Paths to a malaria vaccine illuminated by parasite genomics
Authors: David J. Conway
Journal: Trends in Genetics (2015)

•Discovery of vaccine candidate antigens by parasite genome sequence analyses.•Genetic crosses, linkage group selection, and functional studies on parasites.•Characterizing developmental and epigenetic variation alongside allelic polymorphism.•Selection by naturally acquired immune responses helps to focus vaccine design. More human death and disease is caused by malaria parasites than by all other eukaryotic pathogens combined. As early as the sequencing of the first human genome, malaria parasite genomics was prioritized to fuel the discovery of vaccine candidate antigens. This stimulated increased research on malaria, generating new understanding of the cellular and molecular mechanisms of infection and immunity. This review of recent developments illustrates how new approaches in parasite genomics, and increasingly large amounts of data from population studies, are helping to identify antigens that are promising lead targets. Although these results have been encouraging, effective discovery and characterization need to be coupled with more innovation and funding to translate findings into newly designed vaccine products for clinical trials. More human death and disease is caused by malaria parasites than by all other eukaryotic pathogens combined. As early as the sequencing of the first human genome, malaria parasite genomics was prioritized to fuel the discovery of vaccine candidate antigens. This stimulated increased research on malaria, generating new understanding of the cellular and molecular mechanisms of infection and immunity. This review of recent developments illustrates how new approaches in parasite genomics, and increasingly large amounts of data from population studies, are helping to identify antigens that are promising lead targets. Although these results have been encouraging, effective discovery and characterization need to be coupled with more innovation and funding to translate findings into newly designed vaccine products for clinical trials. The drive to sequence malaria parasite genomes began in the 1990s with a bold vision for 'vaccinomics' which would showcase the benefits of genomics to society [1Hoffman S.L. et al.From genomics to vaccines: malaria as a model system.Nat. Med. 1998; 4: 1351-1353Crossref PubMed Scopus (60) Google Scholar]. This was well conceived, and remains relevant because malaria causes at least half a million deaths and 200 million clinical cases each year, with several species of Plasmodium parasites infecting humans via transmission by Anopheles mosquitoes [2White N.J. et al.Malaria.Lancet. 2014; 383: 723-735Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar]. The disease occurs in most tropical areas of the world, with the greatest burden being on children and pregnant women in Africa where Plasmodium falciparum is most prevalent. Existing interventions need to be more widely applied, including insecticide-treated bed nets to reduce mosquito biting, antimalarial drug prophylaxis targeted to vulnerable groups, and access to prompt diagnosis and treatment. An effective vaccine could be a cost-effective additional means of controlling malaria [3Moorthy V.S. et al.Decision-making on malaria vaccine introduction: the role of cost-effectiveness analyses.Bull. World Health Organ. 2012; 90: 864-866Crossref PubMed Scopus (4) Google Scholar] and might enable its elimination from some areas. A key issue for malaria vaccine design is selecting the most appropriate parasite life-cycle stage to be targeted (Box 1) [4Hill A.V. Vaccines against malaria.Philos. Trans. R. Soc. Lond. B: Biol. Sci. 2011; 366: 2806-2814Crossref PubMed Scopus (63) Google Scholar, 5Birkett A.J. et al.Malaria vaccine R&D in the Decade of Vaccines: breakthroughs, challenges and opportunities.Vaccine. 