MINIREVIEW

Vaccines against malaria an update

Kai Matuschewski1 and Ann-Kristin Mueller1,2
1 Department of Parasitology, Heidelberg University School of Medicine, Germany 2 Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, UK

Keywords attenuated live parasite; malaria; MSP1; Plasmodium; protective immunity; RTS S; severe disease; transmission-blocking antibodies; vaccine; var2CSA Correspondence K. Matuschewski, Department of Parasitology, Heidelberg University School of Medicine, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany Fax: +49 6221 564643 Tel: +49 6221 568284 E-mail: Kai.Matuschewski@med. uni-heidelberg.de (Received 27 May 2007, accepted 19 July 2007) doi:10.1111/j.1742-4658.2007.05998.x

Malaria vaccine discovery and development follow two principal strategies. Most subunit vaccines are designed to mimic naturally acquired immunity that develops over years upon continuous exposure to Plasmodium transmission. Experimental model vaccines, such as attenuated live parasites and transmission-blocking antigens, induce immune responses superior to naturally acquired immunity. The promises and hurdles of the different tracks towards an effective and affordable vaccine against malaria are discussed.

Malaria is a preventable and treatable vector-borne infectious disease that is caused by single-cell eukaryotic parasites of the genus Plasmodium. According to recent estimates by the World Health Organization (WHO), malaria remains one of the major causes of mortality and morbidity, with 3.2 billion people at risk, 300500 million clinical cases and more than one million deaths annually, particularly in young children in sub-Saharan Africa [1]. Plasmodium transmission occurs by the injection of infectious sporozoites during the probing phase for a blood meal by an infected female Anopheles mosquito [2]. Sporozoites actively move away from the site of

injection, enter a capillary and within minutes reach the liver where they transform into liver stages and commit to continuous replication resulting in the generation of tens of thousands of pathogenic merozoites [3]. Malaria-associated pathology is exclusively restricted to the asexual replication of the parasite within erythrocytes, a rather unique environment for an intracellular pathogen. This terminally differentiated host cell offers the advantage of complete absence of MHC I-restricted antigen presentation, and, hence cellular immunity against the host cell. Protective mechanisms operate by neutralizing antibodies against the merozoite surface proteins and surface

Abbreviations CSP, circumsporozoite protein; FMP1, falciparum malaria protein-1; GAP, genetically attenuated parasite; MSP1, merozoite surface protein 1; PfEMP1, Plasmodium falciparum erythrocyte membrane protein 1; Pfs25, Plasmodium falciparum surface protein with apparent molecular mass of 25 kDa; RTS S, recombinant P. falciparum CSP vaccine, which includes the central repeat sequence R and major T-cell epitopes T, fused to the entire hepatitis B surface antigen S and coexpressed in yeast with the S antigen; TRAP, thrombospondin-related anonymous protein; var2CSA, variant surface antigen 2, chondroitin sulphate A-binding.

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proteins of the Plasmodium falciparum-infected erythrocyte (Fig. 1), typically resulting in parasite reduction rather than clearance [4]. A lower parasite burden may then account for some of the antidisease effects. In analogy to virtually any vector-borne disease, vector control and exposure prophylaxis are two basic public health tools that, when combined with clinical management, protect the individual from fatal disease and limit the spread of malaria [5]. However, examples of other arthropod-transmitted infectious diseases, such as the mosquito-transmitted yellow fever virus and tick-borne encephalitis, teach us that for an efcient eradication of the disease a safe vaccine is needed. In the absence of a licensed malaria vaccine, numerous different strategies are currently being tested to develop one [610]. In this review, we highlight some of the most recent developments towards a malaria vaccine.

