Mononuclear cells from patients recovered from cutaneous leishmaniasis respond to Leishmania major amastigote class I nuclease with a predominant Th1-like response

Abstract

The Leishmania major amastigote class I nuclease (LmaCIN) is a developmentally regulated protein that is highly expressed in the amastigote stage of L. major. This protein is homologous to the P4 nuclease of L. pifanoi, which has been shown to induce protective immune response in a murine model. To evaluate LmaCIN as a potential human vaccine candidate, cellular immune responses to recombinant LmaCIN were examined in individuals recovered from Old World cutaneous leishmaniasis. Peripheral blood mononuclear cells (PBMC) from patients recovered from L. major infection were cultured either with recombinant LmaCIN or autoclaved L. major (ALM) as control. rLmaCIN induced significant proliferation of PBMC from 90% of recovered patients. Phenotypic analysis of proliferating cells showed that CD8(+) cells were the predominant cell type proliferating in response to rLmaC1N. Screening of culture supernatants for cytokines showed that rLmaCIN induced high levels of interferon (IFN)-gamma (mean +/- s.e.m.: 1398 +/- 179 pg/ml) associated with little interleukin (IL)-10 and little or no IL-5 production. These findings show that LmaCIN is immunogenic in humans during L. major infection and that it can elicit immunological responses relevant to immunoprophylaxis of leishmaniasis.

Fig. 1

Expression and purification of rLmaCIN. E. coli (BL21/DE3) transformed with the expression vector PET 22b harbouring LmaCIN gene was grown and induced with IPTG. Lysates of non-induced (lane 1) and induced (lane 2) cultures of E. coli and recombinant LmaCIN protein purified by Ni-NTA affinity chromatography (lane 3) were separated by SDS-PAGE and stained with Coomassie blue.

Fig. 2

Proliferative responses of PBMC from individuals with a history of cutaneous leishmaniasis (R) and healthy controls (H) to recombinant LmaCIN, in comparison to ALM and PHA. Proliferative responses were determined by incubating 2 × 10⁵ cells for 3 days with either rLmaCIN (5 µg/ml), ALM (25 µg/ml) or PHA (15 µg/ml). Cultures were pulsed with [³H]thymidine for the last 18 h. The results shown are the mean of triplicate measurements at each data point and the s.d. was less than 10% of the mean.

Fig. 3

Phenotype of proliferating T cells in response to rLmaCIN (a), and ALM (b) in cultures of peripheral blood mononuclear cells from recovered cases. PBMC (1 × 10⁶ cell/ml) were cultured with recombinant LmaCIN or ALM for 5 days and stained with monoclonal antibody for CD3, CD4 and CD8 markers prior to analysis by flow cytometry. The results shown are the mean of triplicate measurements at each data point and the s.d. was less than 10% of the mean.

Fig. 4

Levels of IFN-γ (a), IL-10 (b) and IL-5 (c) in culture supernatants of PBMC from recovered individuals in response to recombinant LmaCIN and ALM antigens. Cytokine production was determined by incubating 1 × 10⁶ cell/ml with rLmaCIN (5 µg/ml) or ALM (25 µg/ml) for 5 days by ELISA. The results shown are the mean of triplicate determinations at each data point and the s.d. was less than 10% of the mean.