2013; 31: B233-B243Crossref PubMed Scopus (11) Google Scholar]. A vaccine to prevent blood-stage infection from occurring could target the parasite at the initial pre-erythrocytic stage, but would need to be fully effective because even a small number of parasites emerging from the liver can initiate a severe blood-stage infection. By contrast, a vaccine targeting the asexual blood-stage directly could be effective in suppressing the replicating parasites and thereby preventing most disease, even if it did not achieve absolute sterile immunity. As a potential complement to either of these, vaccination against parasite sexual stages would not prevent disease directly but might reduce transmission to mosquitoes, thereby possibly having a beneficial effect at the population level. Molecular characterization of these developmentally differentiated stages was an early goal of genomic and transcriptomic studies.Box 1Malaria parasites – species and life-cycle targets for vaccinationThe major malaria parasites of humans are P. falciparum and P. vivax. Vaccine development is generally considered separately for each of these because they are distantly related and have only limited antigenic cross-reactivity. Other human malaria parasites P. malariae, P. ovale (comprising biological species P. o. curtisi and P. o. wallikeri), and P. knowlesi (a zoonosis from macaque monkeys) are less common globally and are not considered for vaccine development. Malaria parasites are haploid throughout the cycle except for a brief diploid zygote stage after fertilization in the midgut of the mosquito. Phases of the parasite life cycle that are candidate targets for vaccination are:Pre-erythrocytic stage. Invasive sporozoites inoculated by mosquitoes and subsequent development to large schizonts within the liver (taking ∼6 days in P. falciparum, and 10 days or longer for P. vivax) which do not cause disease but are a potential target to prevent blood stage infection.Asexual blood stage. Causes all the symptoms of malaria as a result of significant parasitaemia achieved by repeated multiplication cycles (every ∼48 h for P. falciparum and P. vivax), with merozoite invasion followed by growth and replication of the parasite within the erythrocyte (forming early trophozoite 'ring' stages, late trophozoites, and schizonts) before the release of more merozoites.Sexual stage. Male and female gametocytes in the blood are ingested by mosquitoes and transform into gametes and ensuing motile zygotes (ookinetes) which might be targeted by antibodies taken with the parasites into the blood meal. The major malaria parasites of humans are P. falciparum and P. vivax. Vaccine development is generally considered separately for each of these because they are distantly related and have only limited antigenic cross-reactivity. Other human malaria parasites P. malariae, P. ovale (comprising biological species P. o. curtisi and P. o. wallikeri), and P. knowlesi (a zoonosis from macaque monkeys) are less common globally and are not considered for vaccine development. Malaria parasites are haploid throughout the cycle except for a brief diploid zygote stage after fertilization in the midgut of the mosquito. Phases of the parasite life cycle that are candidate targets for vaccination are: Pre-erythrocytic stage. Invasive sporozoites inoculated by mosquitoes and subsequent development to large schizonts within the liver (taking ∼6 days in P. falciparum, and 10 days or longer for P. vivax) which do not cause disease but are a potential target to prevent blood stage infection. Asexual blood stage. Causes all the symptoms of malaria as a result of significant parasitaemia achieved by repeated multiplication cycles (every ∼48 h for P. falciparum and P. vivax), with merozoite invasion followed by growth and replication of the parasite within the erythrocyte (forming early trophozoite 'ring' stages, late trophozoites, and schizonts) before the release of more merozoites. Sexual stage. Male and female gametocytes in the blood are ingested by mosquitoes and transform into gametes and ensuing motile zygotes (ookinetes) which might be targeted by antibodies taken with the parasites into the blood meal. The first genome sequence of a malaria parasite was published in 2002 for a cultured strain of P. falciparum containing approximately 5500 genes encoded on 14 chromosomes with a complete haploid genome size of ∼23 Mb [6Gardner M.J. et al.Genome sequence of the human malaria parasite Plasmodium falciparum.Nature. 