TonB-dependent receptor epitopes expressed in M. bovis BCG induced significant protection in the hamster model of leptospirosis

TonB-dependent receptor epitopes expressed in M. bovis BCG induced significant protection in the hamster model of leptospirosis | 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 Research Article TonB-dependent receptor epitopes expressed in M. bovis BCG induced significant protection in the hamster model of leptospirosis Everton Burlamarque Bettin, Jessica Dorneles, Amanda Silva Hecktheuer, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-996824/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Dec, 2021 Read the published version in Applied Microbiology and Biotechnology → Version 1 posted You are reading this latest preprint version Abstract Leptospirosis is an emerging infectious disease caused by pathogenic Leptospira spp. A universal vaccine against leptospirosis is likely to require highly conserved epitopes from pathogenic leptospires, that are exposed on the bacterial surface, and that generate a protective and sterilizing immune response. Our group recently identified several genes predicted to encode TonB-dependent receptors (TBDR) in Leptospira interrogans using a reverse vaccinology approach. Three leptospiral TBDRs were previously described and partially characterized as ferric-citrate, hemin, and cobalamin transporters. In the current study, we designed a fusion protein composed of predicted surface-exposed epitopes from three conserved leptospiral TBDRs. Based on their three-dimensional structural models and the prediction of immunogenic regions, nine putative surface-exposed fragments were selected to compose a recombinant chimeric protein. A Mycobacterium bovis BCG strain expressing this chimeric antigen encoded in the pUP500/P pAN mycobacterial expression vector was used to immunize Syrian hamsters. All animals (20/20) vaccinated with recombinant BCG survived infection with a endpoint dose of L. interrogan s ( P < 0.001). No animal survived in the negative control group. Immunization with our recombinant BCG elicited a humoral immune response against leptospiral TBDRs, as demonstrated by ELISA and immunoblot. No leptospiral DNA was detected by lipL32 qPCR in the kidneys of vaccinated hamsters. Similarly, no growth was observed in macerated kidney cultures from the same animals, suggesting the induction of a sterilizing immune response. Design of new vaccine antigens based on the structure of outer membrane proteins is a promising approach to overcome the impact of leptospirosis by vaccination. Applied & Industrial Microbiology Biotechnology and Bioengineering Leptospira reverse and structural vaccinology beta-barrel transmembrane protein epitope-based vaccines chimeric protein Figures Figure 1 Figure 2 Figure 3 Key points Predicted surface-exposed epitopes were identified in three leptospiral TBDRs; A M. bovis BCG strain expressing a chimeric protein (rTBDRchi) was constructed; Hamsters vaccinated with rBCG:TBDRchi were protected from lethal leptospirosis. Introduction Leptospirosis is a major zoonosis worldwide in terms of morbidity and mortality rates. The global incidence is estimated to be over one million cases every year, resulting in ~60,000 deaths (Costa et al. 2015 ). Rodents are the primary reservoir hosts of pathogenic Leptospira spp., while humans are incidental hosts. The disease also affects several wild and domestic animals (Ellis 2015 ; Haake and Levett 2015 ). Leptospirosis in humans can vary from mild symptoms, such as fever and myalgia to severe outcomes, with the development of acute kidney injury and pulmonary hemorrhage syndrome (Haake and Levett 2015 ). Leptospiral vaccines currently available are inactivated whole-cell preparations (bacterins) with widespread veterinary applications, but limited use in humans. Bacterins confer a short-term immune response that is protective only against the serovars included in the vaccine formulation (Adler 2015 ). Several recombinant proteins have been evaluated towards the development of new and improved leptospiral vaccines, with variable success (Felix et al. 2020 ). While some experimental recombinant vaccines were protective against lethal challenge, the majority failed to achieve heterologous and/or sterilizing protection, highlighting the need to evaluate novel vaccination strategies (Grassmann et al. 2017c ; Felix et al. 2020 ). A new and effective vaccine against leptospirosis will likely include surface-exposed epitopes conserved among pathogenic Leptospira spp., allowing bacterial recognition by humoral immune response (Grassmann et al. 2017c ; Grassmann et al. 2017a ; Dellagostin et al. 2017 ). Our group previously used a reverse and structural vaccinology approach to identify leptospiral beta-barrel outer membrane proteins (βb-OMP), including six TonB-dependent receptors (TBDR) (Grassmann et al. 2017a ). TBDRs are outer-membrane proteins that promote the uptake of essential nutrients such as iron, nickel, vitamins and carbohydrates (Noinaj et al. 2010 ). Since iron has limited bioavailability, its acquisition by bacteria is facilitated by the formation of complexes with other transport molecules, such as heme and siderophores, which are then internalized by specific TonB-dependent transport systems (Noinaj et al. 2010 ). Pathogenic and saprophytic Leptospira spp. require iron for in vitro growth(Faine 1959 ) and evolved mechanisms for iron sensing, acquisition and utilization (Louvel et al. 2006 ). Two leptospiral TBDRs, encoded by LIC10896 and LIC10964, were described as homologs to the ferric citrate transporter FecA and the PhuR hemin receptor (Nascimento et al. 2004 ; Louvel et al. 2006 ), respectively. Another TBDR, LIC12374, is annotated as a homolog of the cobalamin transporter BtuB (Nascimento et al. 2004 ; Louvel et al. 2006 ). Cobalamin (or vitamin B12) is a cofactor for enzymes related to a variety of essential functions, including those related to carbon metabolism and biosynthesis of nucleotides (Shelton et al. 2019 ). Similarly to iron, cobalamin is a small nutrient required for Leptospira spp. survival (Louvel et al. 2006 ). While saprophytic species rely on external sources, pathogenic leptospires encode a pathway for de novo production of cobalamin from L-glutamate (Fouts et al. 2016 ). Due to their important role in bacterial metabolism and nutrient acquisition, several TBDRs have been evaluated as vaccine candidates against many pathogens, resulting in successful protection (Alteri et al. 2009 ; Hu et al. 2012 ; Hubert et al. 2013 ). Epitope mapping in vaccine candidates permits the identification of regions with increased probability to elicit an immune response, consequently improving the antigenicity and immunogenicity of vaccine formulations and avoiding production of undesirable or ineffective responses (Oyarzún and Kobe 2016 ; Parvizpour et al. 2020 ). The rational selection of conserved epitopes also contributes to cross-protection (Liu et al. 2017 ; Nosrati et al. 2019 ; Xiao et al. 2020 ), a particularly important aspect to consider for the development of leptospirosis vaccines, where the achievement of heterologous, long-term protection are major goals. Combined with protein structural information, epitope selection is a powerful tool to identify immunogenic surface-exposed fragments required for bacterial opsonization (Grassmann et al. 2017a ). Opsonization of leptospires by specific immunoglobulins improved bacterial phagocytosis and clearance (Gomes-Solecki et al. 2017 ; Santecchia et al. 2020 ). Even though the immune response required for protection against leptospirosis is not yet fully understood, induction of a balanced cellular/humoral response is believed to be key for a protective immune response (Gomes-Solecki et al. 2017 ; Grassmann et al. 2017c ; da Cunha et al. 2019 ). Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is an attenuated strain widely used as a vaccine against human tuberculosis (TB) (Dockrell and Smith 2017 ). In use for a hundred years, BCG vaccination is a highly cost-effective intervention against tuberculosis, and is recommended for all newborns in countries with a high burden of TB (Trunz et al. 2006 ; Lobo et al. 2021 ). BCG triggers a long-lasting immunity after a single dose, presenting several adjuvant properties and a strong cellular response (Dockrell and Smith 2017 ). BCG-induced adaptative immune response include the expression of several interleukins, such as TNF-α and IFN-γ, and consequently, the proliferation and activation of macrophages (Dockrell and Smith 2017 ; Covián et al. 2019 ). Several studies have demonstrated the potential of recombinant BCG (rBCG) to enhance the immune response towards heterologous antigens (Gröschel et al. 2017 ; Soto et al. 2018 ), including those from L. interrogans (Seixas et al. 2007 ; Oliveira et al. 2019a ; Dorneles et al. 2020 ), and represents a promising alternative for antigen delivery (Oliveira et al. 2017 ). In this study, we identified highly conserved surface-exposed regions containing B and T CD4+ cell epitopes in three leptospiral TBDRs: LIC10896, LIC10964, and LIC12374. These fragments were combined on a multi-epitope fusion protein and evaluated using BCG as a vectorized vaccine carrier. Our preparation induced a protective immune response in the hamster model of acute leptospirosis, generating specific antibodies and sterilizing immunity. Materials And Methods Bacterial strains and growth conditions Escherichia coli strains BL21 (DE3) Star and TOP10 (Invitrogen, São Paulo, SP, Brazil) were grown at 37°C in Lysogeny Broth (LB) medium, with 100 µg.ml −1 ampicillin when required. M. bovis BCG (Pasteur and recombinant strains) were cultivated at 37°C in Middlebrook 7H9 broth (Difco, BD, São Paulo, SP, Brazil) supplemented with 0.5% glycerol, 0.05% Tween 80 and 10% Oleic acid, Albumin, Dextrose Complex (OADC) or in 7H10 agar (Difco, BD, São Paulo, SP, Brazil) with 10% OADC. The BCG media was supplemented with kanamycin, 25 µg.ml −1 , when needed. Low-passage virulent L. interrogans serogroup Icterohaemorrhagiae serovar (sv.) Copenhageni strain (st.) Fiocruz L1–130 (WDCM1012) was cultivated at 30°C in Ellinghausen–McCullough–Johnson–Harris (EMJH) medium supplemented with Leptospira EMJH enrichment (Difco, BD, São Paulo, SP, Brazil). In silico structure predictions and epitope selection Three proteins (LIC10896, LIC10964 and LIC12374) from L. interrogans sv. Copenhageni st. Fiocruz L1-130 (Taxid:267671), previously identified as leptospiral TonB-Dependent receptors, were selected for evaluation in this study. Three-dimensional (3D) structures were predicted by threading using the I-TASSER server (Yang and Zhang 2015 ). Models with the highest C-Score were selected for epitope prediction. The 3D protein structures were visualized using USCF Chimera 1.12 (Pettersen et al. 2004 ). Sequences for the CD4+ T cell epitopes, with high affinity to 14 different human major histocompatibility complex class II (MHC-II) alleles, were predicted using NetMHCII 2.2(Nielsen and Lund 2009 ) at the default settings. For linear (continuous) B cell epitope prediction, amino acid sequences were analyzed using BepiPred 1.0 (Larsen et al. 2006 ). Only T cell epitopes with a strong binding affinity (IC50 < 50 nM) and amino acid residues with a score higher than 0.35 in the B cell epitope analysis were considered. Epitopes were manually mapped onto the predicted 3D models of each βb-OMP in USCF Chimera 1.12. These models were aligned with their best structural template obtained from the Orientations of Proteins in Membranes (OPM) database (Lomize et al. 2012 ). All surface-exposed fragments containing B and T CD4+ cell epitopes were used for antigen construction. Orthologs for each L. interrogans TBDR were identified by BlastP (NCBI) in 10 pathogenic (P1 clade) Leptospira spp. proteomes (Online Resource 1, Table S1). Protein sequences with >70% identity and 40% query coverage were considered orthologs. Identity between the L. interrogans polypeptides and their orthologs was determined after multiple sequence alignments (MSA) using MUSCLE (EMBL-EBI) (Edgar et al. 2004 ). Recombinant protein expression and purification Selected surface-exposed fragments were assembled in silico using Vector NTI Advance® 11.5 (Invitrogen) without linkers, to construct the fusion proteins rTBDRchi, rLIC10896chi, rLIC10964chi and rLIC12374chi (Online Resource 2). E. coli codon optimized coding sequences (CDS) for each fusion protein were synthesized by Epoch Life Science (Texas, USA) and cloned into E. coli expression vector pAE (Ramos et al. 2004 ) using Bam HI and Hind III restriction sites. Recombinant plasmids were used to transform E. coli BL21 (DE3) C41 strain (Invitrogen, São Paulo, SP, Brazil) by heat-shock. When cultures reached mid-log phase (0.6-0.8 absorbance at OD 600 ), expression was induced by adding 0.5 mM IPTG (isopropyl-β-D-thiogalactopyranoside) and incubated at 37°C with agitation. After 4h, cells were harvested by centrifugation (7,000 × g , 15 min, 4°C). Pellets were washed in phosphate-buffered saline (PBS) and resuspended in lysis buffer (20 mM sodium phosphate, 0.5 M NaCl and 20 mM Imidazole, pH 8.0) before sonication with six 30-s pulses on ice. After lysis, insoluble proteins were harvested by centrifugation (10,000 × g , 1 h, 4˚C), and resuspended in denaturing binding buffer (8M urea, 20 mM sodium phosphate, 0.5 M NaCl, and 20 mM Imidazole, pH 8,0) and incubated under constant agitation for 16 h. After a final centrifugation (10,000 × g , 1 h, 4˚C), recombinant proteins were purified using the AKTA Start automated chromatography system (GE Healthcare). Briefly, the supernatant was applied to a nickel-charged HisTrap FF column (GE Healthcare, São Paulo, SP, Brazil), and after washing with 20 column volumes, recombinant fusion proteins were eluted over a total of 20 ml using a 0-100% gradient of elution buffer (8M urea, 20 mM sodium phosphate, 0.5 M NaCl, and 0.5 M Imidazole, pH 8,0). Purified proteins were then dialyzed against PBS at 4°C for 24 h and stored at -80˚C or 4°C. Production of anti-rTBDRchi hyperimmune sera Four-week-old female Wistar rats ( Rattus norvegicus ) were injected intraperitoneally with 50 µg rTBDRchi emulsified in complete Freund adjuvant for the first dose and incomplete Freund adjuvant for the remaining two doses. Two weeks after the second boost, animals were euthanized by exsanguination, and hyperimmune sera were obtained by centrifugation (3,500 × g , 15 min, 4°C) and stored at -20°C. Construction of recombinant M. bovis BCG and expression analysis Using pAE/rTBDRchi as template, the rTBDRchi CDS was amplified by PCR using primers described in Online Resource 1 (Table S2). The PCR product was digested using Xba I and Pst I restriction enzymes (New England BioLabs) and cloned into the BCG expression vector pUP500/PpAN (Oliveira et al. 2019b ), previously digested with Spe l e Pst I (New England BioLabs), generating pUP500/PpAN:rTBDRchi plasmid. Recombinant pUP500/PpAN:rTBDRchi was transformed into M. bovis BCG Pasteur cells by electroporation, as previously described (Parish and Stoker 1995 ). Cells were plated onto 7H10 agar (Difco, BD, São Paulo, SP, Brazil) containing 25 µg.ml −1 of kanamycin and incubated at 37°C for 21 days. Resistant colonies were inoculated in selective Middlebrook 7H9 broth (Difco) and cultivated at 37ºC to evaluate recombinant expression by immunoblot. Briefly, whole-cell lysates were prepared from rBCG:TBDRchi grown to mid-logarithmic phase at 37°C (OD 600 = 0.8), when cells were collected by centrifugation (4,000 × g , 15 min) and resuspended in Tris-HCl (pH 8), adjusting the cell density to 10 7 CFU.ml −1 . rBCG:TBDRchi whole-cell lysates were separated in 12% polyacrylamide gels and transferred to nitrocellulose membranes (Hybond ECL, GE Healthcare, Illinois, United States). Membranes were incubated overnight with rat anti-rTBDRchi sera at 1:100 dilution, followed by anti-rat HRP-conjugated secondary antibodies (Sigma Aldrich, Missouri, United States) diluted 1:5,000. Immunoblots were developed using Pierce ECL Western Blotting Substrate (Thermo-Fisher, Illinois, USA). Vaccination and challenge of hamsters rBCG:TBDRchi and BCG Pasteur (negative control) strains were cultivated in vitro and collected by centrifugation (4,000 × g , 15 min). Cells were resuspended in PBS, adjusting concentration to 10 7 cells/ml (considering 1 OD 600 = 1 x 10 8 cells/ml), and immediately used. Male Syrian hamsters ( Mesocricetus auratus) , 4–6 weeks old, were subcutaneously inoculated with 10 6 cells of BCG Pasteur (n=13 per experiment) or recombinant BCG:TBDRchi (n=10 per experiment). An additional control group (n=4 per experiment) was immunized (intramuscular) with a leptospiral whole-cell bacterin (10 8 heat-inactivated leptospires in 100 µl PBS). Two doses of each formulation were administered 21 days apart. Thirty days after the second boost, hamsters were infected intraperitoneally with 10 3 leptospires ( L. interrogans sv. Copenhageni st. Fiocruz L1-130), equivalent to five times the average 50% endpoint dose (ED50), as previously determined (Oliveira et al. 2019a ). Hamsters were monitored daily for clinical signs of acute leptospirosis for 30 days after challenge. Criteria for humane euthanasia (CO 2 narcosis) was established as previously described (Coutinho et al. 2011 ), and includes: >10% weight loss, prostration, bristling, apathy, and lack of appetite. Blood samples were collected one day before the first immunization (day zero) and before challenge (day 51). A total of two independent experiments were performed. All animal procedures were conducted according to the rules and regulations of the Animal Experimentation Ethics Committee at UFPel. Analysis of the leptospiral burden in hamster kidneys post-challenge At the end of the experiment, kidneys were collected aseptically in order to evaluate the leptospiral burden post-challenge. Kidney samples were macerated in 10 ml EMJH medium supplemented with 10% Leptospira enrichment supplement. After one hour at 30°C, 500 µl of the macerate was inoculated into 10 ml of fresh EMJH and incubated at 30ºC for up to 8 weeks. Cultures were