The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 4193 -4201
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bejon, P.
Right arrow Articles by Hill, A. V. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bejon, P.
Right arrow Articles by Hill, A. V. S.

The Induction and Persistence of T Cell IFN-{gamma} Responses after Vaccination or Natural Exposure Is Suppressed by Plasmodium falciparum1

Philip Bejon2,*, Jedidah Mwacharo*, Oscar Kai*, Stephen Todryk{dagger}, Sheila Keating{dagger}, Brett Lowe*, Trudie Lang{dagger}, Tabitha W. Mwangi*, Sarah C. Gilbert{ddagger}, Norbert Peshu*, Kevin Marsh*,§ and Adrian V. S. Hill{dagger},{ddagger}

* Kenya Medical Research Institute, Centre for Geographical Medicine Research (Coast) Kilifi, Kenya; {dagger} Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford, Oxford, United Kingdom; {ddagger} Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom; and § Nuffield Department of Clinical Medicine, Oxford University, John Radcliffe Hospital, Oxford, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Epidemiological observations suggest that T cell immunity may be suppressed in malaria-endemic areas. In vitro studies, animal models, and limited data in humans link immunosuppression with malaria, malnutrition, and other parasitic infections. However, there are no data to determine whether malaria-induced immunosuppression is significant in the long-term, or relative data comparing it with other factors in malaria-endemic areas, so as to measure the impact of malaria, other parasitic disease, nutritional status, age. and location on the acquisition and longevity of IFN-{gamma} responses in children in Kenya. We studied these factors in two cohorts of 1- to 6-year-old children in a malaria-endemic area. T cell responses were induced by vaccination in one cohort, and acquired as a result of natural exposure in a second cohort. Serial ELISPOT assays conducted over a 1-year period measured the induction and kinetics of IFN-{gamma} production in response to the malaria Ag thrombospondin-related adhesion protein. Induced responses in both cohorts and the longevity of response in the vaccinated cohort were fitted to potential explanatory variables. Parasitemia was prospectively associated with reduced IFN-{gamma}-producing T cells in both cohorts (by 15–25%), and both parasitemia and episodes of febrile malaria were associated with 19 and 31% greater attrition of T cell responses, respectively. Malaria may reduce the efficacy vaccinations such as bacillus Calmette-Guérin and investigational T cell-inducing vaccines, and may delay the acquisition of immunity following natural exposure to malaria and other pathogens.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Some epidemiological observations suggest that cellular responses may be suppressed in malaria-endemic areas (1, 2), but a number of environmental factors might be responsible. Malaria-infected erythrocytes modulate dendritic cell function in vitro, inhibiting Th cell induction (3, 4) and stimulating T regulatory cells (5), but the evidence that associates malaria with immunosuppression in vivo is limited. Although routine childhood Ab-inducing vaccinations appear to be effective in malaria-endemic areas, and are mostly unaffected by chemoprophylaxis (6), there may be a transient suppression of cellular responses during acute infection in Thai adults (7, 8). Acute infection has also been associated with a loss of T cell inhibition of EBV-infected cells (9), and there is a transient suppression of vaccine-induced Ab responses after inpatient treatment for malaria (10). However, the long-term in vivo impact of malaria-induced immunosuppression in the populations at risk has not been determined.

Furthermore, other factors might also suppress cellular responses in malaria-endemic areas. Severely malnourished children have a suppressed cellular response to mycobacterial Ags (11). Mild malnutrition is common in malaria-endemic areas and might also lead to depressed cellular immunity. Concurrent helminth infections suppress T cell responses by inducing a TH2 cytokine profile (12). T cell responses to tetanus toxoid (13, 14) and bacillus Calmette-Guérin (BCG)3 (15) may be inhibited by helminth infection.

We measured T cell responses by IFN-{gamma} production in two cohorts of children in rural Kenya, for whom data on malaria infection, helminth infection, nutritional status, age, and village was available. We used both ex vivo and cultured ELISPOT assays. Responses detected by cultured ELISPOTs are sustained for at least 6 mo after vaccination of naive subjects, but responses detected by ex vivo ELISPOT begin to fall soon after induction (16).

The vaccination regimen induces T cell responses by sequential immunization with the attenuated fowlpox strain, FP9, and modified virus Ankara, to deliver a multiple epitope (ME) string (17) coupled to the pre-erythrocytic malaria Ag thrombospondin-related adhesion protein (TRAP) (18).

Vaccine efficacy, safety, and immunogenicity are described elsewhere (19). The study presented here examines whether malaria, other parasitic infections, or malnutrition influence natural or vaccine-induced acquisition of T cell responses. The impact of these exposures is studied on three different data sets.

The first data set comprises T cell responses measured in a cross-sectional bleed of children 1 wk after the last of three vectored ME-TRAP vaccinations (the second cross-sectional bleed). These responses were adjusted by T cell responses at the first cross-sectional bleed, taken before vaccination. The second data set comprises naturally acquired T cell responses measured in rabies (i.e., control) vaccine recipients at the end of the malaria season (the third cross-sectional bleed). This analysis was adjusted by T cell responses at the cross-sectional bleed before the start of the malaria season (the first cross-sectional bleed). The third data set comprises T cell responses measured in ME-TRAP vaccinees 9 mo after the last vaccination (the fourth cross-sectional bleed). Analysis was adjusted by responses immediately after vaccination (the second cross-sectional bleed). Thus, the analysis in each data set is adjusted by T cell responses at a prior cross-sectional bleed. Parasitemia is considered as a potential factor, identified on the cross-sectional bleed analyzed (referred to as "concurrent" parasitemia), and also on the prior bleed (referred to as "previous").


