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

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/PpAN mycobacterial expression vector was used to immunize Syrian hamsters. All animals (20/20) vaccinated with recombinant BCG survived infection with an endpoint dose of L. interrogans (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. KEY POINTS: • Predicted surface-exposed epitopes were identified in three leptospiral TBDRs. • An M. bovis BCG strain expressing a chimeric protein (rTBDRchi) was constructed. • Hamsters vaccinated with rBCG:TBDRchi were protected from lethal leptospirosis.

Protection against leptospirosis conferred by Mycobacterium bovis BCG expressing antigens from Leptospira interrogans.

Leptospirosis is a zoonotic disease worldwide and caused by the pathogenic spirochetes of the genus Leptospira. Bacterins make up the vaccines used against leptospirosis, but they only succeed in providing short-term and serovar-specific protection. The use of Mycobacterium bovis BCG as a live vaccine vector expressing leptospiral antigens is a promising alternative, particularly due to its adjuvant properties. Four distinct portions P1 (lipL32), P2 (ligAni), P3 (lemA:ligAni) and P4 (lipL32:lemA) of a recombinant chimera composed of the lipL32, lemA and ligANI genes from Leptospira interrogans were cloned individually according to the BioBricks® strategy in the plasmid pUP500/PpAN. These constructs were individually transformed into a BCG Pasteur strain, and protein expression was detected by Western blot. For vaccination, 5 groups of 10 Golden Syrian hamsters were used, aged 4-6 weeks - group 1, rBCG (LipL32); group 2, rBCG (LigAni); group 3, rBCG (LemA:LigAni); group 4, (LipL32:LemA); group 5, wild-type BCG Pasteur (negative control). Two doses containing 106 CFU of rBCG were administered subcutaneously, the challenge was performed with 5 × LD50 of Leptospira interrogans serovar Copenhageni L1-130, and the animals were observed for a 30-day period until the endpoint was reached. Humoral immunity was assessed via indirect ELISA, while renal colonisation was assessed by culture and quantitative real-time PCR. All vaccinated groups were protected against lethal challenge and renal colonisation, in comparison with negative control group (P < 0.05). Recombinant vaccines were not effective at inducing significant humoral immunity, which suggests the induction of cellular immunity - a characteristic of M. bovis BCG. In conclusion, all formulations provide 100% significant protection against leptospirosis in hamsters with no renal colonisation. The use of rBCG as a vaccine vector represents a promising alternative for the control of animal leptospirosis, allowing for protection against clinical signs of leptospirosis and renal colonisation.

Recombinant BCG strains expressing chimeric proteins derived from Leptospira protect hamsters against leptospirosis.

Leptospirosis is a zoonosis that is responsible for one million human cases per year. Fusing multiple immunogenic antigens represents a promising approach to delivering an effective vaccine against leptospirosis. Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a potential vaccine vector due to its adjuvant properties and safety. Two chimeric genes based on genic sequences of ligANI, ligBrep, lipL32, and lemA, were individually cloned into five BioBrick vectors with different promoters (pAN, Hsp60, 18 kDa, Ag85B and Ag85B plus signal sequence) for antigen expression in BCG. Groups of ten hamsters were vaccinated with recombinant BCG (rBCG) strains in two doses of 106 CFU and challenged with 5 × LD50 of L. interrogans serovar Copenhageni. All rBCG vaccines expressing chimera 1, based on antigens LipL32, LigANI, and LemA, under the control of any promoter, protected 80-100% of the hamsters from challenge (P < 0.05) and four of them also protected from renal carrier status; for chimera 2, based on LigANI and LigBrep antigens, the only vaccine that afforded survival rates statistically different from the control was the vaccine that incorporated the pAN promoter (60% of survival). A single vaccine dose was sufficient to induce significant IgG levels by all vaccine compositions evaluated; however, humoral response was not related to protection. These findings suggest that the combination of potential vaccine candidates in chimeric antigens and the use of BCG as a live vector are promising strategies by which it is possible to obtain an effective and sterilizing vaccine against leptospirosis.

A standardized BioBrick toolbox for the assembly of sequences in mycobacteria.

For more than 25 years, recombinant Mycobacterium bovis BCG has been genetically engineered for use as a vehicle for antigen expression and immunomodulation, typically through introducing or deleting a gene from BCG genome. However, BCG transformation efficacy is still unpredictable, and cloning and expression of sequences from mycobacteria is difficult to predict due to the lack of standardization. To overcome such limitations, we have employed the BioBrick format to construct a toolbox of several mycobacterial parts, including coding sequences, reporter genes, selective markers, promoters, and other regulatory sequences. Additionally, we have developed and characterized BioBrick-compatible episomal vectors that are able to replicate in M. bovis BCG to enable expression of heterologous antigens. The availability of a BCG Biobrick toolbox will enable any coding sequence to be optimally expressed in BCG. We believe that this mycobacterial toolbox represents a standardized and useful kit to enhance the efficacy and use of recombinant BCG.