Evaluation of protective efficacy, serological responses, and cytokine modulation induced by polyvalent Leptospira vaccines in hamsters.

Whole-cell inactivated vaccines (bacterins) are the only licensed vaccines available for leptospirosis prevention and control, especially in domestic and farm animals. However, despite their widespread use, inconsistencies in their efficacy have been reported. Because immunity induced by bacterins is mainly mediated by antibodies against leptospiral lipopolysaccharides, the involvement of cellular responses is not well-known. The aim of this study was to investigate the efficacy and characterize the humoral and cellular immune responses induced by whole-cell inactivated leptospirosis bacterin formulations containing serovars Bratislava, Canicola, Copenhageni, Grippotyphosa, Hardjoprajitno, and Pomona. For the potency test, hamsters were immunized with one dose of polyvalent bacterins (either commercial or experimental) and then challenged with a virulent Pomona strain. Serological (MAT and IgM and IgG-ELISA) and cellular (cytokine transcription in blood evaluated by RT-qPCR) analyses were performed. The results revealed that vaccination with either bacterin formulation was able to protect 90-100% of the hamsters infected with the Pomona serovar, although most of the surviving animals remained as renal carriers. Specific agglutinating antibodies and significant levels of IgM, IgG, and IgG2 (P < 0.05) that were able to react with the six serovars present in the vaccine formulations were produced, indicating that the vaccines can potentially provide immunity against all strains. The protective immunity of these vaccines was mainly mediated by balanced a Th1/Th2 response, characterized by increased IFN-γ, IL-10 and IL-α transcription. These data support the importance of characterizing immunological responses involved in bacterin efficacy and investing in the improvement of these vaccine formulations.

Characterization of a Molecularly Engineered Banlec-Type Lectin (rBTL).

Lectins are proteins that reversibly bind to carbohydrates and are commonly found across many species. The Banana Lectin (BanLec) is a member of the Jacalin-related Lectins, heavily studied for its immunomodulatory, antiproliferative, and antiviral activity. In this study, a novel sequence was generated in silico considering the native BanLec amino acid sequence and 9 other lectins belonging to JRL. Based on multiple alignment of these proteins, 11 amino acids of the BanLec sequence were modified because of their potential for interference in active binding site properties resulting in a new lectin named recombinant BanLec-type Lectin (rBTL). rBTL was expressed in E. coli and was able to keep biological activity in hemagglutination assay (rat erythrocytes), maintaining similar structure with the native lectin. Antiproliferative activity was demonstrated on human melanoma lineage (A375), evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT). rBTL was able to inhibit cellular growth in a concentration-dependent manner, in an 8-h incubation, 12 µg/mL of rBTL led to a 28.94% of cell survival compared to cell control with 100%. Through a nonlinear fit out log-concentration versus biological response, an IC50% of 3.649 µg/mL of rBTL was determined. In conclusion, it is possible to state that the changes made to the rBTL sequence maintained the structure of the carbohydrate-binding site without changing specificity. The new lectin is biologically active, with an improved carbohydrate recognition spectrum compared to nBanLec, and can also be considered cytotoxic for A375 cells.

Characterization of cellular immune response in hamsters immunized with recombinant vaccines against leptospirosis based on LipL32:LemA:LigAni chimeric protein.

In the last 20 years, various research groups have endeavored to develop recombinant vaccines against leptospirosis to overcome the limitations of commercially available bacterins. Numerous antigens and vaccine formulations have been tested thus far. However, the analysis of cellular response in these vaccine formulations is not commonly conducted, primarily due to the scarcity of supplies and kits for the hamster animal model. Our research group has already tested the Q1 antigen, a chimeric protein combining the immunogenic regions of LipL32, LemA, and LigANI, in recombinant subunit and BCG-vectored vaccines. In both strategies, 100 % of the hamsters were protected against clinical signs of leptospirosis. However, only the recombinant BCG-vectored vaccine provided protection against renal colonization. Thus, the objective of this study is to characterize the cellular immune response in hamsters immunized with different vaccine formulations based on the Q1 antigen through transcriptional analysis of cytokines. The hamsters were allocated into groups and vaccinated as follows: recombinant subunit (rQ1), recombinant BCG (rBCG:Q1), and saline and BCG Pasteur control vaccines. To assess the cellular response induced by the vaccines, we cultured and stimulated splenocytes, followed by RNA extraction from the cells and analysis of cytokines using real-time PCR. The results revealed that the recombinant subunit vaccine elicited a Th2-type response, characterized by the expression of cytokines IL-10, IL-1α, and TNF-α. This pattern closely resembles the cytokines expressed in severe cases of leptospirosis. On the other hand, the rBCG-vectored vaccine induced a Th1-type response with significant up-regulation of IFN-γ. These findings suggest the involvement of the cellular response and the IFN-γ mediated inflammatory response in the sterilizing immunity mediated by rBCG. Therefore, this study may assist future investigations in characterizing the cellular response in hamsters, aiming to elucidate the mechanisms of efficacy and establish potential correlates of protection.

Challenges and Strategies for Developing Recombinant Vaccines against Leptospirosis: Role of Expression Platforms and Adjuvants in Achieving Protective Efficacy.

