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.

Evaluation of different strategies to promote a protective immune response against leptospirosis using a recombinant LigA and LigB chimera.

Leptospirosis is a zoonosis of worldwide distribution, caused by infection with pathogenic Leptospira species. The vaccines that are currently available are bacterins, with limited human use, that confer short-term, serovar-specific immunity. Lig proteins are considered to be the best vaccine candidates to date. Here, we aimed to construct a recombinant Lig chimera (LC) comprised of LigAni and LigBrep fragments, and to evaluate it as subunit or DNA vaccine using different administration strategies. Vaccines were formulated with 50 µg of recombinant LC associated with different adjuvants or with 100 µg of pTARGET/LC. Four-week-old hamsters received two doses of vaccine with different strategies and were challenged with 5 × DL50Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130. The immune response generated by Lig chimera conferred 100% protection to hamsters treated with at least one dose of recombinant LC. Despite the high levels of antibodies that vaccinated animals produced, a sterilizing immunity was not achieved. The lack of a sterilizing immunity could indicate the importance of a mixed humoral and cellular immune response. The present study generated insights that will be useful in the future development of improved subunit vaccines against leptospirosis.

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.

Human and animal leptospirosis in Southern Brazil: A five-year retrospective study.

BACKGROUND: Leptospirosis is an emerging zoonosis attributed to multiple reservoirs. Climatic conditions influence the transmission of pathogenic leptospires, which require warm and humid conditions for survival. The influence of seasonality in human and animal leptospirosis in the subtropical region of Brazil remains poorly understood. METHODS: We performed a retrospective study to describe the patterns of human and animal exposure to leptospirosis and their association with precipitation events in Southern Brazil. Rainfall data were obtained from satellite images. Serum samples were tested using the microscopic agglutination test (MAT); samples with titer ≥ 100 were defined as seroreactive. Linear regression and Pearson's correlation were performed to assess whether there is a relationship between these variables. RESULTS: We found that precipitation events were not significantly associated with the exposure to leptospirosis in humans or animal species, except for dogs. The interspecies analysis revealed an association between canine and human exposure to leptospirosis. Leptospira kirschneri serovar Butembo (serogroup Autumnalis) presented the highest seroreactivity in humans. CONCLUSION: This study provides valuable insights in human and animal leptospirosis in Southern Brazil. These insights will be essential to design intervention measures directed to reduce disease dissemination.

Saccharomyces boulardii improves humoral immune response to DNA vaccines against leptospirosis.

PURPOSE: Saccharomyces boulardii may improve the immune response by enhancing the production of anti-inflammatory cytokines, T-cell proliferation and dendritic cell activation. The immunomodulator effect of this probiotic has never been tested with DNA vaccines, which frequently induce low antibody titers. This study evaluated the capacity of Saccharomyces boulardii to improve the humoral and cellular immune responses using DNA vaccines coding for the leptospiral protein fragments LigAni and LigBrep. BALB/c mice were fed with rodent-specific feed containing 108 c.f.u. of Saccharomycesboulardii per gram. METHODOLOGY: Animals were immunized three times intramuscularly with 100 µg of pTARGET plasmids containing the coding sequences for the above mentioned proteins. Antibody titers were measured by indirect ELISA. Expression levels of IL-4, IL-10, IL-12, IL-17, IFN-γ and TGF-ß were determined by quantitative real-time PCR from RNA extracted from whole blood, after an intraperitoneal boost with 50 µg of the recombinant proteins.Results/Key findings. Antibody titers increased significantly after the second and third application when pTARGET/ligAni and pTARGET/ligBrep were used to vaccinate the animals in comparison with the control group (P<0.05). In addition, there was a significant increase in the expression of the IL-10 in mice immunized with pTARGET/ligBrep and fed with Saccharomyces boulardii. CONCLUSION: The results suggested that Saccharomyces boulardii has an immunomodulator effect in DNA vaccines, mainly by stimulating the humoral response, which is often limited in this kind of vaccine. Therefore, the use of Saccharomyces boulardii as immunomodulator represents a new alternative strategy for more efficient DNA vaccination.

Protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D.

Botulinum neurotoxin (BoNT) serotypes C and D are responsible for cattle botulism, a fatal paralytic disease that results in great economic losses in livestock production. Vaccination is the main approach to prevent cattle botulism. However, production of commercially available vaccines (toxoids) involves high risk and presents variation of BoNT production between batches. Such limitations can be attenuated by the development of novel nontoxic recombinant vaccines through a simple and reproducible process. The aim of this study was to evaluate the protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D. Bivalent vaccines containing 200 µg rHCC and rHCD each were formulated in three different ways: (1) purified antigens; (2) recombinant Escherichia coli bacterins; (3) recombinant E. coli cell lysates (supernatant and inclusion bodies). Guinea pigs immunized subcutaneously with recombinant formulations developed a protective immune response against the respective BoNTs as determined by a mouse neutralization bioassay with pooled sera. Purified recombinant antigens were capable of inducing 13 IU/mL antitoxin C and 21 IU/mL antitoxin D. Similarly, both the recombinant bacterins and the cell lysate formulations were capable of inducing 12 IU/mL antitoxin C and 20 IU/mL antitoxin D. These values are two times as high as compared to values induced by the commercial toxoid used as control, and two to ten times as high as the minimum amount required by the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA), respectively. Therefore, we used a practical, industry-friendly, and efficient vaccine production process that resulted in formulations capable of inducing protective immune response (neutralizing antitoxins) against botulism serotypes C and D.

Immunogenicity of a Trivalent Recombinant Vaccine Against Clostridium perfringens Alpha, Beta, and Epsilon Toxins in Farm Ruminants.

Clostridium perfringens is an anaerobic bacterium that produces several toxins. Of these, the alpha, beta, and epsilon toxins are responsible for causing the most severe C. perfringens-related diseases in farm animals. The best way to control these diseases is through vaccination. However, commercially available vaccines are based on inactivated toxins and have many production drawbacks, which can be overcome through the use of recombinant antigens. In this study, we produced recombinant alpha, beta, and epsilon toxins in Escherichia coli to formulate a trivalent vaccine. Its effectiveness was evaluated through a potency test in rabbits, in which the vaccine generated 9.6, 24.4, and 25.0 IU/mL of neutralizing antibodies against the respective toxins. Following this, cattle, sheep, and goats received the same formulation, generating, respectively, 5.19 ± 0.48, 4.34 ± 0.43, and 4.70 ± 0.58 IU/mL against alpha toxin, 13.71 ± 1.17 IU/mL (for all three species) against beta toxin, and 12.74 ± 1.70, 7.66 ± 1.69, and 8.91 ± 2.14 IU/mL against epsilon toxin. These levels were above the minimum recommended by international protocols. As such, our vaccine was effective in generating protective antibodies and, thus, may represent an interesting alternative for the prevention of C. perfringens-related intoxications in farm animals.

Infection with Leptospira kirschneri Serovar Mozdok: First Report from the Southern Hemisphere.

Leptospirosis is a global zoonosis caused by pathogenic Leptospira spp. In this study, we characterized two Leptospira kirschneri serogroup Pomona serovar Mozdok isolates, one obtained from a dog and the other from a patient with severe leptospirosis, 4 years later. Histopathological analysis showed that both isolates caused severe tissue damage when used to infect hamsters. While L. kirschneri serogroup Pomona serovar Mozdok is endemic in animals in Europe, there is only one report of human leptospirosis in the literature. Although strains belonging to L. kirschneri serogroup Pomona have been identified in cases of human leptospirosis in Europe, serovar Mozdok has not yet been implicated. The 4-year interval between isolations and the fact that this is the first report of serovar Mozdok as the causative agent of human leptospirosis in the southern hemisphere, demonstrates its epidemiological importance to public health. Moreover, the presence of serovar Mozdok in Brazil has the potential to affect vaccine and diagnostic test development.