monitored weekly by dark-field microscopy. DNA extraction from kidney samples, followed by qPCR was performed as previously described (Dorneles et al. 2020 ). Briefly, DNA was extracted from approximately 40 mg of kidney tissue using SV Genomic DNA Purification Kit (Promega, Brazil) and quantified using Qubit 2.0 fluorometer (Thermo Fisher). Quantitative real-time PCR (qPCR) detection of leptospiral DNA was performed in a LightCycler 96 system (Roche, Basel, Switzerland), using 200 ng of total DNA, 0.4 µM of each primer specific to lipL32 (Online Resource 1, Table S2) and SYBR Green PCR Master Mix (Applied Biosystems, São Paulo, SP, Brazil). Reactions were performed in triplicate as previously described (Dorneles et al. 2020 ). A standard curve was generated from 10 6 to 10 copies of lipL32 and samples were considered negative when their qPCR quantitation cycle (Cq) were higher than that obtained for the lowest concentration of 10 copies. Absolute quantification analysis using LightCycler 96 software (Roche) was performed to determine the number of leptospire genome equivalents per reaction, and thereafter converted to copies per µg of total DNA. Evaluation of the humoral immune response Humoral immune response elicited after vaccination was evaluated by indirect ELISA (Enzyme-Linked Immunosorbent Assay), as previously described (da Cunha et al. 2019 ). First, 96 well microtiter plates (Polysorp, Nunc, São Paulo, Brazil) were coated with 1 µg of purified rTBDRchi per well at 4ºC overnight, in 0.1 M carbonate-bicarbonate buffer, pH 9.6. Plates were washed three times with PBS-0.05% Tween 20 (PBST) and then blocked with 200 µl of 5% non-fat dry milk in PBS-T, for 1 h at 37ºC. Hamsters’ sera were diluted 1:50 in PBST and evaluated in triplicate. After incubation for 1h at 37ºC, plates were washed and HRP-conjugated anti-Syrian hamster IgG (Sigma Aldrich, Missouri, United States) was added at 1:5,000 dilution for 1 h at 37°C. After a final wash, reactions were developed using o-phenylenediamine dihydrochloride (Sigma-Aldrich, Missouri, United States) and hydrogen peroxide, and stopped after 15 min with 4N H 2 SO 4 . Absorbance was read at 492 nm wavelength in a microplate reader (Mindray MR-96A, São Paulo, Brazil). Immunoblots were performed as previously described, with small changes (Groshong et al. 2021 ). Briefly, samples containing 1 µg of each recombinant protein (rTBDRchi, rLIC10896, rLIC10964, and rLIC12374) or L. interrogans st. Fiocruz L1-130 whole-cell lysate (10 8 /lane), were analyzed by SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare, Illinois, United States) at 20 V for 25 min using a semi-dry transfer (Bio-Rad). Membranes were blocked for 1 h at room temperature with 5% non-fat dried milk diluted in PBS-T, and probed overnight at 4°C with a pool of hamster sera obtained after immunization, at a 1:200 dilution in blocking buffer. After washing 5 times with PBS-T, membranes were incubated for 1 h at RT with an HRP-conjugated goat anti-hamster IgG antibody (Sigma Aldrich, Missouri, United States) diluted in blocking buffer (1:6,000). Following six washes with PBS-T, immunoblots were developed using the Pierce ECL Western Blotting Substrate (Thermo-Fisher, Illinois, USA) detection reagent, and results were visualized using C-Digit Blot Scanner (LI-COR, Nebraska, USA). Statistical analysis Significant protection against lethal leptospirosis was evaluated using Fisher’s exact test (two-tailed). Comparison of humoral immune response elicited by different experimental groups was performed using analysis of variance (one way-ANOVA with Tukey multiple comparison). For all analyses, P -values < 0.05 were considered significant. Graphical representations and statistical analysis were performed using GraphPad Prism 8. Results Structural modeling, sequence analysis and design of rTBDRchi Our group previously described leptospiral TonB-dependent receptor (TBDR) orthologs based on a reverse and structural vaccinology approach (Grassmann et al. 2017a ). In this study, we selected the three TBDRs for evaluation as vaccine candidates against leptospirosis, LIC10896, LIC10964, and LIC12374, for which important nutrient transport functions were previously suggested (Nascimento et al. 2004 ; Louvel et al. 2006 ). 3D models were generated using the standard threading assembly method by I-TASSER. All retrieved models with the best confidence score (C-score) were identified as structural analogs to TonB-dependent receptors with resolved structures, deposited on the Protein Data Bank (PDB) (Online Resource 1, Table S3). The I-TASSER server was also used to perform structural alignments between the leptospiral queries and their PDB templates using the TM-align program, with reported values varying from 0 to 1, where 1 indicates a perfect superposition of two structures (Zhang and Skolnick 2005 ). Predicted structures for the leptospiral TBDRs LIC10896, LIC10964, and LIC12374 presented TM-Align values varying from 0.724 to 0.842 (Online Resource 1, Figure S1). Next, we analyzed each of the selected leptospiral proteins in order to predict T CD4+ and linear B cell epitopes along their sequences. Several T CD4+ and linear B-cell epitopes were identified along the full-length leptospiral TBDR sequences (Online Resource 3). Membrane-embed templates for each generated model were obtained from the Orientations of Proteins in Membranes (OPM) database (Lomize et al. 2012 ), and structural alignments were performed to identify the predicted surface-exposed fragments (Online Resource 1, Figure S1). Each identified T CD4+ and linear B cell epitope was mapped onto 3D structures allowing the selection of all predicted surface-exposed regions containing antigenic determinants. The three TBDRs analyzed contained nine surface-exposed loops with T and B cell epitopes (Figure 1 a). Antigenic loops were selected and combined to construct a 41 kDa recombinant protein, named rTBDRchi (Figure 1 b). Five fragments were selected from LIC10896, and two fragments from LIC10964 and LIC12374 (Figure 1 , Table 1 ). In silico approaches for the T CD4+ cell epitope prediction focused on the identification of core binding regions composed of nine residues (9-mer), which largely determined binding affinity and specificity for human leukocyte antigens (HLA) presentation (Sanchez-trincado et al. 2017 ). A total of 24 strong binder 9-mers (IC50 <50 nM) were identified in the exposed loops. Thirteen different HLA alleles (out of 14 alleles analyzed) were predicted to bind to the rTBDRchi epitopes (Online Resource 1, Table S4). rTBDRchi also included promiscuous T cell epitopes, with predicted binding to several HLAs, from all selected leptospiral TBDRs. Table 1 Sequences of the selected fragments predicted to contain surface-exposed regions, and B and T cell epitopes. Protein sequences were submitted to BepiPred and NetMHCII for prediction of linear B cell (bold) and T CD4+ cell (underlined) epitopes, respectively. All surface-exposed loops containing identified epitopes were selected to compose the fusion protein and are listed. Protein Fragment Sequence LIC10896 3 YN RSYNYREEANARFQASNPISIYLKDSNMLRPLN QNNLKIYGEE VLWGNNLNL AYEPK IGQQFFIKTLYSVQSDK IVRE GDGANYI DN FNFKSTNLNFI 4 WSQGGTDLAN G Y RRLGNNPD GTR 5 REIAQR NFTGSDRDV IYPIPGEVIYNPL AYANGNRK IYER 6 S WNGFNTSYGCKTNSE EER LLLVRANICDAT 9 DSK IYGLIST GQVDPLSTYAA YSPTTLNRPLQGQSD LIC10964 3 D LLDNPNENPGATV SQKLLHE KSHFHSFFIFSAGNLELDFSYQR 8 IQN FIYAASIAQIDLD SGLPK YEYKQGN LIC12374 2 DQNFS YKNDHGTVVLNTLDD TIDRRKNAS 6 FPSEEPW YRRQDPLSG DIK rTBDRchi fragments are conserved among pathogenic Leptospira spp. The selected fragments were evaluated for amino acid conservation between L. interrogans and the orthologs from ten additional pathogenic (P1 clade) Leptospira spp. (Vincent et al. 2019 ). By performing a MSA, all sequences showed high conservation levels with identities varying from 73.7 to 100% for different rTBDRchi fragments (Online Resource 4). We were not able to identify a LIC10896 ortholog in L. kmetyi , although fragments from LIC10964 and LIC12374 from L. interrogans , were highly conserved (84.21 – 93.10%) with their orthologs in L. kmetyi . As expected, due to their close phylogenetic relationship, L. kirschneri and L. noguchii showed the highest conservation with the L. interrogans TBDRs, with 7/9 fragments presenting more than 95% identity. BCG-vectored rTBDRchi protects hamsters against L. interrogans challenge The rTBDRchi CDS was synthesized and cloned into pUP500/P pAN vectors (Oliveira et al. 2019b ), allowing its heterologous expression in M. bovis BCG, as demonstrated by immunoblot (Online Resource 1, Figure S2). Subcutaneous immunization of hamsters with 10 6 live rBCG:TBDRchi mycobacteria, elicited 100% protection against homologous L. interrogans challenge in two independent experiments (20/20 animals, P < 0.001) with animals surviving for 30 days post-challenge (Table 2 , Figure 2 ). In contrast, all animals inoculated with wild-type BCG Pasteur (26/26) developed endpoint criteria, with clinical signs and weight-loss observed for all animals 13-15 days post-challenge (Figure 2 ). Leptospira burden in kidneys was evaluated by culture and lipL32 qPCR using tissues from animals immunized with rBCG-TBDRchi or from the control groups (Table 2 ). Cultures from macerated kidneys from the negative control group (BCG Pasteur) were positive for leptospires following two weeks of incubation. No growth was detected after eight weeks in the cultures from the rBCG:TBDRchi and bacterin groups. Similarly, lipL32 qPCR analysis was negative for leptospiral DNA in the kidneys of animals vaccinated with recombinant BCG (Table 2 ). The leptospiral burden in the negative control group ranged from 1.08x10 1 – 6.97x10 4 genome equivalents per µg of total extracted DNA, with an average of 5.03 x 10 3 (Table 2 , Online Resource 1 – Table S5). Table 2 Protection against lethal leptospirosis elicited by immunization with rBCG:TBDRchi. Syrian hamsters were immunized with 10 6 cells of BCG Pasteur wild-type or recombinant BCG:TBDRchi. Thirty days after the second dose, animals were injected intraperitoneally with 5x median endpoint dose (ED50) of L. interrogans serovar Copenhageni st. Fiocruz L1-130. Data from two independent experiments are presented. Vaccine % Protection a P value b Leptospires in kidneys c % Positive Culture % Positive qPCR rBCG:TBDRchi 100 (20/20) < 0.001 0 (0/20) 0 (0/20) BCG Pasteur 0 (0/26) ns 100 (26/26) 100 (26/26) Bacterin 100 (8/8) < 0.001 0 (0/8) 0 (0/8) a in parenthesis: survivors/total hamsters from two independent experiments. b P values were calculated by Fisher’s exact test (two-tailed) for each experiment. c in parenthesis: number of positive kidneys/total samples. rBCG:TBDRchi elicits a humoral immune response in hamsters The immunogenicity of rBCG:TBDRchi was evaluated by indirect ELISA using rTBDRchi as the antigen. Hamsters vaccinated with rBCG:TBDRchi produced a significant anti-rTBDRchi IgG response ( P < 0.05), detected 30 days after the second dose (Figure 3 a), demonstrating the potential of rBCG:TBDRchi to promote a humoral immune response. In contrast, hamsters injected with BCG-Pasteur were negative for rTBDRchi IgG in the same ELISA. The presence of antibodies against each leptospiral TBDR in immunized hamsters was assessed by Western blotting (WB). Sera obtained from hamsters after immunization with rBCG:TBDRchi (1:200) detected two proteins in L. interrogans lysates, with apparent molecular weight of ~100 and ~85 kDa, likely corresponding to LIC10896 (99 kDa) and LIC10964 (87 kDa) (Figure 3 b). Discussion Predicted surface-exposed fragments from three leptospiral TonB-dependent receptors were used in this work to construct the chimeric protein rTBDRchi. L. interrogans proteins LIC10896, LIC10964 and LIC12374 are putative TBDRs homologs (Nascimento et al. 2004 ; Louvel et al. 2006 ; Grassmann et al. 2017b ). These observations were further supported in the current study, the 3D models of LIC10896, LIC10964 and LIC12374 presented high similarity to previously described TBDRs from other bacteria (Online Resource 1, Table S3). Among the three structural models, LIC12374 is the model with the highest RMSD when aligned to its closest structural analog in PDB, the cobalamin transporter BtuB from E. coli (Chimento et al. 2003 ). Previously reported as a leptospiral FecA ortholog, LIC10896 was modeled with high structural similarity to the FpvA pyoverdine-Fe transporter from Pseudomonas aeruginosa (Cobessi et al. 2005 ). Similarly, LIC10964 was previously associated with hemin metabolism in Leptospira spp., and our 3D model was highly similar to the N. meningitidis zinc transporter, ZnuD (Calmettes et al. 2015 ). Identification of specific functions for TBDRs through structural modelling is difficult and should be determined experimentally. Independently of the specific substrate for a TBDR, the 22 transmembrane β-strand barrel conformation is highly conserved among known TBDR structures, supporting our approach for the identification of surface-exposed regions. While LIC10896 and LIC12374 have orthologs conserved in all pathogenic and saprophyte species, LIC10964 is present only in pathogenic species, as previously noted (Grassmann et al. 2017a ). Host-adapted leptospires, cultivated within dialysis membrane chambers (DMCs), expressed higher levels of LIC10964 compared to those cultivated in vitro (Caimano et al. 2014 ), suggesting a potential role during infection, in agreement with its predicted function as hemin transporter. The high level of sequence conservation of TBDRs across pathogenic leptospires makes this class of proteins an attractive target for development of cross-protective leptospirosis vaccines. In agreement with this notion, the TBDR TbpA from Haemophilus parasuis was able to induce a cross-protective immune response against three different serovars in a guinea pig model of Glasser’s disease, with protection varying between 60 and 80% (Huang et al. 2013 ). Similarly, hyperimmune sera generated in mice and guinea pigs against N. meningitidis outer membrane vesicles overexpressing the TBDR ZnuD, presented a neutralizing effect on 14 different N. meningitidis strains in vitro (Hubert et al. 2013 ). Although we did not evaluate rBCG:TBDRchi in a heterologous challenge, all of the epitopes in the recombinant vaccine antigen were highly conserved in other pathogenic Leptospira spp. (Online Resource 3), enhancing the possibility of heterologous protection after immunization, a requisite for a universal leptospiral vaccine (Grassmann et al. 2017c ). Multi-epitope constructs based on different proteins have been reported for leptospirosis diagnosis and vaccine development. (Lin et al. 2016 )) evaluated a recombinant protein containing four repeats of T and B cell epitopes in the leptospiral proteins OmpL1, LipL32 and LipL21. That chimeric protein also stimulated significant protection (4/5) in a guinea pig model of leptospirosis and induced a cross-reactive antibody response against various L. interrogans serogroups. (Fernandes et al. 2017 ), designed a chimeric protein based on sequences from five leptospiral proteins, however, this construction failed to induce significant protection in hamsters, where 50% survived, despite the presence of epitopes from LigA, a well-known protective antigen (Yan et al. 2009 ; Coutinho et al. 2011 ). To the best of our knowledge, this study pioneered the use of protein structural information on leptospiral βb-OMPs to construct a recombinant molecule for use in an experimental vaccine against leptospirosis. In the current study, the humoral response generated by rBCG:TBDRchi was predominantly directed against two (LIC10896 and LIC10964) of the three proteins used in the antigen construction (Figure 3 b). However, this does not rule out additional antibodies against LIC12374 that were below the limit of detection for the Western blot and might have contributed to the protection observed, given the presumable importance of LIC12374 epitopes in rTBDRchi. Different antigen presentation strategies for the selected epitopes should be assessed in the future, towards the induction of strong antibody responses against all epitopes in rTBDRchi. Our approach for designing rTBDRchi antigen proved to be successful, given the protection we observed in the hamster model of acute leptospirosis. Recombinant BCG vaccines have been shown to elicit both cellular and humoral immunity (Oliveira et al. 2017 ). In the current study, immunization with rBGG:TBDRchi induced a robust humoral immune responses against highly conserved surface-exposed epitopes from leptospiral TBDRs, as demonstrated by ELISA and Western blot. In contrast, in our previous study, we were unable to detect significant levels of antibodies by ELISA, in sera from animals immunized with a chimeric antigen containing fragments from the leptospiral lipoproteins LipL32, LigAni and LemA (Oliveira et al. 2019a ; Dorneles et al. 2020 ). The rationale of using epitope-rich fragments may have contributed to the induction of humoral response obtained by rBGG:TBDRchi. As an extracellular bacterium, clearance of leptospires by the immune system seems to be related to bacterial opsonization followed by phagocytosis (Gomes-Solecki et al. 2017 ; Santecchia et al. 2020 ). Different studies have shown that opsonization is a requirement for efficient bactericidal activity of macrophages in leptospirosis (Johnson and Muschel 1966 ; Banfi et al. 1982 ; Wang et al. 1984 ; Santecchia et al. 2020 ). Studies on phagocytosis without opsonization showed that pathogenic leptospires have several mechanisms to survive and escape killing by phagocytic cells (Zhang et al. 2012 ; Hu et al. 2013 ; Zhao et al. 2013 ; Toma et al. 2014 ). It is accepted that opsonization with immune serum enhances Leptospira uptake and killing by human macrophages; the role of phagocytic cells during infection was recently reviewed (Santecchia et al. 2020 ). These observations support the importance of the humoral response promoted by rBCG:TBDRchi immunization in the clearance of virulent leptospires during early stages of infection. The apparent absence of leptospires in the