immunity (CMI), as evidenced by increased Th1 (IFN-γ) and Th2 (IL-4) cytokine levels. While pro-inflammatory cytokines such as TNF-α were not significantly elevated compared to controls, the vaccines did induce significant IL-10 activation, supporting a balanced immune response. These findings,aligned with previous studies,show that excessive TNF-α levels are associated with severe leptospirosis\u003csup\u003e29\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe histopathological analysis revealed that rLigA-C and mRNA immunizations provided substantial protection, as immunized animals displayed minimal lung lesions and absence of severe kidney damage, in contrast to severe pathology observed in control animals. While the heat-killed vaccine also elicited some protection, its immune response was insufficient to completely eliminate bacterial colonization. Notably, mRNA and rLigA-C vaccines achieved 100% survival in vaccinated hamsters, with no detectable lesions in vital organs, further supporting their superior efficacy. Alhydrogel, an adjuvant commonly used in animal immunization and approved for human use\u003csup\u003e30\u003c/sup\u003eplayed a crucial role in enhancing immune responses in this study. The significant reduction in histopathological damage and the robust immune responses observed highlight the potential of LigA-C mRNA vaccines as a viable strategy for preventing leptospirosis. Although current adjuvants and delivery systems could further optimize these vaccines, LigA demonstrates a distinct advantage over other OMPs by inducing substantial protection in both recombinant protein and mRNA vaccine formats.\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrates that rLigA-C-based protein and LigA-C-mRNA vaccines elicit robust antibody responses and provide sterilizing immunity against lethal leptospiral infections in a hamster model. The conserved region of LigA-C effectively activates humoral and cell-mediated immunity, suggesting its potential for developing a universal leptospirosis vaccine for humans and animals.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLeptospiral bacterins\u003c/h2\u003e \u003cp\u003eThe leptospiral bacterins (heat-killed antigens) used for immunization were prepared following previously described protocols\u003csup\u003e31\u003c/sup\u003e. In brief, \u003cem\u003eLeptospira interrogans\u003c/em\u003e serovar Australis strain Ballico was subjected to centrifugation at 12,000 \u0026times; g, washed four times with phosphate-buffered saline (PBS), and then heat-inactivated by incubation at 100\u0026deg;C for 2 hours. The inactivated cells were washed four additional times with PBS and resuspended in PBS to achieve a final concentration of approximately 10⁸ leptospires per 0.1 ml. Sterility was confirmed by the absence of bacterial growth when cultured on EMJH medium and blood agar at both 30\u0026deg;C and 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCloning and expression of LigA-C\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eL. interrogans\u003c/em\u003e serovar Australis strain BallicoligA-C terminus gene was cloned into the pET15b vector as previously described\u003csup\u003e9\u003c/sup\u003e and provided by the Medical Microbiology Laboratory, Department of Microbiology, Bharathidasan University, Tiruchirappalli-24. Recombinant clones were validated through colony PCR and restriction endonuclease analysis (REA). The LigA-C recombinant protein was overexpressed and purified using the \u003cem\u003eEscherichia coli\u003c/em\u003e expression system,following established protocols\u003csup\u003e9\u003c/sup\u003e.Recombinant plasmids were transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) cells (Novagen, Madison, WI), and expression of the His₆-tagged LigA-C protein was induced by adding 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to LB medium (pH 7.5) once the cultures reached an optical density (OD₆₀₀) of 0.6. Cells were harvested 2 to 3 hours post-induction. The recombinant His₆-tagged protein was purified using immobilized metal affinity chromatography (IMAC) with a Ni\u0026sup2;⁺ resin column (Bio-Rad, USA) under denaturing conditions in a buffer containing 8 M urea, as per the manufacturer\u0026rsquo;s protocol. The protein was then extensively dialyzed, and