The first leptospiral recombinant vaccine was developed in the late 1990s. Since then, progress in the fields of reverse vaccinology (RV) and structural vaccinology (SV) has significantly improved the identification of novel surface-exposed and conserved vaccine targets. However, developing recombinant vaccines for leptospirosis faces various challenges, including selecting the ideal expression platform or delivery system, assessing immunogenicity, selecting adjuvants, establishing vaccine formulation, demonstrating protective efficacy against lethal disease in homologous challenge, achieving full renal clearance using experimental models, and reproducibility of protective efficacy against heterologous challenge. In this review, we highlight the role of the expression/delivery system employed in studies based on the well-known LipL32 and leptospiral immunoglobulin-like (Lig) proteins, as well as the choice of adjuvants, as key factors to achieving the best vaccine performance in terms of protective efficacy against lethal infection and induction of sterile immunity.

Anthelmintic effect of Cassia fistula and Combretum leprosum protein fractions against goat gastrointestinal nematodes.

In this study, we evaluated the ovicidal and larvicidal activity of protein preparations obtained from Cassia fistula L. and Combretum leprosum Mart. leaves on the gastrointestinal parasites of goats. Protein preparations were obtained after the extraction of C. fistula L. and C. leprosum Mart. leaves, followed by protein fractionation (with ammonium sulfate saturation percentages of 30%, 30%-60%, and 60%-90%) and dialysis, which resulted in protein fractions (called F1, F2, and F3, respectively). The fractions were evaluated by egg hatching (the eggs were recovered in stool samples from naturally infected goats) and larval development tests. The results reveled that the inhibition of hatching of eggs caused by the protein fractions of C. fistula (38%) were similar to that of the control drug, thiabendazole. In addition, the fractions of C. fistula caused significant inhibition (61-69%) of larval development also. However, C. leprosum did not reveal significant inhibition of egg hatching and larval development. We conclude that C. fistula L. showed better ovicidal and larvicidal activity against endoparasites.

Anthelmintic effect of Cassia fistula and Combretum leprosum protein fractions against goat gastrointestinal nematodes / Efeito antihelmíntico das frações proteicas de Cassia fistula e Combretum leprosum contra nematodeos gastrintestinais de caprinos

Abstract In this study, we evaluated the ovicidal and larvicidal activity of protein preparations obtained from Cassia fistula L. and Combretum leprosum Mart. leaves on the gastrointestinal parasites of goats. Protein preparations were obtained after the extraction of C. fistula L. and C. leprosum Mart. leaves, followed by protein fractionation (with ammonium sulfate saturation percentages of 30%, 30%-60%, and 60%-90%) and dialysis, which resulted in protein fractions (called F1, F2, and F3, respectively). The fractions were evaluated by egg hatching (the eggs were recovered in stool samples from naturally infected goats) and larval development tests. The results reveled that the inhibition of hatching of eggs caused by the protein fractions of C. fistula (38%) were similar to that of the control drug, thiabendazole. In addition, the fractions of C. fistula caused significant inhibition (61-69%) of larval development also. However, C. leprosum did not reveal significant inhibition of egg hatching and larval development. We conclude that C. fistula L. showed better ovicidal and larvicidal activity against endoparasites.

Resumo Neste estudo, foram avaliadas as atividades ovicida e larvicida de preparações proteicas de Cassia fistula L. e Combretum leprosum Mart. em parasitas gastrointestinais de caprinos. As preparações proteicas foram obtidas por extração das folhas de C. fistula L. e C. leprosum Mart. seguido pelo fracionamento proteico (com porcentagens de saturação de sulfato de amônio de 30%, 30-60%, 60-90%) e diálise, resultando nas frações proteicas (intituladas F1, F2 e F3, respectivamente). As frações foram avaliadas nos testes de eclosão de ovos (os ovos foram recuperados em amostras de fezes de cabras naturalmente infectadas) e de desenvolvimento larvar. Os resultados revelaram que a inibição da eclosão de ovos causada pelas frações proteicas de C. fistula (38%) foi semelhante à do fármaco controle, o tiabendazol. Além disso, as frações de C. fistula também causaram inibição significativa (61-69%) do desenvolvimento larvar. No entanto, C. leprosum não revelou inibição significativa na eclosão dos ovos e no desenvolvimento larvar. Concluiu-se que C. fistula L. mostrou uma melhor atividade ovicida e larvicida contra endoparasitas.

Gene expression and activity of antioxidant enzymes in rice plants, cv. BRS AG, under saline stress.

The rice cultivar (Oryza sativa L.) BRS AG, developed by Embrapa Clima Temperado, is the first cultivar designed for purposes other than human consumption. It may be used in ethanol production and animal feed. Different abiotic stresses negatively affect plant growth. Soil salinity is responsible for a serious reduction in productivity. Therefore, the objective of this study was to evaluate the gene expression and the activity of antioxidant enzymes (SOD, CAT, APX and GR) and identify their functions in controlling ROS levels in rice plants, cultivar BRS AG, after a saline stress period. The plants were grown in vitro with two NaCl concentrations (0 and 136 mM), collected at 10, 15 and 20 days of cultivation. The results indicated that the activity of the enzymes evaluated promotes protection against oxidative stress. Although, there was an increase of reactive oxygen species, there was no increase in MDA levels. Regarding genes encoding isoforms of antioxidant enzymes, it was observed that OsSOD3-CU/Zn, OsSOD2-Cu/Zn, OsSOD-Cu/Zn, OsSOD4-Cu/Zn, OsSODCc1-Cu/Zn, OsSOD-Fe, OsAPX1, OsCATB and OsGR2 were the most responsive. The increase in the transcription of all genes among evaluated isoforms, except for OsAPX6, which remained stable, contributed to the increase or the maintenance of enzyme activity. Thus, it is possible to infer that the cv. BRS AG has defense mechanisms against salt stress.