Abstract
BackgroundLive attenuated Leishmania donovani parasites as LdCen−/− were shown to confer protective immunity against Leishmania infection in mice, hamsters, and dogs. Strong immunogenicity in dogs vaccinated with LdCen−/− has been previously reported, including increased antibody response favoring Th1 response lymphoproliferative responses, CD4+ and CD8+ T-cells activation, increased levels of Th1 and reduction of Th2 cytokines, in addition to a significant reduction in parasite burden after 18 and 24 months post virulent parasite challenge.MethodsAimed at validating a new method using in vitro co-culture systems with macrophages and purified CD4+ or CD8+ or CD4+:CD8+ T-cells of immunized dogs with both LdCen−/− and Leishmune® to assess microbicide capacity of macrophages and the immune response profile as the production of IFN-γ, TNF-α, IL-12, IL-4 and IL-10 cytokines.Results and discussionOur data showed co-cultures of macrophages and purified T-cells from dogs immunized with LdCen−/− and challenged with L. infantum were able to identify high microbicidal activity, especially in the co-culture using CD4+ T-cells, as compared to the Leishmune® group. Similarly, co-cultures with CD8+ T-cells or CD4+:CD8+ T-cells in both experimental groups were able to detect a reduction in the parasite burden in L. infantum infected macrophages. Moreover, co-cultures using CD4+ or CD8+ or CD4+:CD8+ T-cells from immunized dogs with both LdCen−/− and Leishmune® were able to identify higher levels of IFN-γ and IL-12 cytokines, reduced levels of IL-4 and IL-10, and a higher IFN-γ/IL-10 ratio. While the highest IFN-γ levels and IFN-γ/IL-10 ratio were the hallmarks of LdCen−/− group in the co-culture using CD4+ T-cells, resulting in strong reduction of parasitism, the Leishmune® immunization presented a differential production of TNF-α in the co-culture using CD4+:CD8+ T-cells.ConclusionThe distinct conditions of co-culture systems were validated and able to detect the induction of immune protection. The method described in this study applied a new, more accurate approach and was able to yield laboratory parameters useful to test and monitor the immunogenicity and efficacy of Leishmania vaccines in dogs.
Highlights
Live attenuated Leishmania donovani parasites as LdCen−/− were shown to confer protective immunity against Leishmania infection in mice, hamsters, and dogs
Researchers need to develop methodological strategies that enable a more rapid and accurate evaluation of the dog’s immune system. Such tests could be included in clinical trials of vaccines against canine visceral leishmaniasis (CVL), so that the time needed for the experiments could be reduced
The results suggest that this in vitro co-culture system allows rapid efficacy analysis of vaccines against canine visceral leishmaniasis
Summary
Live attenuated Leishmania donovani parasites as LdCen−/− were shown to confer protective immunity against Leishmania infection in mice, hamsters, and dogs. The impact of controlling the infected dog population to reduce human visceral leishmaniasis prevalence in these endemic areas has been debated [2,3,4]. Researchers need to develop methodological strategies that enable a more rapid and accurate evaluation of the dog’s immune system. Such tests could be included in clinical trials of vaccines against CVL, so that the time needed for the experiments could be reduced.
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