INTRODUCTION

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Epidemics of malaria in the highlands of Uasin Gishu district of Kenya have been reported intermittently since 1902 (1, 3, 8, 12, 17, 18). These outbreaks usually occur during the rainy season (generally between April and September), when the number of Anopheles mosquitoes increases (17). Since the late 1980s, highland malaria epidemics have occurred more frequently and caused significant morbidity and mortality during the rainy season (37). The partial immunity to malaria that develops in adults living in areas where malaria is holoendemic is associated with repeated and frequent exposure to infective mosquitoes (36). In highland areas, prolonged periods of low or no exposure to infective mosquitoes during the dry season presumably results in reduction in the number of parasites that become established in the liver. This may in turn lead to diminished antigen-specific immunity to pre-erythrocytic and blood-stage Plasmodium falciparum with increased susceptibility to malaria infection when transmission rises during the rainy season.

To date, the only longitudinal study of immune responses in epidemic highland malaria has been done in Madagascar and was concerned with a blood-stage antigen. Examination of adult residents during an epidemic in 1986 to 1987 showed that antibody levels and lymphocyte proliferation to Pf155 ring-infected erythrocyte surface antigen peptides decreased after the outbreak was controlled. Cytokine responses were not reported (27). Studies on immune responses in areas of Sudan with unstable malaria transmission have focused primarily on antibody responses (4, 9, 10, 39). Prospective studies of seasonal changes in malaria antigen-specific immune responses in areas where malaria is holoendemic have also focused primarily on antibody and lymphocyte proliferation responses (31, 32). The importance of cytokines in mediating resistance against pre-erythrocytic malaria infection has been documented in animal models (21, 34, 35, 38) and suggested by observations of naturally infected and irradiated-sporozoite-immunized humans (23, 25, 26). We therefore examined antigen-specific cytokine responses of residents of a village in the highlands of western Kenya during the rainy and dry seasons.

This study focused on P. falciparum liver-stage antigen 1 (LSA-1), an ~200-kDa molecule expressed exclusively during hepatic schizogony (11, 42). Previous studies of cytokine responses to LSA-1 have been conducted in residents of areas of Africa and Papua New Guinea where malaria is holoendemic. These have documented gamma interferon (IFN-), tumor necrosis factor alpha (TNF-), interleukin-10 (IL-10), and cytotoxic T-lymphocyte (CTL) responses to polypeptides encoded by the N- and C-terminal nonrepeat regions of LSA-1 (5, 7, 13, 23, 25, 26). Recent observations of Gabonese children have shown a decreased rate of reinfection in individuals whose peripheral blood mononuclear cells (PBMC) made IFN- in response to LSA-1 (25). In addition, examination of residents of an area of Kenya where malaria is holoendemic, located approximately 50 miles from the current study site, showed that IL-10 responses to recombinant LSA-1 proteins correlated with a delayed rate of reinfection following radical cure with chemotherapy (23). These and other studies of CTL responses to LSA-1 (13) support its inclusion in a multistage malaria vaccine.

	MATERIALS AND METHODS

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Human subjects. Volunteers were residents of the village of Kabobo in Uasin Gishu district of Kenya. Kabobo is situated at an elevation of approximately 7,000 feet (2,134 m). The nearest paved road is located 10 miles from the village. P. falciparum transmission is unstable, and epidemics of malaria with high morbidity and mortality have been described (37). The regular outbreaks of malaria that occur in this area every rainy season may be indicative of a gradual change in malaria endemicity in this area from epidemic to seasonally endemic. Only P. falciparum and Plasmodium malariae species have been documented. The great majority of malaria is due to P. falciparum (17).

Seasonal changes in malaria prevalence. Thick and thin smears of fingerprick blood samples from 6- to 12-year-old schoolchildren were examined for P. falciparum in October 1996, at the end of the rainy season, and in March 1997, at the end of the dry season. One hundred eighty children were tested in October, and 201 children were tested in March.