its purity was confirmed by SDS-PAGE analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSDS-PAGE and Immunoblotting\u003c/h2\u003e \u003cp\u003eSDS-PAGE was conducted using 10% polyacrylamide gels and a discontinuous buffer system as previously described\u003csup\u003e32\u003c/sup\u003e. Affinity-purified proteins were mixed with 2\u0026times; SDS-PAGE sample buffer (125 mM Tris-HCl, 4% SDS, 2% glycerol, 1% β-mercaptoethanol, 0.5% bromophenol blue), boiled for 5 minutes, and loaded onto the gels. Electrophoresis was performed in a Mini-PROTEAN tetra cell apparatus (Bio-Rad, USA) using Tris-glycine running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3) at 100 V for 2 hours.The separated proteins were transferred onto nitrocellulose membranes (Bio-Rad, USA) using transfer buffer (20 mM Tris, 190 mM glycine, 20% methanol, pH 8.3). Membranes were then blocked with 4% non-fat dry milk in Tris-buffered saline (20 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5). Hyperimmune rabbit sera were used as the primary antibody, followed by incubation with a secondary antibody (anti-rabbit IgG conjugated with horseradish peroxidase [HRP], Sigma, St. Louis, MO). Detection was carried out using the West Pico Signal chemiluminescence kit (Thermo Fisher Scientific, USA), and the results were documented using the Fusion Solo STM imaging system (VilberLourmat, Paris, France).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro transcription of mRNA and purification\u003c/h2\u003e \u003cp\u003eThe plasmid DNA (pDNA) was linearized using restriction endonuclease digestion (REA) and used as a template for in vitro transcription. The mRNA was synthesized in a 50 \u0026micro;L reaction mixture containing 5\u0026times; transcription buffer, 2 mM rNTP mix, 40 U RNase H, 30 U T7 RNA polymerase, 0.5 \u0026micro;g linearized pDNA template, and DEPC-treated Milli-Q water. The reaction was incubated at 37\u0026deg;C for 5 hours. Following transcription, the pDNA template was digested with 2 U DNase I at 37\u0026deg;C for 30 minutes, and the reaction was terminated by adding 0.5 M EDTA (pH 8.0) and heating at 65\u0026deg;C for 10 minutes.The synthesized mRNA was purified using the ethanol precipitation method with 3 M sodium acetate (pH 5.2) as described previously\u003csup\u003e33\u003c/sup\u003e. The quality and quantity of the purified mRNA was checked by agarose gel electrophoresis and mRNA quantification using a Biophotometer (Eppendorf, Hamburg, Germany)\u003csup\u003e34\u003c/sup\u003e.The purified mRNA was stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eConventional PCR and Real-time qPCR\u003c/h2\u003e \u003cp\u003eThe specificity and quantity of the gene were assessed using conventional PCR and qRT-PCR with the LigA-C terminus forward primer (5\u0026rsquo;-CCGCTCGAGACAGAGCAAGTCACCTGGA-3\u0026rsquo;) and reverse primer (5\u0026rsquo;-CGCGGATCCTATGGCTCCGTTTTAATAGAGGC-3\u0026rsquo;).For conventional PCR, the 50 \u0026micro;L reaction mixture consisted of 1\u0026times; Taq buffer, 0.2 mM dNTPs, 10 pM of each primer, 1.25 U of Taq DNA polymerase, 40 ng of cDNA as a template, and Milli-Q water to make up the final volume.Quantification of mRNA was performed using LigA-C gene-based real-time PCR on the CFX96 Touch\u0026trade; Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmunization and challenge experiments\u003c/h2\u003e \u003cp\u003eGolden Syrian hamsters, aged 5\u0026ndash;6 weeks, were divided into six groups and each group contains 5\u0026ndash;6 animals (n\u0026thinsp;=\u0026thinsp;5\u0026ndash;6) and immunized intramuscularly. One group received 100 \u0026micro;g of naked mRNA dissolved in ringer\u0026rsquo;s buffer solution, while another group received 100 \u0026micro;g of recombinant LigA-C (rLigA-C) protein formulated with Alhydrogel. A negative control group was immunized with an emulsion of Alhydrogel and PBS, and a positive control group was immunized with 10⁸ heat-killed whole leptospires. Immunizations were administered at 14-day intervals.Twenty-eight days after the initial immunization, all hamsters were challenged intraperitoneally with 5\u0026times; the median lethal dose (LD₅₀) of \u003cem\u003eL. interrogans\u003c/em\u003e serovar Australis strain Ballico (1.3 \u0026times; 10\u0026sup3; leptospires), following the protocol described in previous studies\u003csup\u003e35\u003c/sup\u003e. The animals were monitored daily for survival, and those surviving were euthanized on day 28 post-challenge.Blood samples were collected from the jugular vein on day 0 (pre-immune), day 14 (post-first dose), and day 28 (post-second dose). Serum samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent analysis.The study protocols were approved by the Institutional Ethics Committee of Bharathidasan University (BDU/IAEC/2011/29).