MHC and malaria: the relationship between HLA class II alleles and immune responses to Plasmodium falciparum.

© 1992 Oxford University Press

International Immunology, Vol. 4, No. 9, pp. 1055- 1063

MHC and malaria: the relationship between HLA class II alleles and immune responses to Plasmodium falciparum E. M. Riley5, 0 . Olerup1, S. Bennett2, P. Rowe2, S. J. Allen, M. J. Blackman3, M. Troye-Blomberg4, A. A. Holder3, and B. M. Greenwood

5

Present address: Institute of Cell, Animal and Population Biology, Division of Biological Sciences, University of Edinburgh, Ashworth Laboratory, West Mains Road, Edinburgh EH9 3JT, UK

Key words: genetic restriction, immune response, malaria, Plasmodium falciparum, ring-infected erythrocyte surface antigen, vaccine Abstract In mice, immune responses to subunits of defined malaria antigens are regulated by genes mapping within the MHC and it has been suggested that such genetic restriction will be a major obstacle in the development of a human malaria vaccine. The relationship between class II human leukocyte antigen (HLA) genes and immune recognition of three candidate antigens for a vaccine against Plasmodium falciparum malaria has been investigated in a human population living in a malaria endemic area of West Africa. The study population was shown to be extremely heterogeneous for HLA class II alleles and marked differences in allelic frequency were detected between members of different ethnic groups. One class II DQA-DQB combination (serological specificity DQw2) was particularly common among members of the Fula ethnic group. This haplotype was significantly associated with higher than average levels of antibody to a peptide epitope, (EENV)6, of the malaria antigen PM55/RESA. There was little evidence of association between HLA class II genotype and cellular proliferative or interferon y responses to the antigens tested. Overall, the number of significant associations between immune responses and specific HLA class II haplotypes was greater than would be expected by chance but less than would be expected if class ll-dependent genetic restriction were a major factor governing human immune responses to malaria antigens. Thus, although some qualitative variation in the immune response to vaccine antigens may occur in ethnically different target populations, widespread HLAassociated nonresponsiveness to a multivalent subunit malaria vaccine is unlikely. Introduction Current attempts to develop vaccines against complex parasitic organisms depend on the identification of immunogenic subunits of parasite proteins which can be produced in vitro by recombinant DNA or synthetic peptide technologies (1). One of the most widespread and potentially fatal human parasitic diseases is Plasmodium falciparum malaria. Several synthetic vaccines against P. falciparum have now reached the stage of preliminary human trials (2-4) and it is likely that an effective malaria vaccine for use in endemic countries will be a multivalent cocktail of defined peptides and/or recombinant proteins.

One potential limitation of small subunit vaccines is that the vaccine peptides may be unable to bind to certain human leukocyte antigen (HLA) class I or class II molecules and that the vaccine may therefore be nonimmunogenic in a proportion of the target population. Immunization studies in mice have shown that the immune response to certain epitopes of the circumsporozoite (CS) protein and the ring-infected erythrocyte surface antigen (Pf 155/RESA) is limited to strains of mouse with particular MHC class II phenotypes (5-7). Furthermore, it has been suggested that MHC-mediated restriction of responses to

Correspondence to: E. M. Riley, Institute of Cell, Animal and Population Biology, Division of Biological Sciences, University of Edinburgh, Ashworth Laboratory, West Mains Road, Edinburgh EH9 3JT, UK Transmitting editor: P. Kourilsky

Received 3 March 1992, accepted 10 June 1992

Downloaded from http://intimm.oxfordjournals.org/ at Russian Archive on January 19, 2014

Medical Research Council Laboratories, Fajara, The Gambia 1 Center for BioTechnology, Karolinska Institute, Novum, Huddinge, Sweden ^Tropical Health Epidemiology Unit, London School of Hygiene and Tropical Medicine, London, UK 3 Division of Parasitology, National Institute for Medical Research, Mill Hill, UK "Department of Immunology, University of Stockholm, Stockholm, Sweden

1056

HLA alleles and immune responses to malaria

Humoral and cellular immune responses to a number of potential P. falciparum vaccine antigens have previously been examined in a malaria endemic population in the Gambia and compared with subsequent susceptibility to malaria infection (14-16). The HLA class II (DR and DO) genotypes of the subjects included in these studies have now been determined by restriction fragment length polymorphism (RFLP) analysis (17). In this study the relationship between HLA class II genotype and immune responses [serology, lymphocyte proliferation, and interferon y (IFN-7) release] to three defined P. falciparum antigens—the CS protein, Pf155/RESA, and the major merozoite surface protein (PfMSPI)—are examined and the implications for vaccine development discussed. Methods Study population

Table 1. Antigens used in cellular and serological assays Antigen

Epitope

Assaya

PfCSPb

Th2R Th3R repeats

PSDQHIEKYLKTIQNSLSTE IKPGSANKPKDQLDYANDIE (NANP)40

LP/IFN LP/IFN ELISA

MSP1C

pME1 pME2 pME6 pME7 pME11 pME14 MSP 1 8 3 S42DA

aa aa aa aa aa aa aa aa

LP/IFN LP/IFN LP/IFN LP/IFN LP/IFN LP/IFN ELISA ELISA

Pf155/RESAd

P1 P2 P3 P4 P5 P6 P7

TVAEEHVEEPTVAEE SLRWIFKHVAKTHLK DWGYIMHGISTINKEMK EENVEHDAEENVEENV (EENV)6 (DDEHVEEPTVA)2 (EENVEHDA)3

154-1047 1046-1639 181 - 3 8 8 348-494 1209-1614 902-1499 20 - 668 1348-1620

LP/IFN LP/IFN LP/IFN LP/IFN ELISA ELISA ELISA

a

LP - lymphoproliferation; IFN - interferon gamma. According to the sequence published by De la Cruz er al. (44). c Numbers refer to the sequence of the Wellcome (Lagos) strain (45). d According to the sequence published by Favoloro et al. (46). b