Preparation of PBMC and cytokine assays. Blood was anticoagulated with heparin and transported from the field to the laboratory within 4 h of venipuncture. PBMC were separated from whole blood by Hypaque-Ficoll density gradient centrifugation. For cytokine assays, 10⁶ PBMC with or without peptide, antigen, or mitogen were incubated for 5 days in culture medium, and supernatants were stored at 70°C before transport to Cleveland. IL-5, IL-10, IFN-, and TNF- were measured by two-site enzyme-linked immunosorbent assay as previously described (19). IFN- production was measured prior to testing for other cytokines. The limited amount of supernatant precluded testing all samples for all cytokines from each time period. Cytokine concentrations in supernatants of unstimulated (control) cultures were subtracted from the values of peptide or antigen/mitogen-stimulated cultures.

LSA-1 peptides, antigens, and mitogens. Five peptides, one from the N-terminal and four from the C-terminal nonrepeat regions of LSA-1 (NF54 strain of P. falciparum, GenBank accession no. X56203) were used. The amino acid sequences were residues 84 to 107 (LTMSNVKNVSQTNFKSLLRNLGVS), 1742 to 1760 (HTLETVNISDVNDFQISKY), 1813 to 1835 (NENLDDLDEGIEKSSEELSEEKI), 1836 to 1849 (KKGKKYEKTKDNNF), and 1888 to 1909 (DNEILQIVDELSEDITKYFMKL). Peptides were synthesized by 9-fluorenylmethoxy carbonyl chemistry (15) (kindly supplied by Nga Nguyen, Food and Drug Administration) and used at a concentration of 10 µg/ml with the exception of peptide 1836-1849, which was used at 2 µg/ml. Peptides 84-107, 1813-1835, and 1888-1909 have been shown to stimulate proliferation of PBMC from North Americans inoculated with irradiated P. falciparum sporozoites (22) and IFN- production by adults living in an area of Papua New Guinea where malaria is holoendemic (5). Phytohemagglutinin (1 µg/ml) was used as a mitogen control, and streptolysin O (10 µg/ml) and/or Mycobacterium tuberculosis purified protein derivative (10 µg/ml) served as nonmitogen antigen controls. Only those PBMC preparations that produced cytokines in response to these three controls were included in the analysis.

Statistics. Differences in the frequency of positive cytokine responses to LSA-1 peptides were compared by the ² test. Quantitative differences in the level of cytokine production were evaluated by the nonparametric Mann-Whitney U test. The correlation between the levels of two cytokines was assessed by the Spearman rank test. Association of positive responses between two cytokines was assessed by contingency table analysis (² test). Quantitative differences in the level of cytokine production for the paired PBMC and CD8⁺ cell-depleted PBMC samples were evaluated by the nonparametric Wilcoxon matched-pairs signed-rank test.

	RESULTS

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Prevalence of blood-stage P. falciparum infection during the rainy and dry seasons. In October 1996, near the end of the rainy season, 82 of 180 schoolchildren (45.5%) had P. falciparum parasitemia. In March 1997, at the end of the dry season, only 18 of 201 schoolchildren (8.9%) were parasitemic (P < 0.001). No other Plasmodium species were observed. Among the study subjects, 47 of 80 (58.7%) and 18 of 112 (16.1%) individuals were parasitemic in the rainy and dry seasons, respectively (P < 0.001).

Cytokine responses by North Americans. PBMC from North Americans were examined because some malaria antigens have been found to stimulate lymphocyte responses by persons who have never been infected or exposed to the parasite (41). Cytokine production of >20 pg/ml in response to one or more LSA-1 peptides was observed for PBMC from 3 of 14 individuals for IL-5, 5 of 13 for IL-10, 5 of 14 for IFN-, and 6 of 14 for TNF-. The cutoff value for a positive response by Kenyan study subjects was defined as greater than the mean plus 2 standard deviations (SD) of the North American controls. Positive responses were defined as follows: for IL-5, >72 pg/ml; for IL-10, >132 pg/ml; for IFN-, >214 pg/ml; and for TNF-, >188 pg/ml.