Naturally acquired immunity ) imitate nature to advance the immune responses of malaria-nave individuals
A key observation in malaria-endemic regions is the gradual acquisition of protective immune responses as infants grow older and continue to be exposed to Plasmodium transmission. By the time children reach adolescence they typically mount strong antibody responses against the surface proteins of merozoites and infected erythrocytes. Indeed, an inuential study demonstrated early on that the passive transfer of immunoglobulins from semi-immune adults cures the clinical complications of malaria [11]. This nding provided the conceptual framework for malaria vaccine development, i.e. to speed up the generation of protective immune responses by active immunization with protective Plasmodium antigens. Apart from the undesirable slow kinetics of immune acquisition observed in endemic areas, the fundamental limitation of this strategy is the identication of protective as opposed to immunogenic antigens. Assays that permit the identication of correlates of protection are largely limited to cytoadhesion of P. falciparum-infected erythrocytes. Therefore, malaria vaccine research in the past was largely empirical and driven by vaccine development rather than by vaccine discovery, which typically comes rst. Moreover, as candidate formulations progress to larger clinical studies, another critical obstacle emerges: subunit vaccines typically have little, if any, impact on overall parasitemia [12,13], which would constitute an ideal endpoint being both highly desirable and easy to measure. Another endpoint that does matter is disease severity,

which serves as a predictor for morbidity and mortality. However, severe malaria is a very complex multisystem disorder [14] and remains an ill-dened endpoint. Moreover, vaccine trials differ fundamentally from clinical management studies in their study population, i.e. healthy individuals versus patients. Because the incidence of severe malaria is relatively low in any given study population, most studies remain considerably underpowered for this outcome, except for large cohort studies such as the recent successful recombinant P. falciparum circumsporozoite protein (CSP) vaccine (RTS S) trial in Mozambique [13]. The rst subunit vaccine, which was rapidly accelerated to phase III clinical trials, was SPf66 [12]. This vaccine, which consists of short peptide sequences of the two major glycosylphosphatidyl inositol (GPI)-anchored surface proteins of the invasive stages, merozoite protein 1 (MSP1) and CSP (Fig. 1), together with two uncharacterized peptide fragments, initially showed promising protection in an open human challenge study with P. falciparum-infected erythrocytes [15] and rst eld trials in South America, yet failed to confer substantial protection against natural malaria transmission in subsequent clinical trials in other endemic countries [12]. The two parasite surface proteins remain the major candidate antigens that are being developed and tested in various formulations. Strong support for these antigens comes from two landmark studies that addressed the relative importance of CSP and MSP1 in protective immunity. In an engineered PyCSP-tolerant mouse system Plasmodium yoelii CSP was shown to contribute to protection in the irradiated sporozoite vaccine model (see below) [16]. These PyCSP-transgenic mice can now be explored to identify additional protective pre-erythrocytic antigens that together with CSP confer sterile long-lasting protection in the rodent malaria model system. Using a population genetics approach a small N-terminal oligomorphic region of MSP1, termed block 2, was identied as a likely target of acquired immunity in endemic populations [17]. Antibody responses against this region appeared to be strongly associated with protection against clinical malaria.

The RTS S vaccine ) leading present subunit vaccine research

Currently, the RTS S vaccine ) a CSP fragment covering the central repeat peptide and the C-terminal T-cell epitope fused to the hepatitis B surface antigen (Fig. 1) formulated in the proprietary adjuvant AS02A ) is the most advanced candidate [18,19]. RTS S was moved on to proof-of-concept eld trials
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after phase I IIa trials, where initially 6 of 7 and, more recently, 40% of malaria-na ve individuals remained protected against a single mosquito challenge [2022]. Moreover, a consistent delay in patency in those individuals that became infected indicates that the vaccine eliminates 90% of the sporozoite inoculum. The anticipated outcomes of two large phase IIb trials, one in adult men in Gambia [23] and one in young children in Mozambique [13,24], were partial delay of infection and a trend towards reduction of clinical malaria. Unexpectedly, the vaccine also showed a 58% efcacy
Plasmodium life cycle stage Sporozoite invasion
CSP TRAP

in reducing the incidence of severe disease, an important nding that awaits conrmation in other epidemiological settings. Together the outcomes were interpreted as indicating signicant protection against natural P. falciparum infection and the vaccine was advanced to a large-scale, multicenter phase III trial [19]. As expected, RTS S did not induce T-cell epitope selection indicating that cell-mediated immunity may not be the major protective mechanism [25]. Somewhat uncommonly, the elementary ndings of corresponding phase I studies on the safety and immunogenicity of
Vaccine
RTS/S
HbS HbS

