Identification of new antigens in visceral leishmaniasis by expression cloning and immunoblotting with sera of kala-azar patients from Bihar, India

Abstract

Sera of kala-azar patients from Bihar, India, were used to identify Leishmania donovani antigens encoded by a phage expression library. Ten antigens were identified, five of which have not been described as leishmania antigens before. The antigens specifically react with sera of leishmania-infected patients but not of toxoplasma- or plasmodium-infected patients.

FIG. 1.

Analysis of the frequencies of seropositivity in kala-azar patients from the Kala-Azar Medical Research Center in Muzaffarpur, Bihar, India. The recombinant λ phages for the 10 antigens identified in this study were spotted onto E. coli layers according to the scheme in panel A. After the induction of antigen expression, the antigens were blotted onto nitrocellulose and probed with the sera for antibodies with the corresponding specificities. (A) Examples of the nitrocellulose filters. Replicas of these filters were prepared for and probed with the sera of the patients and healthy controls. The upper filter shown is for patient P14, the lower for patient P15. The charts give the positions of the antigens on the filter. Serodetected antigens are highlighted. For HASPB1, LDHSP70, and ARP-1, two independently identified phage clones were employed. WT, wild-type phages included as negative control; C01 through C03, phages with undefined polyclonal cDNA inserts. (B) Frequencies of seropositivity for the indicated antigens in seroreactive patients. (C) Seroreactivity of the Indian kala-azar patient sera against the identified antigens. Black fields indicate that the patient is seropositive for the corresponding antigen. Only the results for patients who are seropositive for at least 1 of the 10 antigens are shown. Fifteen patients, the 10 healthy relatives of patients, and 11 outgroup individuals (1 from India and 10 from Germany), were seronegative for the antigens.

FIG. 2.

Serological responses of antisera from healthy donors and patients infected with Toxoplasma or Plasmodium to the 10 identified Leishmania donovani antigens. (A) The proteins of Leishmania donovani AG83 lysate were separated by SDS-PAGE and probed with eight sera each of Plasmodium-infected patients (PM01 through PM06, PM08, and PM09) or Toxoplasma-infected patients (TX01 through TX08) by using a serum dilution of 1:60. (B through D) Lysates of wild-type E. coli (WT) or E. coli overexpressing the antigens were probed with nine control sera from healthy relatives of VL patients (H01 and H03 through H10) and one serum from a healthy donor from a nonendemic area in India (H11) (panel B), sera of Toxoplasma-infected patients (panel C) or sera of Plasmodium-infected patient (panel D), as well as two kala-azar patients (P14 and P15) in all panels B through D at dilutions of 1:60. The antigens were expressed from full-length expression clones amplified from Leishmania donovani DNA. Exceptions were EF1B and a second expression clone of LDHSP70 that were expressed from the original phage expression clone insert (both are in italics). The lysates of the bacteria that overexpress the antigens were colored with bromophenol blue and blotted as stripes onto nitrocellulose. The sera were incubated with these blots in stripes perpendicular to the antigen stripes resulting in the grid pattern in panels C and D. The light blue horizontal stripes indicate the distribution of the antigens, the dark purple stripes the Western blot stain of Nitro Blue Tetrazolium-BCIP (5-bromo-4-chloro-3-indolylphosphate).