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

Abstract

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.

INTRODUCTION

Malaria due to Plasmodium falciparum is a major public health concern in tropical areas, causing more than 200 million episodes of febrile illness and 655,000 deaths (1). P. falciparum-specific cytokine responses have been associated with protection against clinical malaria, including gamma interferon (IFN-γ) responses to the vaccine candidate antigens circumsporozoite protein (CSP) (2), liver-stage antigen 1 (LSA-1) (3–5), thrombospondin-related adhesive protein (TRAP) (3, 6, 7), apical membrane antigen (AMA)-1 (8, 9) and merozoite surface protein 1 (MSP-1) (10), and interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-α) responses to LSA-1 (11, 12). Conversely, some P. falciparum-specific cytokine responses, such as IL-6 responses to P. falciparum-parasitized red blood cells (pRBC), are associated with an increased risk of clinical malaria (13). Serum levels of other cytokines, including IL-5, and the chemokine RANTES, appear to be important in the pathogenesis of severe malaria (13–15).

Clinical immunity to malaria appears to wane quickly in the absence of active malaria transmission (16, 17), but the reasons for this loss of clinical immunity are not well characterized. One potential reason for loss of clinical immunity to malaria is the loss of cellular immune responses associated with protection against malaria in the absence of repeated exposure. A highland study site setting in Kenya in which we have conducted studies for the last decade provided a unique opportunity to assess how cellular immune responses to P. falciparum infection change in the absence of malaria transmission. From 2007 to 2008, there was a 14-month period of interrupted transmission in this area (18), and subsequently there has been very low-level seasonal transmission at the site. We conducted a longitudinal cohort study from April 2008 to April 2009, a period of very low malaria incidence (<1 case per 1,000 persons/month), to assess the effect of absent or minimal P. falciparum exposure for a 1-year period on P. falciparum antigen-specific cytokine responses that may be important to protection against uncomplicated or severe malaria.

MATERIALS AND METHODS

Study area and participants.

The study was conducted in two adjacent highland locations, Kipsamoite and Kapsisiywa, located in Nandi County in the highland areas of Kenya, with a total population of approximately 8,100 persons in 2008. The area is characterized by low and unstable malaria transmission with seasonal peaks typically occurring from May to August, although aberrations in the months of peak incidence can occur when unusual weather events such as an El Niño southern oscillation occur (19, 20). The estimated entomological inoculation rate for this area is <1 infectious bites/person/year (21). The main vector of malaria is Anopheles gambiae. For the present study, a cohort of 300 persons were randomly selected from the community and actively monitored for clinical malaria starting in August 2007. Venous blood samples were obtained from the cohort in April 2008, October 2008 and April 2009. From this cohort, samples from individuals who had sufficient peripheral blood mononuclear cells (PBMCs) for testing at each time point were randomly selected for the present study of 100 individuals. Hemoglobin values were tested for all study participants at each time point by photometry using the Hemo Control photometer (EKF Diagnostics, Magdeburg, Germany). All individuals in the cohort were tested by microscopy for P. falciparum infection at each study time point, using Giemsa staining as previously described (22). Three drops of blood from each sample were spotted onto filter paper for parasite by nested PCR as previously described (22, 23). Sample collection was done after a 14-month period from April 2007 to May 2008 when no clinical cases of malaria occurred in the entire area (18, 24). Blood samples were also collected for the isolation of PBMCs from 10 North American individuals never exposed to malaria. Written informed consent was obtained from the study participants. The study was approved by the ethical and scientific review committees at the Kenya Medical Research Institute and the Institutional Review Board at the University of Minnesota.

Isolation and culture of PBMCs.

Venous blood (3 to 5 ml from children and 10 to 15 ml from adults) was collected in sodium heparinized Vacutainer tubes (BD Biosciences, San Jose, CA). PBMCs were separated from whole blood by Histopaque (Sigma-Aldrich, St. Louis, MO, USA) density gradient centrifugation and resuspended in complete media (RPMI 1640; Sigma, St. Louis, MO) supplemented with 10% human type AB heat-inactivated serum (Sigma), 10 μg/ml gentamicin (Amresco, Solon, OH), 10 mM HEPES (Gibco/Invitrogen, Paisley, Scotland, United Kingdom) and 10 mM l-glutamine (Sigma, St. Louis, MO)). A total of 2 × 10⁵ PBMCs were added to each well of the 96-well round-bottom plates and stimulated with P. falciparum antigens at 10 μg/ml. Phytohemagglutinin (PHA) at 5 μg/ml and 1× phosphate-buffered saline (PBS) were used as positive and negative controls, respectively. After a 5-day incubation at 37°C with humidity and 5% CO₂, the supernatants were removed and stored at −80°C for cytokine and chemokine testing. A 5-day incubation period was used based on prior experiments showing the strongest IFN-γ and IL-10 responses at this time point compared to 16- or 72-h incubations (3) and to compare with multiple prior studies in this population in which we used the 5-day incubation period to test IFN-γ, IL-10. and TNF-α responses (3, 5, 11).