has been described previously (14 -16). In brief, microtitre plates were coated with purified recombinant protein or with synthetic peptides conjugated to bovine serum albumin (BSA) using glutaraldehyde. Control plates were coated with coating buffer or with glutaraldehyde-treated BSA. After washing, test sera (diluted in PBS containing BSA and Tween 20) were added to duplicate wells. After incubation at 37°C, the plates were washed and developed with alkaline phosphatase or horseradish peroxidase-conjugated anti-human Ig and the appropriate enzyme substrate. Results are given as antibody units (AU)/ml l of Ig calculated by titration of a known standard serum.

The study subjects were children and adults who were permanent residents of a group of villages on the north bank of the Gambia river - 1 5 0 km from the coast (14). Thirty per cent of the population belong to the Mandinka ethnic group, 30% are Wollof, and the remainder are Fula. Malaria transmission in this region is seasonal with most new infections occurring in the period July-November (18). At the beginning of the malaria transmission season (May), venous blood samples were collected from 355 children aged 3 - 8 years (group A). At the end of the transmission season (November), samples were collected from an additional 283 subjects aged 9 - 8 6 years; immunological data were obtained from only 141 of the 283 adult subjects (group B). The ethnic composition of the two groups varied, with very few Fula people included in group B. Control subjects were 30 adult Europeans and 30 European children with no prior exposure to malaria. Serum was separated from heparinized blood samples and stored at - 2 0 ° C until required. Mononuclear cells were separated by buoyant density centrifugation. Haemoglobin was extracted from the red cell pellet and used for detection of carriage of the sickle cell trait by electrophoresis.

HLA class II typing was performed by Southern blot analysis of Taq 1 -cleaved DNA obtained from peripheral blood leukocytes (20). Allelic RFLP patterns at each locus (DRB, DOA, and DOB) were designated by Roman numerals (17,20,21). For the purpose of this communication, Arabic numerals have also been ascribed to individual DRB-DOA-DOB haplotypes (Table 2).

Immunological assays

Statistical methods

Serology. Antibodies to a number of defined antigens were measured by ELISA (Table 1). The methodology for each assay

Because the potential number of comparisons which could be made on this data set was enormous, the following strategy was

7 cell responses. Lymphocyte proliferation and IFN-7 production were measured in 7 day cultures of peripheral blood mononuclear cells as described previously (14). The antigens used for these assays are given in Table 1. Lymphocyte proliferation was assessed by incorporation of [3H]thymidine. The geometric mean of triplicate antigen-stimulated cultures was divided by the geometric mean of control cultures to give a stimulation index (SI) (14). IFN-7 production was measured by a two site ELISA using two murine mAbs to recombinant human IFN-7 as described previously (19). IFN-7 levels were calculated by comparison with the international human IFN-7 standard Gg23-901-530 (NIAID, Bethesda, MD). HLA class II typing


immunodominant epitopes in man may explain the apparent lack of response to defined malaria antigens seen in a proportion of individuals living in malaria endemic areas (8,9). The role of the MHC in determining resistance to malaria in man under conditions of natural exposure to infection is unclear (10). Variations in the distribution of MHC alleles between different populations have been attributed to the selective pressure exerted by malaria (11,12) and it has been suggested that this selection operates at the level of the immune system. A recent case-control study in the Gambia has indicated that certain HLA class I and class II alleles (serological specificities Bw52 and DRw13 -DQw5) may protect against the development of severe malaria (cerebral malaria and severe anaemia) but the mechanisms by which such protection might operate are unclear (13). Furthermore, preliminary data from studies of monozygous and dizygous African twins suggests that antibody responses to synthetic peptides representing the primary sequence of Pf155/RESA may be regulated by non-MHC-associated genes (30).

HLA alleles and immune responses to malaria 1057 Table 2. Distribution of HLA class II haplotypes in the Gambian population8 RFLP pattern DRB

DQA

DQB

Associated serologic specificity DQ DR

1 2 7 8 9 11 12 14 15 17 18 21 22 23 26 29 34 35 37 38 39

I II VIII XV XXIII XII XII XIII XIV V V XVII III VI XX XXI IX IX XI X XV

I II IV VI VI V V V V VI IV V I IV IV IV III III III II IV

I II III IV IV IV VI VI VI V V IX I V V V II I II I V

1 w15 w17 w18 3 4 4 7 7 w8 w8 9 w10 w11 w11 w11 w13 w13 w13 w13 w13

w5 w6 w2 w4 w4 w8 w2 w2 w2 w7 w7 w2 w5 w7 w7 w7 w6 w1 w6 w6 w7

Ethnic group Fula (n = 590)

Mandinka (n = 382)

Wollof (n = 304)

Caucasian (n = 500)

0.019 0.012 0.048 0.014 N 0.036 0.071 0.024 0.115 0.068 0.019 0.048 0.097 0.017 0.025 0.131 0.038 0.029 0.029 0.054 0.015

0.024 0.013 0.034 0.016 0.034 0.018 0.002 0.044 0.024 0.024 0.013 0.045 0.047 0.031 0.005 0.359 0.008 N 0.005 0.115 0.031