kidneys of rBCG:TBDRchi-immunized hamsters agrees with previous studies using BCG vectorized vaccines against leptospirosis (Oliveira et al. 2019a ; Dorneles et al. 2020 ). In contrast, several subunit vaccines against leptospirosis have failed to induce sterilizing immunity (Felix et al. 2020 ). It is believed that a robust and effective cell-mediated response may contribute to limit the initial tissue colonization by Leptospira spp. and contribute towards bacterial clearance (Zuerner 2015 ; Santecchia et al. 2019 ). A BCG-induced trained immunity has been shown to decrease host susceptibility against different pathogens (Covián et al. 2019 ). Trained immunity is a proposed term to describe the enhancement of a non-specific immune response generated against unrelated infections after previous exposure to microbial fragments (Netea et al. 2011 ). This phenomenon has been observed after inoculation of mycobacterial components such as muramyl di-peptide, complete Freund’s adjuvant and live BCG (Netea et al. 2011 ; Kleinnijenhuis et al. 2012 ). BCG vaccination increases IFN-γ production and other monocyte-derived cytokines, such as tumor necrosis factors (TNF) and IL-1β, through an NOD2 pathway (Kleinnijenhuis et al. 2012 ). Interestingly, the effects of trained immunity on leptospirosis were previously described using CL429, a TLR2 and NOD2 pathways synthetic agonist (Santecchia et al. 2019 ). CL429-trained macrophages produced significantly more pro-inflammatory cytokines in response to pathogenic Leptospira than naïve macrophages, independently of B and T cells. The same trained macrophages also produced increased amounts of nitric oxide (Santecchia et al. 2019 ), a potent antimicrobial compound previously related to leptospiral clearance (Prêtre et al. 2011 ). Unfortunately, the lack of immunological reagents to characterize cellular immunity in the hamster model for acute leptospirosis limited in-depth analyses of cellular responses elicited by rBCG:TBDRchi immunization. In conclusion, using a structural vaccinology approach, we designed a chimeric antigen that included predicted surface-exposed B and T cell epitopes from L. interrogans TBDRs. We used a BCG delivery system targeting robust immune response, resulting in production of a humoral immune response specific to the selected epitopes in leptospires. This vaccine formulation induced a protective and sterilizing immune response in the hamster model of lethal leptospirosis following a homologous challenge with L. interrogans . Declarations Author contributions: EB, JD, AG, AM, TO and OD contributed to conceive and design the study. EB, JD, AH, AM performed research throughout the work. JD, AH, AM, ASN performed animal experimentations. EB, AH, JD analyzed the data. EB, AG, AM wrote the paper. All authors contributed for manuscript review. Acknowledgements: We are grateful to our lab technician Michele dos Santos and the UFPel’s animal facility staff for the assistance provided during this study. Funding: Execution of this work was possible thanks to the support of CAPES, CNPq and FAPERGS through grants and scholarships. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 Conflicts of interest: The authors declare no conflict of interest. Data availability: Leptospira interrogans Fiocruz L1–130 strain is available as described in the Fiocruz/CLEP collection (WDCM 1012). Raw data from epitope mapping are available as Online Resource (ESM_3). Any additional data and materials used in this article are available upon request. Ethics approval: All procedures involving manipulations of animals were approved by the Ethics Committee in Animal Experimentation (CEEA) of Universidade Federal de Pelotas (UFPel) under protocol number 4646-2015. CEEA-UFPel is accredited by the Brazilian National Council for Animal Experimentation Control (CONCEA). References Adler B (2015) Vaccines against leptospirosis. Curr Top Microbiol Immunol 387:251–272. doi: 10.1007/978-3-662-45059-8_10 Alteri CJ, Hagan EC, Sivick KE, Smith SN, Mobley HLT (2009) Mucosal Immunization with Iron Receptor Antigens Protects against Urinary Tract Infection. PLoS Pathogen 5:e1000586. doi: 10.1371/journal.ppat.1000586 Banfi E, Cinco M, Bellini M, Soranzo MR (1982) The role of antibodies and serum complement in the interaction between macrophages and leptospires. J Gen Microbiol 128:813–816. doi: 10.1099/00221287-128-4-813 Caimano MJ, Sivasankaran SK, Allard A, Hurley D, Hokamp K, Grassmann A, Hinton JCD, Nally JE (2014) A Model System for Studying the Transcriptomic and Physiological Changes Associated with Mammalian Host-Adaptation by Leptospira interrogans Serovar Copenhageni. PLoS Pathog 10:e1004004. doi: 10.1371/journal.ppat.1004004 Calmettes C, Ing C, Buckwalter CM, Bakkouri M, el, Lai CC, Moraes TF, Pogoutse A, Gray-owen SD (2015) The molecular mechanism of Zinc acquisition by the neisserial outer-membrane transporter ZnuD. Nat Commun 6:1–11. doi: 10.1038/ncomms8996 Chimento DP, Kadner RJ, Wiener MC (2003) The Escherichia coli Outer Membrane Cobalamin Transporter BtuB: Structural Analysis of Calcium and Substrate Binding, and Identification of Orthologous Transporters by Sequence/Structure Conservation. J Mol Biol 332:999–1014. doi: https://doi.org/10.1016/j.jmb.2003.07.005 Cobessi D, Celia H, Folschweiller N, Schalk IJ, Abdallah MA, Pattus F (2005) The Crystal Structure of the Pyoverdine Outer Membrane Receptor FpvA from Pseudomonas aeruginosa at 3.6 A Resolution. J Mol Biol 347:121–134. doi: 10.1016/j.jmb.2005.01.021 Costa F, Hagan JE, Calcagno J, Kane M, Torgerson P, Martinez-silveira MS, Stein C, Abela-ridder B, Ko AI (2015) Global Morbidity and Mortality of Leptospirosis: A Systematic Review. PLoS Negl Trop Dis 9:e0003898. doi: 10.1371/journal.pntd.0003898 Coutinho ML, Choy HA, Kelley MM, Matsunaga J, Babbitt JT, Lewis MS, Aleixo JAG, Haake DA (2011) A LigA Three-Domain Region Protects Hamsters from Lethal Infection by Leptospira interrogans . PLoS Negl Trop Dis 5:1–10. doi: 10.1371/journal.pntd.0001422 Covián C, Fernández-Fierro A, Retamal-Díaz A, Díaz FE, Vasquez AE, Lay MK, Riedel CA, González PA, Bueno SM, Kalergis AM (2019) BCG-Induced Cross-Protection and Development of Trained Immunity: Implication for Vaccine Design. Front Immunol 10:1–14. doi: 10.3389/fimmu.2019.02806 da Cunha CEP, Bettin EB, Bakry AFAAY, Seixas Neto ACP, Amaral MG, Dellagostin OA (2019) Evaluation of different strategies to promote a protective immune response against leptospirosis using a recombinant LigA and LigB chimera. Vaccine 37:1844–1852. doi: 10.1016/j.vaccine.2019.02.010 Dellagostin OA, Grassmann AA, Rizzi C, Schuch RA, Jorge S, Oliveira TL, Mcbride AJA, Hartwig DD (2017) Reverse Vaccinology: An Approach for Identifying Leptospiral Vaccine Candidates. Int J Mol Sci 18:158. doi: 10.3390/ijms18010158 Dockrell HM, Smith SG (2017) What have we learnt about BCG vaccination in the last 20 years? Front Immunol 8. doi: 10.3389/fimmu.2017.01134 Dorneles J, Bonemann A, Clair A, Seixas P, Rizzi C, Burlamarque É, Silva A, Caetano C, Castro D, Gevehr C, Larré T, Antonio O (2020) Protection against leptospirosis conferred by Mycobacterium bovis BCG expressing antigens from Leptospira interrogans. Vaccine 38:8136–8144. doi: 10.1016/j.vaccine.2020.10.086 Edgar RC, Drive RM, Valley M (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. doi: 10.1093/nar/gkh340 Ellis WA (2015) Animal leptospirosis. Curr Top Microbiol Immunol 387:99–137. doi: 10.1007/978-3-662-45059-8_6 Faine S (1959) Iron as a Growth Requirement for Pathogenic Leptospira . J Gen Microbiol 20:246–251. doi: 10.1099/00221287-20-2-246 Felix CR, Siedler BS, Barbosa LN, Timm GR, McFadden J, McBride AJA (2020) An overview of human leptospirosis vaccine design and future perspectives. Expert Opin Drug Discov 15:179–188. doi: 10.1080/17460441.2020.1694508 Fernandes LGV, Teixeira AF, Filho AFS, Souza GO, Vasconcellos SA, Heinemann MB, Romero EC, Nascimento ALTO (2017) Immune response and protective profile elicited by a multi-epitope chimeric protein derived from Leptospira interrogans . International Journal of Infectious Diseases 57:61–69. doi: 10.1016/j.ijid.2017.01.032 Fouts DE, Matthias MA, Adhikarla H, Adler B, Amorim-Santos L, Berg DE, Bulach D, Buschiazzo A, Chang YF, Galloway RL, Haake DA, Haft DH, Hartskeerl R, Ko AI, Levett PN, Matsunaga J, Mechaly AE, Monk JM, Nascimento ALT, Nelson KE, Palsson B, Peacock SJ, Picardeau M, Ricaldi JN, Thaipandungpanit J, Wunder EA, Yang XF, Zhang JJ, Vinetz JM, Derrick E, Michael A, Fouts DE, Matthias MA, Adhikarla H, Adler B, Amorim- L (2016) What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira . PLoS Negl Trop Dis 10:1–57. doi: 10.1371/journal.pntd.0004403 Gomes-Solecki M, Santecchia I, Werts C (2017) Animal models of leptospirosis: Of mice and hamsters. Front Immunol 8. doi: 10.3389/fimmu.2017.00058 Grassmann AA, Kremer FS, dos Santos JC, Souza JD, Pinto L, McBride S, Gomes-solecki AAJA, John M, McBride A (2017a) AAJA Discovery of Novel Leptospirosis Vaccine Candidates Using Reverse and Structural Vaccinology. Frontiers in Immunology 8:463. doi: 10.3389/fimmu.2017.00463 Grassmann AA, Kremer FS, dos Santos JC, Souza JD, Pinto L, da McBride S, Gomes-solecki AJAAJA, John M, McBride A, Santos AJAAJA, Souza JC, Pinto JD, da McBride L (2017b) AJAAJA Discovery of Novel Leptospirosis Vaccine Candidates Using Reverse and Structural Vaccinology. Frontiers in Immunology 8:463. doi: 10.3389/fimmu.2017.00463 Grassmann AA, Souza JD, John A, Mcbride AJA, John A, Mcbride AJA (2017c) A universal vaccine against leptospirosis: Are we going in the right direction? Front Immunol 8:1–11. doi: 10.3389/fimmu.2017.00256 Gröschel MI, Sayes F, Shin SJ, Frigui W, Pawlik A, Orgeur M, Canetti R, Honoré N, Simeone R, van der Werf TS, Bitter W, Cho SN, Majlessi L, Brosch R (2017) Recombinant BCG Expressing ESX-1 of Mycobacterium marinum Combines Low Virulence with Cytosolic Immune Signaling and Improved TB Protection. Cell Reports 18:2752–2765. doi: 10.1016/j.celrep.2017.02.057 Groshong AM, Grassmann AA, Luthra A, McLain MA, Provatas AA, Radolf JD, Caimano MJ (2021) PlzA is a bifunctional c-di-GMP biosensor that promotes tick and mammalian hostadaptation of Borrelia burgdorferi . PLoS Pathog 17:1–31. doi: 10.1371/journal.ppat.1009725 Haake DA, Levett PN (2015) Leptospirosis in Humans. Curr Top Microbiol Immunol 387:65–97. doi: 10.1007/978-3-662-45059-8 Hu W, Ge Y, Ojcius DM, Sun D, Dong H, Yang XF, Yan J (2013) p53 controls apoptosis in Leptospira-infected macrophages. Cell Microbiol 15:1642–1659. doi: 10.1111/cmi.12141 Hu Y, Dang W, Sun L (2012) A TonB-dependent outer membrane receptor of Pseudomonas fluorescens : virulence and vaccine potential. Arch Microbiol 194:795–802. doi: 10.1007/s00203-012-0812-3 Huang X, Li Y, Fu Y, Ji Y, Lian K, Zheng H, Wei J, Cai X (2013) Cross-Protective Efficacy of Recombinant Transferrin-Binding Protein A of Haemophilus parasuis in Guinea Pigs. Clin Vaccine Immunol 20:912–919. doi: 10.1128/CVI.00621-12 Hubert K, Devos N, Mordhorst I, Tans C, Baudoux G, Feron C, Goraj K, Tommassen J, Vogel U, Poolman JT (2013) ZnuD, a Potential Candidate for a Simple and Universal Neisseria meningitidis Vaccine. Infect Immun 81:1915–1927. doi: 10.1128/IAI.01312-12 Johnson RC, Muschel LH (1966) Antileptospiral activity of serum. I. Normal and immune serum. J Bacteriol 91:1403–1409. doi: 10.1128/jb.91.4.1403-1409.1966 Kleinnijenhuis J, Quintin J, Preijers F, Joosten LAB, Ifrim DC, Saeed S, Jacobs C, Van Loenhout J, De Jong D, Hendrik S, Xavier RJ, Van Der Meer JWM, Van Crevel R, Netea MG (2012) Bacille Calmette-Guérin induces NOD2-dependent nonspecific protection from reinfection via epigenetic reprogramming of monocytes. Proc Natl Acad Sci USA 109:17537–17542. doi: 10.1073/pnas.1202870109 Larsen JEP, Lund O, Nielsen M (2006) Improved method for predicting linear B-cell epitopes. Immunome Research 2:2. doi: 10.1186/1745-7580-2-2 Lin X, Xiao G, Luo D, Kong L, Chen X, Sun D, Yan J (2016) Chimeric epitope vaccine against Leptospira interrogans infection and induced specific immunity in guinea pigs. BMC Microbiol 16. doi: 10.1186/s12866-016-0852-y Liu J, Ma Q, Yang F, Zhu R, Gu J, Sun C, Feng X, Du C, Langford PR, Han W, Yang J, Lei L (2017) B cell cross-epitope of Propionibacterium acnes and Actinobacillus pleuropneumonia selected by phage display library can efficiently protect from Actinobacillus pleuropneumonia infection. Vet Microbiol 205:14–21. doi: 10.1016/j.vetmic.2017.04.026 Lobo N, Brooks NA, Zlotta AR, Cirillo JD, Boorjian S, Black PC, Meeks JJ, Bivalacqua TJ, Gontero P, Steinberg GD, McConkey D, Babjuk M, Alfred Witjes J, Kamat AM (2021) 100 years of Bacillus Calmette–Guérin immunotherapy: from cattle to COVID-19. Nature Reviews Urology 18:611–622. doi: 10.1038/s41585-021-00481-1 Lomize MA, Pogozheva ID, Joo H, Mosberg HI, Lomize AL (2012) OPM database and PPM web server: Resources for positioning of proteins in membranes. Nucleic Acids Res 40:370–376. doi: 10.1093/nar/gkr703 Louvel H, Bommezzadri S, Zidane N, Boursaux-Eude C, Creno S, Magnier A, Rouy Z, Médigue C, Saint Girons I, Bouchier C, Picardeau M (2006) Comparative and functional genomic analyses of iron transport and regulation in Leptospira spp. J Bacteriol 188:7893–7904. doi: 10.1128/JB.00711-06 Nascimento ALTO, Ko AI, Martins EAL, Monteiro-Vitorello CB, Ho PL, Haake DA, Verjovski-Almeida S, Hartskeerl RA, Marques MV, Oliveira MC, Menck CFM, Leite LCC, Carrer H, Coutinho LL, Degrave WM, Dellagostin OA, El-Dorry H, Ferro ES, Ferro MIT, Furlan LR, Gamberini M, Giglioti EA, Góes-Neto A, Goldman GH, Goldman MHS, Harakava R, Jerônimo SMB, Junqueira-De-Azevedo ILM, Kimura ET, Kuramae EE, Lemos EGM, Lemos MVF, Marino CL, Nunes LR, De Oliveira RC, Pereira GG, Reis MS, Schriefer A, Siqueira WJ, Sommer P, Tsai SM, Simpson AJG, Ferro JA, Camargo LEA, Kitajima JP, Setubal JC, Van Sluys MA (2004) Comparative Genomics of Two Leptospira interrogans Serovars Reveals Novel Insights into Physiology and Pathogenesis. J Bacteriol 186:2164–2172. doi: 10.1128/JB.186.7.2164-2172.2004 Netea MG, Quintin J, Van Der Meer JWM (2011) Trained immunity: A memory for innate host defense. Cell Host and Microbe 9:355–361. doi: 10.1016/j.chom.2011.04.006 Nielsen M, Lund O (2009) NN-align. An artificial neural network-based alignment algorithm for MHC class II peptide binding prediction. BMC Bioinformatics 10:296. doi: 10.1186/1471-2105-10-296 Noinaj N, Guillier M, Barnard TJ, Buchanan SK (2010) TonB-Dependent Transporters: Regulation, Structure, and Function. Annu Rev Microbiol 43–60. doi: 10.1146/annurev.micro.112408.134247 Nosrati M, Hajizade A, Nazarian S, Amani J, Namvar Vansofla A, Tarverdizadeh Y (2019) Designing a multi-epitope vaccine for cross-protection against Shigella spp: An immunoinformatics and structural vaccinology study. Mol Immunol 116:106–116. doi: 10.1016/j.molimm.2019.09.018 Oliveira TL, Rizzi C, da Cunha CEP, Dorneles J, Seixas Neto ACP, Amaral MG, Hartwig DD, Dellagostin OA (2019a) Recombinant BCG strains expressing chimeric proteins derived from Leptospira protect hamsters against leptospirosis. Vaccine 37:776–782. doi: 10.1016/j.vaccine.2018.12.050 Oliveira TL, Rizzi C, Dellagostin OA (2017) Recombinant BCG vaccines: molecular features and their influence in the expression of foreign genes. Appl Microbiol Biotechnol 101:6865–6877. doi: 10.1007/s00253-017-8439-6 Oliveira TL, Stedman A, Rizzi C, Dorneles J, da Cunha CEP, Junior ASV, Dellagostin OA, McFadden J (2019b) A standardized BioBrick toolbox for the assembly of sequences in mycobacteria. Tuberculosis 119:101851. doi: 10.1016/j.tube.2019.07.002 Oyarzún P, Kobe B (2016) Recombinant and epitope-based vaccines on the road to the market and implications for vaccine design and production. Human Vaccines & Immunotherapeutics 12:763–767. doi: 10.1080/21645515.2015.1094595 Parish T, Stoker NG (1995) Electroporation of mycobacteria. Methods in molecular biology. (Clifton, NJ) 47:237–252. doi: 10.1385/0-89603-310-4:237 Parvizpour S, Pourseif MM, Razmara J, Rafi MA, Omidi Y (2020) Epitope-based vaccine design: a comprehensive overview of bioinformatics approaches. Drug Discovery Today 25:1034–1042. doi: 10.1016/j.drudis.2020.03.006 Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera — A Visualization System for Exploratory Research and Analysis. J Comput Chem 1605–1612. doi: 10.1002/jcc.20084 Prêtre G, Olivera N, Cédola M, Haase S, Alberdi L, Brihuega B, Gómez RM (2011) Role of inducible nitric oxide synthase in the pathogenesis of experimental leptospirosis. Microb Pathog 51:203–208. doi: 10.1016/j.micpath.2011.03.011 Ramos CRR, Abreu PAE, Nascimento ALTO, Ho PL (2004) A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal his-tagged fusion peptide. Braz J Med Biol Res 37:1103–1109. doi: 10.1590/S0100-879X2004000800001 Sanchez-trincado JL, Gomez-perosanz M, Reche PA (2017) Fundamentals and Methods for T- and B-Cell Epitope Prediction. Journal of Immunology Research 2017. doi: doi.org/10.1155/2017/2680160 Santecchia I, Ferrer F, Vieira ML, Mart R (2020) Phagocyte Escape of Leptospira : The Role of TLRs and NLRs. Front Immunol 11. doi: 10.3389/fimmu.2020.571816 Santecchia I, Vernel-Pauillac F, Rasid O, Quintin J, Gomes-Solecki M, Boneca IG, Werts C (2019) Innate immune memory through TLR2 and NOD2 contributes to the control of Leptospira interrogans infection. PLoS Pathog 15:1–26. doi: 10.1371/journal.ppat.1007811 Seixas F, Drawanz D, Maia G, Amaral M, Quevedo M (2007) Recombinant Mycobacterium bovis BCG expressing the LipL32 antigen of Leptospira interrogans protects hamsters from challenge. Vaccine 26:88–95. doi: 10.1016/j.vaccine.2007.10.052 Shelton AN, Seth EC, Mok KC, Han AW, Jackson SN, Haft DR, Taga ME (2019) Uneven distribution of cobamide biosynthesis and dependence in bacteria predicted by comparative genomics. ISME J 13:789–804. doi: doi.org/10.1038/s41396-018-0304-9 Soto JA, Gálvez NMS, Rivera CA, Palavecino CE, Céspedes PF, Rey-Jurado E, Bueno SM, Kalergis AM (2018) Recombinant BCG Vaccines Reduce Pneumovirus-Caused Airway Pathology by Inducing Protective Humoral Immunity. Front Immunol 9:2875. doi: 10.3389/fimmu.2018.02875 Toma C, Murray GL, Nohara T, Mizuyama M, Koizumi N, Adler B, Suzuki T (2014) Leptospiral outer membrane protein LMB216 is involved in