2002; 419: 498-511Crossref PubMed Scopus (2257) Google Scholar] (Figure 1). Genome sequences of diverse malaria parasite species, including P. vivax, the second most important cause of human malaria [7Carlton J.M. et al.Comparative genomics of the neglected human malaria parasite Plasmodium vivax.Nature. 2008; 455: 757-763Crossref PubMed Scopus (319) Google Scholar], the zoonotic P. knowlesi [8Pain A. et al.The genome of the simian and human malaria parasite Plasmodium knowlesi.Nature. 2008; 455: 799-803Crossref PubMed Scopus (156) Google Scholar], other primate malaria parasites [9Tachibana S. et al.Plasmodium cynomolgi genome sequences provide insight into Plasmodium vivax and the monkey malaria clade.Nat. Genet. 2012; 44: 1051-1055Crossref PubMed Scopus (26) Google Scholar, 10Otto T.D. et al.The genomes of chimpanzee malaria parasites reveal possible pathways of adaptation to human hosts.Nat. Commun. 2014; 5: 4754Crossref PubMed Scopus (1) Google Scholar], and parasites of rodents which can be studied in laboratory mice [11Hall N. et al.A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses.Science. 2005; 307: 82-86Crossref PubMed Scopus (429) Google Scholar], show similar core genome organization. Each of the chromosomes shows a largely conserved syntenic gene order [12Kooij T.W. et al.A Plasmodium whole-genome synteny map: indels and synteny breakpoints as foci for species-specific genes.PLoS Pathog. 2005; 1: e44Crossref PubMed Scopus (78) Google Scholar], except in the subtelomeric regions which contain large gene families that have diverged substantially between species [10Otto T.D. et al.The genomes of chimpanzee malaria parasites reveal possible pathways of adaptation to human hosts.Nat. Commun. 2014; 5: 4754Crossref PubMed Scopus (1) Google Scholar, 12Kooij T.W. et al.A Plasmodium whole-genome synteny map: indels and synteny breakpoints as foci for species-specific genes.PLoS Pathog. 2005; 1: e44Crossref PubMed Scopus (78) Google Scholar, 13Frech C. Chen N. Variant surface antigens of malaria parasites: functional and evolutionary insights from comparative gene family classification and analysis.BMC Genomics. 2013; 14: 427Crossref PubMed Scopus (4) Google Scholar]. Within each species, meiotic recombination occurs in a brief diploid stage within the mosquito midgut after fertilization between haploid male and female gametes in the blood meal. Recombination occurs at an average of 1 cM per 10–30 kb in experimental crosses of P. falciparum [14Jiang H. et al.High recombination rates and hotspots in a Plasmodium falciparum genetic cross.Genome Biol. 2011; 12: R33Crossref PubMed Scopus (24) Google Scholar], although effective recombination rates in natural populations can be lower due to self-fertilization [15Nkhoma S.C. et al.Close kinship within multiple-genotype malaria parasite infections.Proc. Biol. Sci. 2012; 279: 2589-2598Crossref PubMed Scopus (17) Google Scholar]. The diversity of parasites within infections directly influences the rate of outcrossing, which is greatest in highly-endemic populations where superinfection by unrelated genotypes commonly occurs [16Manske M. et al.Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing.Nature. 2012; 487: 375-379Crossref PubMed Scopus (84) Google Scholar]. Subtelomeric chromosomal regions can additionally undergo recombination during mitotic replication in the asexual blood stage of infection, with crossing-over occurring between non-homologous chromosomes in the same genome [17Bopp S.E. et al.Mitotic evolution of Plasmodium falciparum shows a stable core genome but recombination in antigen families.PLoS Genet. 