Avaliação do potencial imunoterapêutico e imunoprofilático de formulações contendo antígenos de Leishmania infantum associados a antígenos de Lutzomyia longipalpis contra a leishmaniose visceral canina

Abstract

Visceral leishmaniasis is a severe disease caused by protozoa of the Leishmania genus. Dogs are known to be the domestic reservoirs of the parasite, becoming infected during the blood meal of female sandflies from the Lutzomyia and Phlebotomus genera. In Brazil, the species Leishmania infantum (synonym Leishmania chagasi) is primarily responsible for causing canine visceral leishmaniasis (CVL). Currently, the control and treatment of the disease present several challenges. Given these limitations, the development of new, effective alternatives for the treatment and prophylaxis of CVL is of utmost importance. Immunotherapy, as well as vaccination, are relevant strategies. The aim of this study was to evaluate the therapeutic and prophylactic potential of formulations composed of antigens from L. infantum and Lutzomyia longipalpis. To this end, clinical trials were conducted on dogs naturally infected with L. infantum, and preclinical trials were performed using hamsters and mice. The dogs and hamsters were used in immunotherapy trials, while mice were evaluated in preclinical vaccine trials. In the clinical trial with dogs, the immunotherapeutic formulation included the L22.4_CHP protein combined with the vector antigen and the adjuvant saponin. The immunotherapy regimen consisted of six doses of the formulation, administered subcutaneously, with a 15-day interval between each dose. The dogs received 100μg of the protein associated with 2x10⁻¹¹ TU of phage and 300μg of saponin per dose. In the preclinical immunotherapy trial, the therapeutic regimen was similar to the clinical one, but the doses consisted of 50μg of recombinant P1 or P3 proteins, associated with 50μg of saponin or Montanide at 60% of the dose. For this trial, the hamsters were infected with 10⁷ L. infantum promastigotes in the stationary phase. Treatment began two months after infection, and euthanasia occurred 30 days after treatment completion. Regarding the vaccination protocol, the formulations used were the same as those used in the preclinical immunotherapy protocol. The vaccination schedule consisted of 3 doses, administered subcutaneously, with a 15-day interval between doses. Half of the animals were euthanized at the end of the protocol, and the other half was infected with 10⁷ L. infantum promastigotes in the stationary phase, 15 days later. Euthanasia for these animals occurred 30 days post-infection. The main results obtained showed that in all the models evaluated, the immunobiologics used were safe, not causing alterations in hematological and biochemical parameters, and not presenting any severe adverse effects. Furthermore, they exhibited high antigenicity, producing elevated levels of total IgG against the antigens of the tested formulations as well as against L. infantum antigens. In the vaccination protocol, an increase in CD4+ and CD8+ T cells was also observed, with a concurrent induction of IgG1 and IgG2a, indicating a mixed type 1 and 2 immune response. These results corroborated the parasitological findings, which demonstrated a significant reduction in parasitic load in the vaccinated mice group treated with the P3 protein associated with saponin adjuvant. In dogs, the immunotherapeutic treatment was able to improve clinical signs, with a reduction in parasitic load to nearly undetectable levels, while in hamsters, the treatment showed a trend toward decreased parasitic load in the spleen and liver (especially for the P1 protein associated with saponin or Montanide adjuvant). Regarding the inhibition of L. infantum binding to Lulo cells, a trend toward reduced parasite binding was observed in the immunotherapeutic formulation tested in dogs, as well as in the vaccine study in mice for the P1SAP, P1MONT, and P3MONT formulations. Therefore, it was concluded that collectively, the data indicate that the evaluated immunobiologics have potential as both a vaccine and immunotherapy for visceral leishmaniasis.