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHumoral immune response\u003c/h2\u003e \u003cp\u003eELISA was conducted to evaluate the humoral immune response. Polystyrene 96-well microtiter plates (Nunc; Thermo Scientific, USA) were coated overnight at 4\u0026deg;C with 0.2 \u0026micro;g/well of recombinant LigA (rLigA) antigen in carbonate coating buffer (pH 9.6). The plates were then washed three times with PBS containing 0.05% Tween 20 (PBST) and blocked with 4% non-fat milk in PBST.Hamster serum samples, diluted 1:100, were added to the wells and incubated at 37\u0026deg;C for 1 hour, followed by three washes with PBST. Goat anti-hamster IgG conjugated to peroxidase (Sigma-Aldrich, St. Louis, MO) was diluted 1:4000, added to the wells, and incubated at 37\u0026deg;C for 1 hour. The plates were then washed five times with PBST.The immune reaction was visualized using o-phenylenediamine dihydrochloride (Sigma-Aldrich, St. Louis, MO) as the substrate. The reaction was terminated by adding 0.1 M sulfuric acid (Merck, Germany), and the optical density (OD) was measured at 490 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Sera samples were assayed in triplicate, and mean values were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation, cDNA synthesis, and qRT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from the blood samples of immunized hamsters using the RNeasy Mini Kit (Qiagen, USA) following the manufacturer's protocol. Complementary DNA (cDNA) synthesis was performed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) as per the manufacturer\u0026rsquo;s instructions.Quantitative PCR (qPCR) was conducted with SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA) in a 20 \u0026micro;l reaction volume containing 50 ng of cDNA, 10 \u0026micro;l of Master Mix, and 0.2 \u0026micro;M of each primer. The qPCR cycling conditions included an initial denaturation step at 95\u0026deg;C for 3 minutes, followed by 39 amplification cycles (95\u0026deg;C for 5 seconds, 58\u0026deg;C or 60\u0026deg;C for 30 seconds, and a variable extension time at 72\u0026deg;C).Melting curve analysis was performed immediately after amplification at a linear temperature transition rate of 0.1\u0026deg;C/s from 70\u0026deg;C to 95\u0026deg;C, with continuous fluorescence detection. Cytokine gene expression was quantified using the relative CT (ΔΔCT) method. Briefly, the fold change of each target gene was normalized to the CT value of the GAPDH housekeeping gene (ΔCT) and compared to a calibrator sample, represented by the same normalized gene in the PBS or Alhydrogel-immunized hamster group (ΔΔCT). The final values, representing the relative fold change between immunized and non-immunized hamsters, were calculated as the mean of triplicate measurements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology\u003c/h2\u003e \u003cp\u003eSurviving hamsters were euthanized on day 28 post-challenge. To euthanize the hamsters, the animals were exposed to inhalation anesthesia using an open drop jar method. Isoflurane (1\u0026ndash;3%) was administered via the respiratory route at a dose of 0.05 ml/L; the volume in the chamber was vaporized by placing it on cotton at the bottom of the jar. The hamster was then placed inside a closed chamber for induction. The animal was closely monitored in the jar, focusing on its respiration rate. Once the animal lost the righting reflex and its breathing slowed, it was removed from the jar for further assessment process\u003csup\u003e36\u003c/sup\u003e. Kidney and lung tissues were harvested and fixed in 10% neutral buffered formalin (pH 7.0). The fixed tissues were then sectioned into 5\u0026ndash;6 \u0026micro;m slices, stained with hematoxylin and eosin, and examined under a light microscope for histopathological analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eThe Fisher exact test and log-rank test were employed to assess significant differences in mortality and survival, respectively, between the experimental groups. Cytokine and antibody levels were analyzed using the paired t-test and two-way ANOVA to compare group differences. A p-value of \u0026le;\u0026thinsp;0.05 was considered statistically significant in all analyses. Data were analyzed using GraphPad Prism 8.0 (GraphPad Software, Inc.).