We examined immunity induced by subpatent blood-stage malaria (undetectable by microscopy)

We examined immunity induced by subpatent blood-stage malaria (undetectable by microscopy) using the rodent malaria parasite, malaria transmission is high, people eventually acquire nonsterile immunity, where low parasitemia may occur in the absence of clinical symptoms. studies show that malaria-specific effector and helper CD4+ T cells are deleted by apoptosis during contamination (12, 41, 42), and this may also be the mechanism in humans (39). We postulated that limited exposure to blood-stage malaria may allow the growth of helper and effector CD4+ T cells, which are normally eliminated during untreated contamination. Recently, we showed that repeated exposure of human volunteers to extremely low doses of induced immunity to a low-dose challenge with homologous parasites in the absence of detectable malaria-specific antibodies (31), consistent GSK1292263 with this hypothesis. For ethical reasons, we were unable to challenge human volunteers with realistic parasite doses or to challenge with heterologous parasite strains or variants to determine the specificity of this immunity. In the present study, we have used the rodent malaria model in a resistant mouse strain to clearly demonstrate that repeated subpatent contamination with blood-stage malaria, drug cured before parasites were detectable by microscopy, could induce effective immunity against high-dose challenge with homologous or heterologous parasites. Mice exposed to subpatent contamination lacked variant-specific antibodies but experienced antibodies to merozoite antigens and prominent cell-mediated immune responses, and this was associated with protection of CD4+ and CD8+ splenic lymphocytes from apoptosis that occurred during untreated patent parasitemia. MATERIALS AND METHODS Mice. Female C57BL/6j mice, 8 to 12 weeks aged, were obtained from the Animal Resources Centre (Willeton, Australia). Experiments were approved by the Institute Ethics Committee. Parasites. CB so that as had been given by Richard Carter, School of Edinburgh (4, 24, 36). Parasites had been cryopreserved in glycerolyte 57 (Baxter Health care Corp.). Parasitemias had been monitored by executing Giemsa-stained slim tail bloodstream smears. Infection process. Mice received three intravenous (i.v.) attacks with 105 parasitized crimson bloodstream cells (pRBC) of AS at 4-every week intervals to permit drug clearance. Parasites were derived from a single collection during maximum parasitemia in one mouse. For each subpatent illness, mice were drug cured 48 h after illness (0.2 mg of atovaquone and 0.08 mg of proguanil in 100 l of water by oral gavage daily for 4 days). Initial experiments confirmed that this protocol was highly effective in preventing the development of microscopically detectable parasitemia. Control patently infected mice self-cured. Naive mice were injected with phosphate-buffered saline (PBS) and received drug treatment at the same time as subpatently infected mice. All mice were drug treated 15 to 20 days before being subjected to challenging illness with 106 pRBC. ELISA. To prepare crude parasite antigen for enzyme-linked immunosorbent assay (ELISA), blood from mice infected with AS or CB (30 to 40% parasitemia) was collected, washed in PBS, and incubated with 0.01% (wt/vol) saponin (Aldrich) at 37C for 20 min. The pellet was washed in PBS, resuspended in 1.5 ml of PBS, and sonicated. MaxiSorp Nunc immuno plates, with 96 GSK1292263 wells (Nalge Nunc Int.), were GSK1292263 coated over night at 4C with 5 or 10 g of parasite antigen per ml in bicarbonate covering buffer (pH 9.6). The details of the assay have been explained previously (13). Staining variant antigens on the surface of pRBC. The staining process was based on previously explained methods (8, 37). Mice were kept inside a reverse light cycle (20:00 h to 08:00 h) for at least 1 week before an infection in order that late-stage parasites expressing variant surface area antigens could possibly GSK1292263 be collected each day. The mice had been contaminated from iced aliquots of AS (the same stabilate utilized through the subpatent an infection process) or CB. At 10 to 20% parasitemia, the mice were sacrificed at 10:00 blood and h was collected. After two washes, bloodstream was resuspended at 5% hematocrit in RMPI-HEPES supplemented with 0.2% (wt/vol) NaHCO3 and 10% fetal leg serum (FCS) and cultured in 37C for three to four 4 h in 5% CO2-5% O2 until past due trophozoites were evident. After three washes in PBS-1% FCS, a 100-l level of cells (0.2% Elf3 hematocrit) was stained utilizing a three-step method and GSK1292263 sequentially incubated with check serum (1/10 dilution), goat anti-mouse immunoglobulin G (IgG) (1/50 dilution; Caltag), and fluorescein isothiocyanate (FITC)-conjugated swine anti-goat IgG (1/20 dilution; Caltag) plus ethidium bromide (20 g/ml). Incubations had been completed for 30 min at area heat range, and cells had been cleaned in PBS-1% FCS between each stage. Fluorescence was assessed on the FACSCalibur (BD), and data had been examined using CellQuest software program (BD). Identification.