0.016 0.010 0.030 0.020 0.016 0.023 0.003 0.016 0.010 0.016 0.040 0.102 0.115 0.033 0.020 0.211 0.020 N 0.003 0.165 0.033

0.096 0.172 0.004 Nc N 0.064 N 0.012 0.O22 0.002 N N 0.010 0.070 N N 0.050 N N 0.056 0.008

a

Genotype frequencies based on RFLP typing of 638 Gambian samples. This data has previously been presented in a slightly different format (17). Recognized Caucasian haplotypes that are present in the Gambian population at a frequency of 2 0 5 d.f. (group A) or 8 and > 119 d.f. (group B). IFN = Interferon gamma. Table shows x 2 statistic o n 8 d.f. (both groups). ELISA = antibody levels measured by ELISA. Table shows F statistic for MSP1 proteins and x 2 statistic (9 d.f. for Group A and 8 d.f. for Group B) for all other peptides. n 0 . 0 5 < P < 0.10; 'P < 0.05; "P < 0 . 0 1 ; * * * P < 0.001. b

C


as a discontinuous variable in group A and a continuous variable in group B), sex, and previous malaria control interventions (14). The percentage of the total variance explained by the confounding variables ranged from < 1 to 24%. For group A (children) allowance was also made for carriage of the sickle cell trait and concurrent parasitaemia. Ethnic group was not included as a confounder because its causal association with haplotype would have led to a masking of the association between haplotype and disease. The effect of individual haplotypes was evaluated by comparing the ratio of the parameter estimate to its SE with a normal distribution. Gambian villages are made up of compounds which accommodate large extended families. Thus, in addition to sharing HLA haplotypes and a variable number of non-HLA (background) genes, related individuals also share the same environment. The effect of shared environment and, to a large extent, the effect of shared background genes (insofar as the compound is identical with the family) were removed by stratifying on compound using conditional logistic regression (25), which

HLA alleles and immune responses to malaria the subjects in group B. Thus, these data were analysed using the haplotypes appropriate to group B. Associations between specific immune responses and HLA class II haplotype

Analysis of responses to the antigens identified by the omnibus test revealed the individual haplotypes that were associated with significantly increased or decreased cellular or antibody responses to the CS protein, MSP1, and Pf 155/RESA (Table 6). After adjusting for the effects of multiple comparisons (i.e. that the likelihood of obtaining a statistically significant result increases in proportion to the number of comparisons being made) only six associations between individual alleles and particular immune responses remained significant. Five of the associations were shown in the children (group A) and only one in the smaller and less diverse sample of adults (group B). Haplotypes 12 and 15, which are common only in members of the Fula ethnic group, were positively associated with the

presence of antibodies to one repetitive epitope of Pf155/RESA, (EENV)6. These two haplotypes were also associated with higher than average titres of antibody to the 83 kDa N-terminal fragment of MSP1 (haplotype 15) and the 42 kDa C-terminal sequence of the same protein (haplotype 12). Stratified analysis, conditional on compound of residence, confirmed the overall association of HLA type with antibody response to (EENV)6 (x2 = 19.5, 8 d.f., P < 0.02) and the individual association with both haplotypes 12 and 15 with this response (P < 0.05 in each case) independent of the effects of shared background genes or shared environment. The associations between responses to the 83 and 42 kDa fragments of MSP1 and HLA type (either overall or for individual haplotypes) became non-significant following such a conditional analysis (x2 = 6.5 and 9.7 respectively on 9 d.f.). Associations were also observed between lymphoproliferative responses to two recombinant proteins representing regions of MSP1 (Table 6). Among the children, haplotype 7 was associated with lower than average responses to pME7 and among the adults, haplotype 22 was associated with higher than average responses to pME14. These associations were only just significant after adjusting for multiple comparisons and, given the very large number of comparisons made, it is possible that they represent chance events. Association between HLA - DQw2 and antibody responses to P1155/RESA and MSP1 Because haplotypes 12 and 15 differ in their DRB genes but share the same Taq1 DQA and DQB RFLPs with the serological specificity of DQw2 (Table 2), the possibility that the association between these haplotypes and higher than average antibody titres might be mediated by this shared DOA/DQB specificity was examined. Several Gambian haplotypes (7,12,14, and 15) bear the serological specificity of DQw2 but the particular allelic RFLP pattern DQA-V/DQB-VI is observed only in haplotypes 12, 14,

Table 6. Associations between class II haplotypes and increased or decreased immune responses to defined antigensa Protein

Epitope

Assayb

CSP

Th2R

LP LP IFN

MSP1

pME1

LP LP IFN IFN IFN LP LP LP LP ELISA ELISA ELISA ELISA

pME2 pME6 pME7 pME11 pME14 MSP1fj3 Pf155/RESA

S42AA P5

Association0

+ _ + + + + + _ + — + +

Haplotyped Group A

Group B

12,22 21 29 29 15,38 9 39 9,22 7* 21" 12 12,15*",17

22 22" 9,29**

7,11,12*",15 12"*, 1 5 ' "

a AII associations shown, P < 0.05 except for " (P < 0.01) and * " (P < 0.001) (these associations remain significant after correction for multiple comparisons). b LP = lymphoproliferation; IFN = interferon gamma. c + and - indicate that these haplotypes are associated with either an increased ( + ) or a decreased (-) immune response. d Haplotype numbers refer to those given in Table 2.


