Human T-cell activation by 14- and 18-kilodalton nuclear proteins of Leishmania infantum

Abstract

Leishmanial antigens which stimulate T lymphocytes from primed individuals may be candidates for a vaccine. We recently found a significant concordance between the humoral response specific for two proteins from Leishmania infantum promastigotes, p14 and p18, and a positive leishmanin delayed-type hypersensitivity reaction, testifying to the occurrence of cell-mediated immunity. In this communication, we describe a partial characterization of these antigens and an in vitro analysis of their capacity to activate primed human T cells. We showed, by immunofluorescent staining and through analysis of subcellular fractions by Western immunoblotting, that in stationary-phase promastigotes, p14 and p18 were located only in the parasite nuclei; in the middle of the log phase, a transitory and only weak expression outside the nucleus was detected. We then showed that p14 and p18 antigens shared a common epitope(s). Finally, we analyzed the in vitro proliferation and interleukin-2 production induced by leishmanial proteins in human peripheral blood mononuclear cells from sensitized subjects. We showed that in some individuals who have been exposed to L. infantum the specific response to the whole lysate was mostly due to the nuclear antigens. We demonstrated directly the capacity of nitrocellulose-bound p14 and p18 to activate in vitro all of the tested primed peripheral blood mononuclear cells, which contrasted with a lack of stimulatory activity of other membrane-bound leishmanial proteins. Taken together, our results suggest that an antigenic determinant(s) dominant for some individuals might exist on both antigens.

Short- and Long-Term Efficacy of Hexadecylphosphocholine against EstablishedLeishmania infantumInfection in BALB/c Mice

“…L. infantum MON1 (MHOM/FR/94/LPN101), isolated from a patient with VL, was maintained by serial passages in Syrian hamsters. The promastigote form was cultured under usual conditions (38,42), and after three in vitro passages, stationary-phase cells from 7-day-old cultures were used at a concentration of 2.5 ϫ 10 7 promastigotes/ml to infect BALB/c mice. For infection the parasites were washed twice by sedimentation at 2,500 ϫ g at 4°C for 15 min each time, resuspended in 0.9% NaCl at 2 ϫ 10 8 cells/ml, and injected into caudal vein at 10 8 promastigotes/mouse.…”

Occurrence ofLeishmania infantumParasitemia in Asymptomatic Blood Donors Living in an Area of Endemicity in Southern France

“…Blood from 565 donations was screened over a period of 12 months (April 1996 to March 1997). On the day of donation, sera were analyzed by Western blotting, and the buffy coats corresponding to the sera which revealed the typical 14-and/or 18-kDa leishmanial bands (21,33) were further examined. Three aliquots (3 ml each) of the 1-day-old (stored at 4°C) buffy coats were stored at Ϫ20°C in EDTA containing vacutainer tubes (Becton Dickinson, Meylan, France) until DNA extraction and amplification by PCR.…”

“…Western blot analysis. The antigen preparations were electrophoresed on sodium dodecyl sulfate-14% polyacrylamide gel and transferred and immunodetected with human sera as described previously (33).…”

“…screening. The bands at 14 and/or 18 kDa, indicative of a previous asymptomatic infection with L. infantum(14,21,33), were revealed by the sera from 76 donors(13.4%) to Monaco Blood Bank. Antibodies detecting both bands were found in 25 serum samples, and antibodies detecting one band at 14 or 18 kDa were found in 20 or 31 serum samples, respectively.Figure 1 shows the characteristic Western blot profiles obtained with the sera of 16 donors found to be Leishmania positive.…”

Progression of visceral leishmaniasis due to Leishmania infantum in BALB/c mice is markedly slowed by prior infection with Trichinella spiralis

“…L. infantum MON1 (MHOM/FR/94/LPN101) was maintained by serial passages in Syrian hamsters. The promastigote form was cultured under standard conditions (39) and used for infection after three in vitro passages as stationary-phase cells from 7-day-old cultures (2.5 ϫ 10 7 promastigotes/ml). After two washings (15-min sedimentation at 2,500 ϫ g and 4°C), promastigotes were resuspended in 0.9% NaCl at 2 ϫ 10 8 cells/ml and injected into the caudal vein at 10 8 promastigotes/mouse.…”