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Study design

The study was conducted in the context of a randomized, controlled, and double-blind vaccine trial. Ethical approval was obtained from the Kenyan Medical Research Institute National Ethics Committee, the Central Oxford Research Ethics Committee, and the London School of Hygiene and Tropical Medicine Ethics Committee. Five milliliters of blood was taken by venipuncture for immunology and safety; cross-sectional assessments of malaria parasitemia were conducted prevaccination, at screening, 1 wk after the third vaccination, then at 3 mo and at 9 mo later. Children were screened in February 2005, immunized between March 2005 and May 2005, and followed up until February 2006.

Participants

The participating children were aged 1–6 years old (inclusive), healthy, and resident in the Junju sublocation, Kilifi District. After a series of public meetings and individual discussions, parents were invited to bring their children to a screening visit. Recruiting continued until the target sample size was reached.

Children were screened by history, examination, and blood tests (full blood count, creatinine, alanine transaminase). Subjects with clinically significant illness were excluded. HIV prevalence among children is low in Kilifi, at <2% (20), and screening did not include HIV serology. Clinically evident immunosuppression was an exclusion criteria, but no children were excluded on that basis. The mid upper arm circumference (MUAC) was measured to determine nutritional status. The median MUAC was 15 cm among the children recruited (95% range 13–17 cm). A cutoff of 13 cm determines severe malnutrition (21), and the median MUAC is 16 cm in nonaffluent populations in the age-group studied (22). Eligible children were invited to attend vaccination in the order in which they were screened.

Location

The study was conducted in the Junju sublocation in the Kilifi District, on the Kenyan coast. Junju contains a group of five closely related villages within the Chonyi area of the Kilifi District. Kilifi is malaria endemic, with all-year-round transmission and two high transmission seasons (23), with a transmission intensity of 22–53 infective bites per year (24).

Vaccinations

Recombinant viral vectored vaccinations were used. The Ag insert was TRAP, joined to a ME string from six Plasmodium falciparum pre-erythrocytic Ags (17). The ME string contains 14 pre-erythrocytic MHC class I epitopes from six P falciparum pre-erythrocytic Ags, three class II epitopes, two pre-erythrocytic B cell epitopes, and pb9 (a Plasmodium berghei T cell epitope that allows preclinical potency and stability testing). The vectors were an attenuated fowlpox virus, FP9, and modified vaccinia virus Ankara. The trial vaccination regimen was two sequential FP9 ME-TRAP vaccinations (5 x 107 PFU) followed by modified virus Ankara ME-TRAP vaccination (1.5 x 108 PFU), given intradermally. The control was rabies vaccine (WISTAR strain; Aventis Pasteur), also given intradermally. Vaccinations were spaced 4 wk apart (acceptable range 3–5 wk). Curative antimalarial treatment was given 1 wk after the final vaccination, using 7 days of directly observed dihydroartemisinin monotherapy (2 mg/kg on the first day, followed by 1 mg/kg for 6 days).

Follow-up

Children were monitored for episodes of malaria, defined as an axillary temperature >37.5 degrees centigrade, with a P. falciparum parasitemia >2500 parasites per microliter. Children were visited every week by field workers. When the temperature was >37.5 degrees, a blood film was made and a rapid near-patient test (Optimal, which detects parasite lactate dehydrogenase) for malaria was conducted. Treatment for episodes of malaria was with the Government of Kenya recommended first line treatment, artemether-lumefantrine.

Four cross-sectional bleeds were conducted to measure T cell responses and to examine blood films on all children. Results were not available for several weeks, during which monitoring of febrile illnesses continued. Asymptomatic parasitemia was therefore not treated unless the child developed a fever. Samples of urine and stool were collected during screening, and examined by direct, wet microscopy for OVA, cysts, and parasites.

ELISPOTs

PBMC were separated at screening, 1 wk after the last vaccination, then at 3 mo and at 9 mo. PBMCs incubations were in RPMI 1640 medium (Sigma-Aldrich) with 10% human AB serum. ELISPOTs used Millipore MAIP S45 plates and MabTech Abs according to manufacturer’s instructions. A total of 4 x 105/well of freshly isolated PBMC were incubated in 100 µl with 10 µg/ml peptides for 18–20 h before developing. Individual 8–17 residue epitopes were pooled for the ME string. Twenty residue peptides overlapping by 10 residues were used for TRAP. TRAP peptides were pooled according to region. 20 µg/ml PHA (Sigma-Aldrich) was used as positive control, and PBMC cultured in medium alone as negative control. Spot-forming cell numbers were counted by ELISPOT plate reader (Autoimmun Diagnostika, version 3.0).

For cultured ELISPOTs, 1 x 106 PBMC were incubated in 0.5 ml of 10 µg/ml/peptide of pooled TRAP and ME peptides in a 24-well plate. On days 3 and 7, 250 µl of culture supernatant was replaced with 250 µl of culture medium containing 20 IU/ml rIL 2. On day 9, the cells were washed three times and left overnight before the standard ELISPOT assay.

Analysis

The origin of data sets used in analysis is shown in Fig. 1. ELISPOT wells were assayed in duplicate, taking the mean and subtracting the negative control well result. Assays were discounted if the positive control was <50 spots, or the negative control >20 spots for ex vivo ELISPOTs, or 40 spots for cultured ELISPOTs. Results are presented per million incubated PBMC. Geometric means and 95% confidence intervals (CI) are reported, substituting 1 spot/well above background for blank wells (the lower limit of detection).


