We found that in two independent studies conducted in both high-transmission (Chonyi cohort) and low-transmission (case-control study) settings at different times, both the breadth of specificity for distinct merozoite antigens and the magnitude of antibody responses to these antigens provide robust predictors of the immune status of children. High titers of antibodies to combinations of three merozoite antigens in particular (AMA1, MSP-2, and MSP-3) were more strongly predictive of protection from clinical episodes of malaria compared to other putative “protective” merozoite antigens (MSP-1 or EBA-175).
Out of the panel of malaria vaccine candidate antigens studied here, high levels of antibodies to combinations that included AMA1, MSP-2, and MSP-3 were the most strongly associated with protection. This is consistent with other studies in which naturally acquired antibodies to each of the three antigens individually have been associated with protection from clinical malaria in this and other populations (1
). Recently, long-term clinical protection was associated with IgG3 isotype antibodies to MSP-3 in Senegalese children (58
). In contrast, antibodies to MSP-1 block 2, which have been associated with protection in two cohorts in West Africa (10
), were not similarly protective in the two cohorts we studied from Kilifi, Kenya. Antibodies to MSP-119
have been associated with protection from clinical malaria in some studies, but not in others (5
). This may be explained in part by the finding that the fine specificity of anti-MSP-119
antibodies appears to be more important with regards to protection (17
). A separate study found that individuals with high-titer anti-MSP-119
-specific invasion-inhibitory antibodies were protected from infection (33
) and underscored the importance of developing robust functional assays for malaria. Antibodies to the F2 subdomain of EBA-175 were not associated with protection from clinical disease in our studies, as has been found in other parts of Africa where this (49
) and other subdomains of EBA-175 (32
) have been studied. To date, only one study has reported significantly higher antibody levels to EBA-175 peptide 4 (residues 1062 to 1103, within region V) in children protected from clinical attacks of malaria compared to susceptible children (68
The importance of allele-specific immunity was highlighted in the Combination B malaria vaccine trial in Papua New Guinea. Children who received this vaccine (containing a combination of P. falciparum
ring-infected erythrocyte surface antigen, MSP-1, and the 3D7 allele of MSP-2) were less likely to be infected with parasites bearing the homologous allele of MSP-2 (28
), suggesting (as was later confirmed) that the vaccine had induced primarily allele-specific MSP-2 antibodies (26
). In the context of naturally acquired infections, while some data suggest that parasites bearing specific genotypes induce allele-specific antibodies (11
), to our knowledge no studies have examined the protective effects of preexisting allele-specific antibodies on subsequent disease caused by parasites bearing homologous alleles. We found that for most antigens tested, responses to allelic forms of each antigen had similar effects on the probability of mild or more severe malaria, suggesting the possibility that there may be significant cross-allele protection to clinical episodes.
In a study conducted in The Gambia, Gray et al. (29
) found that while antibodies to a similar panel of individual antigens were only weakly correlated with protection, those to the combinations of AMA1 and MSP-2 were significantly associated with protection from clinical malaria. There are two important differences between this Gambian study and the results reported here from Kenya. First, k
-means clustering and phylogenetic networks were used to investigate associations between antibody reactivity profiles and clinical status in the Gambian cohort. These methods independently identified the group of children who were asymptomatic (asymptomatic parasitemia, splenomegaly, or both) at the end of the study and who had not apparently experienced clinical disease. That end point differs from that of the studies reported here, in which outcome was simply defined as mild (Chonyi cohort) or severe (case-control study) malaria during the period of observation. Second, the magnitude of responses was not taken into account, mainly because this generates increased individual differences, impairing cluster analysis. One other longitudinal study, carried out among children in Burkina Faso, examined antibodies to a different set of blood-stage malaria antigens (glutamate-rich protein, P. falciparum
exported protein 1, and MSP-3) and, like our studies, they found that the simultaneous presence of antibodies to more than one antigen was associated with a lower frequency of malaria episodes (41
). However, in a separate study on protection from malaria infection as opposed to clinical episodes in Kenyan adults, John et al. (32
) found that high antibody titers to multiple blood-stage antigens were not protective (though there was evidence of protection for responses to preerythrocytic antigens). Our data suggest that the combination of blood-stage antigens analyzed in these Kenyan adults (AMA1, EBA-175, and MSP-119
) may not have been optimal. While these studies are difficult to compare directly due to differences in study design, study populations and end points, antigens tested, and analytical methodologies, the picture that nevertheless emerges clearly is that antibodies to key combinations of multiple parasite targets are more strongly associated with protection from clinical malaria than are antibodies to individual antigens.
With the completion of the P. falciparum
genome, numerous new (and old) antigens of the parasite have been identified and are being characterized. High-throughput assays employing suspension array technology (27
) or microarrays (29
) now allow for simultaneous analysis of antibodies to multiple antigens using minimal amounts of sera. This technology has not been matched with equivalently efficient tools for identifying protective immune responses. Robust concurrent analyses of numerous responses in relatively small studies, where children have been monitored longitudinally over a limited time period for disease episodes, remain challenging. The pair-wise analyses of combinations of high-titer antibody responses as presented here have obvious limitations when numerous antibodies are to be analyzed. Other analytical techniques, such as clustering and the use of phylogenetic networks (29
), while attractive for screening of potential vaccine candidates, similarly become more complex when increasing numbers of responses are analyzed and may well obscure genuinely “protective” responses. New strategies to identify protective responses in humans are urgently needed.
Studies of associations between immune responses and clinical malaria need to take into account the possibility that any given response is merely a marker of cumulative exposure (which is itself necessary to induce immunity) or of a response to an as-yet-unidentified antigen(s) that elicits strongly protective immunity. In our study, the fact that antibodies to specific antigens were more strongly predictive of protection than those to whole-schizont extract (containing all the specific antigens and many other blood-stage antigens) (Fig. ) suggests that specific responses do not merely reflect exposure. The finding that protective efficacy increased with increasing breadth of antibody specificity indicates that the effect of any one apparently protective response does not simply result from correlation with responses to other antigens (Fig. ) and argues for the interpretation that these are truly protective responses. Ultimately, the critical test of any such hypotheses will be to achieve equivalent protection through vaccination. Our demonstration of strong protection against malaria associated with high antibody levels to AMA1, MSP-2, and MSP-3 lends support to the development of vaccines based on combinations of these key malaria antigens.