Frequency of PBMC cytokine responses to various LSA-1 peptides by residents of the Kenyan highlands. IL-5, IL-10, IFN-, and TNF- were produced in response to one or more LSA-1 peptides (Table 1). IL-10 responses to the N-terminal 84-107 peptide were less frequent than to any of the C-terminal peptides (P < 0.01). Similarly, a lower frequency of TNF- responses to the N-terminal 84-107 than the C-terminal 1836-1849 peptide was observed (P = 0.01). The frequencies of responses to the other C-terminal peptides were similar to that of the N-terminal peptide. No significant differences between the rates of responses to the N-terminal and C-terminal peptides were noted for IFN- or IL-5.

Relationship of age to cytokine responses. Table 2 describes the frequencies and levels of cytokine responses for persons 17 and those 18 years old. A pattern similar to that of the entire population was observed; i.e., for both children and adults, the frequencies and median levels of IL-10 and TNF- responses were greater in the rainy than dry season, whereas IL-5 and IFN- responses did not change. For example, the median levels of IL-10 produced by children during the rainy and dry seasons were, respectively, 201 and 37 pg/ml (P < 0.001). The frequencies and levels of cytokine responses were similar in children and adults for all cytokines except IFN-, for which the frequency of responses was lower in children than adults (e.g., during the dry season, 31% of children responded, compared with 53% of adults; P = 0.028).

Cytokine production and resistance to reinfection with P. falciparum. Kaplan-Meier survival analysis demonstrated a trend toward prolonged time to reinfection for IL-10 responders to LSA-1 compared with nonresponders (Fig. 1). The difference did not reach statistical significance at the 10-week follow-up (P = 0.15). A lower percentage of IL-10 responders than nonresponders also developed blood-stage infection detectable by inspection of thick smears (25.0% versus 49.2%). This difference approached statistical significance (P = 0.083) (Table 3). There was no difference in the period of time to development of P. falciparum parasitemia when subjects were grouped according to whether or not their PBMC produced IL-5, IFN-, or TNF- in response to LSA-1 peptides (data not shown). The percentages of IL-5, IFN-, and TNF- responders and nonresponders who developed parasitemia in the 10-week follow-up period also did not differ significantly (Table 3). There was no difference in the level of parasitemia between responders and nonresponders for any cytokine tested (data not shown).

View larger version (11K): [in this window] [in a new window]   FIG. 1.   LSA-1-induced IL-10 production and time to reinfection with P. falciparum (Kaplan-Meier analysis).

Correlation between production of various cytokines. LSA-1-stimulated PBMC production of all four cytokines was measured in 56 subjects during the dry season. (This correlation was not examined during the rainy season since PBMC from only 18 subjects studied at this time had a sufficient amount of culture supernatant to measure all four cytokines.) A positive correlation was observed between all pairs of cytokines evaluated except TNF- and IFN- (Table 4).

Contribution of CD8⁺ cells to LSA-1-stimulated IFN- and IL-10 production. The effect of CD8⁺ cell depletion on LSA-1 peptide-stimulated IFN- production was studied in six individuals whose PBMC produced this cytokine in response to one or more LSA-1 peptides. The level of IFN- decreased by 85% following depletion of CD8⁺ cells (median level for nonfractionated PBMC = 266 pg/ml [range, 224 to 2,184 pg/ml] versus 40 pg/ml [range, 1 to 944 pg/ml] for CD8⁺ cell-depleted PBMC; P = 0.028) (Fig. 2). The effect of CD8⁺ cell depletion on IL-10 production was studied in 11 individuals. IL-10 production increased following depletion of CD8 cells from PBMC of nine subjects (Fig. 3). The median level of IL-10 for the depleted population was 66 pg/ml (range, 25 to 170 pg/ml) versus 15 pg/ml (range, 1 to 70 pg/ml) for nonfractionated PBMC (P = 0.007).

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Peak IFN- production by nonfractionated PBMC and CD8+ cell-depleted PBMC in response to one or more LSA-1 peptides. Lines connecting two points correspond to values for cells from one person. IFN- values are expressed on a logarithmic scale.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Peak IL-10 production by nonfractionated PBMC and CD8+ cell-depleted PBMC in response to one or more LSA-1 peptides. Lines connecting two points represent values for cells from one person.