enhancement of phagocytic uptake by macrophages. Cell Microbiol 16:1366–1377. doi: 10.1111/cmi.12296 Trunz BB, Fine P, Dye C (2006) Effect of BCG vaccination on childhood tuberculous meningitis and miliary tuberculosis worldwide: a meta-analysis and assessment of cost-effectiveness. Lancet 367:1173–1180. doi: 10.1016/S0140-6736(06)68507-3 Vincent AT, Schiettekatte O, Goarant C, Neela VK, Bernet E, Thibeaux R, Ismail N, Khalid MKNM, Amran F, Masuzawa T, Nakao R, Korba AA, Bourhy P, Veyrier FJ, Picardeau M (2019) Revisiting the taxonomy and evolution of pathogenicity of the genus Leptospira through the prism of genomics. PLoS Negl Trop Dis 13. doi: 10.1371/journal.pntd.0007270 Wang B, Sullivan JA, Sullivan GW, Mandell GL (1984) Role of specific antibody in interaction of leptospires with human monocytes and monocyte-derived macrophages. Infect Immun 46:809–813. doi: 10.1128/iai.46.3.809-813.1984 Xiao J, Liu J, Bao C, Zhu R, Gu J, Sun C, Feng X, Du C, Han W, Li Y, Lei L (2020) Recombinant tandem epitope vaccination provides cross protection against Actinobacillus pleuropneumoniae challenge in mice. AMB Express 10:1–13. doi: 10.1186/s13568-020-01051-1 Yan W, Faisal SM, Mcdonough SP, Divers TJ, Barr SC, Chang C, Pan M, Chang Y (2009) Immunogenicity and protective efficacy of recombinant Leptospira immunoglobulin-like protein B (rLigB) in a hamster challenge model. Microbes Infect 11:230–237. doi: 10.1016/j.micinf.2008.11.008 Yang J, Zhang Y (2015) I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Res 43:174–181. doi: 10.1093/nar/gkv342 Zhang L, Zhang C, Ojcius DM, Sun D, Zhao J, Lin X, Li L, Li L, Yan J (2012) The mammalian cell entry (Mce) protein of pathogenic Leptospira species is responsible for RGD motif-dependent infection of cells and animals. Mol Microbiol 83:1006–1023. doi: 10.1111/j.1365-2958.2012.07985.x Zhang Y, Skolnick J (2005) TM-align: A protein structure alignment algorithm based on the TM-score. Nucleic Acids Res 33:2302–2309. doi: 10.1093/nar/gki524 Zhao J, Chen H, Ojcius DM, Zhao X, Sun D, Ge Y, Zheng L, Lin X, Li L, Yan J (2013) Identification of Leptospira interrogans Phospholipase C as a Novel Virulence Factor Responsible for Intracellular Free Calcium Ion Elevation during Macrophage Death. PLoS ONE 8:e75652. doi: 10.1371/journal.pone.0075652 Zuerner RL (2015) Host response to Leptospira infection. Curr Top Microbiol Immunol 387:223–250. doi: 10.1007/978-3-662-45059-8_9 Supplementary Files GraphicalAbstract.tiff ESM1.pdf ESM2.pdf ESM3.xlsx ESM4.pdf Cite Share Download PDF Status: Published Journal Publication published 10 Dec, 2021 Read the published version in Applied Microbiology and Biotechnology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-996824","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":59890976,"identity":"e4f96d17-2c9b-4db2-8000-68120aead8e5","order_by":0,"name":"Everton Burlamarque Bettin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYHACNmYwycB8AEhJyBBUzwPRYiDBxsCWANLCQ7wWINMAKkAA2LN3pz0uqPhTxyfd8/nVjRoLHgb2w0c34LWF5+x24xlngA6TObvNOucY0GE8aWk38GqRyN0mzdsG1AJkGOewAbVI8JgRoeUfSEvOM+Ocf0RraQBrYX6c20aMljMgvxwzlmyTSDNjzu2T4GEj5Bf29t5tjwtq5PjlZyQ//pzzrU6On/3wMbxakAGbBJgkVjkIMH8gRfUoGAWjYBSMHAAAyIU89IBsYSQAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-5095-2198","institution":"Universidade Federal de Pelotas","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Everton","middleName":"Burlamarque","lastName":"Bettin","suffix":""},{"id":59890977,"identity":"dfc0d710-c1c2-409a-aff3-ae14a94d6712","order_by":1,"name":"Jessica Dorneles","email":"","orcid":"","institution":"Universidade Federal de Pelotas","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"","lastName":"Dorneles","suffix":""},{"id":59890978,"identity":"dc27337f-a1dc-4488-8b7a-65fb3523c483","order_by":2,"name":"Amanda Silva Hecktheuer","email":"","orcid":"","institution":"Universidade Federal de Pelotas","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amanda","middleName":"Silva","lastName":"Hecktheuer","suffix":""},{"id":59890979,"identity":"79d717a3-d157-437f-8d97-6b604be108de","order_by":3,"name":"Andriele Bonemann Madruga","email":"","orcid":"","institution":"Universidade Federal de Pelotas","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Andriele","middleName":"Bonemann","lastName":"Madruga","suffix":""},{"id":59890980,"identity":"506a5bd9-27f9-440f-86c6-f0843e065cf7","order_by":4,"name":"Amilton Clair Pinto Seixas Neto","email":"","orcid":"","institution":"Universidade Federal de Pelotas","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amilton","middleName":"Clair Pinto Seixas","lastName":"Neto","suffix":""},{"id":59890981,"identity":"384c3d4e-7b75-42d3-a9b1-6ac454865ac6","order_by":5,"name":"Alan John Alexander McBride","email":"","orcid":"","institution":"Universidade Federal de Pelotas","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alan","middleName":"John Alexander","lastName":"McBride","suffix":""},{"id":59890982,"identity":"b6a1d567-8bc9-4061-8f4a-e12e1e0d49f4","order_by":6,"name":"Thais Larré Oliveira","email":"","orcid":"","institution":"Universidade Federal de Pelotas","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Thais","middleName":"Larré","lastName":"Oliveira","suffix":""},{"id":59890983,"identity":"f12037f2-1c8b-4ae0-944a-b52ea55d35cd","order_by":7,"name":"André Alex Grassmann","email":"","orcid":"","institution":"University of Connecticut Health Center: UConn Health","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"André","middleName":"Alex","lastName":"Grassmann","suffix":""},{"id":59890984,"identity":"088e9d91-22df-409c-972a-df78a49c5c9c","order_by":8,"name":"Odir Antônio Dellagostin","email":"","orcid":"","institution":"Universidade Federal de Pelotas","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Odir","middleName":"Antônio","lastName":"Dellagostin","suffix":""}],"badges":[],"createdAt":"2021-10-19 20:14:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-996824/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-996824/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00253-021-11726-9","type":"published","date":"2021-12-11T03:49:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":15143364,"identity":"353d7a3a-3687-4cd5-a82c-d81adadae9f0","added_by":"auto","created_at":"2021-11-02 14:33:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2808546,"visible":true,"origin":"","legend":"Epitope-mapping of leptospiral TBDRs and in silico construction of rTBDRchi. (a) CD4+ T cell epitopes and linear B cell epitopes were identified by NetMHCII and BepiPred servers, respectively. Epitopes were mapped on 3D structures generated by I-TASSER. Surface-exposed fragments containing identified epitopes (colored) were used to construct the recombinant fusion protein. (b) Nine identified fragments were selected to construct a 41 kDa recombinant fusion protein (rTBDRchi). rTBDRchi contains 11 CD4+ T-cell (black dashes) and 10 linear B-cell (blue dashes) epitopes throughout its sequence. ","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-996824/v1/ef3259f27ffa6ded90cf0344.png"},{"id":15143361,"identity":"224bd3db-9fdb-47ea-a897-9041d8bf9bab","added_by":"auto","created_at":"2021-11-02 14:33:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":159427,"visible":true,"origin":"","legend":"Protection against lethal leptospirosis elicited by immunization with rBCG:TBDRchi. (a) Survival data from vaccinated hamster after challenge with an endpoint dose of L. interrogans sv. Copenhageni st. Fiocruz L1-130 in two independent experiments. All animals that were immunized subcutaneously with 106 recombinant BCG (rBCG:TBDRchi, n = 10), or 107 leptospires (bacterin, n = 4) survived (p \u003c 0.001). Animals from the negative control group (BCG Pasteur, n = 13 per experiment) reached endpoint criteria for euthanasia between days 12-15 post-infection. ","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-996824/v1/c90aac214021437bb0998658.png"},{"id":15143360,"identity":"9f85d299-8e9e-4f76-8639-71453a80de78","added_by":"auto","created_at":"2021-11-02 14:33:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":61962,"visible":true,"origin":"","legend":"IgG antibody response in hamsters immunized with rBCG:TBDRchi evaluated by ELISA and immunoblot. (a) ELISA plates were coated with 1 µg of rTBDRchi. Sera from hamsters immunized with BCG Pasteur (negative control) and rBCG:TBDRchi, collected at day 0 (pre-immune) and 51 (pre-challenge) were evaluated at 1:50 dilution, in triplicate, with an anti-hamster IgG used as secondary antibody. Results are presented as mean absorbance ± standard deviation. Significant differences were determined by one-way ANOVA, followed by Tukey multiple comparison. Asterisk indicates a significance difference (P \u003c 0.05) to other groups. (b) Immunoblot with rTBDRchi, recombinant TBDR proteins and Leptospira lysate using sera obtained from hamsters after immunization with rBCG:TBDRchi (1:200). Humoral immune response was primarily direct against two of the leptospiral TBDRs, LIC10896 and LIC10964.","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-996824/v1/613ecb5bc0f431c3e6ad8052.png"},{"id":16364097,"identity":"2f3d8ff1-ff3e-4f64-9800-2ad4da7ad61f","added_by":"auto","created_at":"2021-12-11 03:49:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2810672,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-996824/v1/b612d88f-09a2-44b7-bbda-cbc1b2be251f.pdf"},{"id":15143367,"identity":"a1135bc8-568b-47e1-a8fb-d08a40ac1c3c","added_by":"auto","created_at":"2021-11-02 14:33:06","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":26055450,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.tiff","url":"https://assets-eu.researchsquare.com/files/rs-996824/v1/113f241669376e911f09b8ce.tiff"},{"id":15143366,"identity":"d1418d81-6772-4b41-8788-e0f9dddcccc7","added_by":"auto","created_at":"2021-11-02 14:33:05","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":904246,"visible":true,"origin":"","legend":"","description":"","filename":"ESM1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-996824/v1/5a01c35b0b17b4a9037c62cf.pdf"},{"id":15143513,"identity":"95a6a337-bf7b-43e4-9269-6d8586f49bd9","added_by":"auto","created_at":"2021-11-02 14:36:05","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":80775,"visible":true,"origin":"","legend":"","description":"","filename":"ESM2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-996824/v1/5ba246b35d84724af2ff84c2.pdf"},{"id":15143365,"identity":"4a5c14e2-4151-4782-aa2f-a22b525ccef7","added_by":"auto","created_at":"2021-11-02 14:33:05","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":184261,"visible":true,"origin":"","legend":"","description":"","filename":"ESM3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-996824/v1/2f659d26d2767eea95773dbf.xlsx"},{"id":15143363,"identity":"06417428-fd8e-4faa-8af8-836fd4651c3f","added_by":"auto","created_at":"2021-11-02 14:33:05","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":206778,"visible":true,"origin":"","legend":"","description":"","filename":"ESM4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-996824/v1/c6d766c84b6d8c5f79c48121.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eTonB-dependent receptor epitopes expressed in \u003cem\u003eM. bovis\u003c/em\u003e BCG induced significant protection in the hamster model of leptospirosis\u003c/p\u003e","fulltext":[{"header":"Key points","content":"\u003cul\u003e\n \u003cli\u003ePredicted surface-exposed epitopes were identified in three leptospiral TBDRs;\u003c/li\u003e\n \u003cli\u003eA \u003cem\u003eM. bovis\u003c/em\u003e BCG strain expressing a chimeric protein (rTBDRchi) was constructed;\u003c/li\u003e\n \u003cli\u003eHamsters vaccinated with rBCG:TBDRchi were protected from lethal leptospirosis.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eLeptospirosis is a major zoonosis worldwide in terms of morbidity and mortality rates. The global incidence is estimated to be over one million cases every year, resulting in ~60,000 deaths (Costa et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Rodents are the primary reservoir hosts of pathogenic \u003cem\u003eLeptospira\u003c/em\u003e spp., while humans are incidental hosts. The disease also affects several wild and domestic animals (Ellis \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Haake and Levett \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Leptospirosis in humans can vary from mild symptoms, such as fever and myalgia to severe outcomes, with the development of acute kidney injury and pulmonary hemorrhage syndrome (Haake and Levett \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Leptospiral vaccines currently available are inactivated whole-cell preparations (bacterins) with widespread veterinary applications, but limited use in humans. Bacterins confer a short-term immune response that is protective only against the serovars included in the vaccine formulation (Adler \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Several recombinant proteins have been evaluated towards the development of new and improved leptospiral vaccines, with variable success (Felix et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). While some experimental recombinant vaccines were protective against lethal challenge, the majority failed to achieve heterologous and/or sterilizing protection, highlighting the need to evaluate novel vaccination strategies (Grassmann et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017c\u003c/span\u003e; Felix et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). A new and effective vaccine against leptospirosis will likely include surface-exposed epitopes conserved among pathogenic \u003cem\u003eLeptospira\u003c/em\u003e spp., allowing bacterial recognition by humoral immune response (Grassmann et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017c\u003c/span\u003e; Grassmann et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e; Dellagostin et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our group previously used a reverse and structural vaccinology approach to identify leptospiral beta-barrel outer membrane proteins (βb-OMP), including six TonB-dependent receptors (TBDR) (Grassmann et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTBDRs are outer-membrane proteins that promote the uptake of essential nutrients such as iron, nickel, vitamins and carbohydrates (Noinaj et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Since iron has limited bioavailability, its acquisition by bacteria is facilitated by the formation of complexes with other transport molecules, such as heme and siderophores, which are then internalized by specific TonB-dependent transport systems (Noinaj et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Pathogenic and saprophytic \u003cem\u003eLeptospira\u003c/em\u003e spp. require iron for \u003cem\u003ein vitro\u003c/em\u003e growth(Faine \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1959\u003c/span\u003e) and evolved mechanisms for iron sensing, acquisition and utilization (Louvel et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Two leptospiral TBDRs, encoded by LIC10896 and LIC10964, were described as homologs to the ferric citrate transporter FecA and the PhuR hemin receptor (Nascimento et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Louvel et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), respectively. Another TBDR, LIC12374, is annotated as a homolog of the cobalamin transporter BtuB (Nascimento et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Louvel et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Cobalamin (or vitamin B12) is a cofactor for enzymes related to a variety of essential functions, including those related to carbon metabolism and biosynthesis of nucleotides (Shelton et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Similarly to iron, cobalamin is a small nutrient required for \u003cem\u003eLeptospira\u003c/em\u003e spp. survival (Louvel et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). While saprophytic species rely on external sources, pathogenic leptospires encode a pathway for \u003cem\u003ede novo\u003c/em\u003e production of cobalamin from L-glutamate (Fouts et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Due to their important role in bacterial metabolism and nutrient acquisition, several TBDRs have been evaluated as vaccine candidates against many pathogens, resulting in successful protection (Alteri et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Hubert et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEpitope mapping in vaccine candidates permits the identification of regions with increased probability to elicit an immune response, consequently improving the antigenicity and immunogenicity of vaccine formulations and avoiding production of undesirable or ineffective responses (Oyarz\u0026uacute;n and Kobe \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Parvizpour et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The rational selection of conserved epitopes also contributes to cross-protection (Liu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Nosrati et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Xiao et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), a particularly important aspect to consider for the development of leptospirosis vaccines, where the achievement of heterologous, long-term protection are major goals. Combined with protein structural information, epitope selection is a powerful tool to identify immunogenic surface-exposed fragments required for bacterial opsonization (Grassmann et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e). Opsonization of leptospires by specific immunoglobulins improved bacterial phagocytosis and clearance (Gomes-Solecki et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Santecchia et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Even though the immune response required for protection against leptospirosis is not yet fully understood, induction of a balanced cellular/humoral response is believed to be key for a protective immune response (Gomes-Solecki et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Grassmann et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017c\u003c/span\u003e; da Cunha et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eMycobacterium bovis\u003c/em\u003e Bacillus Calmette-Gu\u0026eacute;rin (BCG) is an attenuated strain widely used as a vaccine against human tuberculosis (TB) (Dockrell and Smith \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In use for a hundred years, BCG vaccination is a highly cost-effective intervention against tuberculosis, and is recommended for all newborns in countries with a high burden of TB (Trunz et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Lobo et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). BCG triggers a long-lasting immunity after a single dose, presenting several adjuvant properties and a strong cellular response (Dockrell and Smith \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). BCG-induced adaptative immune response include the expression of several interleukins, such as TNF-α and IFN-γ, and consequently, the proliferation and activation of macrophages (Dockrell and Smith \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Covi\u0026aacute;n et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Several studies have demonstrated the potential of recombinant BCG (rBCG) to enhance the immune response towards heterologous antigens (Gr\u0026ouml;schel et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Soto et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), including those from \u003cem\u003eL. interrogans\u003c/em\u003e (Seixas et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Oliveira et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e; Dorneles et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and represents a promising alternative for antigen delivery (Oliveira et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we identified highly conserved surface-exposed regions containing B and T CD4+ cell epitopes in three leptospiral TBDRs: LIC10896, LIC10964, and LIC12374. These fragments were combined on a multi-epitope fusion protein and evaluated using BCG as a vectorized vaccine carrier. Our preparation induced a protective immune response in the hamster model of acute leptospirosis, generating specific antibodies and sterilizing immunity.