Prior studies have provided substantial evidence supporting the importance of antibodies to IE surface antigens in protection against clinical malaria in humans (5
). However, the relative importance of each of the several candidate VSAs to the overall antibody response and protective immunity has not yet been determined, due in part to technical constraints and lack of tools to dissect specific responses. Using approaches with transgenic parasites, we have quantified the importance of PfEMP1, relative to other VSAs, as a target of acquired human antibodies. Our striking findings reveal, for what we believe to be the first time, that the majority of the antibody response targets PfEMP1 and that this holds true for 2 genetically different parasite lines, 3D7 and E8B. We also found that PfEMP1 was the major target of antibodies to IE surface antigens in the great majority of the samples that we tested that were positive for IgG binding to 3D7 parental or E8B parental IEs. In most of these samples, antibodies to PfEMP1 accounted for more than 80% of the antibody binding, suggesting that it is the dominant VSA of P. falciparum–
The proposed importance of PfEMP1 in immunity was further emphasized by showing that it is the key target of antibodies to the surface of IEs that are associated with protective human immunity. Individuals with high levels of antibodies to 3D7 parental or 3D7-PfEMP1–specific antibodies had a significantly reduced risk of symptomatic malaria. In contrast, antibodies to 3D7vpkd showed no significant association with malaria risk, suggesting that non-PfEMP1 antigens are either not an important component of the protective response or that they play a minor role compared with that of antibodies to PfEMP1. The development of immunity to malaria in our study population occurs during childhood (43
). The levels and prevalence of PfEMP1-specific and non-PfEMP1 antibodies showed a clear increase with age during childhood, consistent with the acquisition of immunity in this population. This finding also suggests that repeated infections over time are required to generate antibody responses toward both PfEMP1 and non-PfEMP1 surface antigens. Limiting our survival analysis to children aged 1–10 years helped reduce the potential confounding effects of age. After further adjusting the survival analysis for the age of children, a strong association between antibodies and reduced malaria risk remained (HR = 0.47 for 3D7 parental and HR = 0.49 for 3D7-PfEMP1), although the level of statistical significance was weakened. Higher antibody levels among individuals with parasitemia suggest antibody boosting occurred with infection or that this group had a higher level of exposure. A protective association between antibodies and malaria was only observed among children with active parasitemia at the time of enrollment when antibodies were measured. The lower incidence of malaria among children who were aparasitemic greatly reduced our statistical power to detect associations between antibodies and malaria in that group. It is possible that active parasitemia boosted antibody responses that contribute to protection. Further studies in other populations will be valuable to address these questions and understand their significance.
If immunity to malaria is mediated by antibodies to PfEMP1, it may be that a broad repertoire of antibodies to different variants is required, given that PfEMP1 is a highly polymorphic protein; however, some data suggest that this antibody repertoire may not need to be extensive, as there may be a restricted number of common PfEMP1 variants that are important targets of immunity (47
). Alternatively, protective antibodies may target cross-reactive, or even conserved, epitopes that give broad protection against the diversity of infecting variants (12
). However, very little is currently known about the extent of antigenic diversity or relatedness between different PfEMP1 variants and the extent to which human antibodies target conserved and polymorphic epitopes. This is an important issue for future research. Our finding that antibodies to 3D7-PfEMP1 are associated with protective immunity in the study population may reflect the presence of antibodies that have broad cross-reactivity against different isolates or antibodies to PfEMP1 variants that are common in the study population and similar to those expressed by 3D7. Alternatively, antibodies to 3D7-PfEMP1 may be a marker of a broad repertoire of antibodies to different PfEMP1 variants that are present in protected children. Antibodies to IE surface antigens are thought to act in part by opsonizing IEs for clearance by monocytes and macrophages in the circulation and spleen (19
). Importantly, we showed that antibody-mediated opsonic phagocytosis was significantly reduced in vpkd parasites of both 3D7 and E8B lines, suggesting that PfEMP1 is the major target of these functional antibodies. Together with findings on associations with protection, these results provide further evidence of an important role for PfEMP1 as a target of protective immunity. Interestingly, there was some measurable opsonic phagocytic activity with vpkd parasites. This suggests that the low level of IgG reactivity seen to the vpkd parasites, which may represent antibodies to non-PfEMP1 antigens, may still contribute to clearance of IEs and protective immunity.
