Changes in antigen-specific cytokine and chemokine responses to Plasmodium falciparum antigens in a highland area of Kenya after a prolonged absence of malaria exposure.

Individuals naturally exposed to Plasmodium falciparum lose clinical immunity after a prolonged lack of exposure. P. falciparum antigen-specific cytokine responses have been associated with protection from clinical malaria, but the longevity of P. falciparum antigen-specific cytokine responses in the absence of exposure is not well characterized. A highland area of Kenya with low and unstable malaria transmission provided an opportunity to study this question. The levels of antigen-specific cytokines and chemokines associated in previous studies with protection from clinical malaria (gamma interferon [IFN-γ], interleukin-10 [IL-10], and tumor necrosis factor alpha [TNF-α]), with increased risk of clinical malaria (IL-6), or with pathogenesis of severe disease in malaria (IL-5 and RANTES) were assessed by cytometric bead assay in April 2008, October 2008, and April 2009 in 100 children and adults. During the 1-year study period, none had an episode of clinical P. falciparum malaria. Two patterns of cytokine responses emerged, with some variation by antigen: a decrease at 6 months (IFN-γ and IL-5) or at both 6 and 12 months (IL-10 and TNF-α) or no change over time (IL-6 and RANTES). These findings document that P. falciparum antigen-specific cytokine responses associated in prior studies with protection from malaria (IFN-γ, TNF-α, and IL-10) decrease significantly in the absence of P. falciparum exposure, whereas those associated with increased risk of malaria (IL-6) do not. The study findings provide a strong rationale for future studies of antigen-specific IFN-γ, TNF-α, and IL-10 responses as biomarkers of increased population-level susceptibility to malaria after prolonged lack of P. falciparum exposure.

Standardization and validation of a cytometric bead assay to assess antibodies to multiple Plasmodium falciparum recombinant antigens.

BACKGROUND: Multiplex cytometric bead assay (CBA) have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA. METHODS: Antibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared. RESULTS: Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from <0.0001 - 0.004), and antibodies to variants of the same antigen were accurately distinguished within a multiplex reaction. Plasma dilutions of 1:100 or 1:200 were optimal for all antigens for CBA testing. Plasma diluted in a buffer containing 0.05% sodium azide, 0.5% polyvinylalcohol, and 0.8% polyvinylpyrrolidone had the lowest background activity. CBA median fluorescence intensity (MFI) values with 1,000 antigen-conjugated beads/well did not differ significantly from MFI with 5,000 beads/well. CBA and ELISA results correlated well for all antigens except apical membrane antigen-1 (AMA-1). CBA testing produced a greater range of values in samples from malaria endemic areas and less background reactivity for blank samples than ELISA. CONCLUSION: With optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in positive samples and lower background readings for blank samples than ELISA.

The Plasmodium falciparum Antigen MB2 Induces Interferon-γ and Interleukin-10 Responses in Adults in Malaria Endemic Areas of Western Kenya.

BACKGROUND: MB2 is a novel Plasmodium falciparum antigen of unknown function expressed in pre-erythrocytic and blood stages of infection in the human host. Interferon-gamma (IFN-γ) and interleukin (IL)-10 responses to other P. falciparum antigens have been associated with protection from clinical malaria, but these responses have not been studied for MB2. The present study was undertaken to characterize IFN-γ and IL-10 responses to P. falciparum MB2 antigen in adults living in areas of differing malaria transmission in Western Kenya. MATERIALS AND METHODS: Cytokine responses to two 9-mer MB2 peptides predicted to be human leukocyte antigen (HLA) class I restricted T-cell epitopes were measured by enzyme-linked immunosorbent assay (ELISA) (IFN-γ and IL-10) and enzyme-linked immunosorbent spot (ELISPOT) (IFN-γ) in adults (n = 228) in areas of unstable and stable malaria transmission. HLA class I restriction of responses was assessed in a sub-group of the study population. RESULTS: IFN-γ and IL-10 responses to MB2 peptides by ELISA were observed in both sites with no significant difference in prevalence (IFN-γ, unstable transmission area, 18.8%, stable transmission area, 27.5%, P = 0.33; IL-10, unstable transmission area, 22.5%, stable transmission area, 25.0%, P = 0.78). Prevalence of IFN-γ responses by ELISPOT was also similar in both areas (unstable, 10.8%, stable, 10.9%, P = 0.98). Neither IFN-γ nor IL-10 responses showed evidence of HLA class I restriction. CONCLUSIONS: MB2 induces IFN-γ and IL-10 responses in adults living in both stable and unstable malaria transmission areas. Future studies should assess if these responses are associated with protection from clinical malaria.