Mitogen and antigens.

Antigens included CSP peptide cs22 (amino acids [aa] 378 to 392; DIEKKICKMEKCSSV) (2), AMA-1 peptide PL171 (aa 348 to 366; DQPKQYEQHLTDYEKIKEG) (25, 26), TRAP peptide tp6 (aa 51 to 70; LLMDCSGSIRRHNWVNHAVP) (27), LSA-1 peptide T3 (aa 1813 to 1835; NENLDDLDEGIEKSSEELSEEKI) (4), MSP-1 peptide M1 (aa 20 to 39; VTHESYQELVKKLEALEDAV) (28) and peptide M2 (aa 1467 to 1483; GISYYEKVLAKYKDDLE) (29), and MB2 peptide MB1 (aa 191 to 199; SVSSINTNL) (30) and peptide MB2 (aa 119 to 127; KPKKKYYEV) (30), all of which have been documented to produce antigen-specific cytokine responses in persons in areas of malaria endemicity, were used as P. falciparum antigens. Conserved regions of vaccine candidate P. falciparum antigens, strains 3D7 and/or FVO, were used for these peptides. The MSP-1 peptides, M1 and M2, were pooled and used in a single well. Similarly, the MB2 peptides, MB1 and MB2, were pooled and used in a single well. Peptides were synthesized and purified by high-performance liquid chromatography to >95% purity (Sigma Genosys, St. Louis, MO). All peptides were tested at a concentration of 10 μg/ml. PHA at 10 μg/ml was used as the positive mitogen control.

Determination of cytokine and chemokine levels.

The levels of IL-5, IL-6, IL-10, IFN-γ, RANTES, and TNF-α (in malaria-exposed individuals) and of all of these cytokines/chemokines except IL-5 (in malaria-nonexposed individuals) were measured in culture supernatants using customized cytometric bead assay (CBA) kits according to the manufacturer's instructions (Bio-Rad Laboratories, Hercules, CA). IFN-γ, IL-10, and TNF-α were chosen because P. falciparum-specific responses to the cytokines have been correlated with protection from clinical disease (3, 5, 11), while IL-6 was chosen because P. falciparum-specific response have been associated with an increased risk of disease (13), and IL-5 and RANTES were chosen because serum levels have been associated with increased (31) and decreased (14) risk of severe disease, respectively. IL-5 was not measured in malaria-nonexposed individuals due to a technical problem with IL-5 measurement during CBA testing of these individuals. The plates were run on a Luminex 100 instrument, and data acquisition and analysis were performed using BioPlex manager software version 5.0 (Bio-Rad Laboratories). Samples from the three time points for each individual were tested on the same plate to minimize plate-to-plate variability in readings. A minimum of 100 beads per region in a volume of 25 μl was analyzed. A five-parameter curve fit was applied to each standard curve to obtain sample concentration values. The data points below the detection limit were assigned the value of the lowest standard, while those above the detection limit were assigned the value of the highest standard. Due to plate space constraints, 70 individuals were tested for cytokine responses to MB2 and MSP-1 peptides, while the full cohort of one hundred individuals was tested for cytokine responses to all other peptides. Values from unstimulated (PBS) culture supernatants were subtracted from antigen- or PHA-stimulated supernatant levels to provide antigen-specific cytokine levels.

Statistical analyses.

Differences in the levels of cytokine production for paired samples between any two time points were evaluated by the nonparametric Wilcoxon signed-rank test with Bonferroni correction. Correlations between antigen-specific cytokine levels and age were evaluated by Spearman's rank. GraphPad version 5 (GraphPad Software, Inc., La Jolla, CA) was used to generate all graphs. The data were analyzed using Stata 12 (Stata Corp., College Station, TX). P values of <0.05 were considered significant for all tests.

RESULTS

Demographic characteristics and malaria incidence in study participants.

