Cellular and humoral immune responses of healed cutaneous leishmaniasis and Mediterranean visceral leishmaniasis patients were evaluated against results for Leishmania major virulence proteins L. major protein disulfide isomerase (LmPDI) and mitogen-activated protein kinase kinase (MAPKK). Only MAPKK induces significant peripheral blood mononuclear cell proliferation with gamma interferon production as well as antibody responses. Thus, MAPKK may be of interest in Leishmania vaccination and serodiagnosis.The leishmaniases are diseases caused by vector-borne pathogens that represent a major public health problem affecting the lives of millions of people worldwide (http://www.who .int/whr/en). Depending on the parasite species and on the immunological response of the human host, leishmaniasis ranges from an asymptomatic infection to a self-limiting cutaneous lesion(s) or a fatal visceral form.No anti-Leishmania vaccine is available at the moment. Different studies showed that development of Th1-and Leishmania-specific cytotoxic immune responses correlate with healing of patients with cutaneous leishmaniasis (CL) (3,12). An intense effort is being made to identify antigens that could induce an immune state similar to that developed by individuals who recover from symptomatic infection and are resistant to a subsequent natural challenge. Such antigens may contribute to the development of an anti-Leishmania vaccine.Diagnostic tools targeting leishmaniasis are available. However, parasite detection is invasive and poorly sensitive. Serological diagnosis using various techniques based on detection of Leishmania-specific antibodies that are developed during acute disease are often more sensitive, less time-consuming, and more user friendly (4,11,15).In an attempt to identify new antigens to be used as vaccines or for serodiagnosis, we focused on Leishmania virulence factors. Indeed, several studies described these factors as potentially immunogenic in humans, mice, and, more recently, dogs (6,8,9,13). Here, cellular and humoral immune responses of healed CL (hCL) and Mediterranean visceral leishmaniasis (MVL) patients were evaluated against results for Leishmania major protein disulfide isomerase (LmPDI) and mitogen-activated protein kinase kinase (MAPKK), which we and others previously described as potential virulence factors (2, 10).We produced LmPDI (55 kDa) and MAPKK (40 kDa) in the prokaryotic expression pET system (Novagen, Gibbstown, NJ) and then purified them by affinity chromatography (Fig. 1). Lymphoproliferative responses (Fig. 2) and gamma interferon (IFN-␥) and interleukin-10 (IL-10) production (Fig. 3) induced by recombinant LmPDI and MAPKK (10 g/ml) were characterized in vitro using peripheral blood mononuclear cells (PBMC) from two groups. The first group consisted of 18 hCL patients (age range, 17 to 42 years; mean Ϯ standard deviation [SD], 28 Ϯ 10.9 years) living in an area of L. major infection endemicity (the governorate of Sidi Bouzid, Tunisia). Diagnosis of leishmaniasis was based on the presence o...