Figure 1
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 1. The cross-sectional bleeds used for each data set and their relationships to malaria transmission and vaccination are shown. Each data set uses two cross-sectional bleeds, modeling the impact of T cell responses at the earlier time point, parasitemia at both time points, and malaria episodes during the intervening time on T cell responses seen at the later time point.

 
To examine the impact of various exposures on T cell responses, multiple linear regression analysis was conducted, using log-transformed spot numbers for three peptide pools per individual. Significance testing was adjusted with peptide pool as a factor to account for the multiple measures (one for each pool) per individual. Independent variables for nutritional status, gastrointestinal helminth infection, urinary schistosomiasis, eosinophilia (as a categorical variable with three levels), malaria parasitemia (concurrent and previous), and episodes of febrile malaria (binary variables, present, or absent) were examined for their impact on T cell response. Analysis was adjusted by village and age (as a categorical variable). Because longitudinal data was available, T cell responses in each data set were adjusted by previous T ELISPOT responses (rather than being analyzed as a cross-section in isolation). Log-likelihood testing was used to derive a single p value for categorical variables with more than two levels.

The coefficients derived from regression models were transformed to percentages by the formula 10(coefficient)/10 x 100%. This expresses the increase or decrease in T cell responses as a percentage of the responses seen without that factor. The baseline factor, with which others are compared, is given as 100% in the results of all regression models (see Tables II–IV). Where data was not available from both time points used in the data set, children were excluded from the analysis.


View this table:
[in this window]
[in a new window]

  		Table II. Results of multiple regression model to examine factors influencing the acquisition of T cell responses after vaccination (data set 1)a

 

View this table:
[in this window]
[in a new window]

  		Table III. Results of multiple regression model to examine factors influencing the acquisition of T cell responses after natural exposure (data set 2)a

 

View this table:
[in this window]
[in a new window]

  		Table IV. Results of multiple regression model to examine factors influencing the attrition of T cell responses after vaccination (data set 3)a

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Study profile

Numbers lost to follow-up are shown in Fig. 2, and the numbers contributing to each analysis are marked in Figs. 3 and 4.


Figure 2
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 2. The study profile for the 405 subjects recruited is shown. ELISPOT data was missing from the four time points because of assay failure in 51, 8, 45, and 7 instances, and because of nonattendance in 0, 33, 57, and 102 instances, respectively. The high number of nonattendances at the last time point appeared to be related to seasonal traveling patterns.

 

Figure 3
View larger version (39K):
[in this window]
[in a new window]

  		FIGURE 3. T cell responses to vaccination identified by both ex vivo and cultured ELISPOT are displayed over time for each data set analyzed. Median, 25th and 75th quartile, 5th and 95% quartile and outlying results are given by box and whisker plots. Vaccination with FFM ME-TRAP induced ex vivo ELISPOT and cultured responses (p < 0.0005, p < 0.0005, data set 1). There was also a significant rise in T cell responses during the malaria season among control vaccinees (data set 2, p < 0.0005, p = 0.001 for ex vivo and cultured responses, respectively). Vaccine-induced ex vivo response fell over the next 9 mo (data set 3, p < 0.0005), but cultured responses were sustained (p = 0.51). The geometric mean responses are shown below each box and whisker plot.

 

Figure 4
View larger version (48K):
[in this window]
[in a new window]

  		FIGURE 4. IFN-{gamma}-producing T cells are compared by malaria parasitemia at the cross-sectional bleed before immunological studies (as paired box and whisker plots). The first two box and whisker pairs in each data set show the absolute values seen at the cross-sectional bleed that was analyzed as the outcome data in each data set. The second two box and whisker pairs show the fold change in T cell numbers when the outcome data is divided by T cell numbers seen at a previous cross-sectional bleed. Hence, x1 indicates no change in T cell responses over time. Values of p for each comparison (malaria parasites positive vs malaria parasites negative) are shown under each pair.

 
Kinetics of responses

Vaccination with sequential FP9 virus vaccinations (twice) FFM then modified Vaccinia Ankara virus (once), all encoding Ag construct ME-TRAP induced both ex vivo ELISPOT measured responses (from a geometric mean of 9 spots/million, CI 7–11 to 55/million, CI 45–67, p < 0.0005) and cultured ELISPOT measured responses (from a geometric mean of 55 spots/million, CI 44–68 to 240/million, CI 200–290, p < 0.0005) (Fig. 3, data set 1).

There was also a significant rise in IFN-{gamma}-producing T cells during the malaria season among control vaccinees. Ex vivo responses rose from 10 spots/million (95% CI 8–12) before the malaria season (January 2005), to 23 spots/million (95% CI 18–28) in the malaria season (August 2005), p = 0.0005. Resting memory (cultured) responses also rose significantly during the malaria season, from 53/million (CI 42–66) in January 2005, to 97/million (CI 79–120) in August 2005, p = 0.0001 (Fig. 3, data set 2).

Responses that had been induced by vaccination then fell over the following 9 mo. Ex vivo responses fell from a geometric mean of 55 spots/million (CI 43–66) in early May 2005 (1 wk after vaccination) to 19 spots/million (CI 16–24) in January 2006 (9 mo after vaccination), p < 0.00005. However, resting memory responses were preserved, from 234 spots/million in May 2005 (CI 194–281) to 213 (CI 169–269) in January 2006, p = 0.51. The attrition of responses during the 9 mo after vaccination is shown in Fig. 3, data set 3.

Multiple regression models to determine factors associated with variation of responses

The responses at the second time point for each of the three data sets are modeled as outcomes, using the earlier T cell responses and blood film results at both time points as explanatory variables. The frequencies of other covariates in the population are given in Table I, and the results of multiple regression models to identify explanatory variables for T cell responses are shown in Table II–IV.


