Scientific Publications by FDA Staff

Parasit Vectors 2016 Apr 30;9(1):250

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-/-).

Viana KF, Fiuza JA, Gannavaram S, Dey R, Selvapandiyan A, Bartholomeu DC, Silveira-Lemos DD, Bueno LL, Dutra WO, Fujiwara RT, Nakhasi HL, Giunchetti RC

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-¿, TNF-a, 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-¿ and IL-12 cytokines, reduced levels of IL-4 and IL-10, and a higher IFN-¿/IL-10 ratio. While the highest IFN-¿ levels and IFN-¿/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-a 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.