The omnibus test for overall association between class II haplotype and immune response indicated that immune responses to a number of antigens varied between individuals bearing different class II haplotypes (Table 5). Among children in group A, nine out of the 36 comparisons made showed a significant association (at the 5% level) between an immune response and an HLA class II haplotype; the corresponding figure for group B was four out of 38 comparisons. Although this is more than would be expected by chance if there was no HLA-related variation in immune responses, it is less than would have been expected in such a large sample if class ll-mediated genetic restriction was the overriding factor in determining the immune response to a defined antigen. In fact, the multiple regression analyses showed that HLA-related variation accounted for only between 1 and 14% (Rz values) of the total variation in immune response observed in this population.

1059

1060

a.


too

0-4

5-9

10-19

20-39

> "10

Antibody concentration (jig/ml)

Discussion

30

20

10

< I

2-10

11-20 2 1 - 5 0 51-100 > 100

Antibody concentration (AU/ml)

c.

40

~

30

20

I

io

I I 100

Antibody concentration (AU/ml) Fig. 1. Antibody responses to potential malaria vaccine antigens in Gambian children (Group A) aged 3 - 8 years: (a) Antibodies to the (EENV)6 epitope of PM55/RESA; (b) antibodies to MSPIga; and (c) antibodies to S42AA. Frequency (%) of response at different antibody levels. Hatched columns represent children possessing DOA-V/DOB-VI (DQw2) (heterozygous or homozygous); open columns represent children without this haplotype.

The detection of MHC-dependent genetic restriction of immune responses to defined peptide epitopes of malaria antigens in mouse models has led to the fear that genetically regulated non-responsiveness to malaria antigens may be widespread in human populations and could be a major obstacle in the development of an effective subunit malaria vaccine (8,26). This fear has been reinforced by data from epidemiological studies which indicate that serological non-responsiveness to some defined malaria antigens—including the (NANP)40 epitope of the P. falciparum CS protein, peptide epitopes of Pf155/RESA, and the gametocyte surface antigens—is common in endemic populations (9,27,28). This has been interpreted as evidence for genetic regulation of the human immune response to malaria. However, under conditions of natural exposure to polymorphic parasite antigens, there are many explanations for individual variation in serological responses that are independent of host genetic make-up (10). In an effort to determine the practical importance of genetic constraints in the development of antimalarial immunity in endemic human populations, naturally acquired cellular and humoral immune responses to defined P. falciparum antigens were examined in individuals of differing HLA class II genotype. Detection of such genetic influences in an outbred population, most of whom are heterozygous at each class II locus (17) and therefore express two complete sets of class II antigens, requires a large study population. For example, in this study an association between the possession of DQA-VIDQB-VI(DQw2) and the high frequency of antibodies to the (EENV)6 peptide of Pf155/RESA has been demonstrated. This association was not apparent in a smaller study with a less diverse range of class II haplotypes (29). It is possible that study of a larger cohort than that described here would reveal additional associations between HLA antigens and defined immune responses. Also, a different pattern of class II associations may be seen in studies conducted in other malaria-endemic populations with different ethnic origins. Genetic regulation of antibody responses to Pf155/RESA has previously been inferred from longitudinal studies which showed that, under conditions of continual year-round malaria transmission, individuals tend to have consistently low or consistently high titres of antibody to Pf155/RESA-derived peptides (28). A previous study of the association between HLA class II genotype and responses to Pf155/RESA-derived peptides, and a study of


b.

and 15. One hundred and three of the children in Group A were either heterozygous or homozygous for DQA-V/DOB-VI. Twentysix of these 103 DQA-VIDQB-VI-bearing children (25.2%) possessed antibodies to (EENV)6 while only 19 of 247 (7.7%) DQ/4-WDQB-W-negative age-matched children possessed these antibodies (Fig. 1a). This difference was highly significant (x2 = 18.4, 1 d.f., P < 0.0001). Children bearing DQA-V/DOB-VI also had higher titres of antibody to MSPI^ (Fig. 1 b; Wilcoxon test: Z = 2.63, P < 0.01). The prevalence of antibodies to MSP142 was higher amongst children bearing the DOA-V/DOB-VI haplotype than amongst children without this haplotype [32/103 (31%) versus 45/246 (18%) respectively; x2 = 6.2, 1 d.f., P < 0.05] but the titres of antibody-positive children were not significantly different between the two groups (Wilcoxon test: Z = 1.40, NS).

HLA alleles and immune responses to malaria 1061

Associations were also observed between possession of certain DRB - DOA - DQB haplotypes and immune responses to various regions of PfMSPI. However, in this case, the associations became nonsignificant after conditional stratified analysis. This, together with the fact that, as for Pf155/RESA, all the apparent associations were with haplotypes that are relatively more common among Fulas than among Mandinka or Wollof people, suggests that associations between immune responses to PfMSPI and HLA class II haplotypes may in fact reflect the influence of shared background, non-MHC genes, or shared environment. Ethnic differences in susceptibility to malaria infection have previously been recorded in this population (33) and it has been suggested that Fula people are genetically predisposed to the development of hyper-reactive malarial splenomegaly (HMS) (33,34). HMS is believed to be the result of abnormal regulation of the humoral immune response to malaria antigens; family studies and ethnic group comparisons

