The goals of this study were to investigate whether HLA-A2 allelic subtypes might effectively present malaria peptides eliciting a T-cell-mediated immune response and to evaluate whether there are differences in these responses in matched volunteers with severe or uncomplicated malaria and healthy controls. After determining HLA haplotypes by sensitive PCR techniques, we demonstrated for the first time that alleles of HLA-A2-restricted PBMC are capable of recognizing HLA-A2 preerythrocytic malaria antigenic epitopes. We found that 43% of Malian individuals have an HLA-A2 supertype phenotype (A*0201, A*0202, A*0205, or A*6802 allele), similar to estimated frequencies of 36 to 63% in other African ethnicities (6
). We observed IFN-γ responses to at least one peptide pool in 42 to 67% of all volunteers tested (depending upon disease state) after stimulating PBMC from HLA-A2-positive children with HLA-A2-restricted epitope pools of CSP, TRAP, and Exp-1 malaria proteins. Although proliferative responses were observed to all peptide pools, these responses were limited. These results might have important implications in that they demonstrate that a larger segment of the population than previously thought could be responsive to malaria subunit vaccines.
After stratifying participants according to the four HLA-A2 subtype alleles and analyzing CMI responses, no measurable differences were noted between children with different subtypes. Due to the limited number of subjects with A*0205 and A*6802 alleles, the effect of structural allelic differences may not be clearly illustrated. The specificity of the responses to malaria peptide pools was confirmed by the absence of CMI to malaria epitopes when PBMC from four malaria-naive HLA-A*0201 controls and eight HLA-A2-negative study participants were tested (data not shown).
We were encouraged to observe that subtype alleles could effectively present HLA-A2-restricted peptides and anticipated that a high proportion of individuals would have demonstrable CMI responses to malaria antigens. However, despite stimulating known HLA-A2 PBMC with HLA-A2-restricted malaria peptides, the majority of children did not mount a detectable immune response either during the acute illness (wet season) or after disease recovery (dry season). Among the enrollment groups, IFN-γ production as determined by ELISPOT was highest in healthy volunteers. When allelic subtypes were combined, healthy volunteers demonstrated 33% reactivity to TRAP (versus 41% in Kenyan studies [14
]), 31% to CSP, and 20% to Exp-1. In contrast, significantly lower reactivity rates were observed in children with uncomplicated or severe malaria. Similarly, we found that PBMC from healthy controls had increased proliferative responses upon malaria peptide stimulation compared to matched cases of severe and uncomplicated malaria in combined wet- and dry-season analysis. We observed limited overall proliferative responses (0 to 23%) when allelic subtypes were combined into one group and analyzed as a whole, complicating conclusions made regarding differences among individual allelic subtypes. The few severe cases that did proliferate (to any peptide pool) had a vigorous response. The finding of increased immunoreactivity among healthy volunteers may reflect blunted immune responses in patients with malaria, as has been reported in cases of severe disease (4
) and/or indirect evidence for stronger host responsiveness in healthy controls endowing them with the ability to mount these, as well as yet-undefined, immune responses. No changes were observed in the magnitude of responses to control stimulant (anti-CD3/CD28 beads) among enrollment groups to suggest immunologic blunting. Of interest, all of the ELISPOT responses that remained positive from the wet to the dry season were documented in healthy volunteers (n
Of the peptide pools tested, TRAP consistently elicited the greatest IFN-γ responses in all study groups while Exp-1 elicited the least. These findings were nearly identical during the dry season follow-up. This contrasts with proliferation results where Exp-1 elicited the most activity in all enrollment groups in both seasons with almost no detectable proliferation to CSP, suggesting that different effector immune responses are elicited by different malarial proteins. Studies have demonstrated little correlation between cellular IFN-γ secretion and lymphoproliferation in response to malaria peptides (13
). This is likely due to the fact that these assays measure different CMI responses, i.e., effector T-cell function for ex vivo IFN-γ production and largely memory T cells for lymphoproliferative responses.
A number of factors may have influenced the low percentage of immune responses detected in this study. The mechanism of preerythrocytic protective immunity in natural malaria remains unclear, hampered by the inability to examine liver CD8+
T cells directly. As a result, the immunologic assay(s) that best assesses host response to malaria infection and its relevance in measuring host protection from disease is uncertain. The low frequency of Plasmodium
T cells, the relatively immature immune systems of these very young children (mean age 41 months), antigenic variation, and short-lived immunity may have affected our ability to detect malaria-induced immune responses. We found that the majority of positive CMI responses documented during active infection reverted to negative during a follow-up period with negligible transmission. This compares with Kenyan results in which short-lived IFN-γ immune responses to TRAP were documented when ELISPOT assays were performed at 1-year intervals (14
). Moreover, peptides capable of inducing proliferative responses in one setting do not necessarily elicit similar responses in another (13
), nor does binding affinity necessarily correlate with IFN-γ responses (15
). We did not perform longitudinal follow-up to quantify malaria exposure following enrollment, so it is unclear whether the immune responses present during the low-transmission season were due to long-lived immunity or to ongoing “low-level” antigenic exposure.
We wish to emphasize that, due to the small quantity of blood collected from severely ill children, immunologic studies could only be performed once with cryopreserved PBMC. This limited our analysis to the measurement of immune responses to pools of epitopes rather than individual epitopes. Variations in ELISPOT technique (ex vivo versus cultured PBMC) and the use of cryopreserved versus fresh cells could alter or limit the ability to detect low-level CD8+
T-cell secretion of IFN-γ (13
). To maximize the detection of ex vivo IFN-γ responses, we added anti-CD28 and anti-CD49d mAbs to the cultures. Anti-CD28 and -CD49d mAbs have been shown to increase the measured effector frequency of peripheral blood-derived CD4+
T cells (34
). Anti-CD28 has recently been validated with peripheral CD8+
T cells (22
). In extensive control experiments, the addition of these mAbs did not increase IFN-γ SFC in media control cultures from HLA-A2- and HLA-A3-positive children (results not shown). To our knowledge, this is the first time that an ex vivo IFN-γ ELISPOT assay optimized by the addition of costimulation has been utilized in PBMC from malaria-endemic areas.
In summary, we have demonstrated for the first time that HLA-A2 allelic subtypes can effectively present malaria peptides eliciting a T-cell-mediated immune response. Despite demonstrating allelic cross-reactivity, only a minority of children, regardless of disease state, exhibited detectable immune responses to malaria peptide pools. Additional research is needed to identify the individual epitopes responsible for eliciting detectable CMI responses and to determine whether these immune responses are critical in mediating protection from P. falciparum infection. These results suggest that immune responses to other epitopes in TRAP, CSP, Exp-1 or other uncharacterized proteins might be required to endow protection from infection and/or disease.