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Indian Council of Medical Research (ICMR) grant IIRP-2023-6075/F1. We also acknowledge the funding support of RashtriyaUchchatar Shiksha Abhiyan (RUSA 2.0). We also acknowledge UGC-Non-SAP, DST-FIST, and DST-PURSE for the common instrumentation facility at Bharathidasan University. We acknowledge Rowan-Virtua School of Osteopathic Medicine, Rowan University, Stratford, NJ, USA for the startup grants.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang, C., Maruggi, G., Shan, H., \u0026amp; Li, J. (2019). 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Heterologous DNA prime-protein boost immunization with RecA and FliD offers cross-clade protection against leptospiral infection. \u003cem\u003eScientific Reports\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e(1), 6447. https://doi.org/10.1038/s41598-018-24674-8\u003c/li\u003e\n\u003cli\u003eFox, James G., et al. \u0026ldquo;Inhalant Anesthetics.\u0026rdquo; Laboratory Animal Medicine, Third ed., Elsevier/Academic Press, 2015, pp. 1141\u0026ndash;1142.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Leptospirosis, Leptospira interrogans, LigA, subunit vaccine, mRNA vaccine ","lastPublishedDoi":"10.21203/rs.3.rs-6665688/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6665688/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLeptospirosis, caused by the pathogenic\u003cem\u003eLeptospira\u003c/em\u003e species, is one of the most prevalent zoonotic infections worldwide. Developing effective diagnostics and vaccines remains a critical challenge in combating this disease. Traditional bacterin-based vaccines have notable limitations, including short-lived immunity and serovar specificity. This study evaluated the leptospiral immunoglobulin-like protein A (LigA-C) as a vaccine candidate, focusing on its ability to elicit protective immune responses against leptospirosis. Using a hamster model, we demonstrated the protective efficacy of LigA-C mRNA immunization and explored the underlying immune mechanisms.LigA-C/mRNA immunization induced a robust antibody response, with significant increases observed following booster doses. Cytokine profiling revealed that elevated levels of IL-4, TNF-α, and IFN-γ were positively correlated with survival in immunized hamsters, while IL-10 levels were inversely correlated with protection. Immunized groups achieved 100% survival and exhibited minimal histopathological lesions in the kidneys and lungs. In contrast, control animals succumbed to infection within 11\u0026ndash;15 days post-challenge with \u003cem\u003eLeptospira interrogans\u003c/em\u003e serovar Australis strain Ballico.These findings indicate that LigA-C mRNA immunization confers protection through both humoral and cellular immune responses. The high survival rate and reduced pathology highlight the potential of LigA-C mRNA as a promising vaccine candidate for the prevention of leptospirosis.\u003c/p\u003e","manuscriptTitle":"Leptospiral LigA-C mRNA-based immunization protects against Leptospira interrogans Serovar Australis infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-17 15:57:12","doi":"10.21203/rs.3.rs-6665688/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7c44cee9-7439-4b39-81ef-8d84e1ca5b00","owner":[],"postedDate":"June 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50136781,"name":"Biological sciences/Immunology"},{"id":50136782,"name":"Biological sciences/Microbiology"},{"id":50136783,"name":"Health sciences/Diseases"}],"tags":[],"updatedAt":"2025-10-06T09:38:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-17 15:57:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6665688","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6665688","identity":"rs-6665688","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}