The 3D7vpkd and E8Bvpkd lines used here were generated by transfecting parental parasites with a var
promoter construct that lacked any coding sequence for PfEMP1 (40
). Under drug selection, this approach substantially reduced endogenous PfEMP1 production, thus creating a PfEMP1-deficient line. This was supported by the reduced or absent var
gene expression by Northern blots, the lack of detectable PfEMP1 in Western blots of IE membrane extracts, and the markedly reduced adhesion of vpkd parasites to vascular receptors CD36 and ICAM-1. In our study, we demonstrated that other candidate surface proteins, RIFIN and STEVOR, were still expressed by these vpkd-transfected parasites, suggesting that protein trafficking and export was not affected. Furthermore, the expression and trafficking of PfEMP3 and knob-associated histidine-rich protein (KAHRP) (40
) appeared to occur normally. The demonstration that the 3D7vpkd-IEs still express erythrocyte membrane knobs further suggests that the expression and assembly of membrane proteins, other than PfEMP1, occurs normally in the vpkd parasites. While it is possible that there could be disruption of other parasite-derived surface proteins if these normally exist in a complex with PfEMP1, at present, there are no published data to suggest that this is the case (50
), and collectively our data suggest this is an unlikely explanation for our results.
The identity of the targets of antibodies to surface antigens expressed by 3D7vpkd and E8Bvpkd parasites is unclear. These antigens may include RIFIN, STEVOR, and SURFIN proteins, which have been identified on the IE surface (21
). While it remains possible that some of the reactivity to vpkd parasites represents antibodies to residual PfEMP1 on the IE surface (if var
gene expression is not completely inhibited in the vpkd lines), our Western blot analysis suggests that residual PfEMP1 is minimal. The proportion of the IgG response to 3D7vpkd compared with that to 3D7 parental IEs or to E8Bvpkd compared with that to E8B parental IEs varied between sera, therefore suggesting residual PfEMP1 is unlikely to account for all IgG binding to 3D7vpkd or E8Bvpkd parasites, and antibodies to other VSAs are likely explanations. Previous reports indicate that PfEMP1 is highly sensitive to cleavage by trypsin compared with other surface antigens, such as RIFINs that are partially trypsin resistant (11
). We found that IgG reactivity to 3D7 parental parasites was highly trypsin sensitive, consistent with PfEMP1 being the major target of antibodies to the IE surface of these parasites. IgG reactivity to 3D7vpkd was less sensitive to trypsin treatment, and some serum antibodies appeared to target trypsin-resistant epitopes expressed by 3D7vpkd. This finding suggests that non-PfEMP1 antigens may also be targets of human antibodies. Adhesion to ICAM-1 was almost completely absent in E8Bvpkd-IEs, whereas a significant level of adhesion to CD36 was retained in both E8Bvpkd and 3D7vpkd parasite lines, even though there was little or no detectable PfEMP1 or var
expression. Currently, PfEMP1 is thought to be the sole parasite ligand for adhesion to ICAM-1 (50
), and our findings are consistent with that. However, other ligands have been suggested for adhesion to CD36, such as parasite-modified erythrocyte Band 3 (26
). It is possible that the residual CD36 adhesion may represent binding activity of other antigens, or alternatively a very low level of PfEMP1 expression may be sufficient to mediate adhesion to CD36. This issue warrants investigation in future studies.
Our findings represent a major advance in understanding VSAs as vaccine candidates by clearly establishing the importance of PfEMP1 relative to other candidate antigens and developing methods and approaches to more precisely measure antibodies to PfEMP1 and other VSAs that can be used in future immunity studies and vaccine trials. IE surface antigens have long been regarded as attractive vaccine candidates because of their importance as targets of acquired immunity, their key role in disease pathogenesis, and their prominent exposure to the immune system. However, one major roadblock to their development as vaccines has been a lack of understanding of the relative importance of the different candidate antigens, which we address here. A further barrier has been their level of antigenic diversity. In this respect, recent data on PfEMP1 suggest that diversity may not be as great as previously thought and that only a subset of variants may be responsible for causing severe malaria illness (47
). If these variants are found to be antigenically restricted, the prospect of a multivalent vaccine based on PfEMP1 may be possible. Additionally, knowledge of key immune targets is valuable for developing serological approaches for malaria surveillance, identifying populations at risk, and evaluating the impact of malaria control interventions on malarial immunity. There is increasing interest in using serological assays as low-cost tools for surveillance of malaria exposure in populations to guide control efforts (52
). Our data showing the dominance and importance of PfEMP1 as an immune target suggest it would be a valuable antigen for use in serologic assays for malaria surveillance programs.
In conclusion, this study provides major evidence that antibodies to PfEMP1 are the most abundant and functionally important antibodies to VSAs on P. falciparum–IEs. Our findings suggest that PfEMP1 is a major target of antibodies that clear parasitemia and protect from clinical malaria. Furthermore, we have developed powerful approaches which we believe to be novel, which we refer to as transgenic parasite comparison assays, to measure PfEMP1-specific responses and dissect components of protective immunity. Such approaches illustrate the value of translating molecular approaches to clinical immunology. These findings, therefore, have significant implications for understanding and measuring immunity to malaria that are relevant for the development of malaria vaccines or approaches to monitor immunity and malaria exposure in populations.