indicate that the disease may have a genetic basis, but attempts to link this to the HLA region have been inconclusive (35). The increased prevalence and high concentration of antibodies to blood stage malaria antigens such as PfMSPI and Pf155/RESA among members of the Fula population may be an indication of defective immunoregulation rather than enhanced protective immunity to malaria. Interestingly, a parallel study of the relationship between HLA genotype and clinical manifestations of malaria in The Gambia (13) indicates that haplotype 15 (DR7-DQw2) is associated with an increased risk of severe anaemia, a complication of falciparum malaria which is believed to be the result of an inability to clear low levels of parasites from the circulation. Children with severe anaemia tend to have higher levels of antibodies to Pf155/RESA peptides than children with mild malaria or cerebral malaria (36), suggesting that these antibodies may be markers for prolonged parasitaemia. The only evidence of any effect of class II haplotype on cellular responses to malaria antigens came from a rather weak association between low lymphoproliferative responses to the pME7 region of MSP1 and haplotype 7 in children, and between high proliferative responses to pME14 and haplotype 22 in adults. It is possible that these are spurious associations arising as a result of the very large number of comparisons that were made. Processing of large molecules presumably allows a comprehensive array of peptides to be presented to the T cell repertoire, at least some of which are capable of interacting with any particular expressed class II specificity. Thus, given that most individuals are heterozygous, primary class ll-mediated nonresponsiveness to complex antigens is unlikely. However, it is possible that immune responses to short synthetic vaccine peptides may be more obviously restricted to some class II phenotypes. The failure to detect class ll-associated genetic restriction of humoral responses to the CS protein in individuals naturally immunized by exposure to whole live sporozoites confirms previous reports (37,38) and is in accordance with the recent demonstration that intact sporozoites can induce anti-sporozoite antibodies in a T cell independent MHC-unrestricted manner, with the repetitive regions of the CS protein functioning as a classical T-independent antigen (39). Also a 21 amino acid sequence from the C-terminus of the CS protein has been shown to contain multiple overlapping T cell epitopes which can be recognised in the context of a wide range of DR and DQ specificities (40). The association reported previously between lymphoproliferative responses'to the Th2R and Th3R regions of the CS protein and HLA-DRw13 (41) has since been shown to be spurious (42). Indeed, none of the immune responses investigated in this study appeared to be associated (either negatively or positively) with HLA-DRw13, an allele which has been postulated to confer resistance to severe malarial anaemia (13). In conclusion, it would appear from the studies reported here and elsewhere (29,43) that non-responsiveness to defined malaria antigens is not primarily due to HLA class ll-mediated genetic restriction. The only statistically significant association that resulted from this data set was not an absolute association between class II genotype and responsiveness, and it is possible that it may have arisen by chance. However, we did observe clustering of apparent associations within members of one particular ethnic group. Although attempts were made to correct for the effects of shared environment and familial relationships, it remains


dizygous and monozygous twins and HLA-matched siblings, indicated that these responses may be under genetic control but suggested that the controlling genes are not linked to the HLA region (29,30). However, in this much larger study a highly significant association was observed between the antibody response of children to one R155/RESA peptide, (EENV)6, and possession of one particular class II allele, DOA-V/DQB-VI (DQw2). This association persisted after allowance was made for shared environment and other non-MHC genes. This allele(DQAV/ DOB-VI) is found at high frequency in only one of the three ethnic groups studied here (Fula) and was not seen in any of the subjects of the twin study (30). The association was apparent in children (group A) but the number of Fulas in the adult cohort (group B) was too small for the effect to be observed. The association between DQA-V/DQB-VI (DQw2) and antipeptide antibodies was not absolute; some DOA-V/DOB-VInegative individuals were seropositive and many DOA-V/DOBW-positive individuals were seronegative. Many of the subjects in this study were young children and antibody titres were measured at the end of the long dry season, several months after malaria transmission ceased. It is possible that possession of DOA-V/DQB-VI may lead to a superior T helper or memory T cell response, resulting in the production of high titres of high affinity antibodies which persist in the serum from one year to the next. However, none of the lymphoproliferative or IFN-7 responses that were measured appeared to be associated with possession of the DOA-V/DOB-VI haplotype. Thus, if the association between antibodies to (EENV)6 and DQw2 is a true HLA association (and not an association with a closely linked non-HLA locus), the T helper epitope involved does not appear to be any of the epitopes that were investigated in this study. Additional T cell epitopes have been identified within the Pf155/RESA molecule (31) but these were not included in this study. It is possible that the assays of T cell function used in this study were not appropriate for detecting mature T helper cell clones. In general, proliferative and IFN-7 responses to the Pf155/RESA peptides were rather infrequent (see primary data in 15) and a close correlation between the presence of serum antibody to Pf 155/RESA and the production of IL-4 (but not IFN-7) by (EENV)6-activated T cells has recently been shown (15,32). Also, it is possible that T cell help for production of anti-(EENV)6 antibodies could be provided by cells which are specific for other malaria antigens.

1062


possible that genetically determined, but non-MHC-dependent, effects may be responsible for the observed associations. Further studies are required to evaluate the relative importance of MHCand non-MHC-associated genes in the regulation of immune responses to malaria antigens.

