The immunological challenges of malaria vaccine development Jiraprapa Wipasa & Eleanor M Riley † † London School of Hygiene and Tropical Medicine , Immunology Unit , Department of Infectious and Tropical Diseases , Keppel Street , London, WC1E 7HT , UK
Malaria remains an important public health problem throughout the tropical world causing immense human suffering and impeding economic development. Despite extensive research for > 100 years, options for preventing malaria remain limited to vector control and chemoprophylaxis. The complexity of the organism and its life cycle have, thus far, thwarted vaccine development and exacerbated the perennial problems of drug resistance. Nevertheless, development of a vaccine against malaria that reduces morbidity and mortality, and ideally also reduces transmission, has long been seen as an essential component of a sustainable malaria control strategy. In this article the authors review the biological challenges of malaria vaccine development, summarise some of the recent advances and offer some immunological insights which might facilitate further research.
Malaria is endemic in most tropical countries and has the potential to spread to subtropical regions where the climate permits the mosquito vector to become established. It is estimated that each year > 500 million people are affected by malaria and between 1 and 3 million people die from it, mostly in sub-Saharan Africa. Malaria is caused by protozoa of the genus Plasmodium . Among the four species of malaria parasites that infect humans ( P. falciparum , P. vivax , P. malariae and P. ovale) , P. falciparum is the cause of the overwhelming majority of malaria deaths, although the severe morbidity associated with P. vivax infection imposes a considerable economic and social burden.
The life cycle of Plasmodium ( Figure 1 ) comprises two asexual replicative stages, in the liver and blood, respectively, of the vertebrate host, and a sexual replicative cycle which begins with gametocytogenesis in the vertebrate and continues with zygote formation followed immediately by meiosis in the mosquito vector. Infection begins when haploid sporozoites are injected into the skin by the bite of Anopheles mosquito. The sporozoites enter venous capillaries and are carried to the liver where they invade hepatocytes, differentiate and divide to produce tens of thousands of merozoites. After a period of time, which is characteristic for each species, these exoerythrocytic schizonts in the infected liver cell rupture, releasing the merozoites into the circulation. Merozoites attach to specific erythrocyte surface receptors and invade the red blood cells (RBCs) where they differentiate via ring stage trophozoites into mature schizonts. The rupture of schizonts releases more merozoites, perpetuating the blood stage of the life cycle. As the percentage of parasite-infected RBCs (iRBC) increases over several rounds of schizogony, the characteristic clinical symptoms of malaria begin. Eventually, some merozoites differentiate into male and female gametocytes, which can be taken up by mosquitoes during blood feeding. Fertilization of mature female gametes by male microgametes, emerging from the remnants of the host RBC in the midgut of the
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mosquito, produces the motile ookinete, which penetrates the mosquito gut wall and develop into oocysts within which sporozoites are produced that travel to the salivary glands of the mosquito. When the mosquito bites, the sporozoites are inoculated into the skin of the vertebrate host, initiating a new infection.
Only the asexual intra-erythrocytic stage of the malaria life cycle causes any obvious pathology. Periodic fever, headache, nausea, drowsiness and muscular pain occur around the time of iRBC rupture, and are directly linked to schizont rupture and release of glycosylphosphatidylinositol (GPI) and hemozoin, which induce production of inflammatory cytokines such as TNF- α and lymphotoxin- α from macrophages [1,2] . Anaemia results from erythrocyte destruction, sequestration of iron in the insoluble hemozoin pigment and suppression of erythropoiesis. Cerebral malaria is a frequent presentation of P. falciparum infection and is caused by adherence of iRBC to the endothelial lining of small blood vessels in the brain, reducing blood flow and leading to metabolic acidosis and coma (reviewed in [3] ). This process is exacerbated by localised inflammation ( Figure 2 ) which causes upregulation of endothelial receptors
for iRBC, such as intercellular adhesion molecule 1. Sequestration of iRBCs in the lung can exacerbate the acidosis leading to respiratory distress, and sequestration in the placenta during pregnancy can cause miscarriages, fetal death, low birth weight and premature delivery.