2013; 9: e1003293Crossref PubMed Scopus (18) Google Scholar]. For vaccine design, the genetics and development of the parasite need to considered in the context of host immune responses that might suppress or eliminate an infection. A degree of immunity to malaria is generally acquired by experience of the infection, as shown by experimental studies in the first half of the 20th century on patients with neurosyphilis. Fevers induced by administering malaria parasite infections were beneficial to such patients in the age before antibiotics, because elevated temperatures killed spirochetes more effectively than other available treatments, but many patients became immune to repeated malaria infections [18Collins W.E. Jeffery G.M. A retrospective examination of sporozoite- and trophozoite-induced infections with Plasmodium falciparum in patients previously infected with heterologous species of Plasmodium: effect on development of parasitologic and clinical immunity.Am. J. Trop. Med. Hyg. 1999; 61: 36-43Crossref PubMed Google Scholar]. Studies on naturally acquired immunity in endemic populations then showed that malaria parasite levels in children or non-immune adults were reduced by passive transfer of serum Immunoglobulin G (IgG) from relatively immune adults, which has encouraged subsequent focus on the role of antibodies in acquired immunity to blood-stage parasites [19Fowkes F.J. et al.The relationship between anti-merozoite antibodies and incidence of Plasmodium falciparum malaria: a systematic review and meta-analysis.PLoS Med. 2010; 7: e1000218Crossref PubMed Scopus (97) Google Scholar]. More detailed studies over the past 40 years have shown that immunization of human volunteers with attenuated parasites confers strong protection against experimental challenge infections [20Roestenberg M. et al.Long-term protection against malaria after experimental sporozoite inoculation: an open-label follow-up study.Lancet. 2011; 377: 1770-1776Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 21McCarthy J.S. Good M.F. Whole parasite blood stage malaria vaccines: a convergence of evidence.Hum. Vaccin. 2010; 6: 114-123Crossref PubMed Google Scholar]. Parasites may be attenuated at the infective sporozoite stage which invades hepatocytes [20Roestenberg M. et al.Long-term protection against malaria after experimental sporozoite inoculation: an open-label follow-up study.Lancet. 2011; 377: 1770-1776Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar], or at the subsequent blood stage in erythrocytes [21McCarthy J.S. Good M.F. Whole parasite blood stage malaria vaccines: a convergence of evidence.Hum. Vaccin. 2010; 6: 114-123Crossref PubMed Google Scholar]. Intravenous vaccination using sporozoites isolated from irradiated mosquitoes requires a demanding regimen of five doses of >100 000 parasites over a 3 month period to elicit responses that prevent parasites developing beyond the pre-erythrocytic liver stage [22Seder R.A. et al.Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine.Science. 2013; 341: 1359-1365Crossref PubMed Scopus (74) Google Scholar]. It is difficult to standardize the irradiation dose that each individual parasite receives, but alternative possibilities are chemical attenuation (e.g., alkylation of parasite DNA) [23Good M.F. et al.Cross-species malaria immunity induced by chemically attenuated parasites.J. Clin. Invest. 2013; 123: 3353-3362Crossref Scopus (12) Google Scholar], or genetic attenuation to remove the need for treatment of each batch. Multiple gene knockouts have been identified that, in combination, cause parasites to arrest development within the liver and elicit pre-erythrocytic stage immunity [24van Schaijk B.C. et al.A genetically attenuated malaria vaccine candidate based on gene-deficient sporozoites.Elife. 2014; 3 (Published online November 19, 2014. http://dx.doi.org/10.7554/eLife.03582)Crossref PubMed Google Scholar, 25Mikolajczak S.A. et al.A next-generation genetically attenuated Plasmodium falciparum parasite created by triple gene deletion.Mol. Ther. 2014; 22: 1707-1715Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar]. Engineering parasites to attenuate at the blood stage would require modification of metabolism to allow growth in selective laboratory media but not in normal human blood, as illustrated by parasites that have lost the apicoplast organelle under selection in culture [26Yeh E. DeRisi J.L. Chemical rescue of malaria parasites lacking an apicoplast defines organelle function in blood-stage Plasmodium falciparum.PLoS Biol. 2011; 9: e1001138Crossref PubMed Scopus (96) Google Scholar, 27Gisselberg J.E. et al.The suf iron–sulfur cluster synthesis pathway is required for apicoplast maintenance in malaria parasites.PLoS Pathog. 