Review On Blood Stage Vaccine Against Malarai

Immunology and Cell Biology (2009) 87, 377390 & 2009 Australasian Society for Immunology Inc.
All rights reserved 0818-9641/09 $32.00

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REVIEW
The future for blood-stage vaccines against malaria

Jack S Richards and James G Beeson
Malaria is a leading cause of mortality and morbidity globally, and effective vaccines are urgently needed. Malaria vaccine approaches can be broadly grouped as pre-erythrocytic, blood stage and transmission blocking. This review focuses on bloodstage vaccines, and considers the evidence supporting the development of blood-stage vaccines, the advantages and challenges of this approach, potential targets, human vaccine studies and future directions. There is a strong rationale for the development of vaccines based on antigens of blood-stage parasites. Symptomatic malaria is caused by blood-stage parasitemia and acquired immunity in humans largely targets blood-stage antigens. Several candidate vaccines have proved efcacious in animal models and at least one vaccine showed partial efcacy in a clinical trial. At present, all leading candidate blood-stage antigens are merozoite proteins, located on the merozoite surface or within the apical organelles. Major challenges and priorities include overcoming antigenic diversity, identication of protective epitopes, understanding the nature and targets of protective immune responses, and dening antigen combinations that give the greatest efcacy. Additionally, objective criteria and approaches are needed to prioritize the large number of candidate antigens, and strong candidates need to be tested in clinical trials as quickly as possible. Immunology and Cell Biology (2009) 87, 377390; doi:10.1038/icb.2009.27; published online 5 May 2009 Keywords: Plasmodium; malaria; vaccine; blood stage; merozoite
Malaria is an important cause of global mortality and morbidity. There are an estimated 550 million cases of malaria and 1 million deaths each year, and around 2.5 billion people are at risk.14 The malaria eld has seen major achievements over the last decade, including development and implementation of preventive strategies, new treatments and advances in basic research.515 Malaria vaccine development has been encouraged particularly with trials of the preerythrocytic vaccine, RTS,S, showing protective efcacy.1621 Malaria vaccines can be considered in three broad groups: pre-erythrocytic, blood stage and transmission blocking. This review focuses only on blood-stage vaccines, and considers the evidence supporting the development of blood-stage vaccines, the advantages and challenges of this approach, potential targets, human vaccine studies and future directions. BACKGROUND There are ve species of Plasmodium that naturally infect humans: P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. These can cause asymptomatic infection in those with existing immunity, or a spectrum of clinical disease ranging from mild to severe disease and death in those lacking substantial immunity. P. falciparum accounts for the great majority of morbidity and mortality, but P. vivax also causes considerable symptomatic disease. Estimates suggest that P. falciparum may be the largest single cause of mortality in children below 5 years of age.22 Therefore, the efforts to develop vaccines have, to date, largely focused on P. falciparum.
After an initial period of incubation and replication in the liver, the blood-stage of the lifecycle commences when merozoites are released from the infected hepatocytes. The 48 to 72 h-cycle involves merozoites invading erythrocytes, intra-erythrocytic development and asexual reproduction, erythrocyte rupture and the release of new merozoites to continue the cycle (Figure 1). Erythrocyte invasion is thought to occur over several steps involving multiple receptorligand interactions. Initial attachment of the merozoite to the erythrocyte surface is followed by apical reorientation, tight-junction formation, entry into the erythrocyte through an actinmyosin motor, and is completed by resealing of the erythrocyte membrane.23 This occurs over 12 min and is the only time that the parasite is directly exposed to the extracellular environment during the blood stage. For most of the blood-stage cycle, parasites are partially hidden from immune recognition inside the erythrocyte, which lacks major histocompatibility complex molecules. The parasite exports proteins to the erythrocyte surface, enabling cytoadherence to a range of endothelial receptors, facilitating sequestration and reducing splenic clearance, and these seem to be important immune targets. Clinical illness occurs only during blood-stage infection, with the liver stage of the infection being asymptomatic. The pathogenesis of disease is complex and is reviewed elsewhere.24,25 Despite the complexity of the parasite lifecycle, a high level of antigenic diversity and parasite mechanisms of immune evasion, naturally acquired immunity to malaria does develop after repeated exposure. This immunity affords protection against symptomatic disease, high-density
Infection and Immunity Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia Correspondence: Dr JG Beeson, Department of Infection and Immunity, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia. E-mail: beeson@wehi.edu.au Received 6 February 2009; accepted 15 March 2009; published online 5 May 2009
Blood-stage vaccines against malaria JS Richards and JG Beeson 378
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parasitemia and death; however, it is less effective at preventing lowlevel parasitemia.26,27 Naturally acquired immunity seems to predominantly target blood-stage parasites.28 Effective immunity involves both humoral- and cell-mediated immune responses, which gives some cross-species and cross-strain protection, though this seems to be limited.29,30 The importance of antibodies has been supported by the passive transfer of immunoglobulin from immune individuals conferring protective benet to nonimmune individuals.31,32 Immune effector mechanisms are poorly understood and the relative importance of different mechanisms has not been quantied. Antibodies may have a role in preventing merozoite invasion, clearance of infected erythrocytes, prevention of adhesion and sequestration in the vasculature, and prevention of schizont rupture. Cytophilic IgG subclasses seem to be particularly important.3336 They probably facilitate parasite clearance in the spleen through opsonization for phagocytosis, antibody-dependent cell-mediated cytotoxicity, and complement-mediated lysis.26,3739 Antibodies do not act alone and there is increasing evidence of the importance of T cells, not only in providing B cell help, but in activating Th1 responses, which help mediate antibody effector mechanisms, as well as cell-mediated effector mechanisms. ADVANTAGES AND CHALLENGES OF A BLOOD-STAGE VACCINE It is likely that a highly effective malaria vaccine will need to be multivalent and may need to incorporate antigens of several lifecycle stages. There is a strong rationale for developing blood-stage vaccines as part of this strategy. Pathogenesis of malarial disease results from blood-stage infection and studies in humans and animal models have clearly established that immune responses targeting blood-stage antigens can protect against disease or facilitate control of parasitemia.31,32,40,41 Immunization with blood-stage antigens, mainly merozoite antigens, has been shown to be protective in a number of animal models using different antigens and there was some protective effect with one blood-stage vaccine tested in humans.4246 At present, the leading blood-stage vaccine candidates are all merozoite proteins, either located on the merozoite surface or contained within the apical organelles (Figure 2).
Merozoite surface proteins MSP1, 2, 4, 5, 10 (GPI anchored) MSP3, 6, 7, 9 (ABRA) GLURP SERA S-antigen 6-cys family (GPI anchored) Micronemes AMA1 EBA175, 140, 181 MTRAP PTRAMP ASP Rhoptries Rh 1, 2a, 2b, 4, 5 RAP1, 2, 3 RhopH1, 2, 3 RAMA
