Studies on Anti-Leishmania Activity of Immune Rabbit Serum

Abstract

The leishmanial growth-inhibitory substance of immune rabbit serum was analyzed by DEAE-cellulose chromatography and immunoelectrophoresis. This inhibitory activity as well as the agglutinating and complement-fixing antibodies was found to be in the gamma G fraction of immunoglobulins. The antigen responsible for eliciting this anti-leishmanial activity of immune rabbit serum was located in the aqueous insoluble fraction of leishmanial cells. The immune serum had no effect on the course of leishmanial infection in infected mice. In a previous communication (Rezai, Sher, and Gettner, 1969), we showed that after immunization of rabbits with Leishmania tropica, an antibodylike substance is produced in the blood which inhibits the growth of L. tropica, L. enriettii, and L. donovani in vitro. This finding was in contrast to Adler (1964) who found that immune serum did not inhibit the growth of Leishmania. The growth-inhibitory substance could not be adsorbed with the specific antigen, i.e., the leptomonad form of Leishmania, and was not complement-dependent. As both of these characteristics are unusual for a conventional inhibitory antibody, it was one of the purposes of this investigation to characterize further the inhibitory factor of immune rabbit serum. In addition, we have attempted to partially characterize the eliciting immunogen responsible for the production of the inhibitory factor. After finding a leishmanial inhibitory substance in the serum of artificially immunized rabbits, the effect of this factor on the course of infection of L. tropica in mice was also investigated. MATERIALS AND METHODS Preparation of immune serum Rabbits were injected intravenously 8 times with 0.5 ml live Leishmania suspension containing 2 X 107 L. tropica. This serum, with an approximate complement-fixation titer of 1:2,560, was employed for the serum characterization and in vivo studies. Evaluation of inhibitory activity The growth-inhibitory activity was tested in 5 dilutions of the serum using L. tropica as the test Received for publication 29 July 1969. * This research was partially supported by the WHO Grant L3/181/8. organism. For this purpose, a 1-ml volume of diluted serum was used as the liquid phase in the modified NNN medium (Lemma and Schiller, 1964) in screw-capped tubes. Tubes containing 1 ml of saline as the liquid phase served as controls. Each tube was inoculated with 0.06 ml of a suspension of L. tropica containing 2 X 106 organisms and incubated at 26 C. Five days after inoculation, an aliquot from each tube was examined microscopically for leishmanial growth. Each tube was rated according to its content of motile, morphologically normal leptomonad forms of Leishmania in comparison to growth in the control tubes. Fractionation and column chromatography of immune rabbit serum The globulin fraction of immune rabbit serum (10 ml) was precipitated by the addition of 50% NH4SO4 at 0 C. Following dialysis against 0.01 molar pH 8.0 phosphate buffer, the globulin fraction was chromatographed on a DEAE-cellulose column prepared according to the method of Peterson and Sober (1956). The cellulose column (30 by 2 cm) was packed by gravity flow and the gradient elution technique of Fahey and coworkers (1958) for separation of serum proteins was employed. Column elution was carried out at approximately 5 C using 0.01 to 0.3 molar pH 8.0 phosphate buffer. The flow rate was approximately 60 ml per hour. Five-ml aliquots were collected and absorbencies were determined at 280 mt, in a Beckman DU spectrophotometer. Preparation of leishmanial immunogens Four-day cultures of L. tropica were harvested and centrifuged. Following a saline wash, the packed cells were dispersed in 4 volumes of 0.85% NH4C1 and were allowed to stand at room temperature for 20 min to remove the red blood cells. The organisms were washed twice, resuspended in 20 volumes of saline, and disrupted by 10 successive freeze-thaw cycles. Disruption of the cells was verified by microscopic examination. After centrifugation at 37,000 g for 20 min, the supernatant was removed and used as the soluble antigen. The sediment was washed with saline 6 times and centrifuged each time at 37,000 g for 20 min. The resulting pellet was resuspended in 10 volumes of saline and used as the insoluble antigen.