View this table:
[in this window]
[in a new window]

  		Table I. The frequencies of covariates among study subjectsa

 
Covariates

The pool of TRAP peptides that was tested (three pools covering the full length of the molecule), the child’s village and prior immunity were all significant factors in each data set. Neither vaccine-induced nor naturally acquired responses examined in data sets 1 and 2 varied by age. Nine months after vaccination, ex vivo responses in data set 3 were significantly better sustained among older children (2–7 year olds) compared with 1- to 2-year-old children, but cultured responses did not vary.

In data set 1, the ex vivo ELISPOT results measured before vaccination predicted the ex vivo responses after vaccination (p = 0.012). In data set 3, cultured responses 9 mo after vaccination were predicted by cultured responses 1 wk after vaccination (p < 0.0005), and ex vivo responses 9 mo after vaccination were predicted by ex vivo responses 1 wk after vaccination (p = 0.009). However, in data set 2, neither cultured nor ex vivo responses during the dry season predicted naturally acquired responses seen during the rainy season.

Nutrition and helminth infection

ELISPOT results did not vary by nutritional status (as determined by MUAC in January 2005). Parasitic infection was determined by stool and urine microscopy, and peripheral blood eosinophil counts (conducted in January 2005). There were no associations between T cell responses and microscopy results for urinary schistosomiasis or gastrointestinal helminth infections, but eosinophilia was strongly associated with a greater attrition of ex vivo ELISPOT responses, examined in data set 3 (p = 0.006 by log-likelihood ratio testing, Table IV). Responses in moderate and highly eosinophilic subjects were 69 or 79% of the magnitude of responses without eosinophilia (Table IV). However, cultured responses did not vary by eosinophilia, and eosinophilia did not alter vaccine induction of responses (Table II) or naturally acquired responses (Table III), examined by data sets 1 and 2, respectively.

Parasitemia and malaria infection

Asymptomatic malaria parasitemia as vaccination began was associated with a reduction in postvaccination T cell responses (i.e., data set 1) to 85% of the magnitude of nonparasitemic children (p = 0.09) by ex vivo assays and 79% (p = 0.043) by cultured ELISPOT assays (Table II). Naturally acquired responses (data set 2) were also suppressed by previous parasitemia, to 75% (p < 0.0005) by ex vivo assays and 75% (p = 0.032) by cultured assays (Table III). However, parasitemia at the time blood was drawn for ELISPOT assays did not reduce T cell responses for either vaccine-induced or naturally acquired responses.

Ex vivo responses to vaccination with FFM ME-TRAP were less well-sustained among children previously parasitemic (at 81%, p = 0.002), with concurrent parasitemia (69%, p < 0.0005) and with episodes of febrile malaria before the ELISPOT assays (79% p = 0.014). Cultured responses did not vary by asymptomatic parasitemia, but were reduced to 72% by episodes of febrile malaria (p = 0.04).

Fig. 4 shows the frequencies of T cell responses by previous exposure to malaria parasitemia, displayed for each data set studied. The change in ELISPOT results compared with the prior studies used to adjust results is shown for each data set (i.e., for the first data set, the change in response from pre- to postvaccination is shown. For the second data set, the change in response from pre- to post-malaria season, and for the third data set, the change in response from 1 wk to 9 mo postvaccination is shown.

Combined multiple regression analysis for sustained ex vivo responses

In data sets 1 and 2, only one factor (malaria parasitemia) significantly altered responses after adjusting for peptide pool, previous ELISPOT results and village. However, several additional factors were predictors of the attrition of ex vivo responses after vaccination (Table IV, data set 3), and so were entered into a further multiple regression analysis, to examine whether any were confounding factors. The effect of eosinophilia was still highly significant (p = 0.0005, with reductions in response to 63 and 79%, respectively, for moderate and high eosinophilia). Concurrent parasitemia (74%, p = 0.001), previous parasitemia (85%, p = 0.048), and episodes of febrile malaria (81%, p = 0.033) were also independently associated with reduced ex vivo ELISPOT results.

Protection against episodes of malaria

Vaccination with FFM ME-TRAP was not protective, and neither ex vivo nor cultured ELISPOT results predicted protection within FFM ME-TRAP vaccinees (19). The naturally acquired T cell responses were not protective, either for ex vivo (incident rate ratio = 1.17 for each 10-fold rise in T cell numbers, 95% CI 0.7–2) or cultured ELISPOT (incident rate ratio = 0.84, 95% CI 0.5–1.3, p = 0.48).

Relationship of ex vivo to cultured responses

Although cultured and ex vivo responses did not correlate with each other among unvaccinated children (r = –0.017, p = 0.71), there was a significant association between cultured and ex vivo responses 1 wk after vaccination, in May 2005 (r = 0.18, p < 0.00005 unadjusted, coefficient = 0.14, p = 0.006 when adjusted for peptide pool and village).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Parasitemia immediately before vaccination suppressed the acquisition of T cell responses, as measured by IFN-{gamma} production, in data set 1 (where responses were acquired from vaccination) and in data set 2 (where responses were acquired as a result of natural exposure in the unvaccinated group). Parasitemia immediately after vaccination did not suppress responses. The data sets are demonstrated in Fig. 3, and the effect of parasitemia in Fig. 4. This might reflect an effect of parasitemia per se at the time point immediately before vaccination, or simply that more highly exposed children are both parasitemic and immunosuppressed. However, concurrent parasitemia would be equally strongly linked to overall exposure, but did not influence T cell responses. Furthermore, malaria episodes during the time of monitoring were not associated with lower T cell responses in data sets 1 and 2.