Targets
RI RIII TSR GPI

Clinical Phase
Phase III Phase II

A-domainTSR

CTD

AMA-1 Anti-parasite: Inhibitory antibodies reduce inoculation dose

II

III

Phase I

Merozoite invasion
MSP1
bII p83 p30 p38 p42 EGF GPI

FMPI

p42

Phase IIb Phase I

MSP3 AMA-1 Anti-parasite: Inhibitory antibodies prevent high parasitemia

SPAM I II III

Phase I

Cytoadhesion
var2CSA
1x 2x DBL 3x 4 5 6

Research

PfEMP1s

1 DBL CIDR

ATS

Preclinical

Anti-disease: Inhibitory antibodies block sequestration

Liver stage maturation

GAP IFN

Research

Anti-parasite: IFN-secreting T-cells destroy infected hepatocytes

Ookinete penetration
Pfs25 Pfs28
EGF EGF

Research

Altruistic vaccine: Inhibitory antibodies reduce transmission rate

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RTS S AS02A in children were reported only after the phase IIb trial [26,27]. Importantly, the formulation was safe and highly immunogenic for antibody responses against both P. falciparum CSP and the hepatitis B surface antigen. The anticipated continued success of the RTS S AS02A formulation in the induction of signicant protective immune responses will greatly inuence next-generation subunit vaccine developments. Critical issues are: (a) the selection of additional antigens to build on the CSP fragment; (b) adjuvant selection, which made a major contribution to the efcacy of RTS S [19]; and (c) whether a partially protective vaccine would be a valuable public health tool. Notably, there are numerous alternative CSP-based strategies under preclinical and clinical development [7], such as linear peptides that contain minimal T- and B-cell epitopes [28], and Plasmodium vivax long synthetic peptides [29].

Catching up ) MSP1-based vaccines

Prior to formulations with recombinant proteins native, afnity-puried MSP1 was tested in three Aotus monkeys in a pilot challenge study and was shown to confer complete protection against inoculation with the blood stages of a lethal P. falciparum strain [30]. Because of its central function for merozoite invasion MSP1 is under high natural selection resulting in the maintenance of allelic variation [17]. However, MSP1 is composed of modules that constitute the four subunits and most natural variants are derived from two prototypes only. Therefore, a mixture of recombinant codon-optimized full-length MSP1 constructs is

feasible and is currently in the preclinical phase [31]. Using a formulation that is conceptually similar to RTS S AS02, recent progress has been made to advance a vaccine based on the C-terminal p42 fragment, termed falciparum malaria protein-1 (FMP1) [32,33]. The encouraging safety and immunogenicity proles of FMP1 AS02A allowed its entry into proofof-concept phase IIb trials. However, choice of the C-terminal p42 fragment remains problematic in the absence of a clear association with protection. Two additional targets stand out among the numerous potential merozoite surface and secretory proteins and are currently being developed further for vaccine trials: merozoite surface protein 3 (MSP3) fullls many crucial criteria for a potential vaccine candidate: (a) induction of protective immune responses in the Aotus monkey challenge model [34], (b) direct proof of an effector mechanism through a process termed antibody-dependent cellular inhibition [35], and (c) association of allele-specic natural responses with protection from clinical malaria [36]. Apical membrane antigen 1 constitutes a potential multistage vaccine in itself because it appears to play important roles both during merozoite and sporozoite host cell entry (Fig. 1) [37].

var2CSA ) a case for a tailor-made subunit vaccine

A hallmark of P. falciparum blood-stage infections is the presence of parasite-encoded antigens on the surface of infected erythrocytes. These variant surface antigens (VSAs) mediate the adhesion of infected erythrocytes to endothelial cells and cause many of the

