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Visceral leishmaniasis is the most severe form of leishmaniasis. Approximately 20% of zoonotic human visceral leishmaniasis worldwide is caused by Leishmania infantum, which is also known as Leishmania chagasi in Latin America, and disease incidence is increasing in urban and peri-urban areas of the tropics. In this form of disease, dogs are the main reservoirs. Diagnostic methods used to identify Leishmania infected animals are not able to detect all of the infected ones, which can compromise the effectiveness of disease control. Therefore, to contribute to the improvement of diagnostic methods for canine visceral leishmaniasis (CVL), we aimed to identify and test novel antigens using high-throughput analysis. Immunodominant proteins from L. infantum were mapped in silico to predict B cell epitopes, and the 360 predicted peptides were synthesized on cellulose membranes. Immunoassays were used to select the most reactive peptides, which were then investigated with canine sera. Next, the 10 most reactive peptides were synthesized using solid phase peptide synthesis protocol and tested using ELISA. The sensitivity and specificity of these peptides were also compared to the EIE-LVC Bio-Manguinhos kit, which is recommended by the Brazilian Ministry of Health for use in leishmaniasis control programs. The sensitivity and specificity of the selected synthesized peptides was as high as 88.70% and 95.00%, respectively, whereas the EIE-LVC kit had a sensitivity of 13.08% and 100.00% of specificity. Although the tests based on synthetic peptides were able to diagnose up to 94.80% of asymptomatic dogs with leishmaniasis, the EIE-LVC kit failed to detect the disease in any of the infected asymptomatic dogs. Our study shows that ELISA using synthetic peptides is a technique with great potential for diagnosing CVL; furthermore, the use of these peptides in other diagnostic methodologies, such as immunochromatographic tests, could be beneficial to CVL control programs.

Current treatment for visceral leishmaniasis (VL) is associated with toxicity, a high cost, and the emergence of drug-resistant parasite strains. Moreover, no human vaccine is available. In this context, immunotherapeutics combining vaccination and chemotherapy have emerged as a promising alternative. In this study, we combined a recombinant chimeric protein (ChimT) with the adjuvant 3-O-desacyl–monophosphoryl lipid A (MPLA) and the antileishmanial drug miltefosine (Milt) and used it to treat Leishmania infantum-infected mice. Parasitological, immunological, and toxicological assays were performed at 1 and 30 days post treatment. The ChimT/MPLA/Milt combination was the most effective therapeutic regimen, inducing robust Th1-type cellular and humoral immune responses, as demonstrated by increased levels of IFN-γ, IL-12, and TNF-α cytokines, nitrite, and IgG2a antibodies. These responses were associated with significant reductions in parasite load across various organs. Furthermore, the treatment showed low renal and hepatic toxicity at both evaluation time points. By contrast, ChimT alone, ChimT/MPLA, and Milt induced lower immunological and parasitological responses when compared with the ChimT/MPLA/Milt group. The results observed at 1 and 30 days post treatment demonstrated the potential of ChimT/MPLA/Milt combination against VL.

Loxoscelism is a necrotic–hemolytic syndrome caused by bites of brown spiders belonging to the genus Loxosceles. Many approaches for the treatment of Loxosceles poisoning have already been proposed, among which administration of specific antivenom is thought to be the more specific. We have evaluated the use of peptides as immunogen to raise in rabbits an antibody response that could protect animals from a challenge by the Loxtox isoform LiD1, one of the main toxic component of Loxosceles intermedia venom. Six antigenic regions of LiD1 were mapped by using the SPOT method. The corresponding peptides were further chemically synthesized, mixed, and used as immunogens in rabbits. Control animal received recombinant LiD1 alone or together with peptides. We found that the rabbit antibody response to peptides was cross-reactive with LiD1, although only one peptide from the mix of six was immunogenic. The dermonecrotic, hemorrhagic and oedema forming activities induced by LiD1 in naïve rabbits were inhibited by 82%, 35% and 35% respectively, by preincubation of LiD1 with anti-peptide antibodies prepared from immunized rabbits. Animals that were immunized with peptides or LiD1r, were found to be protected from the dermonecrotic, hemorrhagic and oedema forming activities induced by a challenge with LiD1. The protection conferred by peptides was, however, lower than that provided by the peptide protein combination or by the full-length protein. These results encourage us in the utilization of synthetic peptides for therapeutic serum development or vaccination approaches.

AbstractLoxoscelism is a necrotic–hemolytic syndrome caused by bites of brown spiders belonging to the genus Loxosceles. Many approaches for the treatment of Loxosceles poisoning have already been proposed, among which administration of specific antivenom is thought to be the more specific. We have evaluated the use of peptides as immunogen to raise in rabbits an antibody response that could protect animals from a challenge by the Loxtox isoform LiD1, one of the main toxic component of Loxosceles intermedia venom. Six antigenic regions of LiD1 were mapped by using the SPOT method. The corresponding peptides were further chemically synthesized, mixed, and used as immunogens in rabbits. Control animal received recombinant LiD1 alone or together with peptides. We found that the rabbit antibody response to peptides was cross-reactive with LiD1, although only one peptide from the mix of six was immunogenic. The dermonecrotic, hemorrhagic and oedema forming activities induced by LiD1 in naïve rabbits were inhibited by 82%, 35% and 35% respectively, by preincubation of LiD1 with anti-peptide antibodies prepared from immunized rabbits. Animals that were immunized with peptides or LiD1r, were found to be protected from the dermonecrotic, hemorrhagic and oedema forming activities induced by a challenge with LiD1. The protection conferred by peptides was, however, lower than that provided by the peptide protein combination or by the full-length protein. These results encourage us in the utilization of synthetic peptides for therapeutic serum development or vaccination approaches.

Loxoscelism is a necrotic-hemolytic syndrome caused by bites of brown spiders belonging to the genusLoxosceles. Many approaches for the treatment ofLoxoscelespoisoning have already been proposed, among which administration of specific antivenom is thought to be the more specific. We have evaluated the use of peptides as immunogen to raise in rabbits an antibody response that could protect animals from a challenge by the Loxtox isoform LiD1, one of the main toxic component ofLoxosceles intermediavenom. Six antigenic regions of LiD1 were mapped by using the SPOT method. The corresponding peptides were further chemically synthesized, mixed, and used as immunogens in rabbits. Control animal received recombinant LiD1 alone or together with peptides. We found that the rabbit antibody response to peptides was cross-reactive with LiD1, although only one peptide from the mix of six was immunogenic. The dermonecrotic, hemorrhagic and oedema forming activities induced by LiD1 in naïve rabbits were inhibited by 82%, 35% and 35% respectively, by preincubation of LiD1 with anti-peptide antibodies prepared from immunized rabbits. Animals that were immunized with peptides or LiD1r, were found to be protected from the dermonecrotic, hemorrhagic and oedema forming activities induced by a challenge with LiD1. The protection conferred by peptides was, however, lower than that provided by the peptide protein combination or by the full-length protein. These results encourage us in the utilization of synthetic peptides for therapeutic serum development or vaccination approaches.