Evaluation of ex vivo human immune response against candidate antigens for a visceral leishmaniasis vaccine

Abstract

People cured from visceral leishmaniasis (VL) develop protection mediated by Th1-type cellular responses against new infections. We evaluated cytokine responses against 6 defined candidate vaccine antigens in 15 cured VL subjects and 5 healthy endemic controls with no evidence of previous exposure to Leishmania parasites. Of the 6 cytokines examined, only interferon-gamma (IFN-gamma) differentiated cured VL patients from non-exposed individuals, with cured patients mounting a significantly higher IFN-gamma response to a crude parasite antigen preparation. Among candidate vaccine antigens tested, the largest number of cured subjects recognized cysteine proteinase B, leading to heightened IFN-gamma responses, followed by sterol 24-c-methyltransferase. These two antigens were the most immunogenic and protective antigens in a murine VL model, indicating a relationship between T cell recall responses of humans cured from VL and protective efficacy in an experimental model. Further studies may help prioritize antigens for clinical development of a subunit vaccine against VL.

Figure 1.

Phytohaemagglutinin (PHA)-induced production of Th1/Th2 cytokines in cured and non-exposed (NE) subjects. Cytokine production by peripheral blood in response to either phosphate-buffered saline (PBS) or PHA was examined in cured and NE subjects by whole blood assay (WBA)/cytometric bead assay (CBA). Cytokine concentration values of PHA-stimulated sample (minus the individual's non-stimulated value) are shown. Bars represent median values. Dotted lines represent the assay detection limit (20 pg/mL).

Figure 2.

Soluble Leishmania antigen (SLA)-specific Th1/Th2 cytokine production in cured and non-exposed (NE) subjects. Cytokine production by peripheral blood in response to either phosphate-buffered saline (PBS) or SLA was examined in cured and NE subjects by whole blood assay (WBA)/cytometric bead assay (CBA). Cytokine concentration values of SLA-stimulated sample (minus the individual's non-stimulated value) are shown. Bars represent median values. Dotted lines represent the assay detection limit (20 pg/mL).

Figure 3.

Immunogenicity and protective efficacy of vaccine candidate antigens in mice. (A) Spleen cells from mice inoculated with an antigen plus an adjuvant were stimulated in vitro with medium alone, con A, or the antigen. Culture supernatants were collected after 72 hrs stimulation and the levels of IFN-γ in the supernatants were measured by sandwich enzyme-linked immunosorbent assay (ELISA). Mean and SEM of three mice in each group are shown. (B) Mice, either immunized or unimmunized, were challenged with 5 × 10⁶ promastigotes of Leishmania infantum and the numbers of parasites in the liver were measured by limiting dilutions 4 weeks after the infection. Reductions in parasite numbers compared with the unimmunized control are shown here with mean values and SEM of at least three independent experiments for individual antigens.