The cohort consisted of 100 individuals, 52 females and 48 males. Thirty-eight were children (mean [range] of 8.60 [2.07 to 14.69] years) and 62 were adults (36.95 [15.65 to 69.25] years). Asymptomatic P. falciparum infection was not present by microscopy at any sample collection time point and was present in 0, 1, and 2 study participants in April 2008, October 2008, and April 2009, respectively. No episodes of clinical malaria were observed in the study participants during the entire period of the study. Malaria surveillance of the entire study site showed no cases of malaria from April 2007 to May 2008, an incidence of <1 case/1,000 persons/month from May 2008 to March 2009, and a small spike to 2 cases/1,000 persons/month in April 2009.

P. falciparum antigen-specific cytokine and chemokine levels in individuals in an area of historically seasonal malaria transmission during a period of prolonged low transmission.

Two patterns were seen for cytokine and chemokine responses to P. falciparum antigens. The first pattern was a decrease in cytokine levels compared to April 2008 levels at the 6-month follow-up (October 2008) or at the 6- and 12-month follow-up (October 2008 and April 2009). IL-10 levels to all antigens decreased at 6 and 12 months (Fig. 1A); TNF-α levels to four antigens decreased at 6 months and levels to two antigens remained decreased at 12 months (Fig. 1B); IFN-γ levels to four antigens decreased at 6 months and levels to one antigen remained decreased at 12 months (Fig. 2A); IL-5 levels to three antigens decreased at 6 months (Fig. 2B). For IL-10 only, a modest increase was seen in levels from the 6-month to the 12-month follow-up (Fig. 1A). In contrast, IL-6 and RANTES levels were similar for all antigens at all time points, except for an increase in IL-6 levels at 6 months to MB2 (Fig. 3A and B). PHA responses were higher at 6 months than baseline for all cytokines/chemokines, and higher at 12 months than baseline for all cytokines/chemokines except IL-6 (Fig. 4).

FIG 1.

Magnitude of antigen-specific IL-10 and TNF-α response to P. falciparum antigens over time. (A) Levels of IL-10 to AMA-1, CSP, LSA-1, MB2, MSP-1, and TRAP. (B) Levels of TNF-α to AMA-1, CSP, LSA-1, MB2, MSP-1, and TRAP. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (as determined by two-tailed Wilcoxon signed-rank test for matched pairs [with Bonferroni correction]). Bars indicate geometric mean levels of cytokines secreted in April 2008, October 2008, and April 2009.

FIG 2.

Magnitude of antigen-specific IFN-γ and IL-5 response to P. falciparum antigens over time. (A) Levels of IFN-γ to AMA-1, CSP, LSA-1, MB2, MSP-1, and TRAP. (B) Levels of IL-5 to AMA-1, CSP, LSA-1, MB2, MSP-1, and TRAP. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (as determined by two-tailed Wilcoxon signed-rank test for matched pairs [with Bonferroni correction]). Bars indicate geometric mean levels of cytokines secreted in April 2008, October 2008, and April 2009.

FIG 3.

Magnitude of antigen-specific IL-6 and RANTES response to P. falciparum antigens over time. (A) Levels of IL-6 to AMA-1, CSP, LSA-1, MB2, MSP-1, and TRAP. (B) Levels of RANTES to AMA-1, CSP, LSA-1, MB2, MSP-1, and TRAP. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (as determined by two-tailed Wilcoxon signed-rank test for matched pairs [with Bonferroni correction]). Bars indicate geometric mean levels of cytokines secreted in April 2008, October 2008, and April 2009.

FIG 4.

Magnitude of IL-5, IL-6, IL-10, IFN-γ, TNF-α, and RANTES response in PHA-stimulated PBMCs over time. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (as determined by two-tailed Wilcoxon signed-rank test for matched pairs [with Bonferroni correction]). Bars indicate geometric mean levels of cytokines secreted in April 2008, October 2008, and April 2009.

