Application of Rapid in Vitro Co-culture System of Macrophages and T-cell Subsets to Assess the Immunogenicity of Dogs Vaccinated with Live Attenuated Leishmania Donovani Centrin Deleted Parasites (LdCen-/-)

Background: Live 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.

Methods: Aimed 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 Discussion: Our 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.

Conclusion: The 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.