What are the limitations of this study? The link between immunosuppression and parasitemia is made by association. In vitro studies suggest malaria is immunosuppressive (25). However, the association could be the result of T cell responses protecting against infection. But naturally acquired ex vivo responses to TRAP were not associated with protection previously (26), and in this study neither cultured or ex vivo detected responses were protective. Furthermore, the temporal nature of the association is between parasitemia at a previous cross-sectional bleed to vaccination and reduced T cell responses at a later time point. It therefore seems likely that the direction of causation is from the prior event (malaria infection) to the latter outcome (immunosuppression).

Multiple comparisons have been made. Three data sets are analyzed for the impact of nine different variables. One would expect to see some p values <0.05 by chance alone. However, because previous parasitemia was a significant factor on four of six occasions, this suggests the result is unlikely to be due to chance alone, and two of these p values were <0.0005. One should be more cautious in interpreting the single significant association seen for eosinophilia in the third data set.

The study measures responses to one Ag (TRAP), albeit using all potential epitopes (using 57 peptides in three pools), and only used IFN-{gamma} responses. The ELISPOT assay allows reproducible, high throughput measurement of T cell responses (27), but does not allow detailed phenotyping of the cellular response. Although vaccination can produce very diverse cytokine responses (28), IFN-{gamma} responses are strongly predictive of at least one other cytokine response (IL-2) after vaccination with the viral vectors used in our study (29, 30). Furthermore, although we did not conduct magnetic bead depletion studies, previous work shows that natural infection (26) and vaccination (31) induces primarily CD4 responses. Acute malaria induces a monocytosis (32), but the ELISPOT studies were of well, afebrile children, and the monocytosis associated with malaria appears to last only 3–7 days (32). Therefore, despite these limitations, the study allows reasonable conclusions to be drawn from the relative magnitude of cellular responses in the children studied.

Previous studies have identified a temporary cellular immunosuppression in febrile malaria (7, 8), but our data suggest a more long-lasting immunosuppression associated with asymptomatic parasitemia. By what mechanism might early parasitemia but not parasitemia at the time of the assay for cellular responses influence T cell induction? Children received priming vaccinations 2 wk after the earlier blood films, and boosting vaccinations 1 wk before the concurrent blood films. In this vaccinated cohort, parasitemia appeared to inhibit T cell priming, but not the subsequent boosting of responses. In the cohort studied for natural exposure, malaria transmission was high throughout the interval between blood film examinations. Because a single exposure to sporozoites in naive volunteers does not appear sufficient to generate T cell responses (16), it is likely that the T cell responses acquired in this cohort were as a result of multiple exposures. Although this natural exposure is not a heterologous prime boost, it nevertheless seems that parasitemia concurrent with early exposure was associated with suppressed responses, but that parasitemia was concurrent with later exposure. Thus, for vaccination and natural exposure, it appears that parasitemia has an influence on initial priming, but not subsequent recall and/or boosting. The boosting of cultured and ex vivo responses was equally affected.

However, the more rapid attrition of T cell responses associated with parasitemia, episodes of febrile malaria, and eosinophilia was seen only with ex vivo responses. Cultured responses were not affected (data set 3). Furthermore, the variation in attrition of response seen according to village was more pronounced for ex vivo than for cultured responses. These data suggest that the effect of malaria on T cell induction is seen on ex vivo and cultured ELISPOT, but that once induced, cultured responses are relatively durable. Ex vivo responses are susceptible to attrition from a number of environmental factors, of which malaria infection had the greatest impact in our study. This is consistent with previous comparisons of cultured and ex vivo ELISPOTs. Cultured ELISPOT responses appear to identify a more durable T cell response than the ex vivo assay in naive volunteers (16), and did not correlate with ex vivo responses in semi-immune adults in a single cross-sectional survey (33).

The priming of T cells responses depends on dendritic cells, whereas subsequent memory appears to be independent of dendritic cells (34), but is dependent on cytokines such as IL-7 and IL-15 (35, 36). Previous parasitemia may act on dendritic cells to inhibit T cell priming, but intercurrent febrile malaria, parasitemia, and the TH2 responses associated with eosinophilia may impair the cytokine milieu required to support the longevity of responses. T cell responses are depleted but for the inflammatory cytokine response during malaria infection in mice (37). Our data suggest that the cytokine milieu acts differently on T cells seen to respond on ex vivo assays (i.e., with an immediate effector phenotype) than on cultured ELISPOTs (i.e., with a "resting memory" phenotype).

The 15–30% reductions in T cell responses in parasitemic children are likely to be an underestimate of the overall immunosuppression caused by malaria in endemic areas. Parasitemia below the level of blood film detection is frequent in endemic areas (38), and some children may have cleared their parasitemia soon after the film was taken. It is likely that the majority of children in the study (film negative and positive) were exposed to malaria, but those with positive blood films had a greater suppression of cellular responses.

Neither gastrointestinal helminth infection nor urinary schistosomiasis was associated with altered cellular immunity. However, only single specimens of stool and urine were examined, and children with lower parasite burdens might have been identified by repeated specimens or concentration techniques. The only common cause of eosinophilia among children in Africa is helminth infection, and eosinophilia may be a more discriminatory marker for altered immunity, because it is more closely associated with the TH2 cytokine responses than the presence or absence of parasites (39). Because eosinophilia was associated with greater attrition of cellular responses, it is likely that helminths do play a role in the attrition of responses.