P. falciparum antigen-specific cytokine and chemokine levels in malaria-exposed versus nonexposed individuals.

In April 2008, TNF-α levels in malaria–exposed individuals were higher than in the nonexposed individuals for all antigens (Fig. 1B), and IFN-γ levels in malaria-exposed individuals were higher than in nonexposed individuals for three of the six antigens (Fig. 2A). In contrast, IL-10 levels in the malaria-exposed individuals were similar to those in the nonexposed individuals for five antigens (Fig. 1A), and IL-6 and RANTES levels in malaria-exposed individuals were similar for all antigens to those in nonexposed individuals (Fig. 3A and B). TNF-α levels remained higher for five antigens in exposed than nonexposed individuals at 6- and 12-month follow-up (Fig. 1B), while IFN-γ levels were higher in malaria-exposed individuals for two antigens during follow-up, and IL-6 and RANTES levels were higher for three antigens during follow-up (Fig. 3A and B). Conversely, IL-10 levels in malaria-exposed individuals were higher than in the nonexposed individuals for all antigens in the subsequent follow-ups (Fig. 1A). The PHA responses were higher in malaria-nonexposed than malaria-exposed individuals for all cytokines/chemokines tested (Fig. 4).

Associations between antigen-specific cytokine/chemokine responses and age.

Comparing antigens and cytokines with age at all time points, no significant correlations with age were seen (all P > 0.05 when adjusted for multiple comparisons, see Table S1 in the supplemental material).

DISCUSSION

Clinical immunity to malaria appears to wane quickly in the absence of active malaria transmission (16, 17), but the reasons for this loss of clinical immunity are not well characterized. In the present study, we show that P. falciparum-specific cytokine responses associated with protection against clinical malaria in previous studies (IFN-γ, IL-10, and TNF-α) decrease within 24 months of reduction of malaria incidence to low levels (<2 cases/1,000 persons/month), but responses associated with an increased risk of clinical malaria (IL-6) are unchanged over this time period. 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.

Previous studies have shown that responses of the proinflammatory cytokines IFN-γ and TNF-α and the anti-inflammatory cytokine IL-10 to parasitized red blood cells (pRBC) or to specific P. falciparum antigens are associated with protection from clinical malaria. Studies from Kenya (3, 7) and Gabon (5) showed an association of IFN-γ responses to P. falciparum pre-erythrocytic (CSP, LSA-1, and TRAP) and blood-stage (MSP-1) antigens with protection from malaria, while a study from Papua New Guinea showed an association of IFN-γ responses to pRBC (32, 33) with protection from malaria. Similarly, TNF-α responses to LSA-1 (12) and pRBC (13, 34), and IL-10 responses to LSA-1 (11) and pRBC (35) have been associated with protection from malaria. Murine studies and in vitro testing suggest that these cytokines could play a role in host defense against malaria. Murine models show that IFN-γ can work at the sporozoite level by inducing nitric oxide synthase (36), at the liver stage by inhibiting parasite growth and development in hepatocytes (37), and at the blood stage by increasing the phagocytic action of macrophages (38). IL-10 can affect parasite clearance (39), can act on monocytes/macrophages to inhibit antigen presentation and downregulate excessive inflammation (40), or may regulate TNF-α (41), IL-12 (42), or IFN-γ (43), all of which can adversely affect the parasite. TNF-α can inhibit hepatic development of P. falciparum (44, 45), mediate parasite killing by macrophages (46), and inhibit parasite replication (47). Alternatively, antigen-specific IL-10, TNF-α and IFN-γ responses could be surrogate markers for other protective immune responses. In either case, the findings suggest that antigen-specific IL-10, TNF-α, and IFN-γ responses should be further studied as potential markers of loss of clinical protection from malaria in populations that have had a prolonged absence of P. falciparum exposure.

An earlier study documented that IL-6 responses to pRBC were associated with an increased risk of clinical malaria (13), and excess IL-6 has been associated with more severe disease in malaria (48). It is unclear how IL-6 production would lead to increased risk of malaria. One might predict the opposite effect, since high levels of IL-6 can lead to iron deficiency (through induction of hepcidin, which blocks release of iron from macrophages [49]), and iron deficiency is associated with protection from malaria. There have been no studies published to date on P. falciparum antigen-specific RANTES responses in individuals in areas of malaria endemicity, but studies of serum RANTES levels have shown that they are decreased in severe disease (14) and remain decreased in children with prior severe illness (15), suggesting that children with severe malaria have an impaired ability to produce RANTES. In the present study, antigen-specific RANTES levels were maintained in the absence of high-level malaria transmission, suggesting that impaired ability to produce RANTES may not be a primary factor in susceptibility to malaria in this population. We were unable to assess the cellular sources of these cytokines because the number of PBMC and fluorochromes that would be required for flow cytometry testing precluded such testing in the present study. We aim to assess cellular cytokine sources in future studies.