	DISCUSSION

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Malaria epidemics in the highlands of Kenya are characterized by abrupt and transient increases in infection and morbidity that coincide with periods of heightened transmission following prolonged dry spells. The epidemiology of highland malaria differs from that in areas where malaria is holoendemic in that adults as well as children appear to be affected (3, 8, 37). This study describes the temporal changes in antigen-specific T-cell cytokine responses that are believed to contribute to elimination of liver-stage malaria. LSA-1-driven IL-10 and TNF- responses, unlike IFN- and IL-5 responses, were observed to be weaker in the dry, low-transmission season than in the rainy, high-transmission season. The data suggest that lack of frequent and repeated exposure to liver-stage P. falciparum during the dry season leads to decreased immunologic boosting and waning of LSA-1-specific IL-10 and TNF- responses. It is not yet clear why antigen-specific T-cell IFN- and IL-5 responses are not affected in the same way. The results also suggest a trend toward an association between protection from P. falciparum reinfection and LSA-1-driven IL-10 but not TNF-, IFN-, or IL-5 responses. Although these data were obtained from repeated cross-sectional studies of different groups of individuals living in the same area where malaria is endemic, the patterns were similar for IFN- and IL-10 responses of a subset of the same persons evaluated in both seasons. Data comparing the frequency and magnitude of cytokine responses by children and adults suggest that acquisition of T-cell IFN- responses to LSA-1, unlike the other cytokines examined, was related to cumulative and long-term exposure to infection. Once acquired, IFN- responses appear to persist.

Two recent studies have highlighted an association between LSA-1-driven IL-10 responses and P. falciparum infection and morbidity. Luty et al. (26) performed a case-control study of Gabonese children with mild and severe malaria. Parasite clearance times in children with mild malaria were more rapid in those with PBMC IL-10 responses to LSA-1 peptides than in those without IL-10 responses. IL-10 production correlated with higher-level acute-phase antibody responses, which were also associated with rapid parasite clearance. Kurtis et al. (23) demonstrated that IL-10 production in response to recombinant LSA-1 proteins correlated with resistance to reinfection in adults living in an area of western Kenya where malaria is holoendemic. These observations, together with the present data showing that LSA-1-driven IL-10 production diminishes during the dry season, suggest that IL-10 mediates or indirectly contributes to elimination of liver-stage P. falciparum. Ho et al. have suggested that IL-10 down-regulates proinflammatory cytokines such as IFN- and TNF- in acute malaria (14), and the former cytokine has been shown to decrease antigen-specific T-cell cytokine production in general (6). Our observations do not support the notion that IL-10 suppresses LSA-1-driven IFN-, TNF-, or IL-5 since a negative correlation between IL-10 and the latter cytokines was not observed. Rather, the present findings are similar to those of Wenisch et al., who reported a positive correlation between serum IL-10 and IFN- in acute P. falciparum infection (40). More detailed examination of the regulatory role of IL-10 in pre-erythrocytic immunity requires measurement of production of various cytokines in the presence of neutralizing anti-IL-10 antibodies and a better understanding of whether IL-10 influences accumulation and activation of local effector cells, such as CD8⁺ CTL and CD8⁺ IFN--secreting cells in the liver.

The role of TNF- in malaria is complex and incompletely understood. Increased serum TNF- levels have been reported in severe malaria (e.g., cerebral malaria) and uncomplicated morbidity (fever with parasitemia) (24, 28). The relationship between malaria antigen-specific T-cell TNF- responses and liver-stage infection is less well studied. In the present study, the magnitude of the reduction in LSA-1-induced TNF- from rainy to dry season was almost as marked as the decrease in IL-10 responses. The relatively more robust LSA-1-driven TNF- response during the rainy season was not attributable to severe malaria since none of the study subjects had symptoms consistent with this illness. In addition, infection status documented by blood smear did not correlate with TNF- responses, and unlike the case for LSA-1-induced IL-10 responses, there was no trend toward protection from infection with LSA-1-induced TNF- responses. The lower number of individuals tested for TNF- production (n = 54) than for IL-10 (n = 77) may have made correlation between protection and TNF- responses more difficult to detect. Others have reported a lack of correlation between LSA-1-induced TNF- and protection against infection (23, 25). It is possible that LSA-1-driven TNF- responses correlate with relative resistance against severe morbidity rather than infection, although at present morbidity is thought to be associated primarily with immunity to blood-stage antigens.