Acknowledgements

Abbreviations AU BSA CS HLA HMS IFN- 7 LP PfMSPI RESA RFLP SI

antibody units bovine serum albumin circumsporozoite human leukocyte antigen hyper-reactive malarial splenomegaly interferon gamma lymphoproliferation Plasmodium falciparum merozoite surface protein 1 ring-infected erythrocyte surface antigen restriction fragment length polymorphism stimulation index

References 1 Zanetti, M., Sercarz, E., and Salk, J. 1987. The immunology of new generation vaccines. Immunol. Today 8:18. 2 Ballou, W. R., Hoffman, S. L., Sherwood, J. A., Hollingdale, M. R., Neva, F. A., Hockmeyer, W. A., Gordon, D. M., Schneider, I., Wirtz, R. A., Young, J. F., Wasserman, G. F., Reeve, P., Diggs C. L, and Chulay, J. D. 1987. Safety and efficacy of a recombinant DNA. Plasmodium falciparum sporozoite vaccine. Lancet i:1277. 3 Herrington, D. A., Clyde, D. F., Losonsky, G., Cortesia, M., Murphy, J. R., Davis, J., Baqar, S., Felix, A. M., Heimer, E. P., Gillessen, D., Nardin, E., Nussenzweig, R. S., Hollingdale, M. R., and Levine, M. M. 1987. Safety and immunogenicity in man of a synthetic peptide malaria vaccine against Plasmodium falciparum sporozoites. Nature 328:257. 4 Patarroyo, M. E., Amador, R., Clavijo, P., Moreno, A., Guzman, F., Romero, P., Tascon, R., Franco, A., Murillo, L. A., Ponton, G., and Trujillo, G. 1988. A synthetic vaccine protects humans against challenge with asexual blood stages of Plasmodium falciparum malaria. Nature 332:158. 5 Del Giudice, G., Cooper, J. A., Merino, J., Verdini, A. S., Pessi, A., Togna, A. R., Engers, H. D., Corradin, G. P., and Lambert, P. H. 1986. The antibody response in mice to carrier-free synthetic polymers of Plasmodium falciparum circumsporozoite repetitive epitope is I-Ab restricted. Implications for malaria vaccines. J. Immunol. 137:2952. 6 Good, M. F., Berzofsky, J. A., Maloy, W. L., Hayashi, Y., Fujii, N., Hockmeyer, W. T., and Miller, L. H. 1986. Genetic control of the immune response in mice to a Plasmodium falciparum sporozoite vaccine: widespread non-responsiveness to single malaria T epitope in highly repetitive vaccine. J. Exp. Med. 164:655. 7 Lew, A. M., Langford, C. J., Pye, D., Edwards, S., Corcoran, L., and Anders, R. F. 1989. Class II restriction in mice to the malaria candidate


We are grateful to the people of the Kataba and Fula villages for their enthusiastic participation in this programme and to all the staff of the MRC Laboratories at Farafenni and Fajara for their commitment to this project. We would like to thank J. G. Wheeler for assistance with data processing and statistical analysis; W. L. Maloy, A. S. Pessi, and A. Verdini for providing the synthetic CS protein peptides; R. A. Houghten and K. Berzins for the Pf155/RESA peptides; L. Smedman for obtaining the European control blood samples; and G. Andersson for producing the IFN-7 mAbs. This project received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, the Swedish Medical Research Council, and the Swedish Association for Research Co-operation with Developing Countries (SAREC). E.M.R. is a Wellcome Trust Senior Fellow.

vaccine ring-infected erythrocyte surface antigen (RESA) as synthetic peptides or as expressed in recombinant vaccinia. J. Immunol. 142:4012. 8 Good, M. F., Miller, L. H., Kumar, S., Quakyi, I. A., Keister, D., Adams J. H., Moss, B., Berzofsky, J. A., and Carter, R. 1988. Limited immunological recognition of critical malaria vaccine candidate antigens. Science 242:574. 9 Quakyi, I. A., Otoo, L. N., Pombo, D., Sugars, L. Y., Menon, A., De Groot, A. S., Johnson, A., Ailing, D., Miller, L. H., and Good, M. F. 1989. Differential non-responsiveness in humans to candidate Plasmodium falciparum vaccine antigens. Am J. Trop. Med. Hyg. 41:125. 10 Riley, E. M., Olerup, O., and Troye-Blomberg, M. 1991. Immunological recognition of malaria antigens. Parasitol. Today 7:5. 11 Restrepo, M., Rojas, W., Montoya, F., Montoya, A. E., and Dawson, D. 1988. HLA and malaria in four Colombian ethnic groups. Rev. Inst. Med. trop. Sao Paulo 30:323. 12 Piazza, A., Belvedere, M. C , Bernoco, D., Conighi, C , Contu, L., Curton, E. S., Mattiuz, P. L, Mayr, W., Richiardi, P., Scudeller.G., and Ceppellini, R. 1973. HLA variation in four Sardinian villages under differential selective pressure by malaria. In Dausset, J. and Colombani, J., eds, Histocompatibility Testing 1972, pp. 73. Williams and Wilkins, Baltimore, MD. 13 Hill, A. V. S., Allsopp, C. E. M., Kwiatkowski, D., Anstey, N. M., Twumasi, P., Rowe, P., Bennett, S., Brewster, D., McMichael, A. J., and Greenwood, B. M. 1991. Common West African HLA antigens are associated with protection from severe malaria. Nature 352:595. 14 Riley, E. M., Allen, S. J., Bennett, S., Thomas, P. J., O'Donnell, A., Lindsay, S. W., Good, M. F., and Greenwood, B. M. 1990. Recognition of dominant T cell stimulating epitopes from the circumsporozoite protein of Plasmodium falciparum and relationship to malaria morbidity in Gambian children. Trans. R. Soc. Trop. Med. Hyg. 84:648. 15 Riley, E. M., Allen, S. J., Troye-Blomberg, M., Bennett, S., Perlmann, H., Andersson, G., Smedman, L., Perlmann, P., and Greenwood, B. M. 1991. Association between immune recognition of the malaria vaccine candidate antigen PH55/RESA and resistance to clinical disease: a prospective study in a malaria endemic region of West Africa. Trans. R. Soc. Trop. Med. Hyg. 85:436. 16 Riley, E. M., Allen, S., J., Wheeler, J., Blackman, M. J., Bennett, S., Takacs, B., Schonfeld, H.-J., Holder, A. A., and Greenwood, B. M. 1992. Naturally acquired cellular and humoral immune responses to the major merozoite surface antigen of Plasmodium falciparum are associated with reduced malaria morbidity. Parasite Immunol. 14:321. 17 Olerup, O., Troye-Blomberg, M., Schreuder, G. M. T. and Riley, E. M. 1991. HLA-DR and -DO. gene polymorphism of West Africans is twice as extensive as in North European Caucasians: evolutionary implications. Proc. Natl Acad. Sci. USA 88:8480. 18 Greenwood, B. M., Bradley, A. K., Greenwood, A. M., Byass, P., Jammeh, K., Marsh, K., Tulloch, S., Oldfield, F. S. J., and Hayes, R. 1987. Mortality and morbidity from malaria among children in a rural area of the Gambia, West Africa. Trans. R. Soc. Trop. Med. Hyg. 81:478. 19 Andersson, G., Ekre, H.-P. T., Aim, G., and Perlmann, P. 1989. Monoclonal antibody two-site ELISA for human IFN-gamma. Adaptation for determinations in human serum or plasma. J. Immunol. Methods 125:89. 20 Carlsson, B., Wallin, J., Bohme, J., and Moller, E. 1987. HLA-DRDQ haplotypes defined by restriction fragment length analysis: correlation to serology. Hum. Immunol. 20:95. 21 Olerup, O., Hillert, J., Frederikson, S., Olsson, T., Kam-Hansen, S., Moller, E., Carlsson, B., and Wallin, J. 1989. Primarily chronic progressive and relapsing/remitting multiple sclerosis: two immunogenetically distinct disease entities. Proc. Natl Acad. Sci. USA 86:7113. 22 Farewell, V. T. and Dahlberg, S. 1984. Some statistical methodology for the analysis of HLA data. Biometrics 40:547. 23 Thomson, G. 1981. A review of theoretical aspects of HLA and disease associations. Theor. Popul. Biol. 20:168. 24 Bennett, S. and Riley, E. M. 1991. The statistical analysis of lymphocyte proliferation data from immuno-epidemiological studies J. Immunol. Methods 146:229. 25 Breslow, N. E. and Day, N. E. 1980. Statistical methods in Cancer Research, Volume 1: The analysis of case-control studies. International Agency for Research on Cancer, Lyon.