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strains and growth conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e strains BL21 (DE3) Star and TOP10 (Invitrogen, S\u0026atilde;o Paulo, SP, Brazil) were grown at 37\u0026deg;C in Lysogeny Broth (LB) medium, with 100 \u0026micro;g.ml\u003csup\u003e\u0026minus;1\u003c/sup\u003e ampicillin when required. \u003cem\u003eM. bovis\u003c/em\u003e BCG (Pasteur and recombinant strains) were cultivated at 37\u0026deg;C in Middlebrook 7H9 broth (Difco, BD, S\u0026atilde;o Paulo, SP, Brazil) supplemented with 0.5% glycerol, 0.05% Tween 80 and 10% Oleic acid, Albumin, Dextrose Complex (OADC) or in 7H10 agar (Difco, BD, S\u0026atilde;o Paulo, SP, Brazil) with 10% OADC. The BCG media was supplemented with kanamycin, 25 \u0026micro;g.ml\u003csup\u003e\u0026minus;1\u003c/sup\u003e, when needed. Low-passage virulent \u003cem\u003eL. interrogans\u003c/em\u003e serogroup Icterohaemorrhagiae serovar (sv.) Copenhageni strain (st.) Fiocruz L1\u0026ndash;130 (WDCM1012) was cultivated at 30\u0026deg;C in Ellinghausen\u0026ndash;McCullough\u0026ndash;Johnson\u0026ndash;Harris (EMJH) medium supplemented with \u003cem\u003eLeptospira\u003c/em\u003e EMJH enrichment (Difco, BD, S\u0026atilde;o Paulo, SP, Brazil).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn silico\u003c/span\u003e \u003cb\u003estructure predictions and epitope selection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThree proteins (LIC10896, LIC10964 and LIC12374) from \u003cem\u003eL. interrogans\u003c/em\u003e sv. Copenhageni st. Fiocruz L1-130 (Taxid:267671), previously identified as leptospiral TonB-Dependent receptors, were selected for evaluation in this study. Three-dimensional (3D) structures were predicted by threading using the I-TASSER server (Yang and Zhang \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Models with the highest C-Score were selected for epitope prediction. The 3D protein structures were visualized using USCF Chimera 1.12 (Pettersen et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Sequences for the CD4+ T cell epitopes, with high affinity to 14 different human major histocompatibility complex class II (MHC-II) alleles, were predicted using NetMHCII 2.2(Nielsen and Lund \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) at the default settings. For linear (continuous) B cell epitope prediction, amino acid sequences were analyzed using BepiPred 1.0 (Larsen et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Only T cell epitopes with a strong binding affinity (IC50 \u0026lt; 50 nM) and amino acid residues with a score higher than 0.35 in the B cell epitope analysis were considered. Epitopes were manually mapped onto the predicted 3D models of each βb-OMP in USCF Chimera 1.12. These models were aligned with their best structural template obtained from the Orientations of Proteins in Membranes (OPM) database (Lomize et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). All surface-exposed fragments containing B and T CD4+ cell epitopes were used for antigen construction. Orthologs for each \u003cem\u003eL. interrogans\u003c/em\u003e TBDR were identified by BlastP (NCBI) in 10 pathogenic (P1 clade) \u003cem\u003eLeptospira\u003c/em\u003e spp. proteomes (Online Resource 1, Table S1). Protein sequences with \u0026gt;70% identity and 40% query coverage were considered orthologs. Identity between the \u003cem\u003eL. interrogans\u003c/em\u003e polypeptides and their orthologs was determined after multiple sequence alignments (MSA) using MUSCLE (EMBL-EBI) (Edgar et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRecombinant protein expression and purification\u003c/h2\u003e \u003cp\u003eSelected surface-exposed fragments were assembled \u003cem\u003ein silico\u003c/em\u003e using Vector NTI Advance\u0026reg; 11.5 (Invitrogen) without linkers, to construct the fusion proteins rTBDRchi, rLIC10896chi, rLIC10964chi and rLIC12374chi (Online Resource 2). \u003cem\u003eE. coli\u003c/em\u003e codon optimized coding sequences (CDS) for each fusion protein were synthesized by Epoch Life Science (Texas, USA) and cloned into \u003cem\u003eE. coli\u003c/em\u003e expression vector pAE (Ramos et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) using \u003cem\u003eBam\u003c/em\u003eHI and \u003cem\u003eHind\u003c/em\u003eIII restriction sites. Recombinant plasmids were used to transform \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) C41 strain (Invitrogen, S\u0026atilde;o Paulo, SP, Brazil) by heat-shock. When cultures reached mid-log phase (0.6-0.8 absorbance at OD\u003csub\u003e600\u003c/sub\u003e), expression was induced by adding 0.5 mM IPTG (isopropyl-β-D-thiogalactopyranoside) and incubated at 37\u0026deg;C with agitation. After 4h, cells were harvested by centrifugation (7,000 \u0026times; \u003cem\u003eg\u003c/em\u003e, 15 min, 4\u0026deg;C). Pellets were washed in phosphate-buffered saline (PBS) and resuspended in lysis buffer (20 mM sodium phosphate, 0.5 M NaCl and 20 mM Imidazole, pH 8.0) before sonication with six 30-s pulses on ice. After lysis, insoluble proteins were harvested by centrifugation (10,000 \u0026times; \u003cem\u003eg\u003c/em\u003e, 1 h, 4˚C), and resuspended in denaturing binding buffer (8M urea, 20 mM sodium phosphate, 0.5 M NaCl, and 20 mM Imidazole, pH 8,0) and incubated under constant agitation for 16 h. After a final centrifugation (10,000 \u0026times; \u003cem\u003eg\u003c/em\u003e, 1 h, 4˚C), recombinant proteins were purified using the AKTA Start automated chromatography system (GE Healthcare). Briefly, the supernatant was applied to a nickel-charged HisTrap FF column (GE Healthcare, S\u0026atilde;o Paulo, SP, Brazil), and after washing with 20 column volumes, recombinant fusion proteins were eluted over a total of 20 ml using a 0-100% gradient of elution buffer (8M urea, 20 mM sodium phosphate, 0.5 M NaCl, and 0.5 M Imidazole, pH 8,0). Purified proteins were then dialyzed against PBS at 4\u0026deg;C for 24 h and stored at -80˚C or 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eProduction of anti-rTBDRchi hyperimmune sera\u003c/h2\u003e \u003cp\u003eFour-week-old female Wistar rats (\u003cem\u003eRattus norvegicus\u003c/em\u003e) were injected intraperitoneally with 50 \u0026micro;g rTBDRchi emulsified in complete Freund adjuvant for the first dose and incomplete Freund adjuvant for the remaining two doses. Two weeks after the second boost, animals were euthanized by exsanguination, and hyperimmune sera were obtained by centrifugation (3,500 \u0026times; \u003cem\u003eg\u003c/em\u003e, 15 min, 4\u0026deg;C) and stored at -20\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConstruction of recombinant\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eM. bovis\u003c/span\u003e \u003cb\u003eBCG and expression analysis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUsing pAE/rTBDRchi as template, the rTBDRchi CDS was amplified by PCR using primers described in Online Resource 1 (Table S2). The PCR product was digested using \u003cem\u003eXba\u003c/em\u003eI and \u003cem\u003ePst\u003c/em\u003eI restriction enzymes (New England BioLabs) and cloned into the BCG expression vector pUP500/PpAN (Oliveira et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e), previously digested with \u003cem\u003eSpe\u003c/em\u003el e \u003cem\u003ePst\u003c/em\u003eI (New England BioLabs), generating pUP500/PpAN:rTBDRchi plasmid. Recombinant pUP500/PpAN:rTBDRchi was transformed into \u003cem\u003eM. bovis\u003c/em\u003e BCG Pasteur cells by electroporation, as previously described (Parish and Stoker \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Cells were plated onto 7H10 agar (Difco, BD, S\u0026atilde;o Paulo, SP, Brazil) containing 25 \u0026micro;g.ml\u003csup\u003e\u0026minus;1\u003c/sup\u003e of kanamycin and incubated at 37\u0026deg;C for 21 days. Resistant colonies were inoculated in selective Middlebrook 7H9 broth (Difco) and cultivated at 37\u0026ordm;C to evaluate recombinant expression by immunoblot. Briefly, whole-cell lysates were prepared from rBCG:TBDRchi grown to mid-logarithmic phase at 37\u0026deg;C (OD\u003csub\u003e600\u003c/sub\u003e = 0.8), when cells were collected by centrifugation (4,000 \u0026times; \u003cem\u003eg\u003c/em\u003e, 15 min) and resuspended in Tris-HCl (pH 8), adjusting the cell density to 10\u003csup\u003e7\u003c/sup\u003e CFU.ml\u003csup\u003e\u0026minus;1\u003c/sup\u003e. rBCG:TBDRchi whole-cell lysates were separated in 12% polyacrylamide gels and transferred to nitrocellulose membranes (Hybond ECL, GE Healthcare, Illinois, United States). Membranes were incubated overnight with rat anti-rTBDRchi sera at 1:100 dilution, followed by anti-rat HRP-conjugated secondary antibodies (Sigma Aldrich, Missouri, United States) diluted 1:5,000. Immunoblots were developed using Pierce ECL Western Blotting Substrate (Thermo-Fisher, Illinois, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eVaccination and challenge of hamsters\u003c/h2\u003e \u003cp\u003erBCG:TBDRchi and BCG Pasteur (negative control) strains were cultivated \u003cem\u003ein vitro\u003c/em\u003e and collected by centrifugation (4,000 \u0026times; \u003cem\u003eg\u003c/em\u003e, 15 min). Cells were resuspended in PBS, adjusting concentration to 10\u003csup\u003e7\u003c/sup\u003e cells/ml (considering 1 OD\u003csub\u003e600 =\u003c/sub\u003e 1 x 10\u003csup\u003e8\u003c/sup\u003e cells/ml), and immediately used. Male Syrian hamsters (\u003cem\u003eMesocricetus auratus)\u003c/em\u003e, 4\u0026ndash;6 weeks old, were subcutaneously inoculated with 10\u003csup\u003e6\u003c/sup\u003e cells of BCG Pasteur (n=13 per experiment) or recombinant BCG:TBDRchi (n=10 per experiment). An additional control group (n=4 per experiment) was immunized (intramuscular) with a leptospiral whole-cell bacterin (10\u003csup\u003e8\u003c/sup\u003e heat-inactivated leptospires in 100 \u0026micro;l PBS). Two doses of each formulation were administered 21 days apart. Thirty days after the second boost, hamsters were infected intraperitoneally with 10\u003csup\u003e3\u003c/sup\u003e leptospires (\u003cem\u003eL. interrogans\u003c/em\u003e sv. Copenhageni st. Fiocruz L1-130), equivalent to five times the average 50% endpoint dose (ED50), as previously determined (Oliveira et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). Hamsters were monitored daily for clinical signs of acute leptospirosis for 30 days after challenge. Criteria for humane euthanasia (CO\u003csub\u003e2\u003c/sub\u003e narcosis) was established as previously described (Coutinho et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and includes: \u0026gt;10% weight loss, prostration, bristling, apathy, and lack of appetite. Blood samples were collected one day before the first immunization (day zero) and before challenge (day 51). A total of two independent experiments were performed. All animal procedures were conducted according to the rules and regulations of the Animal Experimentation Ethics Committee at UFPel.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of the leptospiral burden in hamster kidneys post-challenge\u003c/h2\u003e \u003cp\u003eAt the end of the experiment, kidneys were collected aseptically in order to evaluate the leptospiral burden post-challenge. Kidney samples were macerated in 10 ml EMJH medium supplemented with 10% \u003cem\u003eLeptospira\u003c/em\u003e enrichment supplement. After one hour at 30\u0026deg;C, 500 \u0026micro;l of the macerate was inoculated into 10 ml of fresh EMJH and incubated at 30\u0026ordm;C for up to 8 weeks. Cultures were monitored weekly by dark-field microscopy. DNA extraction from kidney samples, followed by qPCR was performed as previously described (Dorneles et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Briefly, DNA was extracted from approximately 40 mg of kidney tissue using SV Genomic DNA Purification Kit (Promega, Brazil) and quantified using Qubit 2.0 fluorometer (Thermo Fisher). Quantitative real-time PCR (qPCR) detection of leptospiral DNA was performed in a LightCycler 96 system (Roche, Basel, Switzerland), using 200 ng of total DNA, 0.4 \u0026micro;M of each primer specific to \u003cem\u003elipL32\u003c/em\u003e (Online Resource 1, Table S2) and SYBR Green PCR Master Mix (Applied Biosystems, S\u0026atilde;o Paulo, SP, Brazil). Reactions were performed in triplicate as previously described (Dorneles et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). A standard curve was generated from 10\u003csup\u003e6\u003c/sup\u003e to 10 copies of \u003cem\u003elipL32\u003c/em\u003e and samples were considered negative when their qPCR quantitation cycle (Cq) were higher than that obtained for the lowest concentration of 10 copies. Absolute quantification analysis using LightCycler 96 software (Roche) was performed to determine the number of leptospire genome equivalents per reaction, and thereafter converted to copies per \u0026micro;g of total DNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of the humoral immune response\u003c/h2\u003e \u003cp\u003eHumoral immune response elicited after vaccination was evaluated by indirect ELISA (Enzyme-Linked Immunosorbent Assay), as previously described (da Cunha et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). First, 96 well microtiter plates (Polysorp, Nunc, S\u0026atilde;o Paulo, Brazil) were coated with 1 \u0026micro;g of purified rTBDRchi per well at 4\u0026ordm;C overnight, in 0.1 M carbonate-bicarbonate buffer, pH 9.6. Plates were washed three times with PBS-0.05% Tween 20 (PBST) and then blocked with 200 \u0026micro;l of 5% non-fat dry milk in PBS-T, for 1 h at 37\u0026ordm;C. Hamsters\u0026rsquo; sera were diluted 1:50 in PBST and evaluated in triplicate. After incubation for 1h at 37\u0026ordm;C, plates were washed and HRP-conjugated anti-Syrian hamster IgG (Sigma Aldrich, Missouri, United States) was added at 1:5,000 dilution for 1 h at 37\u0026deg;C. After a final wash, reactions were developed using o-phenylenediamine dihydrochloride (Sigma-Aldrich, Missouri, United States) and hydrogen peroxide, and stopped after 15 min with 4N H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. Absorbance was read at 492 nm wavelength in a microplate reader (Mindray MR-96A, S\u0026atilde;o Paulo, Brazil).\u003c/p\u003e \u003cp\u003eImmunoblots were performed as previously described, with small changes (Groshong et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Briefly, samples containing 1 \u0026micro;g of each recombinant protein (rTBDRchi, rLIC10896, rLIC10964, and rLIC12374) or \u003cem\u003eL. interrogans\u003c/em\u003e st. Fiocruz L1-130 whole-cell lysate (10\u003csup\u003e8\u003c/sup\u003e/lane), were analyzed by SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare, Illinois, United States) at 20 V for 25 min using a semi-dry transfer (Bio-Rad). Membranes were blocked for 1 h at room temperature with 5% non-fat dried milk diluted in PBS-T, and probed overnight at 4\u0026deg;C with a pool of hamster sera obtained after immunization, at a 1:200 dilution in blocking buffer. After washing 5 times with PBS-T, membranes were incubated for 1 h at RT with an HRP-conjugated goat anti-hamster IgG antibody (Sigma Aldrich, Missouri, United States) diluted in blocking buffer (1:6,000). Following six washes with PBS-T, immunoblots were developed using the Pierce ECL Western Blotting Substrate (Thermo-Fisher, Illinois, USA) detection reagent, and results were visualized using C-Digit Blot Scanner (LI-COR, Nebraska, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eSignificant protection against lethal leptospirosis was evaluated using Fisher\u0026rsquo;s exact test (two-tailed). Comparison of humoral immune response elicited by different experimental groups was performed using analysis of variance (one way-ANOVA with Tukey multiple comparison). For all analyses, \u003cem\u003eP\u003c/em\u003e-values \u0026lt; 0.05 were considered significant. Graphical representations and statistical analysis were performed using GraphPad Prism 8.