Figure 1 The blood-stage lifecycle of Plasmodium. (a) Merozoites are an extracellular form of Plasmodium (1) that attach and invade erythrocytes (2). The parasite then matures and divides through asexual replication inside the erythrocyte (3). The expression of parasite proteins on the surface of the infected erythrocyte enables interactions with receptors on the endothelial surface, facilitating sequestration of parasite-infected erythrocytes in various organs. After asexual replication, the schizont form ruptures and releases new merozoites into the circulation (4). (b) Merozoite invasion of erythrocytes. Merozoites are initially free in the intravascular space after schizont rupture (1), and the process of erythrocyte invasion commences when a merozoite binds to the surface of an erythrocyte (2). The merozoite then reorients its apical end to come into contact with the erythrocyte surface (3). A series of irreversible high-afnity ligandreceptor interactions then occur between the parasite and the erythrocyte, enabling tight-junction formation (4) and entry of the erythrocyte through an actinmyosin motor. As it does so, the parasite invaginates the erythrocyte membrane to form the parasitophorous vacuole. Outer surface proteins are partially cleaved as the merozoite enters the erythrocyte (5).
Apical end Rhoptries Apicoplast
Micronemes Merozoite surface
Dense granules Mitochondrion Nucleus
Figure 2 The structure and major antigens of the P. falciparum merozoite. The apical end of the merozoite has specic organelles involved in erythrocyte invasion, including the paired rhoptries and micronemes, which are thought to expel proteins to bind to erythrocyte receptors during invasion. The merozoite also has dense granules, and an apicoplast (a plastid remnant). Merozoite ligands and vaccine candidate antigens are present in the organelles and on the surface of the merozoite. Surface proteins may be GPI anchored or associated with GPI-anchored proteins by molecular interactions. Listed are known proteins of the merozoite surface and organelles, but many others remain to be identied or characterized. Immunology and Cell Biology
One hurdle for the continued development of blood-stage vaccines is the perceived lack of progress despite many years of research. There are many reasons why development of blood-stage vaccines has not progressed more rapidly, but slow progress has been common to all strategies including pre-erythrocytic stage candidates. Clearly greater effort and funding is needed to facilitate the development and evaluation of promising vaccines. The Combination B vaccine, which showed protection against parasitemia, was conducted over 10 years ago. However, no further testing of this vaccine or any of its components in phase II studies has been conducted since. At the time of writing, only four blood-stage vaccines have been tested in phase II trials. Of the two that are published, Combination B showed some degree of efcacy, whereas merozoite surface protein 1 (MSP1)-42 did not. Although RTS,S, which is based on the circumsporozoite protein, has been efcacious in several trials, another pre-erythrocytic vaccine, multi-epitope thrombospondin-related adhesion protein (METRAP)47,48 was not protective. It is likely that few of the vaccines that are eventually tested in clinical trials will prove to be sufciently efcacious; therefore, poor outcomes with specic antigens should not justify abandonment of an overall strategy. Recently, there has been a renewed call for the eradication of malaria.49,50 An effective vaccine will probably be needed to achieve eradication, in addition to other existing measures. Part of the renewed push for eradication of malaria is an emphasis on approaches to reduce malaria transmission and not just clinical illness. Therefore, vaccines inducing sterile immunity and/or transmission-reducing activity would be preferred. The role of blood-stage vaccines as part of this strategy has been questioned, as the objective of blood-stage vaccines is to reduce parasitemia and prevent clinical illness. However, data from animal models and clinical studies suggests that controlling parasite density reduces the generation of gametocytes in the blood stream. Therefore, it seems likely that an effective blood-stage vaccine will have benet in reducing transmission, in addition to preventing clinical illness. Perhaps the greatest challenge in developing a blood-stage vaccine is overcoming antigenic diversity. Most, if not all, important antigens show substantial polymorphism.5154 Antigenic diversity has evolved to facilitate immune evasion and vaccine approaches need to account for this such that they will cover the majority of strains causing infection and disease. This approach may be difcult or impossible if a large number of different allelic forms of an antigen are required in a single vaccine. However, the degree of polymorphism is much less for some antigens than others, and most antigens have domains or regions that are less polymorphic or are conserved. These may be suitable targets of vaccines to overcome the problem of antigenic diversity. Some antigens, such as P. falciparum erythrocyte membrane protein 1 (PfEMP1), are encoded by large multigene families and parasites can switch the expression of different genes to facilitate immune evasion. Recent studies have also shown that some merozoite protein families can be differentially expressed to facilitate immune evasion.5557 Further challenges are presented by potential mechanisms of immune interference seen during natural exposure to Plasmodium infection. As natural immunity is principally directed against bloodstage antigens, this may be more important than for pre-erythrocytic and transmission-blocking vaccines. For example, non-functional antibodies that impair the binding of functionally important antibodies to merozoite antigens have been identied.58,59 There are also likely to be effects of earlier exposure or maternally transferred antibodies in altering immune responses to a vaccine.60,61 Some data suggests that there may be compromised dendritic cell function
and suppression or deletion of memory B cells, and long-lived plasma cells during infection.62 VACCINE APPROACHES Current day immunization evolved from the live-attenuated or wholekilled vaccines of Jenner, Pasteur and Koch. Even today, the majority of licensed vaccines use whole organisms. Recent advances have led to renewed interest in using genetically attenuated parasites as a basis for a malaria vaccine. Most malaria vaccine research, however, has followed subunit approaches in which antigens are identied, puried, characterized and then immunologically evaluated.63 This approach lends itself to using recombinant antigens, peptides and, more recently, viral vectors. Whole blood-stage parasites The whole parasite approach for malaria has partly evolved from studies of radiation-attenuated sporozoite studies, reviewed elsewhere.64 Some information on blood-stage immunity can be gleaned from early studies in which deliberate infection with Plasmodia was used to treat paresis in humans. These studies indicated that infection led to predominantly strain-specic immunity that limited symptomatic disease, but did not prevent re-infection.65 Other studies in animal models also suggest that immunity induced in this way is largely strain-specic.66,67 More recently a study administered live P. falciparum parasites intravenously at low dose to ve patients, followed by early anti-malarial treatment.68 Four patients were rechallenged with live parasites of the same strain, and three did not develop any detectable parasitemia before nal anti-malarial treatment. Surprisingly, these patients developed cell-mediated immune responses, but a limited antibody response. Whole organism approaches have a range of potential advantages and these are more thoroughly reviewed elsewhere.6971 In short, they have the benet of not requiring the precise determination of epitopes or antigens. Rather the whole organism enables a vast array of antigens to be delivered, essentially providing a multi-epitope vaccine, with a high probability of antigens being in their native conformation. There are several important challenges to this approach. Safety is a major concern, as current technology requires that parasites are cultured in human erythrocytes, which is accompanied by a risk of serious bloodborne infections. Large-scale production of whole parasites ensuring consistent quality and dose is more challenging than for recombinant or synthetic vaccines. With live-attenuated parasites there is also the concern that attenuated parasites might revert to a non-attenuated state, although genetic attenuation by targeting multiple genes would make this unlikely. There are likely to be signicant challenges in producing and delivering live blood-stage parasites for widespread use. In vitro culture of P. vivax is difcult to sustain and large-scale production is not currently feasible. Recombinant antigens An advantage of this approach is the ability to direct immune responses to a specied region or epitope. This has the potential to maximize protective responses whilst minimizing undesired responses. A further advantage is the suitability for large-scale antigen production under good manufacturing practice. The inherent difculty is identifying protective target antigens for development, or specic domains of proteins, and the likelihood that multiple antigens would be required to induce an effective response and overcome antigenic diversity. Expression of many Plasmodium proteins is problematic because they are often conformationally complex and/ or large. Most recombinant antigens require some form of adjuvant to
Immunology and Cell Biology
elicit sufcient immune responses. There is much uncertainty about which proteinadjuvant combinations will be well-tolerated, yet elicit an effective immune response with sustained duration. Peptide vaccines Peptide vaccines have the potential to deliver precisely dened epitopes with a large scale of production at relatively low cost.72,73 Using a totally synthetic approach is perhaps the safest manufacturing method, and largely avoids concerns of microbiological contamination. There is also the potential to synthesize or conjugate multiple epitopes into a single construct. However, there are major challenges to synthesizing conformational epitopes; peptide antigens with limited epitopes may not elicit adequate immune responses in children and in certain human leukocyte antigen (HLA) subgroups.74 There are approaches to overcome some of these barriers in which peptides can be conjugated to T-cell epitopes, lipid moieties, or suspended in liposomes or virus-like particles.75 Similarly, peptides are more amenable to engineering to potentially improve immunogenicity and can be built into scaffolds to mimic native conformation.72,76 At present, a lack of knowledge regarding functionally relevant or protective epitopes of major antigens is a major impediment for the development of peptidebased vaccines, and clearly represents an area for further studies. DNA and viral-vectored vaccines The application of DNA and viral-vectored approaches for malaria vaccines is beginning to emerge and is reviewed elsewhere.7781 Most employ prime-boost strategies, but use different viral vectors and a range of different formulations, including liposomes, virosomes, microspheres and nanoparticles.82 They offer the advantage of including multiple antigens and have been shown to effectively induce both cell-mediated immunity and specic antibodies. POTENTIAL TARGETS AND THEIR PRIORITIZATION Prioritizing antigens Until recently, research efforts have focused on a handful of individual antigens. Perhaps the greatest advance for malarial research in the past decade has come from completion of major genome projects, includ-
ing P. falciparum, P. vivax, P. knowlesi, P. berghei, P. chabaudi and P. yoelii.15,8388 Thousands of novel proteins have been identied,5,89 providing new opportunities for vaccine development. However, at present, little is known about their structure and function or their role as targets of protective immunity. Several criteria can be used to objectively prioritize known and predicted antigens for further study.15,85 Table 1 lists some important properties that could be used in this process, and rates ve vaccine candidates against these criteria as an example. Targeting proteins that have an important function seems obvious, but the function is known for very few antigens. In some instances structural features may be suggestive of function (for example, epidermal growth factor domains in MSP1-19), and others have been classied as essential or nonessential on the basis of whether they can be genetically disrupted or not.90 Surface exposure or location within the apical organelles of the merozoite is likely to be highly relevant, but dening surface-exposed epitopes within antigens has proven to be more challenging. An increasing amount of data is emerging about polymorphisms, which may indicate targets under the pressure of immune selection, and regions or proteins of limited diversity. Abundance of proteins varies widely (for example, MSP1 vs MSP4), which may have implications for the extent and nature of immune responses. Structural features and the size of antigens have implications for vaccine manufacture. Studies of naturally acquired immunity can facilitate the identication of antigen-specic immune responses involved in protective immunity and the identication and characterization of immune effector mechanisms. At present, there is an over reliance on using standard immunoassays of uncertain functional signicance, and the development and application of functional assays is greatly needed. Potential targets and results from vaccine trials Human trials of blood-stage malaria vaccines have only been carried out with whole blood-stage parasites and merozoite antigens to date. Table 2 summarizes clinical trials that have been conducted or are in progress. Here, we discuss ndings from these trials and the properties of vaccine antigens. Unfortunately, it was beyond the scope of this review to comprehensively include vaccine testing in animal models.
Table 1 Pre-clinical criteria for prioritizing candidate antigens for vaccine development
Current vaccine candidatesa Criteria MSP1-42 Known functionb Essential or important functionc Location Abundance of antigen Limited diversityd Possible target of protective immunity in humanse Antibodies inhibit parasite growth in vitrof Vaccination induces protective immunity in non-human primate modelsh Phase II vaccination in humansi No Yes Merozoite surface High No Yes Yes Yes Not protective MSP2 No Yes Merozoite surface High No Yes Unknown Unknown Protective MSP3 LSP No No/unknown Merozoite surface High Yes Yes Yesg Yes Trial ongoing AMA1 No Yes Apical Low No Yes Yes Yes Trial ongoing EBA175 RII Yes Yes Apical Low Yes Yes Yes Yes Not tested
Abbreviations: AMA, apical membrane antigen; EBA, erythrocyte-binding antigens; LSP, long synthetic peptide; MSP, merozoite surface protein. aFive leading vaccine antigens were selected as illustrative examples. bEBA175 binds to glycophorin A on the erythrocyte surface. cEssential function dened as an inability to knock out the gene in transfection studies. MSP3 is not essential, and its function is unknown. dMSP3 is polymorphic, but the LSP construct is highly conserved. eStudies of endemic populations have found naturally acquired antibodies associated with protection from symptomatic disease (this does not mean that antibodies are causally responsible for protection). fStandard assay is the growth inhibition assay without immune cells. gMSP3 antibodies do not have direct growth inhibitory activity but show antibody-dependent cellular inhibition. hMSP2 has not been tested in non-human primates. It is not possible to test it in murine models. iPhase II vaccination in humans is meant to serve as a comparison of how the criteria predict the nal outcomes.
Table 2 Summary of vaccine studies in humansa

Stage Antigen Vaccine Trial phase Erythrocytic Whole parasite MSP1-42 Low-dose live parasites/drug cureb FMP1/ASO2 FMP1/ASO2 FMP1/ASO2 FMP1/ASO2 FMP1/ASO2c I/II I I I I II Australia USA Kenya Mali Kenya Kenya ve adults 15 adults 40 adults 40 135 children 14 years 400 children 14 years 60 adults 60 adults 25 adults 45 adults 35 adults 30 adults 45 children 12 years 45 children 12 years 378 children 12 years 30 adults 54 adults 36 children 23 years 900 children 23 years AMA1-C1/alhydrogel (short schedule) C1/alhydrogel +/CPG 7909 C1/alhydrogel +/CPG 7909 C1/alhydrogel +/CPG 7909 C1/alhydrogel +CPG 7909b C1/alhydrogel +/CPG 7909 (high/low) C1/alhydrogel +/CPG 7909 (saline/phosphate) C1/alhydrogel/ISA720 FMP2.1/ASO2 FMP2.1/ASO2 FMP2.1/ASO2 FMP2.1/ASO2c I I I I I/II I I I I I I II USA USA Mali Mali UK USA USA USA USA Mali Mali Mali 18 adults 75 adults 24 adults 200 children 13 years 21 adults No. not stated-adults 24 adults 28 adults 23 adults 60 adults 100 children 16 years 400 children 16 years 35 adults 56 adults 60 adults 80 adults 36 adults Not specied Completed Completed Completed Recruiting Not started Completed Completed Completed Completed Completed Completed Active NP
150
Site
Participants
Status
References
Completed Completed Completed Completed Completed Completed
68
108 109 110 242
NP
MSP1-42 (3D7 or FVO)/alum MSP1 MSP2 MSP3 MSP1-42-C1/alhydrogel (+/CPG 7909) FMP010/ASO1 MSP2 (3D7+FC27) MSP3 LSP/alum/ISA720 MSP3 LSP/alum MSP3 LSP/alum MSP3 LSP/alum MSP3 LSP/alumc
I I I I I I I I II
USA USA USA Australia Switzerland Burkina Faso Tanzania Burkina Faso Mali
Completed Completed Completed Completed Completed Completed Recruiting Recruiting Recruiting
112,243
NP NP NP
131,132 135
NP NP NP
AMA1
AMA1-C1/alhydrogel AMA1-C1/alhydrogel AMA1-C1/alhydrogel AMA1-C1/alhydrogelc
I I I II
USA Mali Mali Mali
Completed Completed Completed Completed
147 148 149
NP
NP NP NP NP NP NP
151
NP NP NP
FMP2.1/ASO2/ASO1 PfAMA1-FVO/alhydrogel/ISA720/ASO2 PfAMA1-FVO/alhydrogel/ISA720/ASO2 EBA175 GLURP SERA5 EBA175 RII-NG GLURP LSP SE36
I I I I I I
USA Netherlands Mali USA Netherlands Japan
Completed Completed Recruiting Completed Completed Completed
NP
155
NP NP
187
244
Table 2 Continued
Stage Antigen Vaccine Trial phase MSP1/MSP2/RESA Combination B/ISA720 Combination B/ISA720 Combination B/ISA720b Combination B/ISA720 Combination B/ISA720c I I I/II I I/II Australia Australia Australia Papua New Guinea Papua New Guinea 32 adults 36 adults 17 adults 12 adults 120 children 59 years 52 adults 70 adults 30 adults 40 adults 30 children 15 years Completed Completed Completed Completed Completed
120 120 121
Site
Participants
Status
References
122 42,123125
AMA1/MSP1-19
PfCP2.9/ISA720 PfCP2.9/ISA720
I I I I I
China China Germany Gabon Gabon
Completed Completed Recruiting Recruiting Recruiting
115 116
MSP3/GLURP
GMZ2/alum GMZ2/alum GMZ2/alum
NP NP NP
SPf66d Multistage Polyprotein (six antigens) FP9-PP, MVA-PP FP9-PP, MVA-PPe AMA1/CSP PEV301 (AMA1), PEV302 (CSP), PEV3A (PEV301+PEV302) PEV3A (AMA1+CSP), FP9 ME-TRAPe AMA1/CSP NYVAC-Pf7 MSP2/CSP AMA1/CSP/Adeno5f CSP, SSP2, LSA1, MSP1, AMA1, SERA, Pfs25e Combination A/alume I II I I/II I/II I/II I/II UK UK Switzerland UK USA USA Switzerland 35 adults 26 adults 46 adults 30 adults 70 adults 59 adults 39 adults Completed Completed Completed Completed Recruiting Completed Completed NP NP
159
158
NP
220
119
Abbreviations: AMA, apical membrane antigen; EBA, erythrocyte-binding antigens; GLURP, glutamate-rich protein; LSP, long synthetic peptide; MSP, merozoite surface protein; NP, Not published; SERA, serine repeat antigen; SSP, sporozoite surface protein; TRAP, thrombospondin-related adhesion protein. aSome of this information is gathered from www.clinicaltrials.gov and is the most accurate publically available data at the time of writing. bBlood stage challenge. cNatural exposure. dSPf66 underwent numerous clinical studies which are comprehensively reviewed elsewhere. eSporozoite challenge.
Merozoite surface antigens. Merozoite surface protein 1 coats the merozoite surface and is GPI (glycosylphosphatidylinositol) anchored.91,92 It undergoes several proteolytic processing steps creating several fragments, of which the 42 kDa (MSP1-42) and 19 kDa fragment (MSP1-19) have been most studied.93 The crystal structure for MSP1-19 has been elucidated.94,95 Its function seems to be essential, probably for initial attachment of merozoites to the erythrocyte surface through a complex with MSP9.96,97 There are two major allelic variants, MAD20 and K1, with highly polymorphic (for example, Block 2) and relatively conserved regions (for example, MSP1-19).98 Antibodies to MSP1-19 have been variably associated with protection from symptomatic disease.99103 Vaccine-induced antibodies have invasion inhibitory activity and may also inhibit MSP1 processing.103,104 Vaccination of mice and primates protected from subsequent challenge in some studies.43,105107 Human vaccine studies include MSP1-42 (3D7) formulated with adjuvant, ASO2A, (GlaxoSmithKline, Brentford, Middlesex, UK) that induced growth inhibitory antibodies.108110 However, a Phase II trial among 12 to 47month-old children in Kenya found no protective effect.111 Phase I studies of MSP1-42 (3D7 and FVO) in Alhydrogel (Sigma-Aldrich,
St Louis, MO, USA) generated moderate antibody responses, but insufcient in vitro inhibitory activity.112 MSP1-19 has only been tested in human vaccine studies as part of the chimeric vaccine, PfCP2.9, which combines domain 3 of apical membrane antigen 1 (AMA1) (3D7).113,114 In phase I studies, adjuvanted with Montanide ISA720, (SEPPIC SA, Paris, France) there was high immunogenicity, but signicant reactogenicity.115,116 Antibody invasion inhibitory activity was weak, as were T-cell responses. Human trials with P. vivax antigens have not yet been carried out. However, immunization using PvMSP1-19 had some protective effect in a non-human primate challenge.117 Merozoite surface protein 2 (MSP2) is also GPI anchored to the merozoite surface. Its function remains unclear, but also seems to be essential.90 There are two major allelic families (FC27 and 3D7) each with a highly variable central region consisting of several repeats and conserved anking regions.54 Natural immunity results in high levels of antibodies, predominantly IgG3 and a moderate association with protection from symptomatic disease.36,118 MSP2 (3D7) was included in the Combination A vaccine, adsorbed onto Alum and found to be safe, but poorly immunogenic.119 Five patients were challenged by
mosquito bite, but no protective benet was detected. Combination B comprised of MSP1 (190LCS.T3, N terminal end, K1 allele), MSP2 (3D7) and a part of the ring-infected erythrocyte surface antigen (RESA) in Montanide ISA720. Phase I/II studies among children in Papua New Guinea reported lower parasite densities in the vaccine recipients.42,120123 Furthermore, the risk of infection with the MSP2 allelic variant included in the vaccine (3D7) was much lower than the risk with the alternate allelic variant (FC27), suggesting that the antigen mediating the major protective effect was MSP2.124,125 Merozoite surface protein 3 (MSP3) has neither a GPI anchor nor typical transmembrane domain, but is non-covalently attached to the merozoite surface. Its function is unknown, but it seems to interact with acidic basic repeat antigen (ABRA, also known as MSP9) and can be genetically disrupted.126 It has allelic dimorphism (K1 and 3D7) with the C-terminal end being conserved and the N terminal end being highly polymorphic.127129 Antibodies have been associated with protective immunity in studies of acquired immunity in humans.130 MSP3 vaccines based on the conserved C-terminal end of the protein expressed as a long synthetic peptide have been tested in clinical trials.131,132 The vaccine was immunogenic, mostly of IgG3 subclass, but antibody titers were not sustained. Antibodies have not been shown to directly inhibit invasion, but instead seem to act in cooperation with monocytes to inhibit parasite replication in vitro.131,133,134 A phase I study in Burkina Faso failed to show any vaccine-related boosting of MSP3 antibody responses above the high level seen at enrollment.135 There are two phase I studies and a phase II study examining this vaccine in children from endemic countries. There are also three phase I studies currently recruiting using a MSP3/ GLURP (glutamate-rich protein) chimera.136 Proteins of the apical organelles of merozoites. The apical organelles of the merozoite contain a number of known or predicted invasion ligands. One of these, AMA1 is thought to play an essential role in erythrocyte invasion, however, its precise function is not known.137 Its crystal structure has recently been solved and it is thought to form a complex with rhoptry neck protein 4 (RON4).138,139 AMA1 is highly polymorphic and allele-specic antibodies are not cross-protective; presenting a major challenge to vaccine development.52,140143 Acquired antibodies have been associated with protection from symptomatic disease in humans.36,144 Antibodies have been shown to inhibit invasion in functional assays, although non-functional antibodies are also prevalent.141,145 Immunization with AMA1 was protective in primate studies.146 There have been numerous AMA1 human vaccine studies, mainly phase I studies to date. The AMA-C1 (3D7 and FVO strains) vaccine, adjuvanted with Alhydrogel, was safe and immunogenic, and generated antibodies that inhibited parasite growth in vitro.141,147 This was conrmed in Malian adults and children, but antibody levels were not maintained.148,149 A phase II study is presently underway. There are a range of studies examining other adjuvants. Adding CPG 7909 to the Alhydrogel formulation led to three- to fourfold higher antibody titers.150 There was signicant invasion inhibition, but this was relatively variant-specic. In phase I trials, the FMP2.1 vaccine (3D7 strain) adjuvanted with ASO2A generated high-titre antibodies that inhibited growth and AMA1 processing.144,151,152 It also resulted in cell-mediated immune responses. Phase II studies are currently underway. ASO1B is also being explored as an alternative adjuvant (unpublished). The PfAMA1-FVO construct has been formulated with a range of adjuvants, including Alhydrogel, Montanide ISO720 and ASO2.153155 Antibody titers were highest with ASO2, and were maintained for longer and inhibited in vitro invasion. This vaccine
is being studied in Malian adults. The NMRC-M3V-Ad-PfCA vaccine contains CSP (circumsporozoite protein) and AMA1 (3D7) using an adenovirus 5 vector. The PEV301, PEV302, PEV3A (AMA1/CSP peptide) vaccines use inuenza virosomes to deliver an AMA1containing peptide from loop I of domain III (K1 strain), as well as a double loop of NPNA repeats of the CSP peptide.156,157 Phase I and II studies showed good safety and immunogenicity with high antibody levels, high avidity and recognition of blood-stage parasites.158,159 Sporozoite challenge failed to afford sterile protection or to prolong time to parasitemia, but vaccine recipients had lower parasite growth rates and had morphologically altered parasites detected. The erythrocyte-binding antigens (EBAs) are a family of antigens, which are orthologues of the Duffy-binding protein of P. vivax and include EBA175, EBA140 (BAEBL), EBA181 (JESEBL), MAEBL, EBL1 and EBA165 (pseudogene). On each of these proteins, there is a receptor-binding domain (region II), which is structurally related between proteins, but differs in sequence; the crystal structure was recently solved for EBA175.160 They are located in the micronemes and are secreted onto the merozoite surface just before invasion.161163 It is known that EBA175 and EBA140 interact with glycophorin A and C, respectively.164167 Vaccine-induced antibodies in animals inhibit invasion.165,168,169 There is limited sequence polymorphism in the receptor-binding domains, but this does not seem to affect the activity of vaccine-induced antibodies.170 Overall, there is little polymorphism throughout the protein. Acquired antibodies among children have been associated with protection from symptomatic disease in some studies.171,172 Parasites can vary the expression and use of these ligands during invasion, which seems to facilitate immune evasion, and the EBAs seem to function in a complementary manner with the reticulocyte binding-like homologs (PfRHs).56 The only human vaccine study is a phase I study of EBA175 region 2 adsorbed onto aluminum phosphate (unpublished). An EBA175 vaccine was shown to have efcacy in a primate challenge.173 Very little work has been carried out on the development of P vivax vaccines. However, the Duffy-binding protein (DBP) is a strong vaccine candidate. DBP binds to DARC (Duffy antigen/receptor for chemokines) during parasite invasion of reticulocytes.174,175 DBP seems essential for invasion, and resistance to P. vivax in humans is conferred by a lack of DARC expression.176,177 DBP is localized in the micronemes,174 and is thought to be essential for the formation of the tight junction. The receptor-binding face of the protein has little polymorphism suggesting that it may be possible to develop a vaccine that predominantly targets the conserved region to block the DBPDARC interaction. Studies have shown that vaccine-induced antibodies in animals and acquired human antibodies can inhibit reticulocyte invasion and inhibit the binding of DBP to DARC.178,179 Furthermore, DBP-binding inhibitory antibodies have been associated with acquired protective immunity in humans and immunization with DBP was partially protective in a primate challenge model.180,181 Other merozoite antigens. Glutamate-rich protein is expressed on the merozoite surface and in liver stage schizonts,182 but its function is unknown. It has two repeat regions (R1 and R2), and an N-terminal non-repeat region which has limited diversity (R0).183 Naturally acquired antibodies of IgG1 and IgG3 subclasses (depending on the antigenic region) are variably associated with protection.184186 Human vaccine studies have focused on a long synthetic peptide derived from the R0 region. A phase I study with glutamate-rich protein long synthetic peptide adjuvanted in alum or Montanide ISA720 generated antibodies that inhibited parasite growth in coopImmunology and Cell Biology
eration with monocytes.187 It has also been examined in combination with MSP3. Future malaria vaccine research will need to assess the potential of other antigens, which have been identied, but have not yet been tested in humans. The reticulocyte binding-like homologs (PfRHs) are a family of invasion ligands that have homology with the reticulocytebinding proteins of P. vivax. They are found in the neck of the rhoptries and are known to bind to erythrocytes, but receptors remain to be identied. Antibodies are acquired naturally and have been strongly associated with protection.188 Antibodies to PfRH1 and PfRH2 have been shown to inhibit invasion, and polymorphism in the PfRH proteins is limited. MSP4 is GPI anchored to the merozoite surface, has limited diversity and elicits naturally acquired antibodies, though these are not associated with protection.189191 Vaccination with the P. yoelii homologue, MSP4/5, is protective in murine challenge.192,193 There many other promising candidates, amongst these are the SERA (serine repeat antigen) family, RhopH1-3, and the 6-cysteine domain family. Variant surface antigens. Antibodies against variant surface antigens (VSAs) expressed on the surface of the infected erythrocyte are thought to contribute to strain-specic protection in humans and primate models.66,194 Studies in monkeys also suggest that VSAs are important targets of protective antibodies. VSAs include PfEMP1, rins, subtelomeric variable open reading frame (STEVOR), and others;25 to date, only PfEMP1 has been established as a major target of antibodies.195,196 PfEMP1 is encoded by the var multigene family, and different var genes encode PfEMP1 variants with different antigenic properties.197 The immune response against VSAs is thought to contribute to strain-specic protection, but only PfEMP1 has been established as a major target of antibodies.194 There is a high level of polymorphism between different var genes within a single genome and between var genes in different genomes. Rins and STEVOR are also encoded by large polymorphic gene families. The major barrier to developing VSAs as vaccines is their very high degree of antigenic diversity and capacity for clonal antigenic variation. Studies are beginning to identify subsets of var genes associated with severe disease that are likely to be important immune targets.198200 Some studies have suggested that cross-reactive antibodies can be generated despite extensive polymorphism and a vaccine trial in primates showed protection against homologous parasites.201,202 A specic variant of PfEMP1 (var2csa) is expressed by P. falciparum-infected erythrocytes that mediates adhesion to the placental lining during pregnancy and seems to be an important target of acquired antibodies.203 Var2csa is less antigenically diverse than other PfEMP1 variants and antibodies that cross-react with different placental-binding isolates have been shown in humans and animal models. Therefore, it may be possible to develop a vaccine inducing broad coverage against different placental-binding variants by targeting conserved and/or common epitopes of var2csa.204206 Other antigens. Glycosylphosphatidylinositol is a glycolipid that anchors a number of erythrocytic stage proteins to the membrane, including MSP1, MSP2 and serine proteases. It has been reported to have toxin-like effects and induce pro-inammatory responses and clinical symptoms in murine models (reviewed in Schoeld and Grau207). Immunization with a synthetic version of the GPI glycan showed some protection against clinical illness in mice, providing a proof-of-concept for this type of approach.208 The vaccine did not have any affect on parasite replication and it is likely that GPI would need to be included in a multivalent vaccine rather than being used alone.
Other human vaccine studies. The SPf66 vaccine consists of a 45 amino-acid peptide, which is a chimera of three merozoite-derived epitopes interspersed with the PNANP sporozoite peptide of CSP. Most studies have examined it adjuvanted with aluminum hydroxide. It showed mild protective efcacy in a South American and a Tanzanian study, but failed to show any benet in the studies conducted in Gambia or Thailand.209217 Much controversy surrounded these studies and it was the subject of a Cochrane review, which concluded that there was no justication for further trials using the same formulation.218 The NYVAC-Pf7 vaccine incorporates seven genes into an attenuated vaccinia virus. These genes included blood-stage antigens, MSP1, AMA1, SERA as well CSP, liver-stage antigens 1 (LSA1), sporozoite surface protein 2(SSP2) and the 25 kDa sexual-stage antigen, SPf25.219 In a human sporozoite challenge model, antibody levels in vaccinated individuals were found to have a slight delay in the prepatent period.220 The FP9-PP, MVA-PP vaccine is principally a pre-erythrocytic vaccine, using the fowlpox 9 (FP9) and the modied vaccinia virus Ankara (MVA) polypeptide (PP) vaccines, which contain proteins that are also expressed during the erythrocytic stage.221 RECENT ADVANCES AND CONTINUING CHALLENGES Determining structure and function A more complete understanding of the structure and function of Plasmodium proteins, particularly those involved in host-cell adhesion and invasion will greatly facilitate the identication, rationalization and evaluation of candidate antigens. As expected, there will be a lag between advances in genomics to that of proteomics, and particularly functional molecular biology. Remarkably little is known about the molecular interactions that occur during merozoite invasion, with the binding partners of most merozoite antigens yet to be identied.222224 This lack of knowledge limits more targeted approaches against functionally important domains, the signicance of sequence polymorphism in antigens and the development of functional immunological assays for vaccine trials. Structural modeling is beginning to inform molecular interactions, including antibodies.95,138,160,225230 This has the potential to assist in epitope mapping and determining the mechanisms of immune evasion.231,232 Identifying correlates of immunity and developing functional assays The identication of robust correlates of immunity and assays that measure functionally relevant immune responses would be of enormous benet for the development of blood-stage vaccines. Unfortunately there is still much to be done to achieve this goal. Studies of acquired human immunity have largely focused on only a small number of antigens using standard immunoassays that provide little indication of the functional relevance of the responses. Very little is known about functional effector mechanisms and there are few assays examining these issues.38,39,131,233 Future studies will need to accommodate the testing of responses to a large number of candidate antigens that are now being identied, and greater effort is required to dene immune effector mechanisms and their contribution to immunity in humans. For example, the predominant assays for merozoite vaccines measure direct antibody growth inhibition or antibodydependent cellular inhibition.38,39 These assays have been carried out for many years, but have rarely been applied to large population studies. The further renement and optimization of these assays and the development of high-throughout methods, such as using ow cytometry, and transgenic parasites to identify and quantify antigenspecic responses will advance our ability to understand and
measure immune responses in studies of acquired and vaccineinduced immunity.56,59,103,180,234240 Pathway to vaccine development: animal models and human challenge In many instances, malaria vaccine research has taken candidate antigens into rodent studies to determine immunogenicity and evaluate efcacy using challenge models. Although this approach has the benets of being readily available and relatively affordable, there are considerable limitations. Human malarias cannot infect mice and rodent malarias lack many important antigens of P. falciparum and P. vivax, which means that murine challenge models cannot be used to assess their efcacy. Additionally, there are important differences between mice and humans in the nature of immune and pathogenic responses to malaria. More recently, there have been efforts to humanize the mouse model to make it more representative of human immune responses.131,241 These models may provide an informative approach to evaluate blood-stage vaccines, but further studies are needed to establish their relevance. Vaccine testing often proceeds to challenge studies in non-human primates. This is expensive, has limited availability, and for P. falciparum, is largely restricted to Aotus and Saimiri models. Given the limitations of animal models for testing malaria vaccines, the safety and immunogenicity of candidate vaccines in humans should be determined as rapidly as possible. It could be argued that vaccine candidates that have a very strong rationale for development, based on a set of widely accepted biological and immunological criteria, could proceed through to human trials without the need for evaluation in animal models. With the need to progress candidate vaccines more quickly into human trials, there is an increasing interest in testing vaccines in humans using a live parasite challenge; this has been carried out either by direct inoculation of blood-stage parasites or with sporozoites through mosquito bite. There are a number of signicant concerns about the relevance of this model for evaluating the efcacy of bloodstage vaccines, and it remains to be established that it is a valid approach. A principle problem with this approach is that participants must be treated as soon as blood-stage parasitemia is detected for ethical reasons. Blood-stage immunity acts primarily by limiting parasite density and protecting against symptomatic illness rather than preventing parasitization per se. However, it is not possible to assess clinical illness or high-density parasitemia as an outcome using this approach, and there is only a limited ability to measure parasite replication rates before treatment. On-going studies might shed light on these issues and clarify whether non-human primate challenge or the human challenge model is more informative for evaluating bloodstage vaccines. Of interest, the Combination B vaccine did not show any effect on blood-stage replication in a challenge carried out in vaccinated malaria-nave individuals, but did show protective effect in a phase II trial in children in Papua New Guinea.42,121 CONCLUSIONS AND PERSPECTIVES In this review, we have highlighted a number of issues surrounding the development of vaccines against malaria based on antigens expressed in the blood-stage, as well as summarized recent progress and the current state of the eld. Effective blood-stage vaccines will almost certainly need to be comprised of several different antigens, or several different alleles of a single antigen, to overcome antigenic diversity and the capacity of P. falciparum for immune evasion or perhaps to achieve more potent anti-parasite activity by combining antigens that induce antibodies with synergistic activities against parasite growth. There are presently a substantial number of blood-stage vaccine candidates and
a multitude of new antigens are likely to emerge from recent genomic and proteomic insights. Therefore, there is a strong need for approaches to validate and prioritize potential vaccine candidates for further development. The recent success of the RTS, S vaccine in several phase II trials conducted in African children has changed the landscape of malaria vaccine development. RTS,S is now entering phase III studies and it seems likely that it will eventually become licensed for broad use. RTS,S represents a rst generation vaccine against malaria, and the challenge ahead will be to develop the next generation of vaccines that have greater efcacy and provide more sustained protection. This may be achieved by combining the RTS,S antigen with other antigens, or the development of vaccines targeting different antigens altogether. There is a strong rationale for the continued development and testing of blood-stage vaccines highlighted in this review, and for investigating the inclusion of blood-stage antigens in combination with preerythrocytic antigens, such as the RTS,S antigen. The importance of blood-stage antigens as targets of acquired immunity in humans together with the demonstrated efcacy of different blood-stage antigens in animal models and the partial efcacy of at least one blood-stage vaccine in humans provides reasonable cause to be optimistic about the development of blood-stage vaccines against malaria. ACKNOWLEDGEMENTS
JGB is supported by a Clinical Career Development Award, and JSR by a Medical Postgraduate Research Scholarship from the National Health and Medical Research Council of Australia. Thanks to Creepy Crawley Cartoons for preparing the gures.
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Review On Blood Stage Vaccine Against Malarai

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Immunology and Cell Biology (2009) 87, 377390 & 2009 Australasian Society for Immunology Inc.

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The future for blood-stage vaccines against malaria

Blood-stage vaccines against malaria JS Richards and JG Beeson 378

Apical end Rhoptries Apicoplast

Micronemes Merozoite surface

Dense granules Mitochondrion Nucleus

Blood-stage vaccines against malaria JS Richards and JG Beeson 379

Blood-stage vaccines against malaria JS Richards and JG Beeson 380

Immunology and Cell Biology

Blood-stage vaccines against malaria JS Richards and JG Beeson 381

Table 2 Summary of vaccine studies in humansa

Completed Completed Completed Completed Completed Completed

108 109 110 242

Completed Completed Completed Completed Completed Completed Recruiting Recruiting Recruiting

AMA1-C1/alhydrogel AMA1-C1/alhydrogel AMA1-C1/alhydrogel AMA1-C1/alhydrogelc

USA Mali Mali Mali

Completed Completed Completed Completed

147 148 149

USA Netherlands Mali USA Netherlands Japan

Completed Completed Recruiting Completed Completed Completed

Immunology and Cell Biology

Blood-stage vaccines against malaria JS Richards and JG Beeson 382

China China Germany Gabon Gabon

Completed Completed Recruiting Recruiting Recruiting

GMZ2/alum GMZ2/alum GMZ2/alum

Blood-stage vaccines against malaria JS Richards and JG Beeson 383

Blood-stage vaccines against malaria JS Richards and JG Beeson 384

Blood-stage vaccines against malaria JS Richards and JG Beeson 385

Immunology and Cell Biology

Blood-stage vaccines against malaria JS Richards and JG Beeson 386

Immunology and Cell Biology

Blood-stage vaccines against malaria JS Richards and JG Beeson 387

Immunology and Cell Biology

Blood-stage vaccines against malaria JS Richards and JG Beeson 388

Immunology and Cell Biology

Blood-stage vaccines against malaria JS Richards and JG Beeson 389

Immunology and Cell Biology

Blood-stage vaccines against malaria JS Richards and JG Beeson 390

Immunology and Cell Biology

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