What are the implications of these data? Malaria parasitemia and helminth infection should be considered in assessing the immunogenicity and efficacy of experimental and routine (i.e., BCG) T cell-inducing vaccinations. The efficacy of BCG is less in malaria-endemic areas (40), and more immunogenic vaccines are in development (41). T cell-inducing vaccines are also in development for HIV (42) and malaria (43). Estimates of immunogenicity and efficacy should be sought in populations chosen for different malaria and helminth endemicities. Age appears to be a less important consideration over the 1- to 6-year range, and mild and moderate malnutrition does not appear to reduce immunogenicity. The clinical development plans of experimental vaccines, intermittent presumptive treatment of malaria, and observational studies of immunity to malaria should consider the immunosuppressive effect of malaria in their designs.


   Acknowledgments
 
We thank the participants and the Junju Dispensary Committee for facilitating the study. The study was performed with the permission of Kenyan Medical Research Institute (KEMRI) National Ethics Committee, and the National Health Service Central Office for Research Ethics Committees, and published with the permission of the director of KEMRI.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 P.B. holds a Wellcome Trust Training Fellowship (073597), K.M. is funded by the Wellcome Trust, and A.V.S.H. is a Wellcome Trust Principal Research Fellow. This work was supported by the Wellcome Trust and the Gates Malaria Partnership at the London School of Hygiene and Tropical Medicine. Back

2 Address correspondence and reprint requests to Dr. Philip Bejon, Kenya Medical Research Institute, Centre for Geographical Medicine Research (Coast). E-mail address: pbejon{at}kilifi.kemri-wellcome.org Back

3 Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; ME, multiple epitope; TRAP, thrombospondin-related adhesion protein; MUAC, mid upper arm circumference; CI, confidence interval. Back