2013; 9: e1003655Crossref PubMed Scopus (8) Google Scholar]. Although there is a precedent for use of live-parasite vaccines to prevent protozoan infections of domestic animals [28McAllister M.M. Successful vaccines for naturally occurring protozoal diseases of animals should guide human vaccine research. A review of protozoal vaccines and their designs.Parasitology. 2014; 141: 624-640Crossref PubMed Scopus (3) Google Scholar], subunit vaccines containing specific antigens are generally considered more feasible for broad delivery to human populations [4Hill A.V. Vaccines against malaria.Philos. Trans. R. Soc. Lond. B: Biol. Sci. 2011; 366: 2806-2814Crossref PubMed Scopus (63) Google Scholar, 5Birkett A.J. et al.Malaria vaccine R&D in the Decade of Vaccines: breakthroughs, challenges and opportunities.Vaccine. 2013; 31: B233-B243Crossref PubMed Scopus (11) Google Scholar]. These may be constructed as recombinant proteins (that are either soluble, or form virus-like particles, or chemically conjugated nanoparticles), long synthetic peptides, recombinant plasmid DNA, or recombinant viral vectors, each potentially offering a product profile that could be incorporated into an Expanded Program for Immunization (EPI) schedule [29Schwartz L. et al.A review of malaria vaccine clinical projects based on the WHO rainbow table.Malar. J. 2012; 11: 11Crossref PubMed Scopus (72) Google Scholar]. The most advanced candidate developed to date is the RTS,S vaccine, which includes a large portion of the primary sequence of P. falciparum CSP (circumsporozoite protein – see Box 2 for abbreviations and names of all malaria antigens subsequently referred to) sequence co-expressed with the hepatitis B surface antigen as a virus-like particle, delivered with a potent proprietary adjuvant (ASO1). This shows safety and short-term protective efficacy of 20–50% against malaria infection and disease in most clinical trial populations [30RTS,S Clinical Trials Partnership Efficacy and safety of the RTS,S/AS01 malaria vaccine during 18 months after vaccination: a phase 3 randomized, controlled trial in children and young infants at 11 African sites.PLoS Med. 2014; 11: e1001685Crossref PubMed Scopus (5) Google Scholar]. The experience of RTS,S and other recombinant protein vaccine development indicates the importance of adjuvant choice to elicit high-titer antibodies. Viral-vectored vaccine delivery may be optimized through prime–boost schedules expressing malaria antigens by one recombinant virus (such as an adenovirus) in the first immunization and by an unrelated viral vector (such as a poxvirus) in boosting immunizations [31Hill A.V. et al.Prime–boost vectored malaria vaccines: progress and prospects.Hum. Vaccin. 2010; 6: 78-83Crossref PubMed Scopus (101) Google Scholar]. Some schedules incorporating particular candidate antigens show potent immunogenicity, although only a small minority of individuals have been protected from challenge infection and the determinants of efficacy need to be resolved [32Tamminga C. et al.Human adenovirus 5-vectored Plasmodium falciparum NMRC-M3V-Ad-PfCA vaccine encoding CSP and AMA1 is safe, well-tolerated and immunogenic but does not protect against controlled human malaria infection.Hum. Vaccin. Immunother. 2013; 9: 2165-2177Crossref PubMed Scopus (4) Google Scholar]. However, for any subunit vaccine, inclusion of the most-effective target antigens is vital, and it is for their identification that parasite genomics is most important.Box 2Malaria parasite antigensAbbreviated and full names of antigens with sequences incorporated in experimental vaccine trials already, and others mentioned in the article.In reported trialsTabled 1AMA1Apical membrane antigen 1CSPCircumsporozoite proteinEBA175Erythrocyte binding antigen 175 kDaEXP-1Exported protein-1GLURPGlutamate-rich proteinLSA-1Liver stage antigen-1LSA-3Liver stage antigen-3STARPSporozoite threonine-asparagine-rich proteinMSP1Merozoite surface protein 1MSP2Merozoite surface protein 2MSP3Merozoite surface protein 3Pfs25P. falciparum sexual stage antigen 25 kDaRESARing-infected erythrocyte surface proteinSERA5Serine-rich antigen 5TRAPThrombospondin-related adhesive protein Open table in a new tab OthersTabled 1CLAGCytoadherence-linked asexual protein, also known as heavy molecular weight