Current knowledge of the merozoite Ags targeted by human immunity is limited, and consequently, data to rationalize candidate Ags for vaccine development or as biomarkers of immunity and exposure are lacking. Very few merozoite Ags have been studied as immune targets in prospective human studies (6
), and few studies compared multiple Ag responses in the same cohort (15
). To address these gaps, we examined a large number of merozoite proteins that are biologically plausible targets of protective Abs, particularly those proteins with a defined role in erythrocyte invasion.
An important finding of our study was that the majority of the MSPs and apical proteins tested in this study were found to be targets of naturally acquired Abs and consistent with the characteristics of naturally acquired immunity (56
). This was reflected by significantly higher reactivity of proteins to Abs from malaria-exposed individuals, but not malaria-naive individuals; Ab prevalence and/or levels being higher in older children; and Ab prevalence and/or levels being higher in children with active infection versus uninfected children. Thirteen Ags were found to have little reactivity with Abs, despite apparently adequate expression and effective coating in ELISA plates. Further studies are required to determine whether these Ags were truly nonimmunogenic, whether folding of these proteins was incorrect, or whether different allelic variants play a particularly important role for these Ags.
When we prospectively assessed Ab associations with protective immunity in our longitudinal cohort, it was striking that Abs to several newly described and understudied merozoite Ags were more strongly associated with protective immunity than were Abs to well-studied Ags or Ags that have already progressed to phase 1 or phase 2 human vaccine trials. Many of the Ags that progressed to clinical trials performed suboptimally or were not efficacious (59
), and there is a strong need to identify and rationalize other Ags for vaccine development to obtain greater efficacy. An additional key finding was that Abs to microneme and rhoptry proteins were generally more strongly associated with protective immunity than were Abs to MSPs. This may reflect the key roles for apical organelle proteins in invasion; defined roles for MSPs are currently lacking (62
). Considering that Ag-specific responses most strongly associated with protective outcomes are more likely to be causally related to protective immunity (63
), Ags highlighted by our study should be considered for further evaluation as immune targets and potential vaccine candidates. High levels of Abs to proteins of the apical organelles, such as PfRh5, Ripr, EBAs, PfRh2, PfRh4, RON2, and GAMA, showed strong associations with protective immunity. The finding of naturally acquired responses to PfRh5 was especially interesting given a recent report suggesting that PfRh5 is not naturally immunogenic (64
). Differences in the protein tested in the assay, expression levels in the parasite populations, or the human populations studied may account for these contrasting findings. A few understudied MSPs also showed strong-intermediate associations with protection (MSPDBL1, MSP9/ABRA, Pf38, MSP6). Overall, we found that responses to almost all of the 46 Ags were associated with protection from symptomatic malaria at some level, and most of these findings remained significant after adjusting for potential confounders. The high proportion of Ags associated with protection may reflect our strategy of selecting Ags that were likely to be targets of protective Abs because of their localization and known function. Protective associations were not adjusted for multiple comparisons for reasons outlined elsewhere (53
). Therefore, associations that were weak or modest in strength or that were of borderline statistical significance should be interpreted with caution.
Of further interest is that the protective association for different Ab levels (high, medium, or low) varied between Ags. These observations are important for future immune-epidemiological studies that examine associations between immune responses and protection from malaria, as well as for identifying potential Ab biomarkers of immunity. These results suggest that analyses that consider Ab levels are important for identifying protective associations, rather than simply classifying Ab responses as present or absent, which was often reported in the literature. It is also likely that Ab concentration plays a critical role in mediating protective immunity (65
). This study provides preliminary evidence that the threshold level of Abs for protection may vary between Ags.
Our study identified combined Ab responses to two or three Ags that were very strongly associated with protective immunity, particularly Ab combinations for microneme and rhoptry Ags. This included combinations of EBA and PfRh Ags, which have emerged as promising vaccine candidates; it is likely that an effective vaccine will need to target multiple members of these invasion ligand families to maximize protective efficacy (40
). Importantly, we found that only a small proportion of Ab combinations showed evidence of an additive effect on protective associations compared with the single-Ag responses, highlighting the need for careful selection of Ags for use as biomarkers of immunity and for vaccine development. It is likely that protective immunity targeting merozoites consists of a repertoire of responses targeting multiple Ags and that a multivalent vaccine will be required to induce an efficacious response. However, very few studies examined this issue in human populations, and only a small number of merozoite Ag combinations has been studied (16
The strength of our approach was that we focused on Ags that are likely to be targets of protective Abs because of their localization on the merozoite surface or in the apical organelles and exposure during or before erythrocyte invasion. This approach was adopted rather than taking a genome-wide approach, which predominantly includes intracellular proteins, because the role of intracellular proteins as targets of protective or functionally active Abs is unclear. Although it was demonstrated that immune responses against intracellular proteins can induce inhibition of in vitro parasite growth by ADCI mechanisms (66
), we sought to focus on Ags that are likely to be direct targets of Abs to intact merozoites. Genome-wide approaches using high-throughput protein expression are also valuable and have identified Ags that may be important immune targets or biomarkers of immunity (15
). An additional strength of our approach is that all recombinant proteins were assessed for quality, coating, and immunoreactivity before being evaluated as potential targets of protective humoral immunity in our cohort of PNG children. Lastly, our prospective cohort study design was important to investigate the temporal relationship between Abs and subsequent malaria risk, thereby allowing us to infer a causal relationship in the protective effects of Abs targeting merozoite Ags. All Ags in this study were based on the 3D7 reference sequence. Many of the Ags or antigenic regions that we used are highly conserved in sequence, but some are known to be polymorphic. Although it is likely that different allelic variants of Ags influence the protective activity of Abs to specific Ags, we did not find that assessing different allelic variants for the same Ag led to major variations in the protective association observed. For example, we found that protective associations were very similar for different alleles of MSP2, AMA1, and EBA175 (data not shown). This may due to the coacquisition of responses to multiple allelic variants of these Ags or to the presence of Abs targeting conserved epitopes.