In the present study, malaria was interrupted for approximately a year prior to the first collection and was present subsequently at very low levels (<1 malaria case per 1,000 persons/month) until the month of final collection. In addition, within the cohort, rates of asymptomatic parasitemia by microscopy and PCR during collection times were low (<3%). The presence of two individuals with asymptomatic parasitemia at the last collection suggests that at that time the study area had low but not completely absent transmission. This could potentially explain the slight increase in some responses (notably IL-10 responses to some antigens) at the last collection. Antigen-specific cytokine levels over time decreased, but for the two cytokines with the most persistent decreases, the TNF-α levels remained above those of nonexposed individuals, while IL-10 levels were lower than those of the nonexposed individuals, demonstrating that these responses were not nonspecific. The lower IL-10 levels in relation to the TNF-α levels suggests that this population is predisposed to severe disease, as demonstrated by a previous study conducted in Kenyan children (50). The reason for the increase in PHA responses at 6- and 12-month follow-up is unclear. Although the variability in the lot numbers might affect the PHA response, it is possible that the increased responses to PHA in the periods of low transmission represent a lack of ongoing suppression of cellular responses from chronic exposure to P. falciparum. However, in prior studies, we have found similar responses to PHA in individuals from areas of high and low malaria transmission (C. C. John, unpublished data), so there may be additional reasons for the lower PHA responses during periods of low malaria transmission.

An earlier study assessing cellular immune responses after experimental human malaria induced with P. falciparum-infected mosquitoes demonstrated that IFN-γ responses to whole sporozoites or to P. falciparum-infected RBC remained present 14 months after even a single infection (17), while a second study of cellular immune responses in naturally exposed individuals in Thailand documented short-lived IFN-γ effector responses to P. falciparum schizont extract (PfSE) but long-lived IL-10 memory T cell responses (>6 years) to PfSE (51). Differences in study populations, antigens, and design make the findings difficult to compare. Our study assessed responses to peptides from vaccine-candidate antigens, while the experimental human study assessed responses to the whole sporozoite, and the Thai study assessed responses to schizont extract, which also contains many antigens. The cytokine responses in our study were more specific than those in the previous studies but are likely weaker and less frequent, since the number of epitopes presented are far fewer. Our findings are relevant to considerations for multiple epitope or multiple antigen vaccines but are likely not as relevant to, for example, a whole-sporozoite vaccine. In the present study, antigen-specific IFN-γ levels decreased in the absence of transmission, but antigen-specific IL-10 levels also decreased in the absence of transmission. These findings are similar to those of a study we conducted in a different highland area of Kenya (4) but differ from studies we conducted in an area of malaria holoendemicity in Kenya (52) and in the same highland area as in the present study at two time points of much higher malaria incidence (53). In those studies, IL-10 levels did not differ significantly over time. Our combined study findings suggest that some minimum level of malaria transmission is required for persistence of responses.

The level of malaria transmission required to sustain cytokine responses that have correlated with protection from malaria, such as IFN-γ, TNF-α, and IL-10 responses, is not known. Based on previous studies and the present study, it appears that in populations of low transmission, the absence of clinical malaria incidence is a crude indicator that malaria transmission is below the required level, and that malaria incidence at some level between 2 cases/1,000 persons/month (the maximum for this period of study) and 15 cases/1,000 persons/month (the minimum at the times of collection in the past study) reflects the necessary transmission level to sustain IFN-γ and IL-10 responses. Our findings of a lack of correlation of cytokine responses with age contrast with the findings of other studies (54, 55), and studies by our group in this highland area in a period of higher transmission (3, 4), but also likely reflect the extremely low transmission setting of the current study, a setting in which responses are infrequently boosted because there is little or no malaria exposure. The study was limited by its sampling period: samples from the earlier time period would have provided valuable information about whether antigen-specific cytokine levels were higher at the start of interrupted transmission, and what the rate of decrease was if so.

In summary, our study findings demonstrate that P. falciparum-specific cytokine responses, previously documented to be correlates of protection against clinical malaria (IFN-γ, IL-10, and TNF-α), decrease after a prolonged period of very low malaria transmission, while responses associated with increased risk of clinical malaria (IL-6) are unchanged. The findings suggest that antigen-specific cytokines responses that correlate with protection against P. falciparum diminish on a population level within 18 months after malaria incidence decreases below a critical threshold, likely between 2 and 15 cases/1,000 persons/month, and decrease on an individual level after absence of exposure to P. falciparum for >18 months. Future studies will investigate the specific cellular contributions to this reduction in antigen-specific cytokine production and how such decreases may relate to loss of clinical protection from malaria.