rhoptry protein family 1 (RhopH1)DBPDuffy-binding protein in P. vivax (homologous to P. falciparum EBA proteins)EBA140Erythrocyte binding antigen 140 kDaEBL-1Erythrocyte-binding-like protein 1MSP6Merozoite surface protein 6 (also known as MSP3.2, paralogous to MSP3 and MSPDBP)MSPDBL1Merozoite surface protein with Duffy-binding-like domain (also known as MSP3.4)MSPDBL2Merozoite surface protein with Duffy-binding-like domain (also known as MSP3.8)MTRAPMerozoite-specific TRAP homologPfEMP1P. falciparum erythrocyte membrane protein 1 encoded by var genesRh4Reticulocyte-binding-like protein homolog 4Rh5Reticulocyte-binding-like protein homolog 5RIFINRepetitive interspersed family, gene productRiprRh5-interacting proteinS-AntigenHeat-stable soluble antigenSTEVORSubtelomeric variant open reading frame, gene productSURFINSurface-associated interspersed gene family productVAR2CSAA particular PfEMP1 that binds chondroitin sulfate A Open table in a new tab Abbreviated and full names of antigens with sequences incorporated in experimental vaccine trials already, and others mentioned in the article. In reported trialsTabled 1AMA1Apical membrane antigen 1CSPCircumsporozoite proteinEBA175Erythrocyte binding antigen 175 kDaEXP-1Exported protein-1GLURPGlutamate-rich proteinLSA-1Liver stage antigen-1LSA-3Liver stage antigen-3STARPSporozoite threonine-asparagine-rich proteinMSP1Merozoite surface protein 1MSP2Merozoite surface protein 2MSP3Merozoite surface protein 3Pfs25P. falciparum sexual stage antigen 25 kDaRESARing-infected erythrocyte surface proteinSERA5Serine-rich antigen 5TRAPThrombospondin-related adhesive protein Open table in a new tab OthersTabled 1CLAGCytoadherence-linked asexual protein, also known as heavy molecular weight rhoptry protein family 1 (RhopH1)DBPDuffy-binding protein in P. vivax (homologous to P. falciparum EBA proteins)EBA140Erythrocyte binding antigen 140 kDaEBL-1Erythrocyte-binding-like protein 1MSP6Merozoite surface protein 6 (also known as MSP3.2, paralogous to MSP3 and MSPDBP)MSPDBL1Merozoite surface protein with Duffy-binding-like domain (also known as MSP3.4)MSPDBL2Merozoite surface protein with Duffy-binding-like domain (also known as MSP3.8)MTRAPMerozoite-specific TRAP homologPfEMP1P. falciparum erythrocyte membrane protein 1 encoded by var genesRh4Reticulocyte-binding-like protein homolog 4Rh5Reticulocyte-binding-like protein homolog 5RIFINRepetitive interspersed family, gene productRiprRh5-interacting proteinS-AntigenHeat-stable soluble antigenSTEVORSubtelomeric variant open reading frame, gene productSURFINSurface-associated interspersed gene family productVAR2CSAA particular PfEMP1 that binds chondroitin sulfate A Open table in a new tab Vaccine candidates based on 15 different antigens of P. falciparum have proceeded to clinical trials that have been published so far (Figure 1 and Box 2). These antigens are diverse proteins, expressed at various stages of the life cycle, and were first described during an early period of parasite molecular characterization; the sequences of these genes were published between 1984 and 2000 when most other genes of the parasite were still unknown (Figure 2). It is remarkable that there have been no reported vaccine trials incorporating any of the thousands of proteins encoded by genes first described in the reference parasite genome sequence published in 2002 [6Gardner M.J. et al.Genome sequence of the human malaria parasite Plasmodium falciparum.Nature. 2002; 419: 498-511Crossref PubMed Scopus (2257) Google Scholar]. It is very improbable that all promising candidate antigens were discovered ahead of the genome sequence, and some potential reasons for the lack of vaccine trials based on genomic findings are discussed below. For the 15 vaccine candidate antigens that have undergone trials to date, there was a mean overall 'lag period' of 10 years from the primary publication of each antigen gene sequence until the first publication of a clinical vaccine trial incorporating a sequence of the respective antigen (Figure 2). There was a wide range in this time-window (from only 3 years for CSP, MSP1, and LSA-1, to 20 years for EBA-175), and for 11 out of the 15 antigens the period was less than 12 