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStructural modeling, sequence analysis and design of rTBDRchi\u003c/h2\u003e \u003cp\u003eOur group previously described leptospiral TonB-dependent receptor (TBDR) orthologs based on a reverse and structural vaccinology approach (Grassmann et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e). In this study, we selected the three TBDRs for evaluation as vaccine candidates against leptospirosis, LIC10896, LIC10964, and LIC12374, for which important nutrient transport functions were previously suggested (Nascimento et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Louvel et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). 3D models were generated using the standard threading assembly method by I-TASSER. All retrieved models with the best confidence score (C-score) were identified as structural analogs to TonB-dependent receptors with resolved structures, deposited on the Protein Data Bank (PDB) (Online Resource 1, Table S3). The I-TASSER server was also used to perform structural alignments between the leptospiral queries and their PDB templates using the TM-align program, with reported values varying from 0 to 1, where 1 indicates a perfect superposition of two structures (Zhang and Skolnick \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Predicted structures for the leptospiral TBDRs LIC10896, LIC10964, and LIC12374 presented TM-Align values varying from 0.724 to 0.842 (Online Resource 1, Figure S1).\u003c/p\u003e \u003cp\u003eNext, we analyzed each of the selected leptospiral proteins in order to predict T CD4+ and linear B cell epitopes along their sequences. Several T CD4+ and linear B-cell epitopes were identified along the full-length leptospiral TBDR sequences (Online Resource 3). Membrane-embed templates for each generated model were obtained from the Orientations of Proteins in Membranes (OPM) database (Lomize et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and structural alignments were performed to identify the predicted surface-exposed fragments (Online Resource 1, Figure S1). Each identified T CD4+ and linear B cell epitope was mapped onto 3D structures allowing the selection of all predicted surface-exposed regions containing antigenic determinants. The three TBDRs analyzed contained nine surface-exposed loops with T and B cell epitopes (Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Antigenic loops were selected and combined to construct a 41 kDa recombinant protein, named rTBDRchi (Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Five fragments were selected from LIC10896, and two fragments from LIC10964 and LIC12374 (Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eIn silico\u003c/em\u003e approaches for the T CD4+ cell epitope prediction focused on the identification of core binding regions composed of nine residues (9-mer), which largely determined binding affinity and specificity for human leukocyte antigens (HLA) presentation (Sanchez-trincado et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). A total of 24 strong binder 9-mers (IC50 \u0026lt;50 nM) were identified in the exposed loops. Thirteen different HLA alleles (out of 14 alleles analyzed) were predicted to bind to the rTBDRchi epitopes (Online Resource 1, Table S4). rTBDRchi also included promiscuous T cell epitopes, with predicted binding to several HLAs, from all selected leptospiral TBDRs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eSequences of the selected fragments predicted to contain surface-exposed regions, and B and T cell epitopes.\u003c/b\u003e Protein sequences were submitted to BepiPred and NetMHCII for prediction of linear B cell (bold) and T CD4+ cell (underlined) epitopes, respectively. All surface-exposed loops containing identified epitopes were selected to compose the fusion protein and are listed.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eFragment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eLIC10896\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eYN\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eRSYNYREEANARFQASNPISIYLKDSNMLRPLN\u003c/span\u003eQNNLKIYGEE\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eVLWGNNLNL\u003c/span\u003eAYEPK\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIGQQFFIKTLYSVQSDK\u003c/span\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eIVRE\u003c/span\u003e\u003cb\u003eGDGANYI\u003c/b\u003eDN\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eFNFKSTNLNFI\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cb\u003eWSQGGTDLAN\u003c/b\u003eG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eY\u003c/span\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eRRLGNNPD\u003c/span\u003e\u003cb\u003eGTR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eREIAQR\u003cb\u003eNFTGSDRDV\u003c/b\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIYPIPGEVIYNPL\u003c/span\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAYANGNRK\u003c/span\u003eIYER\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eS\u003cb\u003eWNGFNTSYGCKTNSE\u003c/b\u003eEER\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eLLLVRANICDAT\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eDSK\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIYGLIST\u003c/span\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eGQVDPLSTYAA\u003c/span\u003e\u003cb\u003eYSPTTLNRPLQGQSD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eLIC10964\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eD\u003cb\u003eLLDNPNENPGATV\u003c/b\u003eSQKLLHE\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eKSHFHSFFIFSAGNLELDFSYQR\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eIQN\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eFIYAASIAQIDLD\u003c/span\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eSGLPK\u003c/span\u003e\u003cb\u003eYEYKQGN\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eLIC12374\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cb\u003eDQNFS\u003c/b\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eYKNDHGTVVLNTLDD\u003c/span\u003e\u003cb\u003eTIDRRKNAS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cb\u003eFPSEEPW\u003c/b\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eYRRQDPLSG\u003c/span\u003e\u003cb\u003eDIK\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003erTBDRchi fragments are conserved among pathogenic\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eLeptospira\u003c/span\u003e \u003cb\u003espp.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe selected fragments were evaluated for amino acid conservation between \u003cem\u003eL. interrogans\u003c/em\u003e and the orthologs from ten additional pathogenic (P1 clade) \u003cem\u003eLeptospira\u003c/em\u003e spp. (Vincent et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). By performing a MSA, all sequences showed high conservation levels with identities varying from 73.7 to 100% for different rTBDRchi fragments (Online Resource 4). We were not able to identify a LIC10896 ortholog in \u003cem\u003eL. kmetyi\u003c/em\u003e, although fragments from LIC10964 and LIC12374 from \u003cem\u003eL. interrogans\u003c/em\u003e, were highly conserved (84.21 \u0026ndash; 93.10%) with their orthologs in \u003cem\u003eL. kmetyi\u003c/em\u003e. As expected, due to their close phylogenetic relationship, \u003cem\u003eL. kirschneri\u003c/em\u003e and \u003cem\u003eL. noguchii\u003c/em\u003e showed the highest conservation with the \u003cem\u003eL. interrogans\u003c/em\u003e TBDRs, with 7/9 fragments presenting more than 95% identity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBCG-vectored rTBDRchi protects hamsters against\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eL. interrogans\u003c/span\u003e \u003cb\u003echallenge\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe rTBDRchi CDS was synthesized and cloned into pUP500/P\u003csub\u003epAN\u003c/sub\u003e vectors (Oliveira et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e), allowing its heterologous expression in \u003cem\u003eM. bovis\u003c/em\u003e BCG, as demonstrated by immunoblot (Online Resource 1, Figure S2). Subcutaneous immunization of hamsters with 10\u003csup\u003e6\u003c/sup\u003e live rBCG:TBDRchi mycobacteria, elicited 100% protection against homologous \u003cem\u003eL. interrogans\u003c/em\u003e challenge in two independent experiments (20/20 animals, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) with animals surviving for 30 days post-challenge (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In contrast, all animals inoculated with wild-type BCG Pasteur (26/26) developed endpoint criteria, with clinical signs and weight-loss observed for all animals 13-15 days post-challenge (Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003cem\u003eLeptospira\u003c/em\u003e burden in kidneys was evaluated by culture and \u003cem\u003elipL32\u003c/em\u003e qPCR using tissues from animals immunized with rBCG-TBDRchi or from the control groups (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Cultures from macerated kidneys from the negative control group (BCG Pasteur) were positive for leptospires following two weeks of incubation. No growth was detected after eight weeks in the cultures from the rBCG:TBDRchi and bacterin groups. Similarly, \u003cem\u003elipL32\u003c/em\u003e qPCR analysis was negative for leptospiral DNA in the kidneys of animals vaccinated with recombinant BCG (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The leptospiral burden in the negative control group ranged from 1.08x10\u003csup\u003e1\u003c/sup\u003e \u0026ndash; 6.97x10\u003csup\u003e4\u003c/sup\u003e genome equivalents per \u0026micro;g of total extracted DNA, with an average of 5.03 x 10\u003csup\u003e3\u003c/sup\u003e (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Online Resource 1 \u0026ndash; Table S5).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eProtection against lethal leptospirosis elicited by immunization with rBCG:TBDRchi.\u003c/b\u003e Syrian hamsters were immunized with 10\u003csup\u003e6\u003c/sup\u003e cells of BCG Pasteur wild-type or recombinant BCG:TBDRchi. Thirty days after the second dose, animals were injected intraperitoneally with 5x median endpoint dose (ED50) of \u003cem\u003eL. interrogans\u003c/em\u003e serovar Copenhageni st. Fiocruz L1-130. Data from two independent experiments are presented.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVaccine\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e% Protection\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eLeptospires in kidneys\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e% Positive Culture\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e% Positive qPCR\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003erBCG:TBDRchi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100 (20/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0/20)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCG Pasteur\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0/26)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ens\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100 (26/26)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100 (26/26)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBacterin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100 (8/8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;\u003c/em\u003e 0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0/8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0/8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003ea\u003c/sup\u003e in parenthesis: survivors/total hamsters from two independent experiments.\u003c/p\u003e \u003cp\u003e\u003csup\u003eb\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e values were calculated by Fisher\u0026rsquo;s exact test (two-tailed) for each experiment.\u003c/p\u003e \u003cp\u003e\u003csup\u003ec\u003c/sup\u003e in parenthesis: number of positive kidneys/total samples.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003erBCG:TBDRchi elicits a humoral immune response in hamsters\u003c/h2\u003e \u003cp\u003eThe immunogenicity of rBCG:TBDRchi was evaluated by indirect ELISA using rTBDRchi as the antigen. Hamsters vaccinated with rBCG:TBDRchi produced a significant anti-rTBDRchi IgG response (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), detected 30 days after the second dose (Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), demonstrating the potential of rBCG:TBDRchi to promote a humoral immune response. In contrast, hamsters injected with BCG-Pasteur were negative for rTBDRchi IgG in the same ELISA. The presence of antibodies against each leptospiral TBDR in immunized hamsters was assessed by Western blotting (WB). Sera obtained from hamsters after immunization with rBCG:TBDRchi (1:200) detected two proteins in \u003cem\u003eL. interrogans\u003c/em\u003e lysates, with apparent molecular weight of ~100 and ~85 kDa, likely corresponding to LIC10896 (99 kDa) and LIC10964 (87 kDa) (Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePredicted surface-exposed fragments from three leptospiral TonB-dependent receptors were used in this work to construct the chimeric protein rTBDRchi. \u003cem\u003eL. interrogans\u003c/em\u003e proteins LIC10896, LIC10964 and LIC12374 are putative TBDRs homologs (Nascimento et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Louvel et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Grassmann et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e). These observations were further supported in the current study, the 3D models of LIC10896, LIC10964 and LIC12374 presented high similarity to previously described TBDRs from other bacteria (Online Resource 1, Table S3). Among the three structural models, LIC12374 is the model with the highest RMSD when aligned to its closest structural analog in PDB, the cobalamin transporter BtuB from \u003cem\u003eE. coli\u003c/em\u003e (Chimento et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Previously reported as a leptospiral FecA ortholog, LIC10896 was modeled with high structural similarity to the FpvA pyoverdine-Fe transporter from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (Cobessi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Similarly, LIC10964 was previously associated with hemin metabolism in \u003cem\u003eLeptospira\u003c/em\u003e spp., and our 3D model was highly similar to the \u003cem\u003eN. meningitidis\u003c/em\u003e zinc transporter, ZnuD (Calmettes et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Identification of specific functions for TBDRs through structural modelling is difficult and should be determined experimentally. Independently of the specific substrate for a TBDR, the 22 transmembrane β-strand barrel conformation is highly conserved among known TBDR structures, supporting our approach for the identification of surface-exposed regions.\u003c/p\u003e \u003cp\u003eWhile LIC10896 and LIC12374 have orthologs conserved in all pathogenic and saprophyte species, LIC10964 is present only in pathogenic species, as previously noted (Grassmann et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e). Host-adapted leptospires, cultivated within dialysis membrane chambers (DMCs), expressed higher levels of \u003cem\u003eLIC10964\u003c/em\u003e compared to those cultivated \u003cem\u003ein vitro\u003c/em\u003e (Caimano et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), suggesting a potential role during infection, in agreement with its predicted function as hemin transporter. The high level of sequence conservation of TBDRs across pathogenic leptospires makes this class of proteins an attractive target for development of cross-protective leptospirosis vaccines. In agreement with this notion, the TBDR TbpA from \u003cem\u003eHaemophilus parasuis\u003c/em\u003e was able to induce a cross-protective immune response against three different serovars in a guinea pig model of Glasser\u0026rsquo;s disease, with protection varying between 60 and 80% (Huang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Similarly, hyperimmune sera generated in mice and guinea pigs against \u003cem\u003eN. meningitidis\u003c/em\u003e outer membrane vesicles overexpressing the TBDR ZnuD, presented a neutralizing effect on 14 different \u003cem\u003eN. meningitidis\u003c/em\u003e strains \u003cem\u003ein vitro\u003c/em\u003e (Hubert et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Although we did not evaluate rBCG:TBDRchi in a heterologous challenge, all of the epitopes in the recombinant vaccine antigen were highly conserved in other pathogenic \u003cem\u003eLeptospira\u003c/em\u003e spp. (Online Resource 3), enhancing the possibility of heterologous protection after immunization, a requisite for a universal leptospiral vaccine (Grassmann et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017c\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMulti-epitope constructs based on different proteins have been reported for leptospirosis diagnosis and vaccine development. (Lin et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e)) evaluated a recombinant protein containing four repeats of T and B cell epitopes in the leptospiral proteins OmpL1, LipL32 and LipL21. That chimeric protein also stimulated significant protection (4/5) in a guinea pig model of leptospirosis and induced a cross-reactive antibody response against various \u003cem\u003eL. interrogans\u003c/em\u003e serogroups. (Fernandes et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), designed a chimeric protein based on sequences from five leptospiral proteins, however, this construction failed to induce significant protection in hamsters, where 50% survived, despite the presence of epitopes from LigA, a well-known protective antigen (Yan et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Coutinho et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). To the best of our knowledge, this study pioneered the use of protein structural information on leptospiral βb-OMPs to construct a recombinant molecule for use in an experimental vaccine against leptospirosis. In the current study, the humoral response generated by rBCG:TBDRchi was predominantly directed against two (LIC10896 and LIC10964) of the three proteins used in the antigen construction (Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). However, this does not rule out additional antibodies against LIC12374 that were below the limit of detection for the Western blot and might have contributed to the protection observed, given the presumable importance of LIC12374 epitopes in rTBDRchi. Different antigen presentation strategies for the selected epitopes should be assessed in the future, towards the induction of strong antibody responses against all epitopes in rTBDRchi. Our approach for designing rTBDRchi antigen proved to be successful, given the protection we observed in the hamster model of acute leptospirosis.