Fig. 1. Vaccine strategies against malaria. Natural transmission to the human host (upper) may be reduced by high titers of sporozoite-neutralizing antibodies that act prior to hepatocyte entry. In addition, vaccination with sporozoite antigens may induce cell-mediated responses to the infected hepatocyte. The progression of pathogenic blood stages can be reduced during the brief phase of merozoite entry into erythrocytes (second row). P. falciparum-infected erythrocytes adhere to endothelial cells (center) in capillaries and the placenta through parasite-encoded surface proteins that eventually lead to antibody recognition. Maturation of liver-stage schizonts (lower center) and ookinete pentration of the mosquito midgut (lower) represent two immunology silent stages of the Plasmodium life cycle. GAPs elicit long-lasting complete protection in experimental models. These genetically dened parasites are inoculated as sporozoites and invade and transform normally, but arrest during subsequent liver-stage development. At this time they likely display protective antigens (yellow) in the context of MHC class I presentation (green) that, in turn, activate interferon-c-secreting effector T cells (blue). Two partially redundant ookinete surface proteins, Pfs25 and Pfs28, constitute attractive targets for the development of transmission blocking vaccines. Targets for vaccine development include surface proteins (red) or adhesion proteins (green) of invasive stages. Shown are the primary structures and known protein domains (colored boxes) for selected vaccine candidates and lead vaccines. Cleavable signal peptides and transmembrane spans are boxed in red and black, respectively. The current developmental status is shown to the right. I-III, AMA-1 domains; A-domain, von Willebrand factor A-domain; AMA-1, apical membrane antigen 1; ATS, acidic terminal segment; bII, block 2 oligomorphic region of MSP1; CIDR, cysteine-rich interdomain region; CSP, circumsporozoite protein; CTD, TRAP-family cytoplasmic tail domain; DBL, Duffy-binding like domain; EGF, epidermal growth factor domain; FMP1, falciparum malaria protein-1; GPI, glycosylphospatidyl inositol anchor; HbS, Hepatitis B surface antigen; MSP, merozoite surface protein; PfEMP1, P. falciparum erythrocyte membrane protein 1; RI, region I; RIII, region III; SPAM, secreted polymorphic antigen associated with merozoites; TSR, thrombospondin type I repeat; var2CSA, variant surface antigen 2, chondroitin sulphate A-binding.

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clinical complications of malaria infection. Nonetheless, they also elicit strong protective immune responses [38]. The most straightforward explanation is that iterative recognition of individual VSAs upon continuous Plasmodium exposure eventually results in naturally acquired immunity to severe disease. The best-characterized family of VSAs is the var gene family, which encodes for  60 different P. falciparum erythrocyte membrane proteins (PfEMP1s; Fig. 1) and undergoes clonal antigenic variation [38]. This remarkable antigenic repertoire partially explains the slow kinetics of naturally acquired immunity and poses tremendous problems for direct vaccine research. Unless a subfraction of the most deleterious PfEMP1s can be identied, mimicking natural immunity with PfEMP1-based subunit vaccines remains a distant vision ) except for one unique, structurally distinct PfEMP1 variant, termed variant surface antigen 2, chondroitin sulphate A-binding (var2CSA) (Fig. 1) [39]. High antibody titers correlate specically with protection against pregnancy-associated malaria [40], a serious complication with poor outcomes such as low birthweight and preterm delivery due to sequestration in the placenta. Although additional VSAs are likely contribute to the pathology, a var2CSA-based vaccine may induce substantial protective maternal immune responses similar to those detected in women after multiple pregnancies [41].