years. This shows that it is possible, as illustrated by most of these examples, to proceed from initial antigen gene discovery to a reported vaccine trial in less time than has already elapsed since the first malaria parasite genome publication in 2002. The absence of any published vaccine trial incorporating a new candidate antigen discovered in the genome sequence is therefore not a trivial observation. Another possible explanation is that most of the limited capacity and funding for malaria vaccine development may be committed to supporting previously identified candidates. Focus on a limited pre-existing portfolio might have unintentionally blocked new candidate development, even without proprietary or other conflicting interests which could exacerbate such an effect. A further possibility is that that the increased scale of discovery promoted by genome sequence analysis has itself inhibited the selection of candidates because there are now so many proteins to investigate that there may be increased uncertainty about the type and amount of data required for prioritization. Clearly, vaccine candidates should not continue to be prioritized for product development solely on the basis of the order in which they were discovered, and there is therefore a need for an evidence-based process to mark the candidacy of an antigen. Given the complexity of the parasite life cycle, and the diverse immune mechanisms that may be relevant, standardization will not be simple, but the following section outlines relevant evidence for inclusion. Studies to identify specific human immune responses associated with protection from malaria are important, particularly when responses to multiple antigens are analyzed in parallel. These involve laboratory assays of antigen-specificity of naturally acquired serum antibodies, or peripheral blood lymphocytes in culture, and correlations with incidence of clinical malaria of the sampled individuals in cohort studies of endemic populations [19Fowkes F.J. et al.The relationship between anti-merozoite antibodies and incidence of Plasmodium falciparum malaria: a systematic review and meta-analysis.PLoS Med. 2010; 7: e1000218Crossref PubMed Scopus (97) Google Scholar, 33Osier F.H. et al.New antigens for a multicomponent blood-stage malaria vaccine.Sci. Transl. Med. 2014; 6: 247ra102Crossref PubMed Google Scholar, 34Richards J.S. et al.Identification and prioritization of merozoite antigens as targets of protective human immunity to Plasmodium falciparum malaria for vaccine and biomarker development.J. Immunol. 2013; 191: 795-809Crossref PubMed Scopus (21) Google Scholar, 35Tetteh K.K. et al.Analysis of antibodies to newly described Plasmodium falciparum merozoite antigens supports MSPDBL2 as a predicted target of naturally acquired immunity.Infect. Immun. 2013; 81: 3835-3842Crossref PubMed Scopus (3) Google Scholar]. The incidence of clinical malaria is more relevant to measure than the incidence of any detected infection by the parasite because naturally acquired immunity mainly acts to reduce the density of parasites in the blood rather than entirely preventing infection [36Stanisic D.I. et al.Immunoglobulin G subclass-specific responses against Plasmodium falciparum merozoite antigens are associated with control of parasitemia and protection from symptomatic illness.Infect. Immun. 2009; 77: 1165-1174Crossref PubMed Scopus (84) Google Scholar, 37Tran T.M. et al.An intensive longitudinal cohort study of Malian children and adults reveals no evidence of acquired immunity to Plasmodium falciparum infection.Clin. Infect. Dis. 2013; 57: 40-47Crossref PubMed Scopus (18) Google Scholar]. A separate approach has been to analyze immune responses in individuals protected by experimental immunization with attenuated parasites, screening reactivity against various parasite proteins in an attempt to implicate particular specificities [38Trieu A. et al.Sterile protective immunity to malaria is associated with a panel of novel P. falciparum antigens.Mol Cell Proteomics. 2011; 10: M111007948Crossref Scopus (25) Google Scholar]. The difficulty in producing high-quality recombinant antigen reagents has limited the effectiveness of protein microarrays to scale