The findings of this study are valuable for informing vaccine development in several ways. The demonstration that a merozoite protein is naturally immunogenic in humans and that Ab responses are prospectively associated with protective immunity support these Ags as vaccine candidates. Identifying Ags as potential vaccine candidates should also incorporate data from functional assays; indeed, some of the Ags studied were shown to generate functional Abs (in GIAs or ADCI assays) when used to immunize experimental animals, further supporting their contribution to protective immunity (Supplemental Table I). However, more functional studies using human Abs are urgently needed, because this knowledge is limited to a small number of merozoite Ags (10
). The complementary roles of studies into naturally acquired immunity and functional assays are especially important given the limitations of animal models for P. falciparum
. Many of the key Ags of P. falciparum
are either not present in rodent malaria species or have major differences in structure and sequence, limiting their ability to be studied in animal models of human malaria. Additionally, Abs induced by immunization of animals can have important differences in affinity, specificity, and function from that seen in humans (69
). Therefore, human studies are a crucial part of establishing evidence for a protective role of responses to specific Ags.
Understanding naturally acquired human immunity to malaria is also important for reasons that extend beyond vaccine development. This study identified a broad array of biomarkers of human immunity that can aid in the development of low-cost serosurveillance tools for malaria. Such tools may guide control efforts by identifying populations at risk and evaluating the impact of malaria-control interventions (70
). Furthermore, knowledge of human immunity to complex pathogens is extremely limited, and current knowledge is largely based on studies of much simpler organisms (mainly viruses and bacteria) that have smaller genomes and few target Ags. These studies are also important for contributing valuable reagents, data, and analytic approaches to the research community for defining targets of immunity and vaccine development. The many reagents generated in this work will be made available to other investigators in the field, and the database of Ab responses to multiple Ags will be accessible to other researchers to interrogate specific questions about the acquisition of Abs, associations among Abs and clinical data and outcomes, and relationships between multiple Ab responses. This will enable the standardization of studies across populations and ensure the generalizability of results. We believe that this will help to facilitate progress toward identifying immune targets and biomarkers of immunity, as well as prioritizing candidate Ags for vaccine development.
Major outcomes from this work are to advance specific Ags as candidates for malaria vaccine or biomarker development. Criteria for ranking and prioritizing merozoite vaccine candidates for further development include strong association of Ab responses with protective immunity in humans, relevant protective functional activity of immune responses (in vitro or in animal models), and a demonstrated important function for the protein in erythrocyte invasion (5
). Considering these criteria, our new data presented in this article, and published findings on function, we propose that the following Ags should be prioritized as leading candidates for vaccine evaluation and development: EBA175, PfRh2, PfRh5, PfRh4, Ripr, MSPDBL1, EBA140, GAMA, and RON2. Other candidates may be prioritized as additional data on function and immune responses become available. Selecting Ags for development as biomarkers of immunity is based primarily on the strength of Ab-protective associations (alone or in combination), with consideration given to overall reactivity and established expression systems. Based on this, we propose the following as leading Ags for initial further evaluation in biomarker development: PfRh2, RALP1, PfRh4, EBA140, RON4/RON2, and RhopH1, and combinations of EBA175 with PfRh Ags.
In conclusion, these studies address an important gap in our knowledge of the targets of human immunity, understanding the potential of the many merozoite Ags as vaccine candidates, and identifying Ag-specific responses as biomarkers of immunity for the development of serosurveillance tools for malaria. These studies have identified sets of merozoite proteins of high priority for further evaluation as malaria vaccine candidates and development as biomarkers of immunity to malaria. Our findings help to define immunological principles underlying protective immunity that will facilitate the design and evaluation of new vaccines and malaria-surveillance tools.