Whole-parasite vaccines
The rst, and as yet unsurpassed, success in inducing protective immune responses against malaria was achieved with irradiated sporozoites in a rodent malaria model system [42]. Immunization of mice with three doses of c-irradiated sporozoites results in attenuated liver-stage development and elicits complete sustained protection against sporozoite challenge. Analogous to other live-attenuated vaccines, arrested Plasmodium liver stages likely induce protective cellmediated immune responses against the entire antigenic repertoire of the liver stage and may be the most potent malaria vaccine. But is it worth investing in a complex live, attenuated liver-stage vaccine, as opposed to an economically more viable subunit strategy that is only limited by the number of potential target proteins? Large-scale production of an attenuated parasite vaccine may indeed become feasible, because some challenges, such as sterility, cryopreservation, and route of immunization, have either already been met or are under active investigation [43]. Other roadblocks related to the safety and batch-to-batch variation of genetically undened irradiated sporozoites have recently been removed in the rodent malaria model system by the generation of genetically attenuated parasites (GAPs) [44]. Although translation to the P. falciparum system may take several years, early human challenge studies with irradiated sporozoites indicate that complete attenuation of liver-stage development elicits protection [45] to date the gold-standard in P. falciparum vaccine development. GAPs differ from c-irradiated sporozoites in their consistent production, genetic stability, and higher potency [46]. A fundamental issue is whether natural exposure to Plasmodium transmission would boost GAP-induced immune responses. If this was the case a GAP vaccine would be feasible for individuals from malaria-endemic countries. Otherwise only short-term visitors would benet and GAPs would fall into the category of boutique vaccines. Irrespective of large-scale application, GAPs may also become an excellent model to study sterilizing cellular immunity and may thus lead to the identication of potential protective liver-stage antigens. These antigens could then be delivered intracellularly as DNA or viral vectors. Such a strategy was advanced for thrombospondin-related anonymous protein (TRAP) and tested in proof-of-concept phase IIb trials [47,48]. The observed lack of protection correlates with the rapid decrease of TRAP expression after sporozoite invasion

Composed strategies ) better than nature?

One central obstacle in malaria vaccine discovery is the absence of sterilizing immunity during natural infection. Our current portfolio of successful vaccines acts against acute viral or bacterial infections. The corresponding whole-organism vaccines mimic an acute pathogen infection, which were known to function as a natural vaccination after the host immune system resolved the rst infection. There is no such model of acquired immunity against the Plasmodium parasite. Yet, a malaria vaccine will only become an efcient public health tool if it provides protection for several years with no more than three immunizations. One potential, yet challenging, solution to this problem may be the composition of vaccine strategies that aim at inducing protective immune responses against immunological silent Plasmodium life cycle stages, i.e. those that are not the typical targets of naturally acquired immunity (Fig. 1). Recent insights into the parasite biology and technological advancements open the possibility to explore such alternative vaccine strategies.

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[49] and highlights the need to prioritize vaccine targets based on immunological as well as biological criteria.

triad, i.e. vector-control programs, exposure prophylaxis and clinical management, as suggested by Ronald Ross nearly a century ago, must be supported.

Transmission-blocking antibodies
Induction of neutralizing antibody responses against gametocyte and ookinete surface proteins that can block the obligatory parasite fertilization, zygote transformation and subsequent traversal of the mosquito midgut is an attractive strategy that would result in interruption of the Plasmodium life cycle. Two major ookinete surface proteins, termed Pfs25 and Pfs28 (Fig. 1), together perform essential functions prior to oocyst development [50]. Because these proteins are expressed only during transmission to the mosquito vector, malaria-exposed individuals do not mount Pfs25 28-specic immune responses [51]. The absence of immune pressure correlates with remarkable sequence conservation. However, Pfs25 is an intrinsically poor immunogenic antigen. This hurdle was recently overcome by the generation of proteinprotein conjugates that proved to be highly immunogenic in mice [52]. High antibody levels persisted over months and these antibodies, when fed to mosquitoes, blocked oocyst formation. The small size and conservation of the Pfs25 28 proteins will expedite vaccine development. Such a transmission-blocking vaccine is highly likely to be efcient against malaria transmission and may prove to be an efcient tool in combination with vector control and exposure prophylaxis.

Acknowledgements
We thank two anonymous reviewers for critical and valuable suggestions. The work in the authors laboratory is supported by the research focus Tropical Medicine Heidelberg of the Medical Faculty of Heidelberg University, and in part by grants from the Deutsche Forschungsgemeinschaft (Ma 2161 3-2), the European Commission (BioMalPar, #23), the Grand Challenges in Global Health initiative, the Joachim Siebeneicher Foundation and the Chica and Heinz Schaller Foundation. AKM is a recipient of an EMBO long-term fellowship.

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