IFN- is an important mediator of resistance to liver-stage malaria and the pathogenesis of disease in animal models (29, 30, 35), and LSA-1-specific T-cell IFN- responses develop in North American volunteers in whom sterile, transient immunity to P. falciparum infection has been induced by immunization with radiation-attenuated sporozoites (22). We observed no seasonal changes in the strength of LSA-1-induced IFN- responses in the highland study subjects and no correlation between LSA-1-induced IFN- responses and resistance to reinfection with P. falciparum. In a study conducted in a nearby area where malaria is holoendemic, the findings of Kurtis et al. were very similar: the proportion of persons who made IFN- in response to recombinant LSA-1 protein decreased at the end of the dry season, but there was no association between these responses and the time to reinfection (23). In contrast, Luty et al. observed that LSA-1-driven IFN- production was associated with delayed infection and reduction in the rate of reinfection in children (25). A previous study of adults in Papua New Guinea demonstrated that IFN- responses to the N-terminal 84-107 peptide were associated with repeatedly negative smears for P. falciparum over a 6-month period (5). Since we were able to document a trend toward protection from infection in individuals with IL-10 responses to LSA-1, and since our study numbers were even larger for IFN- (n = 94), the very similar rates of infection in those with and without IFN- responses to LSA-1 suggest that, at least in this population, LSA-1-induced IFN- production is not strongly protective against infection. As with TNF- responses, it is possible that LSA-1-induced IFN- responses relate more to malarial morbidity than to infection.

The most remarkable age-associated difference observed in the present study was related to IFN-, the only cytokine for which there were fewer responses by children than by adults. One explanation is that multiple exposures and greater cumulative experience with liver-stage P. falciparum are required to induce IFN- but not IL-5, IL-10, or TNF- responses to LSA-1. If this is so, the present findings suggest that once a given threshold is reached, LSA-1-induced IFN- responses persist even when transmission intensity decreases. In this context, it will be of interest to determine the phenotypes of the T-cell subsets that produce IFN- (see below), whether they have markers of the memory phenotype (e.g., CD45RO) (2), and whether they differ from those of T cells that secrete other cytokines when stimulated with LSA-1.

Because of limitations in the number of PBMC available from the study subjects, we were not able to perform detailed experiments to determine the subsets of T cells that made each of the cytokines. Based on the length of the peptides used to stimulate cytokine production (14 to 24 amino acids), both CD4⁺ and CD8⁺ T cells restricted by HLA class I and II molecules may have contributed. Results of experiments in which CD8⁺ cells were depleted by immunomagnetic selection suggested that this subset is the major but not only source of IFN-. CD8⁺ T cells were also the predominant source of IFN- following stimulation with the LSA-1 84-107 peptide in studies conducted in Papua New Guinea (K. Bucci, unpublished data). By contrast, LSA-1-driven IL-10 production was enhanced following depletion of CD8⁺ cells (in 9 of 11 individuals). It is not clear whether this modest increase in cytokine production was due to removal of cells that secrete molecules which actively suppress production of IL-10 by the remaining monocytes and CD4⁺ T cells or removal of a source for consumption of IL-10. Since IL-10 is a chemoattractant for CD8⁺ (16), it is possible that this cytokine is involved in elimination of liver-stage parasites by virtue of its effects on the local accumulation of effector cells, such as IFN--secreting CD8 cells.

Our study establishes that select LSA-1-driven cytokine responses in highland residents vary according to season and supports the idea that LSA-1-induced IL-10 production may have a role in protection from P. falciparum infection. Future studies will focus on determining whether seasonal changes in CD4⁺ and CD8⁺ T-cell immunity to LSA-1 (and other pre-erythrocytic or blood-stage malaria antigens) (29, 30, 33) are predictive of the rate and clonality of reinfection, high-density parasitemia, and uncomplicated malaria morbidity in children and adults.