\u003c/p\u003e \u003cp\u003eRecombinant BCG vaccines have been shown to elicit both cellular and humoral immunity (Oliveira et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In the current study, immunization with rBGG:TBDRchi induced a robust humoral immune responses against highly conserved surface-exposed epitopes from leptospiral TBDRs, as demonstrated by ELISA and Western blot. In contrast, in our previous study, we were unable to detect significant levels of antibodies by ELISA, in sera from animals immunized with a chimeric antigen containing fragments from the leptospiral lipoproteins LipL32, LigAni and LemA (Oliveira et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e; Dorneles et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The rationale of using epitope-rich fragments may have contributed to the induction of humoral response obtained by rBGG:TBDRchi. As an extracellular bacterium, clearance of leptospires by the immune system seems to be related to bacterial opsonization followed by phagocytosis (Gomes-Solecki et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Santecchia et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Different studies have shown that opsonization is a requirement for efficient bactericidal activity of macrophages in leptospirosis (Johnson and Muschel \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1966\u003c/span\u003e; Banfi et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Santecchia et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Studies on phagocytosis without opsonization showed that pathogenic leptospires have several mechanisms to survive and escape killing by phagocytic cells (Zhang et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Toma et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It is accepted that opsonization with immune serum enhances \u003cem\u003eLeptospira\u003c/em\u003e uptake and killing by human macrophages; the role of phagocytic cells during infection was recently reviewed (Santecchia et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These observations support the importance of the humoral response promoted by rBCG:TBDRchi immunization in the clearance of virulent leptospires during early stages of infection.\u003c/p\u003e \u003cp\u003eThe apparent absence of leptospires in the kidneys of rBCG:TBDRchi-immunized hamsters agrees with previous studies using BCG vectorized vaccines against leptospirosis (Oliveira et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e; Dorneles et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In contrast, several subunit vaccines against leptospirosis have failed to induce sterilizing immunity (Felix et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It is believed that a robust and effective cell-mediated response may contribute to limit the initial tissue colonization by \u003cem\u003eLeptospira\u003c/em\u003e spp. and contribute towards bacterial clearance (Zuerner \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Santecchia et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A BCG-induced trained immunity has been shown to decrease host susceptibility against different pathogens (Covi\u0026aacute;n et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Trained immunity is a proposed term to describe the enhancement of a non-specific immune response generated against unrelated infections after previous exposure to microbial fragments (Netea et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). This phenomenon has been observed after inoculation of mycobacterial components such as muramyl di-peptide, complete Freund\u0026rsquo;s adjuvant and live BCG (Netea et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kleinnijenhuis et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). BCG vaccination increases IFN-γ production and other monocyte-derived cytokines, such as tumor necrosis factors (TNF) and IL-1β, through an NOD2 pathway (Kleinnijenhuis et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Interestingly, the effects of trained immunity on leptospirosis were previously described using CL429, a TLR2 and NOD2 pathways synthetic agonist (Santecchia et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). CL429-trained macrophages produced significantly more pro-inflammatory cytokines in response to pathogenic \u003cem\u003eLeptospira\u003c/em\u003e than na\u0026iuml;ve macrophages, independently of B and T cells. The same trained macrophages also produced increased amounts of nitric oxide (Santecchia et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), a potent antimicrobial compound previously related to leptospiral clearance (Pr\u0026ecirc;tre et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Unfortunately, the lack of immunological reagents to characterize cellular immunity in the hamster model for acute leptospirosis limited in-depth analyses of cellular responses elicited by rBCG:TBDRchi immunization.\u003c/p\u003e \u003cp\u003eIn conclusion, using a structural vaccinology approach, we designed a chimeric antigen that included predicted surface-exposed B and T cell epitopes from \u003cem\u003eL. interrogans\u003c/em\u003e TBDRs. We used a BCG delivery system targeting robust immune response, resulting in production of a humoral immune response specific to the selected epitopes in leptospires. This vaccine formulation induced a protective and sterilizing immune response in the hamster model of lethal leptospirosis following a homologous challenge with \u003cem\u003eL. interrogans\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e EB, JD, AG, AM, TO and OD contributed to conceive and design the study. EB, JD, AH, AM performed research throughout the work. JD, AH, AM, ASN performed animal experimentations. EB, AH, JD analyzed the data. EB, AG, AM wrote the paper. All authors contributed for manuscript review.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eWe are grateful to our lab technician Michele dos Santos and the UFPel\u0026rsquo;s animal facility staff for the assistance provided during this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eExecution of this work was possible thanks to the support of CAPES, CNPq and FAPERGS through grants and scholarships.\u0026nbsp;This study was financed in part by the\u0026nbsp;Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - Brasil (CAPES) -\u0026nbsp;Finance Code\u0026nbsp;001\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e \u003cem\u003eLeptospira interrogans\u0026nbsp;\u003c/em\u003eFiocruz L1\u0026ndash;130 strain is available as described in the Fiocruz/CLEP collection (WDCM 1012). Raw data from epitope mapping are available as Online Resource (ESM_3). Any additional data and materials used in this article are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eAll procedures involving manipulations of animals were approved by the Ethics Committee in Animal Experimentation (CEEA) of Universidade Federal de Pelotas (UFPel) under protocol number 4646-2015. CEEA-UFPel is accredited by the Brazilian National Council for Animal Experimentation Control (CONCEA).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdler B (2015) Vaccines against leptospirosis. Curr Top Microbiol Immunol 387:251\u0026ndash;272. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-3-662-45059-8_10\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlteri CJ, Hagan EC, Sivick KE, Smith SN, Mobley HLT (2009) Mucosal Immunization with Iron Receptor Antigens Protects against Urinary Tract Infection. PLoS Pathogen 5:e1000586. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.ppat.1000586\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBanfi E, Cinco M, Bellini M, Soranzo MR (1982) The role of antibodies and serum complement in the interaction between macrophages and leptospires. J Gen Microbiol 128:813\u0026ndash;816. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1099/00221287-128-4-813\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaimano MJ, Sivasankaran SK, Allard A, Hurley D, Hokamp K, Grassmann A, Hinton JCD, Nally JE (2014) A Model System for Studying the Transcriptomic and Physiological Changes Associated with Mammalian Host-Adaptation by \u003cem\u003eLeptospira interrogans\u003c/em\u003e Serovar Copenhageni. PLoS Pathog 10:e1004004. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.ppat.1004004\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalmettes C, Ing C, Buckwalter CM, Bakkouri M, el, Lai CC, Moraes TF, Pogoutse A, Gray-owen SD (2015) The molecular mechanism of Zinc acquisition by the neisserial outer-membrane transporter ZnuD. Nat Commun 6:1\u0026ndash;11. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/ncomms8996\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChimento DP, Kadner RJ, Wiener MC (2003) The \u003cem\u003eEscherichia coli\u003c/em\u003e Outer Membrane Cobalamin Transporter BtuB: Structural Analysis of Calcium and Substrate Binding, and Identification of Orthologous Transporters by Sequence/Structure Conservation. J Mol Biol 332:999\u0026ndash;1014. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jmb.2003.07.005\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCobessi D, Celia H, Folschweiller N, Schalk IJ, Abdallah MA, Pattus F (2005) The Crystal Structure of the Pyoverdine Outer Membrane Receptor FpvA from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e at 3.6 A Resolution. J Mol Biol 347:121\u0026ndash;134. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jmb.2005.01.021\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCosta F, Hagan JE, Calcagno J, Kane M, Torgerson P, Martinez-silveira MS, Stein C, Abela-ridder B, Ko AI (2015) Global Morbidity and Mortality of Leptospirosis: A Systematic Review. PLoS Negl Trop Dis 9:e0003898. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pntd.0003898\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoutinho ML, Choy HA, Kelley MM, Matsunaga J, Babbitt JT, Lewis MS, Aleixo JAG, Haake DA (2011) A LigA Three-Domain Region Protects Hamsters from Lethal Infection by \u003cem\u003eLeptospira interrogans\u003c/em\u003e. PLoS Negl Trop Dis 5:1\u0026ndash;10. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pntd.0001422\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCovi\u0026aacute;n C, Fern\u0026aacute;ndez-Fierro A, Retamal-D\u0026iacute;az A, D\u0026iacute;az FE, Vasquez AE, Lay MK, Riedel CA, Gonz\u0026aacute;lez PA, Bueno SM, Kalergis AM (2019) BCG-Induced Cross-Protection and Development of Trained Immunity: Implication for Vaccine Design. Front Immunol 10:1\u0026ndash;14. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2019.02806\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eda Cunha CEP, Bettin EB, Bakry AFAAY, Seixas Neto ACP, Amaral MG, Dellagostin OA (2019) Evaluation of different strategies to promote a protective immune response against leptospirosis using a recombinant LigA and LigB chimera. Vaccine 37:1844\u0026ndash;1852. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vaccine.2019.02.010\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDellagostin OA, Grassmann AA, Rizzi C, Schuch RA, Jorge S, Oliveira TL, Mcbride AJA, Hartwig DD (2017) Reverse Vaccinology: An Approach for Identifying Leptospiral Vaccine Candidates. Int J Mol Sci 18:158. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms18010158\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDockrell HM, Smith SG (2017) What have we learnt about BCG vaccination in the last 20 years? Front Immunol 8. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2017.01134\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDorneles J, Bonemann A, Clair A, Seixas P, Rizzi C, Burlamarque \u0026Eacute;, Silva A, Caetano C, Castro D, Gevehr C, Larr\u0026eacute; T, Antonio O (2020) Protection against leptospirosis conferred by \u003cem\u003eMycobacterium bovis\u003c/em\u003e BCG expressing antigens from Leptospira interrogans. Vaccine 38:8136\u0026ndash;8144. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vaccine.2020.10.086\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdgar RC, Drive RM, Valley M (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792\u0026ndash;1797. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/nar/gkh340\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEllis WA (2015) Animal leptospirosis. Curr Top Microbiol Immunol 387:99\u0026ndash;137. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-3-662-45059-8_6\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFaine S (1959) Iron as a Growth Requirement for Pathogenic \u003cem\u003eLeptospira\u003c/em\u003e. J Gen Microbiol 20:246\u0026ndash;251. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1099/00221287-20-2-246\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFelix CR, Siedler BS, Barbosa LN, Timm GR, McFadden J, McBride AJA (2020) An overview of human leptospirosis vaccine design and future perspectives. Expert Opin Drug Discov 15:179\u0026ndash;188. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/17460441.2020.1694508\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFernandes LGV, Teixeira AF, Filho AFS, Souza GO, Vasconcellos SA, Heinemann MB, Romero EC, Nascimento ALTO (2017) Immune response and protective profile elicited by a multi-epitope chimeric protein derived from \u003cem\u003eLeptospira interrogans\u003c/em\u003e. International Journal of Infectious Diseases 57:61\u0026ndash;69. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ijid.2017.01.032\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFouts DE, Matthias MA, Adhikarla H, Adler B, Amorim-Santos L, Berg DE, Bulach D, Buschiazzo A, Chang YF, Galloway RL, Haake DA, Haft DH, Hartskeerl R, Ko AI, Levett PN, Matsunaga J, Mechaly AE, Monk JM, Nascimento ALT, Nelson KE, Palsson B, Peacock SJ, Picardeau M, Ricaldi JN, Thaipandungpanit J, Wunder EA, Yang XF, Zhang JJ, Vinetz JM, Derrick E, Michael A, Fouts DE, Matthias MA, Adhikarla H, Adler B, Amorim- L (2016) What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus \u003cem\u003eLeptospira\u003c/em\u003e. PLoS Negl Trop Dis 10:1\u0026ndash;57. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pntd.0004403\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGomes-Solecki M, Santecchia I, Werts C (2017) Animal models of leptospirosis: Of mice and hamsters. Front Immunol 8. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2017.00058\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrassmann AA, Kremer FS, dos Santos JC, Souza JD, Pinto L, McBride S, Gomes-solecki AAJA, John M, McBride A (2017a) AAJA Discovery of Novel Leptospirosis Vaccine Candidates Using Reverse and Structural Vaccinology. Frontiers in Immunology 8:463. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2017.00463\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrassmann AA, Kremer FS, dos Santos JC, Souza JD, Pinto L, da McBride S, Gomes-solecki AJAAJA, John M, McBride A, Santos AJAAJA, Souza JC, Pinto JD, da McBride L (2017b) AJAAJA Discovery of Novel Leptospirosis Vaccine Candidates Using Reverse and Structural Vaccinology. Frontiers in Immunology 8:463. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2017.00463\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrassmann AA, Souza JD, John A, Mcbride AJA, John A, Mcbride AJA (2017c) A universal vaccine against leptospirosis: Are we going in the right direction? Front Immunol 8:1\u0026ndash;11. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2017.00256\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGr\u0026ouml;schel MI, Sayes F, Shin SJ, Frigui W, Pawlik A, Orgeur M, Canetti R, Honor\u0026eacute; N, Simeone R, van der Werf TS, Bitter W, Cho SN, Majlessi L, Brosch R (2017) Recombinant BCG Expressing ESX-1 of \u003cem\u003eMycobacterium marinum\u003c/em\u003e Combines Low Virulence with Cytosolic Immune Signaling and Improved TB Protection. Cell Reports 18:2752\u0026ndash;2765. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.celrep.2017.02.057\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGroshong AM, Grassmann AA, Luthra A, McLain MA, Provatas AA, Radolf JD, Caimano MJ (2021) PlzA is a bifunctional c-di-GMP biosensor that promotes tick and mammalian hostadaptation of \u003cem\u003eBorrelia burgdorferi\u003c/em\u003e. PLoS Pathog 17:1\u0026ndash;31. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.ppat.1009725\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaake DA, Levett PN (2015) Leptospirosis in Humans. Curr Top Microbiol Immunol 387:65\u0026ndash;97. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-3-662-45059-8\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu W, Ge Y, Ojcius DM, Sun D, Dong H, Yang XF, Yan J (2013) p53 controls apoptosis in Leptospira-infected macrophages. Cell Microbiol 15:1642\u0026ndash;1659. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/cmi.12141\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu Y, Dang W, Sun L (2012) A TonB-dependent outer membrane receptor of \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e: virulence and vaccine potential. Arch Microbiol 194:795\u0026ndash;802. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00203-012-0812-3\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang X, Li Y, Fu Y, Ji Y, Lian K, Zheng H, Wei J, Cai X (2013) Cross-Protective Efficacy of Recombinant Transferrin-Binding Protein A of \u003cem\u003eHaemophilus parasuis\u003c/em\u003e in Guinea Pigs. Clin Vaccine Immunol 20:912\u0026ndash;919. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/CVI.00621-12\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHubert K, Devos N, Mordhorst I, Tans C, Baudoux G, Feron C, Goraj K, Tommassen J, Vogel U, Poolman JT (2013) ZnuD, a Potential Candidate for a Simple and Universal \u003cem\u003eNeisseria meningitidis\u003c/em\u003e Vaccine. Infect Immun 81:1915\u0026ndash;1927. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/IAI.01312-12\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohnson RC, Muschel LH (1966) Antileptospiral activity of serum. I. Normal and immune serum. J Bacteriol 91:1403\u0026ndash;1409. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/jb.91.4.1403-1409.1966\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKleinnijenhuis J, Quintin J, Preijers F, Joosten LAB, Ifrim DC, Saeed S, Jacobs C, Van Loenhout J, De Jong D, Hendrik S, Xavier RJ, Van Der Meer JWM, Van Crevel R, Netea MG (2012) Bacille Calmette-Gu\u0026eacute;rin induces NOD2-dependent nonspecific protection from reinfection via epigenetic reprogramming of monocytes. Proc Natl Acad Sci USA 109:17537\u0026ndash;17542. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.1202870109\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLarsen JEP, Lund O, Nielsen M (2006) Improved method for predicting linear B-cell epitopes. Immunome Research 2:2. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1745-7580-2-2\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin X, Xiao G, Luo D, Kong L, Chen X, Sun D, Yan J (2016) Chimeric epitope vaccine against \u003cem\u003eLeptospira interrogans\u003c/em\u003e infection and induced specific immunity in guinea pigs. BMC Microbiol 16. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12866-016-0852-y\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Ma Q, Yang F, Zhu R, Gu J, Sun C, Feng X, Du C, Langford PR, Han W, Yang J, Lei L (2017) B cell cross-epitope of \u003cem\u003ePropionibacterium acnes\u003c/em\u003e and \u003cem\u003eActinobacillus pleuropneumonia\u003c/em\u003e selected by phage display library can efficiently protect from \u003cem\u003eActinobacillus pleuropneumonia\u003c/em\u003e infection. Vet Microbiol 205:14\u0026ndash;21. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vetmic.2017.04.026\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLobo N, Brooks NA, Zlotta AR, Cirillo JD, Boorjian S, Black PC, Meeks JJ, Bivalacqua TJ, Gontero P, Steinberg GD, McConkey D, Babjuk M, Alfred Witjes J, Kamat AM (2021) 100 years of Bacillus Calmette\u0026ndash;Gu\u0026eacute;rin immunotherapy: from cattle to COVID-19. Nature Reviews Urology 18:611\u0026ndash;622. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41585-021-00481-1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLomize MA, Pogozheva ID, Joo H, Mosberg HI, Lomize AL (2012) OPM database and PPM web server: Resources for positioning of proteins in membranes. Nucleic Acids Res 40:370\u0026ndash;376. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/nar/gkr703\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLouvel H, Bommezzadri S, Zidane N, Boursaux-Eude C, Creno S, Magnier A, Rouy Z, M\u0026eacute;digue C, Saint Girons I, Bouchier C, Picardeau M (2006) Comparative and functional genomic analyses of iron transport and regulation in \u003cem\u003eLeptospira\u003c/em\u003e spp. J Bacteriol 188:7893\u0026ndash;7904. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/JB.00711-06\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNascimento ALTO, Ko AI, Martins EAL, Monteiro-Vitorello CB, Ho PL, Haake DA, Verjovski-Almeida S, Hartskeerl RA, Marques MV, Oliveira MC, Menck CFM, Leite LCC, Carrer H, Coutinho LL, Degrave WM, Dellagostin OA, El-Dorry H, Ferro ES, Ferro MIT, Furlan LR, Gamberini M, Giglioti EA, G\u0026oacute;es-Neto A, Goldman GH, Goldman MHS, Harakava R, Jer\u0026ocirc;nimo SMB, Junqueira-De-Azevedo ILM, Kimura ET, Kuramae EE, Lemos EGM, Lemos MVF, Marino CL, Nunes LR, De Oliveira RC, Pereira GG, Reis MS, Schriefer A, Siqueira WJ, Sommer P, Tsai SM, Simpson AJG, Ferro JA, Camargo LEA, Kitajima JP, Setubal JC, Van Sluys MA (2004) Comparative Genomics of Two \u003cem\u003eLeptospira interrogans\u003c/em\u003e Serovars Reveals Novel Insights into Physiology and Pathogenesis. J Bacteriol 186:2164\u0026ndash;2172. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/JB.186.7.2164-2172.2004\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNetea MG, Quintin J, Van Der Meer JWM (2011) Trained immunity: A memory for innate host defense. Cell Host and Microbe 9:355\u0026ndash;361. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.chom.2011.04.006\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNielsen M, Lund O (2009) NN-align. An artificial neural network-based alignment algorithm for MHC class II peptide binding prediction. BMC Bioinformatics 10:296. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1471-2105-10-296\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNoinaj N, Guillier M, Barnard TJ, Buchanan SK (2010) TonB-Dependent Transporters: Regulation, Structure, and Function. Annu Rev Microbiol 43\u0026ndash;60. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1146/annurev.micro.112408.134247\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNosrati M, Hajizade A, Nazarian S, Amani J, Namvar Vansofla A, Tarverdizadeh Y (2019) Designing a multi-epitope vaccine for cross-protection against \u003cem\u003eShigella\u003c/em\u003e spp: An immunoinformatics and structural vaccinology study. Mol Immunol 116:106\u0026ndash;116. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.molimm.2019.09.018\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOliveira TL, Rizzi C, da Cunha CEP, Dorneles J, Seixas Neto ACP, Amaral MG, Hartwig DD, Dellagostin OA (2019a) Recombinant BCG strains expressing chimeric proteins derived from \u003cem\u003eLeptospira\u003c/em\u003e protect hamsters against leptospirosis. Vaccine 37:776\u0026ndash;782. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vaccine.2018.12.050\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOliveira TL, Rizzi C, Dellagostin OA (2017) Recombinant BCG vaccines: molecular features and their influence in the expression of foreign genes. Appl Microbiol Biotechnol 101:6865\u0026ndash;6877. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00253-017-8439-6\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOliveira TL, Stedman A, Rizzi C, Dorneles J, da Cunha CEP, Junior ASV, Dellagostin OA, McFadden J (2019b) A standardized BioBrick toolbox for the assembly of sequences in mycobacteria. Tuberculosis 119:101851. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tube.2019.07.002\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOyarz\u0026uacute;n P, Kobe B (2016) Recombinant and epitope-based vaccines on the road to the market and implications for vaccine design and production. Human Vaccines \u0026amp; Immunotherapeutics 12:763\u0026ndash;767. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/21645515.2015.1094595\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParish T, Stoker NG (1995) Electroporation of mycobacteria. Methods in molecular biology. (Clifton, NJ) 47:237\u0026ndash;252. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1385/0-89603-310-4:237\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParvizpour S, Pourseif MM, Razmara J, Rafi MA, Omidi Y (2020) Epitope-based vaccine design: a comprehensive overview of bioinformatics approaches. Drug Discovery Today 25:1034\u0026ndash;1042. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.drudis.2020.03.006\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera \u0026mdash; A Visualization System for Exploratory Research and Analysis. J Comput Chem 1605\u0026ndash;1612. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/jcc.20084\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePr\u0026ecirc;tre G, Olivera N, C\u0026eacute;dola M, Haase S, Alberdi L, Brihuega B, G\u0026oacute;mez RM (2011) Role of inducible nitric oxide synthase in the pathogenesis of experimental leptospirosis. Microb Pathog 51:203\u0026ndash;208. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.micpath.2011.03.011\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamos CRR, Abreu PAE, Nascimento ALTO, Ho PL (2004) A high-copy T7 \u003cem\u003eEscherichia coli\u003c/em\u003e expression vector for the production of recombinant proteins with a minimal N-terminal his-tagged fusion peptide. Braz J Med Biol Res 37:1103\u0026ndash;1109. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1590/S0100-879X2004000800001\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanchez-trincado JL, Gomez-perosanz M, Reche PA (2017) Fundamentals and Methods for T- and B-Cell Epitope Prediction. Journal of Immunology Research 2017. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003edoi.org/10.1155/2017/2680160\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantecchia I, Ferrer F, Vieira ML, Mart R (2020) Phagocyte Escape of \u003cem\u003eLeptospira\u003c/em\u003e: The Role of TLRs and NLRs. Front Immunol 11. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2020.571816\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantecchia I, Vernel-Pauillac F, Rasid O, Quintin J, Gomes-Solecki M, Boneca IG, Werts C (2019) Innate immune memory through TLR2 and NOD2 contributes to the control of \u003cem\u003eLeptospira interrogans\u003c/em\u003e infection. PLoS Pathog 15:1\u0026ndash;26. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.ppat.1007811\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeixas F, Drawanz D, Maia G, Amaral M, Quevedo M (2007) Recombinant \u003cem\u003eMycobacterium bovis\u003c/em\u003e BCG expressing the LipL32 antigen of \u003cem\u003eLeptospira interrogans\u003c/em\u003e protects hamsters from challenge. Vaccine 26:88\u0026ndash;95. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vaccine.2007.10.052\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShelton AN, Seth EC, Mok KC, Han AW, Jackson SN, Haft DR, Taga ME (2019) Uneven distribution of cobamide biosynthesis and dependence in bacteria predicted by comparative genomics. ISME J 13:789\u0026ndash;804. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003edoi.org/10.1038/s41396-018-0304-9\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoto JA, G\u0026aacute;lvez NMS, Rivera CA, Palavecino CE, C\u0026eacute;spedes PF, Rey-Jurado E, Bueno SM, Kalergis AM (2018) Recombinant BCG Vaccines Reduce Pneumovirus-Caused Airway Pathology by Inducing Protective Humoral Immunity. Front Immunol 9:2875. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2018.02875\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToma C, Murray GL, Nohara T, Mizuyama M, Koizumi N, Adler B, Suzuki T (2014) Leptospiral outer membrane protein LMB216 is involved in enhancement of phagocytic uptake by macrophages. Cell Microbiol 16:1366\u0026ndash;1377. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/cmi.12296\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrunz BB, Fine P, Dye C (2006) Effect of BCG vaccination on childhood tuberculous meningitis and miliary tuberculosis worldwide: a meta-analysis and assessment of cost-effectiveness. Lancet 367:1173\u0026ndash;1180. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0140-6736(06)68507-3\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVincent AT, Schiettekatte O, Goarant C, Neela VK, Bernet E, Thibeaux R, Ismail N, Khalid MKNM, Amran F, Masuzawa T, Nakao R, Korba AA, Bourhy P, Veyrier FJ, Picardeau M (2019) Revisiting the taxonomy and evolution of pathogenicity of the genus \u003cem\u003eLeptospira\u003c/em\u003e through the prism of genomics. PLoS Negl Trop Dis 13. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pntd.0007270\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang B, Sullivan JA, Sullivan GW, Mandell GL (1984) Role of specific antibody in interaction of leptospires with human monocytes and monocyte-derived macrophages. Infect Immun 46:809\u0026ndash;813. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/iai.46.3.809-813.1984\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiao J, Liu J, Bao C, Zhu R, Gu J, Sun C, Feng X, Du C, Han W, Li Y, Lei L (2020) Recombinant tandem epitope vaccination provides cross protection against \u003cem\u003eActinobacillus pleuropneumoniae\u003c/em\u003e challenge in mice. AMB Express 10:1\u0026ndash;13. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13568-020-01051-1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan W, Faisal SM, Mcdonough SP, Divers TJ, Barr SC, Chang C, Pan M, Chang Y (2009) Immunogenicity and protective efficacy of recombinant \u003cem\u003eLeptospira\u003c/em\u003e immunoglobulin-like protein B (rLigB) in a hamster challenge model. Microbes Infect 11:230\u0026ndash;237. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.micinf.2008.11.008\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang J, Zhang Y (2015) I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Res 43:174\u0026ndash;181. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/nar/gkv342\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Zhang C, Ojcius DM, Sun D, Zhao J, Lin X, Li L, Li L, Yan J (2012) The mammalian cell entry (Mce) protein of pathogenic \u003cem\u003eLeptospira\u003c/em\u003e species is responsible for RGD motif-dependent infection of cells and animals. Mol Microbiol 83:1006\u0026ndash;1023. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1365-2958.2012.07985.x\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Skolnick J (2005) TM-align: A protein structure alignment algorithm based on the TM-score. Nucleic Acids Res 33:2302\u0026ndash;2309. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/nar/gki524\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao J, Chen H, Ojcius DM, Zhao X, Sun D, Ge Y, Zheng L, Lin X, Li L, Yan J (2013) Identification of \u003cem\u003eLeptospira\u003c/em\u003e interrogans Phospholipase C as a Novel Virulence Factor Responsible for Intracellular Free Calcium Ion Elevation during Macrophage Death. PLoS ONE 8:e75652. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0075652\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZuerner RL (2015) Host response to \u003cem\u003eLeptospira\u003c/em\u003e infection. Curr Top Microbiol Immunol 387:223\u0026ndash;250. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-3-662-45059-8_9\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"Leptospira, reverse and structural vaccinology, beta-barrel transmembrane protein, epitope-based vaccines, chimeric protein","lastPublishedDoi":"10.21203/rs.3.rs-996824/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-996824/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLeptospirosis is an emerging infectious disease caused by pathogenic \u003cem\u003eLeptospira\u003c/em\u003e spp. A universal vaccine against leptospirosis is likely to require highly conserved epitopes from pathogenic leptospires, that are exposed on the bacterial surface, and that generate a protective and sterilizing immune response. Our group recently identified several genes predicted to encode TonB-dependent receptors (TBDR) in \u003cem\u003eLeptospira interrogans\u003c/em\u003e using a reverse vaccinology approach. Three leptospiral TBDRs were previously described and partially characterized as ferric-citrate, hemin, and cobalamin transporters. In the current study, we designed a fusion protein composed of predicted surface-exposed epitopes from three conserved leptospiral TBDRs. Based on their three-dimensional structural models and the prediction of immunogenic regions, nine putative surface-exposed fragments were selected to compose a recombinant chimeric protein. A \u003cem\u003eMycobacterium bovis\u003c/em\u003e BCG strain expressing this chimeric antigen encoded in the pUP500/P\u003csub\u003epAN\u003c/sub\u003e mycobacterial expression vector was used to immunize Syrian hamsters. All animals (20/20) vaccinated with recombinant BCG survived infection with a endpoint dose of \u003cem\u003eL. interrogan\u003c/em\u003es (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). No animal survived in the negative control group. Immunization with our recombinant BCG elicited a humoral immune response against leptospiral TBDRs, as demonstrated by ELISA and immunoblot. No leptospiral DNA was detected by \u003cem\u003elipL32\u003c/em\u003e qPCR in the kidneys of vaccinated hamsters. Similarly, no growth was observed in macerated kidney cultures from the same animals, suggesting the induction of a sterilizing immune response. Design of new vaccine antigens based on the structure of outer membrane proteins is a promising approach to overcome the impact of leptospirosis by vaccination.\u003c/p\u003e","manuscriptTitle":"TonB-dependent receptor epitopes expressed in M. bovis BCG induced significant protection in the hamster model of leptospirosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-11-02 14:33:03","doi":"10.21203/rs.3.rs-996824/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":"67394b69-f0b0-4023-b40a-38cbce7a1a00","owner":[],"postedDate":"November 2nd, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":8266262,"name":"Applied \u0026 Industrial Microbiology"},{"id":8266263,"name":"Biotechnology and Bioengineering"}],"tags":[],"updatedAt":"2021-12-11T03:49:02+00:00","versionOfRecord":{"articleIdentity":"rs-996824","link":"https://doi.org/10.1007/s00253-021-11726-9","journal":{"identity":"applied-microbiology-and-biotechnology","isVorOnly":false,"title":"Applied Microbiology and Biotechnology"},"publishedOn":"2021-12-11 03:49:02","publishedOnDateReadable":"December 11th, 2021"},"versionCreatedAt":"2021-11-02 14:33:03","video":"","vorDoi":"10.1007/s00253-021-11726-9","vorDoiUrl":"https://doi.org/10.1007/s00253-021-11726-9","workflowStages":[]},"version":"v1","identity":"rs-996824","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-996824","identity":"rs-996824","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}