Vacunación y diagnóstico de la leishmaniosis visceral mediante proteínas recombinantes de Leishmania infantum producidas en larvas de insecto

Student thesis: Doctoral thesis


Visceral leishmaniosis is a severe parasitic disease that is usually fatal if left untreated, which has an incidence of more than 500 000 new human cases each year. In zoonotic visceral leishmaniasis (ZVL) caused by Leishmania infantum, dogs are the main domestic reservoirs of the parasite. Identification of the antigens and components involved in Lesihmania-specific immune responses to improve diagnosis and vaccination is a research priority.
Our objective has been to evaluate the utility of four evolutionarily conserved antigens of L. infantum KMPII, TRYP, LACK and papLe22 in ZVL control by using them in diagnostic and vaccine strategies. We used baculovirus-infected Trichoplusia ni larvae to obtain the raw protein extracts containing the recombinant Leishmania proteins. This is a eukaryotic system and generally allows the production of high quality proteins processed with most of the post-translational modifications.
Recombinant KMPII was the most recognized antigen by dogs with clinical signs of leishmaniosis (CanL), with a 75% of seroprevalence. Seroprevalence against rTRYP and rLACK in dogs with CanL, was of 51% and 42%, respectively. The results demonstrate that KMPII, TRYP, and rLACK antigens are B-cell immunogens during CanL. When the three recombinant antigen-based ELISA techniques were evaluated in parallel, almost perfect agreement with CTLA-based ELISA was observed, with a specificity of 97% and a sensitivity of 93% in relation to CTLA-based ELISA, offering a sensitive, specific, reproducible and inexpensive diagnostic tool.
rTRYP induced the highest number of positive DTH responses (50%) in infected dogs from an endemic area, suggesting its role as a T-cell immunogen during natural infection. In contrast, rKMPII (27%), rLACK (18%), and rpapLe22 (18%) induced a lower number of reactions, thus proving to be weak T-cell immunogens. When TRYP-DTH and KMPII-DTH tests were evaluated in parallel, 78% of LST-positive dogs were detected, not differing significantly from LST sensitivity. Our results suggest that TRYP antigen could be a promising vaccine candidate against CanL, and that both rTRYP and rKMPII could be considered as components of a standardized DTH immunodiagnostic tool for infected dogs without clinical signs.
DNA-Protein vaccination in hamsters carrying the four antigens stimulated significant production of NO by macrophages before challenge and showed a decrease in the specific humoral response against the parasite upon in vivo challenge. A significant reduction in parasite loads was observed at 20 weeks post-infection, both in spleen (86 %) and in blood (99 %) when compared with controls. Moreover, this vaccine prevented certain histopathological alterations that have been related to disease. DNA-Protein vaccination enhanced the immunogenicity and protection achieved by the DNA vaccine, which could neither stimulate NO production nor reduce parasitaemia. Recombinant protein vaccine was not protective. The results suggest that a prime-boost strategy with DNA and T. ni-derived rKMPII, rTRYP, rLACK, and rpapLe22 proteins from L. infantum could be a safe, effective and low-cost measure for the control of ZVL, better than strategies based on DNA or protein alone with the same antigens.
Date of Award29 Oct 2009
Original languageSpanish
Supervisor Alheli Rodriguez Cortes (Director) & Jordi Alberola Domingo (Director)

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