Berzins, K., Riley, E. M., and Greenwood, B. M. 1992. Malaria antibody levels in Gambian children with severe or mild malaria. Clin. Exp. Immunol, in press. 37 Graves, P. M., Bhatia, K., Burkot, T. R., Prasad, M., Witz, R. A., and Beckers, P. 1989. Association between HLA type and antibody response to malaria sporozoite and gamete epitopes is not evident in immune Papua New Guineans. Clin. Exp. Immunol. 78:418. 38 Brown, A. E., Chandanayingyong, D., Fuggle, S. V., and Webster, H. K. 1989. Immune responses to the circumsporozoite protein of Plasmodium falciparum in relation to HLA-DR type. Tissue Antigens 34:200. 39 Schofield, L. and Uadia, P. 1990. Lack of Ir gene control in the immune response to malaria. 1. A thymus-independent antibody response to the repetitive surface protein of sporozoites. J. Immunol. 144:2781. 40 Sinigaglia, F., Guttinger, M., Kilgus, J., Doran, D. M., Matile, H., Etlinger, H., Trzeciak, A., Gillessen, D., and Pink, J. R. L. 1988. A malaria T-cell epitope recognised with most mouse and human MHC class II antigens. Nature 336:778. 41 De Groot, A. A., Johnson, A. H., Maloy, W. L, Quakyi, I. A., Riley, E. M., Menon, A., Banks, S. M., Berzofsky, J. A., and Good, M. F. 1989. Human T cell recognition of polymorphic epitopes from malaria circumsporozoite protein. J. Immunol. 142:4000. 42 Bennett, S. 1991. Memorable responses: human T cell recognition of polymorphic epitopes. Parasitol. Today 7:123. 43 Riley, E. M., Ong, C. S. L, Olerup, O., Eida, S., Allen, S. J., Bennett, S., Andersson, G., and Targett, G. A. T. 1990. Cellular and humoral responses to Plasmodium falciparum gametocyte antigens in malariaimmune individuals: limited response to the 48/45 kD surface antigen does not appear to be due to MHC restriction. J. Immunol. 144:4810. 44 De la Cruz, V. F., Lai, A. A., and McCutchan, T. F. 1987. Sequence variation in putative functional domains of circumsporozoite protein of Plasmodium falciparum. Implications for vaccine development. J. Biol. Chem. 262:11935. 45 Holder, A. A., Lockyer, M. J., Odink, K. G., Sandhu, J. S., Riveros-Moreno, V., Nicholls, S. C , Hillman, Y., Davey, L. S., Tizard, M. L. V., Schwartz, R. T., and Freeman, R. R. 1985. Primary structure of the precursor to the three major surface antigens of Plasmodium falciparum merozoites. Nature 317:270. 46 Favoloro, J. M., Coppel, R. L., Corcoran, L. M., Foote, S. J., Brown, G. V., Anders, R. F., and Kemp, D. J. 1986. Structure of the RESA gene of Plasmodium falciparum. Nucleic Acids Res. 14:8265.