Received for publication May 14, 2007. Accepted for publication June 22, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Greenwood, B. M., A. M. Bradley-Moore, A. D. Bryceson, A. Palit. 1972. Immunosuppression in children with malaria. Lancet 1: 169-172. [Medline]
  2. Floyd, S., J. M. Ponnighaus, L. Bliss, P. Nkhosa, L. Sichali, G. Msiska, P. E. Fine. 2002. Kinetics of delayed-type hypersensitivity to tuberculin induced by bacille Calmette-Guérin vaccination in northern Malawi. J. Infect. Dis. 186: 807-814. [Medline]
  3. Urban, B. C., D. J. Ferguson, A. Pain, N. Willcox, M. Plebanski, J. M. Austyn, D. J. Roberts. 1999. Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells. Nature 400: 73-77. [Medline]
  4. Wilson, N. S., G. M. Behrens, R. J. Lundie, C. M. Smith, J. Waithman, L. Young, S. P. Forehan, A. Mount, R. J. Steptoe, K. D. Shortman, et al 2006. Systemic activation of dendritic cells by Toll-like receptor ligands or malaria infection impairs cross-presentation and antiviral immunity. Nat. Immunol. 7: 165-172. [Medline]
  5. Riley, E. M., S. Wahl, D. J. Perkins, L. Schofield. 2006. Regulating immunity to malaria. Parasite Immunol. 28: 35-49. [Medline]
  6. Bradley-Moore, A. M., B. M. Greenwood, A. K. Bradley, A. Bartlett, D. E. Bidwell, A. Voller, J. Craske, B. R. Kirkwood, H. M. Gilles. 1985. Malaria chemoprophylaxis with chloroquine in young Nigerian children. II. Effect on the immune response to vaccination. Ann. Trop. Med. Parasitol. 79: 563-573. [Medline]
  7. Ho, M., H. K. Webster, S. Looareesuwan, W. Supanaranond, R. E. Phillips, P. Chanthavanich, D. A. Warrell. 1986. Antigen-specific immunosuppression in human malaria due to Plasmodium falciparum. J. Infect. Dis. 153: 763-771. [Medline]
  8. Walsh, D. S., S. Looareesuwan, S. Vaniganonta, C. Viravan, H. K. Webster. 1995. Cutaneous delayed-type hypersensitivity responsiveness in patients during and after Plasmodium falciparum and Plasmodium vivax infections. Clin. Immunol. Immunopathol. 77: 89-94. [Medline]
  9. Whittle, H. C., J. Brown, K. Marsh, B. M. Greenwood, P. Seidelin, H. Tighe, L. Wedderburn. 1984. T-cell control of Epstein-Barr virus-infected B cells is lost during P. falciparum malaria. Nature 312: 449-450. [Medline]
  10. Williamson, W. A., B. M. Greenwood. 1978. Impairment of the immune response to vaccination after acute malaria. Lancet 1: 1328-1329. [Medline]
  11. Liebeschuetz, S., S. Bamber, K. Ewer, J. Deeks, A. A. Pathan, A. Lalvani. 2004. Diagnosis of tuberculosis in South African children with a T-cell-based assay: a prospective cohort study. Lancet 364: 2196-2203. [Medline]
  12. Ramalingam, T. R., R. M. Reiman, T. A. Wynn. 2005. Exploiting worm and allergy models to understand Th2 cytokine biology. Curr. Opin. Allergy Clin. Immunol. 5: 392-398. [Medline]
  13. Sabin, E. A., M. I. Araujo, E. M. Carvalho, E. J. Pearce. 1996. Impairment of tetanus toxoid-specific Th1-like immune responses in humans infected with Schistosoma mansoni. J. Infect. Dis. 173: 269-272. [Medline]
  14. Nookala, S., S. Srinivasan, P. Kaliraj, R. B. Narayanan, T. B. Nutman. 2004. Impairment of tetanus-specific cellular and humoral responses following tetanus vaccination in human lymphatic filariasis. Infect. Immun. 72: 2598-2604. [Abstract/Free Full Text]
  15. Malhotra, I., P. Mungai, A. Wamachi, J. Kioko, J. H. Ouma, J. W. Kazura, C. L. King. 1999. Helminth- and bacillus Calmette-Guérin-induced immunity in children sensitized in utero to filariasis and schistosomiasis. J. Immunol. 162: 6843-6848. [Abstract/Free Full Text]
  16. Keating, S. M., P. Bejon, T. Berthoud, J. M. Vuola, S. Todryk, D. P. Webster, S. J. Dunachie, V. S. Moorthy, S. J. McConkey, S. C. Gilbert, A. V. Hill. 2005. Durable human memory T cells quantifiable by cultured enzyme-linked immunospot assays are induced by heterologous prime boost immunization and correlate with protection against malaria. J. Immunol. 175: 5675-5680. [Abstract/Free Full Text]
  17. Gilbert, S. C., M. Plebanski, S. J. Harris, C. E. Allsopp, R. Thomas, G. T. Layton, A. V. Hill. 1997. A protein particle vaccine containing multiple malaria epitopes. Nat. Biotechnol. 15: 1280-1284. [Medline]
  18. Robson, K. J., J. R. Hall, M. W. Jennings, T. J. Harris, K. Marsh, C. I. Newbold, V. E. Tate, D. J. Weatherall. 1988. A highly conserved amino-acid sequence in thrombospondin, properdin and in proteins from sporozoites and blood stages of a human malaria parasite. Nature 335: 79-82. [Medline]
  19. Bejon, P., J. Mwacharo, O. Kai, T. Mwangi, P. Milligan, S. Todryk, S. Keating, T. Lang, B. Lowe, C. Gikonyo, et al 2006. A phase 2b randomised trial of the candidate malaria vaccines FP9 ME-TRAP and MVA ME-TRAP among children in Kenya. PLoS Clin. Trials 1: e29[Medline]
  20. Brent, A. J., I. Ahmed, M. Ndiritu, P. Lewa, C. Ngetsa, B. Lowe, E. Bauni, M. English, J. A. Berkley, J. A. Scott. 2006. Incidence of clinically significant bacteraemia in children who present to hospital in Kenya: community-based observational study. Lancet 367: 482-488. [Medline]
  21. Berkley, J., I. Mwangi, K. Griffiths, I. Ahmed, S. Mithwani, M. English, C. Newton, K. Maitland. 2005. Assessment of severe malnutrition among hospitalized children in rural Kenya: comparison of weight for height and mid upper arm circumference. J. Am. Med. Assoc. 294: 591-597. [Abstract/Free Full Text]
  22. de Onis, M., R. Yip, Z. Mei. 1997. The development of MUAC-for-age reference data recommended by a WHO Expert Committee. Bull. World Health Organ. 75: 11-18. [Medline]
  23. Mwangi, T. W., A. Ross, R. W. Snow, K. Marsh. 2005. Case definitions of clinical malaria under different transmission conditions in Kilifi District, Kenya. J. Infect. Dis. 191: 1932-1939. [Medline]
  24. Mbogo, C. M., J. M. Mwangangi, J. Nzovu, W. Gu, G. Yan, J. T. Gunter, C. Swalm, J. Keating, J. L. Regens, J. I. Shililu, et al 2003. Spatial and temporal heterogeneity of Anopheles mosquitoes and Plasmodium falciparum transmission along the Kenyan coast. Am. J. Trop. Med. Hyg. 68: 734-742. [Abstract/Free Full Text]
  25. Urban, B. C., D. J. Roberts. 2003. Inhibition of T cell function during malaria: implications for immunology and vaccinology. J. Exp. Med. 197: 137-141. [Free Full Text]
  26. Flanagan, K. L., T. Mwangi, M. Plebanski, K. Odhiambo, A. Ross, E. Sheu, M. Kortok, B. Lowe, K. Marsh, A. V. Hill. 2003. Ex vivo interferon-{gamma} immune response to thrombospondin-related adhesive protein in coastal Kenyans: longevity and risk of Plasmodium falciparum infection. Am. J. Trop. Med. Hyg. 68: 421-430. [Abstract/Free Full Text]
  27. Tassignon, J., W. Burny, S. Dahmani, L. Zhou, P. Stordeur, B. Byl, D. De Groote. 2005. Monitoring of cellular responses after vaccination against tetanus toxoid: comparison of the measurement of IFN-{gamma} production by ELISA, ELISPOT, flow cytometry and real-time PCR. J. Immunol. Methods 305: 188-198. [Medline]
  28. De Rosa, S. C., F. X. Lu, J. Yu, S. P. Perfetto, J. Falloon, S. Moser, T. G. Evans, R. Koup, C. J. Miller, M. Roederer. 2004. Vaccination in humans generates broad T cell cytokine responses. J. Immunol. 173: 5372-5380. [Abstract/Free Full Text]
  29. Vuola, J. M., S. Keating, D. P. Webster, T. Berthoud, S. Dunachie, S. C. Gilbert, A. V. Hill. 2005. Differential immunogenicity of various heterologous prime-boost vaccine regimens using DNA and viral vectors in healthy volunteers. J. Immunol. 174: 449-455. [Abstract/Free Full Text]
  30. Bejon, P., S. Keating, J. Mwacharo, O. K. Kai, S. Dunachie, M. Walther, T. Berthoud, T. Lang, J. Epstein, D. Carucci, et al 2006. Early {gamma} interferon and interleukin-2 responses to vaccination predict the late resting memory in malaria-naive and malaria-exposed individuals. Infect. Immun. 74: 6331-6338. [Abstract/Free Full Text]
  31. Moorthy, V. S., E. B. Imoukhuede, S. Keating, M. Pinder, D. Webster, M. A. Skinner, S. C. Gilbert, G. Walraven, A. V. Hill. 2004. Phase 1 evaluation of 3 highly immunogenic prime-boost regimens, including a 12-month reboosting vaccination, for malaria vaccination in Gambian men. J. Infect. Dis. 189: 2213-2219. [Medline]
  32. Abdalla, S. H.. 1988. Peripheral blood and bone marrow leucocytes in Gambian children with malaria: numerical changes and evaluation of phagocytosis. Ann. Trop. Paediatr. 8: 250-258. [Medline]
  33. Flanagan, K. L., E. A. Lee, M. B. Gravenor, W. H. Reece, B. C. Urban, T. Doherty, K. A. Bojang, M. Pinder, A. V. Hill, M. Plebanski. 2001. Unique T cell effector functions elicited by Plasmodium falciparum epitopes in malaria-exposed Africans tested by three T cell assays. J. Immunol. 167: 4729-4737. [Abstract/Free Full Text]
  34. Kassiotis, G., B. Stockinger. 2004. Anatomical heterogeneity of memory CD4+ T cells due to reversible adaptation to the microenvironment. J. Immunol. 173: 7292-7298. [Abstract/Free Full Text]
  35. Geginat, J., F. Sallusto, A. Lanzavecchia. 2001. Cytokine-driven proliferation and differentiation of human naive, central memory, and effector memory CD4+ T cells. J. Exp. Med. 194: 1711-1719. [Abstract/Free Full Text]
  36. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, R. Ahmed. 2003. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4: 1191-1198. [Medline]
  37. Xu, H., J. Wipasa, H. Yan, M. Zeng, M. O. Makobongo, F. D. Finkelman, A. Kelso, M. F. Good. 2002. The mechanism and significance of deletion of parasite-specific CD4+ T cells in malaria infection. J. Exp. Med. 195: 881-892. [Abstract/Free Full Text]
  38. Ndao, M., E. Bandyayera, E. Kokoskin, T. W. Gyorkos, J. D. MacLean, B. J. Ward. 2004. Comparison of blood smear, antigen detection, and nested-PCR methods for screening refugees from regions where malaria is endemic after a malaria outbreak in Quebec, Canada. J. Clin. Microbiol. 42: 2694-2700. [Abstract/Free Full Text]
  39. Akdis, C. A., F. Kussebi, B. Pulendran, M. Akdis, R. P. Lauener, C. B. Schmidt-Weber, S. Klunker, G. Isitmangil, N. Hansjee, T. A. Wynn, et al 2003. Inhibition of T helper 2-type responses, IgE production and eosinophilia by synthetic lipopeptides. Eur. J. Immunol. 33: 2717-2726. [Medline]
  40. Colditz, G. A., T. F. Brewer, C. S. Berkey, M. E. Wilson, E. Burdick, H. V. Fineberg, F. Mosteller. 1994. Efficacy of BCG vaccine in the prevention of tuberculosis: meta-analysis of the published literature. J. Am. Med. Assoc. 271: 698-702. [Abstract/Free Full Text]
  41. McShane, H., A. A. Pathan, C. R. Sander, S. M. Keating, S. C. Gilbert, K. Huygen, H. A. Fletcher, A. V. Hill. 2004. Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired antimycobacterial immunity in humans. Nat. Med. 10: 1240-1244. [Medline]
  42. Mwau, M., I. Cebere, J. Sutton, P. Chikoti, N. Winstone, E. G. Wee, T. Beattie, Y. H. Chen, L. Dorrell, H. McShane, et al 2004. A human immunodeficiency virus 1 (HIV-1) clade A vaccine in clinical trials: stimulation of HIV-specific T-cell responses by DNA and recombinant modified vaccinia virus Ankara (MVA) vaccines in humans. J. Gen. Virol. 85: 911-919. [Abstract/Free Full Text]
  43. Bejon, P., O. K. Kai, J. Mwacharo, S. Keating, T. Lang, S. C. Gilbert, N. Peshu, K. Marsh, A. V. Hill. 2006. Alternating vector immunizations encoding pre-erythrocytic malaria antigens enhance memory responses in a malaria endemic area. Eur. J. Immunol. 36: 2264-2272. [Medline]



