Expressão de antígenos de Leptospira interrogans em Mycobacterium bovis BCG e avaliação como vacina recombinante contra leptospirose

Abstract

Mycobacterium bovis BCG is a live attenuated vaccine, used as a vaccine against human tuberculosis. Among several attractive features, it has adjuvant properties, making it a highly relevant vaccine vector for expression of heterologous antigens from several microorganisms, including Leptospira spp., the etiological agent of leptospirosis. This infectious disease affects animals and humans worldwide. Infection occurs through direct contact with water or soil contaminated with pathogenic leptospiras. Leptospiras are classified into more than 300 serovars. This antigenic diversity makes it impossible for the available bacterins (vaccines produced with inactivated bacteria) to provide broad spectrum protection. Aiming at producing a single protective vaccine against leptospirosis, studies are performed with proteins common to all serovars, are exposed on the outer membrane of pathogenic leptospiras, or are expressed at the time of infection, being LipL32, LemA and LigAni, proteins that have such characteristics. The insertion of genes in an expression vector is facilitated by the BioBricks® Standard, which consists of a strategy that allows the construction of biological molecules compatible with each other, where cloning is performed in a simple, reproducible and standardized way. In this work, four portions - P1 (lipL32), P2 (ligAni), P3 (lemA:ligAni) and P4 (lipL32:lemA) - of a recombinant chimera previously built, were cloned using the BioBricks® strategy in plasmid pUP500/PpAN. These constructs were transformed into M. bovis BCG strain Pasteur, and the expression of the proteins was detected by Western blot, where to detect portions 1 and 4 the primary antibody anti-LipL32 was used, and secondary antibody, anti-hamster IgG. To detect portions 2 and 3, primary anti-LigAni antibody and secondary anti-rabbit IgG antibody were used. For inoculation, 5 groups of ten Syrian hamsters each aged 4-6 weeks were used, being these: group 1, rBCG (LipL32); group 2, rBCG (LigAni); group 3, rBCG (LemA:LigAni); group 4, (LipL32:LemA); group 5, BCG Pasteur unprocessed (negative control). Two doses of each vaccine formulation and negative control were administered, containing 106 CFU with an interval of 21 days, and the challenge was performed with 5 x LD50 of Leptospira interrogans serovar Copenhageni strain Fiocruz L1130. The animals were followed up for 30 days after the challenge. Symptoms of leptospirosis were observed only in the negative control group. Vaccine formulations containing rBCGs provided 100% protection, with sterilizing immunity. Significant levels of antibodies against the proteins LipL32, LemA and LigAni, were not detected in the hamsters' sera. With this work, we conclude that recombinant vaccines of M. bovis BCG have the potential to provide significant protection with sterilizing immunity against leptospirosis in a homologous challenge in hamsters, with BCG being a highly relevant vaccine vector.