26 Good, M. F., Kumar, S., and Miller, L. H. 1988. The real difficulties for malaria sporozoite vaccine development: nonresponsiveness and antigenic variation. Immunol. Today 9:351. 27 Carter, R., Graves, P. M., Quakyi, I. A., and Good, M. F. 1988. Restricted or absent immune responses in human populations to Plasmodium falciparum gamete antigens which are the targets of malaria transmission-blocking antibodies. J. Exp. Med. 169:147. 28 Perlmann, H., Perlmann, P., Berzins, K., Wahlin, B., Troye-Blomberg, M., Hagstedt, M., Andersson, I., Hogh, B., Petersen, E., and Bjorkman, A. 1989. Dissection of the human antibody response to the malaria antigen Pf 155/RESA into epitope specific components. Immunol. Revs. 112:113. 29 Troye-Blomberg, M., Olerup, O., Larsson, A., Sjoberg, K., Perlmann, H., Riley, E., Lepers, J.-P., and Perlmann, P. 1991. Failure to detect MHC class II associations of the human immune response induced by repeated malaria infections to the Plasmodium falciparum antigen Pf155/RESA. Int. Immunol. 3:1043. 30 Sjoberg, K., Lepers, J. P., Raharimalala, L., Larsson, A., Olerup, O., Marbiah, N. T., Troye-Blomberg, M., and Perlmann, P. 1992. Genetic regulation of human anti-malarial antibodies in twins. Proc. NatlAcad. Sci. USA 89:2101. 31 Troye-Blomberg, M., Riley, E. M., Perlmann, H., Andersson, G., Snow, R. W., Allen, S. J., Houghten, R. A., Olerup, O., Greenwood, B. M., and Perlmann, P. 1989. T- and B-cell responses of Plasmodium falciparum malaria immune individuals to synthetic peptides corresponding to epitopes in the conserved repeat regions of the P. falciparum antigen Pf155/RESA. J. Immunol. 143:3043. 32 Troye-Blomberg, M., Riley, E. M., Kabilan, L, Holmberg, M., Perlmann, H., Andersson, U., Heusser, C. H., and Perlmann, P. 1990. Production by activated human T cells of IL-4, but not IFN7, is associated with elevated levels of serum antibodies to the activating malaria antigens. Proc. Natl Acad. Sci. USA 87:5484. 33 Greenwood, B. M., Groenendaal, F., Bradley, A. K., Greenwood, A. M., Shenton, F., and Tulloch, S. 1987. Ethnic differences in the prevalence of splenomegaly and malaria in The Gambia. Ann. Trop. Med. Parasitol. 81:345. 34 Bryceson, A. D. M., Fleming, A. F., and Edington, G. M. 1976. Splenomegaly in Northern Nigeria. Ada Trop. 33:424. 35 Crane, G. G. 1986. Hyperreactive malarious splenomegaly (tropical splenomegay syndrome). Parasitol. Today 2:4. 36 Erunkulu, O. A., Hill, A. V. S., Kwiatkowski, D. P., Todd, J. E., Iqbal, J.,

1063


Association between HLA-Class I and HLA-Class II Alleles and Mycobacterium Tuberculosis Infection in Iraqi Patients from Baghdad City.

The relationships between HLA class II alleles and antigens with gestational diabetes mellitus: A meta-analysis.

Erratum: Mutant MHC class II epitopes drive therapeutic immune responses to cancer.

Morbidity from malaria and immune responses to defined Plasmodium falciparum antigens in children with sickle cell trait in The Gambia.

Relationship between Plasmodium falciparum malaria prevalence, genetic diversity and endemic Burkitt lymphoma in Malawi.

Immune activation during cerebellar dysfunction following Plasmodium falciparum malaria.

The Relationship of HLA Class I and II Alleles and Haplotypes with Autism: A Case Control Study.

Plasmodium attenuation: connecting the dots between early immune responses and malaria disease severity.

Inflammatory cytokine and humoral responses to Plasmodium falciparum glycosylphosphatidylinositols correlates with malaria immunity and pathogenesis.

Antibody responses to Plasmodium falciparum and P. vivax sporozoites in areas with stable and unstable malaria.

Plasmodium vivax infection impersonating Plasmodium falciparum malaria.

Enhanced vaccine-induced CD8+ T cell responses to malaria antigen ME-TRAP by fusion to MHC class ii invariant chain.

Chemokines responses to Plasmodium falciparum malaria and co-infections among rural Cameroonians.

The Association Between HLA Class II Alleles and the Occurrence of Factor VIII Inhibitor in Thai Patients with Hemophilia A.

Artemisinin-Resistant Plasmodium falciparum Malaria.

Studies on the humoral immune response to a synthetic vaccine against Plasmodium falciparum malaria.

Anaemia of Plasmodium falciparum malaria.

Genome content analysis yields new insights into the relationship between the human malaria parasite Plasmodium falciparum and its anopheline vectors.

HLA Class I and Class II Alleles and Haplotypes Confirm the Berber Origin of the Present Day Tunisian Population.

Malaria mimicry with tumor necrosis factor. Contrasts between species of murine malaria and Plasmodium falciparum.

Comparison of class I MHC alleles in humans and apes.

Direct Coombs antiglobulin reactions in Gambian children with Plasmodium falciparum malaria. I. Incidence and class specificity.

Inhibition of HLA-DM Mediated MHC Class II Peptide Loading by HLA-DO Promotes Self Tolerance.

Association and differentiation of MHC class I and II polymorphic Alu insertions and HLA-A, -B, -C and -DRB1 alleles in the Chinese Han population.