This article has been cited by other articles:


Home page
 NEJMHome page
M. Roestenberg, M. McCall, J. Hopman, J. Wiersma, A. J.F. Luty, G. J. van Gemert, M. van de Vegte-Bolmer, B. van Schaijk, K. Teelen, T. Arens, et al.
Protection against a Malaria Challenge by Sporozoite Inoculation
N. Engl. J. Med., July 30, 2009; 361(5): 468 - 477.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
F. C. Hartgers, B. B. Obeng, A. Voskamp, I. A. Larbi, A. S. Amoah, A. J. F. Luty, D. Boakye, and M. Yazdanbakhsh
Enhanced Toll-Like Receptor Responsiveness Associated with Mitogen-Activated Protein Kinase Activation in Plasmodium falciparum-Infected Children
Infect. Immun., November 1, 2008; 76(11): 5149 - 5157.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
E. T. Cadman, A. Y. Abdallah, C. Voisine, A.-M. Sponaas, P. Corran, T. Lamb, D. Brown, F. Ndungu, and J. Langhorne
Alterations of Splenic Architecture in Malaria Are Induced Independently of Toll-Like Receptors 2, 4, and 9 or MyD88 and May Affect Antibody Affinity
Infect. Immun., September 1, 2008; 76(9): 3924 - 3931.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bejon, P.
Right arrow Articles by Hill, A. V. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bejon, P.
Right arrow Articles by Hill, A. V. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS