review on blood stage vaccine against malarai

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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 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. 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 efficacious in animal models

and at least one vaccine showed partial efficacy 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, identification of protective epitopes, understanding the nature and targets of protective immune

responses, and defining antigen combinations that give the greatest efficacy. 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 field 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 pre-

erythrocytic vaccine, RTS,S, showing protective efficacy.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 five 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 mero-

zoites invading erythrocytes, intra-erythrocytic development and

asexual reproduction, erythrocyte rupture and the release of newmerozoites 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 12min 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 histocompat-ibility 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 com-

plexity of the parasite lifecycle, a high level of antigenic diversity and

parasite mechanisms of immune evasion, naturally acquired immu-

nity to malaria does develop after repeated exposure. This immunity

affords protection against symptomatic disease, high-density

Received 6 February 2009; accepted 15 March 2009; published online 5 May 2009

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: [email protected]

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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parasitemia and death; however, it is less effective at preventing low-

level parasitemia.26,27 Naturally acquired immunity seems to predo-

minantly 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 immunoglo-

bulin from immune individuals conferring protective benefit to non-immune individuals.31,32 Immune effector mechanisms are poorly

understood and the relative importance of different mechanisms has

not been quantified. 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 fromblood-stage infection and studies in humans and animal models have

clearly established that immune responses targeting blood-stage anti-

gens can protect against disease or facilitate control of parasite-

mia.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).

1

2 3 4

1

2 3

4

5

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-affinity 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 theparasitophorous vacuole. Outer surface proteins are partially cleaved as the

merozoite enters the erythrocyte (5).

Micronemes

Merozoite surface

Dense granules

Nucleus

Rhoptries

Apicoplast

Mitochondrion

Apical end

MSP1, 2, 4, 5, 10 (GPI anchored)MSP3, 6, 7, 9 (ABRA)GLURPSERAS-antigen6-cys family (GPI anchored)

AMA1

EBA175, 140, 181MTRAPPTRAMPASP

Rh 1, 2a, 2b, 4, 5RAP1, 2, 3RhopH1, 2, 3RAMA

Merozoite surface proteins

Micronemes

Rhoptries

Figure 2 The structure and major antigens of the P. falciparum merozoite. The apical end of the merozoite has specific 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 identified or characterized.

Blood-stage vaccines against malariaJS Richards and JG Beeson

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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 over10 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 efficacy, whereas merozoite surface protein 1 (MSP1)-42 did

not. Although RTS,S, which is based on the circumsporozoite protein,

has been efficacious in several trials, another pre-erythrocytic vaccine,multi-epitope thrombospondin-related adhesion protein (ME-

TRAP)47,48 was not protective. It is likely that few of the vaccines

that are eventually tested in clinical trials will prove to be sufficiently

efficacious; therefore, poor outcomes with specific antigens should not

justify abandonment of an overall strategy.

Recently, there has been a renewed call for the eradication ofmalaria.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 partof 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 benefit in reducing transmission, in addition to preventing

clinical illness.

Perhaps the greatest challenge in developing a blood-stage vaccine isovercoming 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 difficult 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 antigenicdiversity. 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 blood-

stage antigens, this may be more important than for pre-erythrocytic

and transmission-blocking vaccines. For example, non-functionalantibodies that impair the binding of functionally important anti-

bodies to merozoite antigens have been identified.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 whole-

killed 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 fora malaria vaccine. Most malaria vaccine research, however, has

followed subunit approaches in which antigens are identified, purified,

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 else-

where.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-specific immunity that limited sympto-matic disease, but did not prevent re-infection.65 Other studies in

animal models also suggest that immunity induced in this way is

largely strain-specific.66,67 More recently a study administered live P.

falciparum parasites intravenously at low dose to five patients,

followed by early anti-malarial treatment.68 Four patients were rechal-

lenged with live parasites of the same strain, and three did not developany detectable parasitemia before final anti-malarial treatment. Sur-

prisingly, 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 benefit 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 ahigh 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 blood-

borne 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 significant challenges inproducing and delivering live blood-stage parasites for widespread

use. In vitro culture of P. vivax is difficult 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 specified 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 produc-

tion under good manufacturing practice. The inherent difficulty is

identifying protective target antigens for development, or specificdomains 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


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elicit sufficient 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 defined

epitopes with a large scale of production at relatively low cost. 72,73

Using a totally synthetic approach is perhaps the safest manufacturingmethod, and largely avoids concerns of microbiological contamina-

tion. 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 peptidescan be conjugated to T-cell epitopes, lipid moieties, or suspended in

liposomes or virus-like particles.75 Similarly, peptides are more amen-

able 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 peptide-based 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 arange of different formulations, including liposomes, virosomes,

microspheres and nanoparticles.82 They offer the advantage of includ-

ing multiple antigens and have been shown to effectively induce both

cell-mediated immunity and specific antibodies.

POTENTIAL TARGETS AND THEIR PRIORITIZATION

Prioritizing antigens

Until recently, research efforts have focused on a handful of individualantigens. 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 identified,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 five vaccinecandidates 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 classified as essential or non-

essential 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 defining 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 implicationsfor 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 identification

of antigen-specific immune responses involved in protective immunity

and the identification and characterization of immune effector

mechanisms. At present, there is an over reliance on using standardimmunoassays of uncertain functional significance, and the develop-

ment 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 findings from these trials and the propertiesof 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

Criteria

Current vaccine candidatesa

MSP1-42 MSP2 MSP3 LSP AMA1 EBA175 RII

Known functionb No No No No Yes

Essential or important functionc Yes Yes No/unknown Yes Yes

Location Merozoite surface Merozoite surface Merozoite surface Apical Apical

Abundance of antigen High High High Low Low

Limited diversityd No No Yes No Yes

Possible target of protective immunity in humanse Yes Yes Yes Yes Yes

Antibodies inhibit parasite growth in vitrof Yes Unknown Yesg Yes Yes

Vaccination induces protective immunity in non-human primate models h Yes Unknown Yes Yes Yes

Phase II vaccination in humansi Not protective Protective Trial ong oing Trial ong oing No t 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 defined 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 forprotection).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 final outcomes.


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Table 2 Summary of vaccine studies in humansa

Stage Antigen Vaccine Trial

phase

Site Participants Status References

Erythrocytic Whole parasite Low-dose live parasites/drug cureb I/II Australia five adults Completed 68

MSP1-42 FMP1/ASO2 I USA 15 adults Completed 108

FMP1/ASO2 I Kenya 40 adults Completed 109

FMP1/ASO2 I Mali 40 Completed 110

FMP1/ASO2 I Kenya 135 children

14 years

Completed 242

FMP1/ASO2c II Kenya 400 children

14 years

Completed NP

MSP1-42 (3D7 or FVO)/alum I USA 60 adults Completed 112,243

MSP1-42-C1/alhydrogel (+/CPG 7909) I USA 60 adults Completed NP

MSP1 FMP010/ASO1 I USA 25 adults Completed NP

MSP2 MSP2 (3D7+FC27) I Australia 45 adults Completed NP

MSP3 MSP3 LSP/alum/ISA720 I Switzerland 35 adults Completed 131,132

MSP3 LSP/alum I Burkina Faso 30 adults Completed 135

MSP3 LSP/alum I Tanzania 45 children

12 years

Recruiting NP

MSP3 LSP/alum I Burkina Faso 45 children

12 years

Recruiting NP

MSP3 LSP/alumc II Mali 378 children

12 years

Recruiting NP

AMA1 AMA1-C1/alhydrogel I USA 30 adults Completed 147

AMA1-C1/alhydrogel I Mali 54 adults Completed 148

AMA1-C1/alhydrogel I Mali 36 children

23 years

Completed 149

AMA1-C1/alhydrogelc II Mali 900 children

23 years

Completed NP

AMA1-C1/alhydrogel (short schedule) I USA 18 adults Completed NP

C1/alhydrogel +/CPG 7909 I USA 75 adults Completed 150

C1/alhydrogel +/CPG 7909 I Mali 24 adults Completed NP

C1/alhydrogel +/CPG 7909 I Mali 200 children

13 years

Recruiting NP

C1/alhydrogel +CPG 7909b I/II UK 21 adults Not started NP

C1/alhydrogel +/CPG 7909 (high/low) I USA No. not stated-adults Completed NP

C1/alhydrogel +/CPG 7909 (saline/phosphate) I USA 24 adults Completed NP

C1/alhydrogel/ISA720 I USA 28 adults Completed NP

FMP2.1/ASO2 I USA 23 adults Completed 151

FMP2.1/ASO2 I Mali 60 adults Completed NP

FMP2.1/ASO2 I Mali 100 children

16 years

Completed NP

FMP2.1/ASO2c II Mali 400 children

16 years

Active NP

FMP2.1/ASO2/ASO1 I USA 35 adults Completed NP

PfAMA1-FVO/alhydrogel/ISA720/ASO2 I Netherlands 56 adults Completed 155

PfAMA1-FVO/alhydrogel/ISA720/ASO2 I Mali 60 adults Recruiting NP

EBA175 EBA175 RII-NG I USA 80 adults Completed NP

GLURP GLURP LSP I Netherlands 36 adults Completed 187

SERA5 SE36 I Japan Not specified Completed 244


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Merozoite surface antigens. Merozoite surface protein 1 coats

the merozoite surface and is GPI (glycosylphosphatidylinositol)

anchored.91,92 It undergoes several proteolytic processing steps creat-

ing several fragments, of which the 42 kDa (MSP1-42) and 19 kDafragment (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 ery-

throcyte 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 vaccinestudies 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 47-

month-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

insufficient 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 significant reactogenicity.115,116 Antibody invasion inhibitory activ-

ity 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 andconserved flanking 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

Table 2 Continued

Stage Antigen Vaccine Trial

phase

Site Participants Status References

MSP1/MSP2/RESA Combination B/ISA720 I Australia 32 adults Completed 120

Combination B/ISA720 I Australia 36 adults Completed 120

Combination B/ISA720b I/II Australia 17 adults Completed 121

Combination B/ISA720 I Papua New Guinea 12 adults Completed 122

Combination B/ISA720c I/II Papua New Guinea 120 children

59 years

Completed 42,123125

AMA1/MSP1-19 PfCP2.9/ISA720 I China 52 adults Completed 115

PfCP2.9/ISA720 I China 70 adults Completed 116

MSP3/GLURP GMZ2/alum I Germany 30 adults Recruiting NP

GMZ2/alum I Gabon 40 adults Recruiting NP

GMZ2/alum I Gabon 30 children

15 years

Recruiting NP

SPf66d

Multistage Polyprotein

(six antigens)

FP9-PP, MVA-PP I UK 35 adults Completed NP

FP9-PP, MVA-PPe II UK 26 adults Completed NP

AMA1/CSP PEV301 (AMA1), PEV302 (CSP),

PEV3A (PEV301+PEV302)

I Switzerland 46 adults Completed 159

PEV3A (AMA1+CSP), FP9 ME-TRAPe I/II UK 30 adults Completed 158

A MA1/CS P A MA1/CS P/Adeno5f I/II USA 70 adults Recruiting NP

NYVAC-Pf7 CSP, SSP2, LSA1, MSP1, AMA1, SERA, Pfs25e I/II USA 59 adults Completed 220

MSP2/ CSP Combi nation A/alume I/II Switzerland 39 adults Completed 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.


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mosquito bite, but no protective benefit 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 theantigen 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 endbeing 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 beenshown 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 invasionligands. 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 acomplex with rhoptry neck protein 4 (RON4).138,139 AMA1 is highly

polymorphic and allele-specific 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 gener-

ated antibodies that inhibited parasite growth in vitro.141,147 This was

confirmed 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 significant invasion inhibition, but this was

relatively variant-specific. 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 under-way. ASO1B is also being explored as an alternative adjuvant (unpub-

lished). 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 influenza virosomes to deliver an AMA1-

containing 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 areceptor-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 inhibitinvasion.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 ligandsduring 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 vac-

cine study is a phase I study of EBA175 region 2 adsorbed onto

aluminum phosphate (unpublished). An EBA175 vaccine was shown

to have efficacy 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 strongvaccine 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 inhibitreticulocyte 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 immu-

nization 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 theantigenic 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 coop-


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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 identified, 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 reticulocyte-

binding proteins of P. vivax. They are found in the neck of the

rhoptries and are known to bind to erythrocytes, but receptors remainto be identified. 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 murinechallenge.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 arethought to contribute to strain-specific protection in humans and

primate models.66,194 Studies in monkeys also suggest that VSAs are

important targets of protective antibodies. VSAs include PfEMP1,

rifins, 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 anti-

genic properties.197 The immune response against VSAs is thought to

contribute to strain-specific 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 vargenes in different genomes. Rifins 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 generateddespite extensive polymorphism and a vaccine trial in primates

showed protection against homologous parasites.201,202 A specific

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 anti-

bodies.203 Var2csa is less antigenically diverse than other PfEMP1

variants and antibodies that cross-react with different placental-bind-

ing isolates have been shown in humans and animal models. There-

fore, it may be possible to develop a vaccine inducing broad coverageagainst 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-inflammatory responses and

clinical symptoms in murine models (reviewed in Schofield andGrau207). 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 efficacy in a South American and a

Tanzanian study, but failed to show any benefit in the studies

conducted in Gambia or Thailand.209217 Much controversy sur-

rounded these studies and it was the subject of a Cochrane review,which concluded that there was no justification for further trials using

the same formulation.218

The NYVAC-Pf7 vaccine incorporates seven genes into an attenu-

ated 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-erythro-

cytic vaccine, using the fowlpox 9 (FP9) and the modified 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 identification, rationalization

and evaluation of candidate antigens. As expected, there will be a lagbetween 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 identified.222224

This lack of knowledge limits more targeted approaches against

functionally important domains, the significance of sequence poly-

morphism in antigens and the development of functional immuno-

logical assays for vaccine trials. Structural modeling is beginning toinform 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 identification of robust correlates of immunity and assays that

measure functionally relevant immune responses would be of

enormous benefit 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 identified, and greater effort is required to

define immune effector mechanisms and their contribution to immu-

nity in humans. For example, the predominant assays for merozoite

vaccines measure direct antibody growth inhibition or antibody-

dependent cellular inhibition.38,39 These assays have been carriedout for many years, but have rarely been applied to large population

studies. The further refinement and optimization of these assays and

the development of high-throughout methods, such as using flow

cytometry, and transgenic parasites to identify and quantify antigen-

specific responses will advance our ability to understand and


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measure immune responses in studies of acquired and vaccine-

induced 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 efficacy using challenge models. Although this approachhas the benefits 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 efficacy. Additionally, there are important differ-

ences between mice and humans in the nature of immune and

pathogenic responses to malaria. More recently, there have been effortsto 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 largelyrestricted 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 tohuman 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 significant concerns

about the relevance of this model for evaluating the efficacy of blood-

stage vaccines, and it remains to be established that it is a validapproach. 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 orthe human challenge model is more informative for evaluating blood-

stage 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 field. Effective blood-stage vaccines will almost

certainly need to be comprised of several different antigens, or severaldifferent alleles of a single antigen, to overcome antigenic diversity and

the capacity ofP. 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 first generation vaccine against malaria, and the

challenge ahead will be to develop the next generation of vaccines that

have greater efficacy 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 investigatingthe inclusion of blood-stage antigens in combination with pre-

erythrocytic antigens, such as the RTS,S antigen. The importance of

blood-stage antigens as targets of acquired immunity in humans

together with the demonstrated efficacy of different blood-stage

antigens in animal models and the partial efficacy of at least one

blood-stage vaccine in humans provides reasonable cause to beoptimistic about the development of blood-stage vaccines against

malaria.

ACKNOWLEDGEMENTSJGB 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 figures.

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