Leishmania donovani Nucleoside Hydrolase (NH36) Domains Induce T-Cell Cytokine Responses in Human Visceral Leishmaniasis

Micheli Luize Barbosa Santos, Dirlei Nico, Fabrícia Alvisi de Oliveira, Aline Silva Barreto, Iam Palatnik-de-Sousa, Eugenia Carrillo, Javier Moreno, Paula Mello de Luca, Alexandre Morrot, Daniela Santoro Rosa, Marcos Palatnik, Cristiane Bani-Corrêa, Roque Pacheco de Almeida, Clarisa Beatriz Palatnik-de-Sousa

Development of immunoprotection against visceral leishmaniasis (VL) focused on the identification of antigens capable of inducing a Th1 immune response. Alternatively, antigens targeting the CD8 and T-regulatory responses are also relevant in VL pathogenesis and worthy of being included in a preventive human vaccine. We assessed in active and cured patients and VL asymptomatic subjects the clinical signs and cytokine responses to the Leishmania donovani nucleoside hydrolase NH36 antigen and its N-(F1), central (F2) and C-terminal (F3) domains. As markers of VL resistance, the F2 induced the highest levels of IFN-γ, IL-1β, and TNF-α and, together with F1, the strongest secretion of IL-17, IL-6, and IL-10 in DTH(+) and cured subjects. F2 also promoted the highest frequencies of CD3(+)CD4(+)IL-2(+)TNF-α(-)IFN-γ(-), CD3(+)CD4(+)IL-2(+)TNF-α(+)IFN-γ(-), CD3(+)CD4(+)IL-2(+)TNF-α(-)IFN-γ(+), and CD3(+)CD4(+)IL-2(+)TNF-α(+)IFN-γ(+) T cells in cured and asymptomatic subjects. Consistent with this, the IFN-γ increase was correlated with decreased spleen (R = -0.428, P = 0.05) and liver sizes (R = -0.428, P = 0.05) and with increased hematocrit counts (R = 0.532, P = 0.015) in response to F1 domain, and with increased hematocrit (R = 0.512, P 0.02) and hemoglobin counts (R = 0.434, P = 0.05) in response to F2. Additionally, IL-17 increases were associated with decreased spleen and liver sizes in response to F1 (R = -0.595, P = 0.005) and F2 (R = -0.462, P = 0.04). Conversely, F1 and F3 increased the CD3(+)CD8(+)IL-2(+)TNF-α(-)IFN-γ(-), CD3(+)CD8(+)IL-2(+)TNF-α(+)IFN-γ(-), and CD3(+)CD8(+)IL-2(+)TNF-α(+)IFN-γ(+) T cell frequencies of VL patients correlated with increased spleen and liver sizes and decreased hemoglobin and hematocrit values. Therefore, cure and acquired resistance to VL correlate with the CD4(+)-Th1 and Th-17 T-cell responses to F2 and F1 domains. Clinical VL outcomes, by contrast, correlate with CD8(+) T-cell responses against F3 and F1, potentially involved in control of the early infection. The in silico-predicted NH36 epitopes are conserved and bind to many HL-DR and HLA and B allotypes. No human vaccine against Leishmania is available thus far. In this investigation, we identified the NH36 domains and epitopes that induce CD4(+) and CD8(+) T cell responses, which could be used to potentiate a human universal T-epitope vaccine against leishmaniasis.