Effective strategies for prevention of malaria include use of insecticide-treated bednets, protective clothing, insect repellents and indoor residual insecticide spraying; these are now being augmented in children and pregnant women by programmes of intermittent presumptive treatment with antimalarial drugs [4] , but resistance of malaria parasites to drugs and of vectors to insecticides becomes increasingly alarming. A cost-effective vaccine that may protect against infection or disease will greatly benefit hundreds of millions of people by reducing morbidity, mortality and economic loss, and increasing the pace of social and economic development. In this article the authors review the present understanding of immunity to malaria and suggest how this knowledge can be used to develop novel approaches to malaria vaccine design (a series of comprehensive reviews of the status of malaria vaccine development and clinical trials are published in [5] ).
Figure 1 . Plasmodium life cycle, vaccine targets and predicted effector mechanisms. A vaccine against liver stages aims to induce antibodies capable of blocking invasion of sporozoites into liver cells and to induce IFN- γ -producing CD4 + and CD8 + T cells capable of inhibiting parasite growth in the liver. The key to a blood-stage malaria vaccine is to induce antibodies that can block merozoite invasion, prevent sequestration of iRBC and facilitate phagocytosis by macrophages/monocytes. IFN- γ -producing effector CD4 + T cells activate macrophages to phagocytose and kill merozoites and iRBC. Transmission-blocking vaccines aim to interrupt development of the parasite in the mosquito; transmission-blocking immunity is primarily antibody mediated. Antibodies either block essential molecular interactions between gametes or between the zygote and the mosquito gut, or mediate complement-dependent lysis of parasites. iRBC: Infected red blood cell; NK: Natural killer.
During the past two decades, malaria research has moved rapidly – driven in part by technological advances – and there is now a genuine possibility that an effective vaccine can be produced. Several different strategies are being pursued, targeting sporozoite, liver, blood and mosquito stages, and these have recently been extensively reviewed [6-9] . Briefly, a pre-erythrocytic (antisporozoite or liver stage) vaccine would induce antibodies that inhibit sporozoite invasion into hepatocytes and/or induce effector CD4 and CD8 T cells to destroy infected hepatocytes, thus preventing liver-stage parasites from developing to maturity ( Figure 1 ). The feasibility of this approach has been amply demonstrated by numerous clinical trials with irradiated sporozoite vaccines in which sporozoites undergo arrested development in hepatocytes and induce effective antiliver-stage immunity; a similar outcome has now been achieved using genetically attenuated rodent parasites offering the potential to define novel liver-stage antigens (reviewed by [5] ). A vaccine against blood-stage infection would reduce morbidity and mortality by eliminating or reducing the parasite load (antiparasitic immunity) or preventing the pathological consequences of
infection (antidisease immunity). The key to an antiparasite vaccine is to induce antibodies that prevent merozoite invasion of RBCs, opsonise merozoites and iRBCs or block adherence of iRBCs to vascular endothelium, and/or to induce effector CD4 + T cells which, via production of IFN- γ , can activate phagocytosis and killing of iRBC by macrophages. An antidisease vaccine might operate by inducing antibodies to parasite toxins such as hemozoin or GPI [10] . Although mice immunised with synthetic GPI eventually died of hyperparasitaemia they were protected from cerebral symptoms of malaria [11] , indicating that an antidisease vaccine which might significantly reduce malaria pathogenesis could form part of a compound vaccine that also controls parasite replication. Lastly, transmission-blocking vaccines aim to induce, in the human host, antibodies targeting gametocyte, gamete or ookinete antigens which would either induce complement-mediated lysis of gametes or zygotes or block the molecular interactions required for fertilisation or ookinete migration. When used on their own, transmission-blocking vaccines would not protect individuals from malaria infection but would prevent infected individuals from transmitting the parasite to mosquitoes, thereby halting the human-to-vector-to-human cycle.
Figure 2 . Immunology and immunopathogenesis of malaria. Activation of macrophages or DCs via interaction of PRRs and their malarial ligands leads to phagocytosis of infected RBCs and presentation of malaria antigens to T cells as well as production of cytokines. IL-12, IL-18 and IFN- α secreted by these activated antigen-presenting cells induce cytokine production by NK cells. IFN- γ secreted by activated T cells and NK cells in turn enhances phagocytic activity and cytokine production by macrophages. IL-1, IL-6 and TNF- α produced by macrophages induce fever and upregulation of adhesion molecules on endothelial cells. Adherence of iRBCs to vascular endothelial adhesion molecules causes obstruction of small blood vessels leading eventually to cerebral malaria and other clinical symptoms. Infl ammatory responses are downregulated by TGF- β and/or IL-10. DC: Dendritic cell; iRBC: Infected red blood cell; NK: Natural killer; NO: Nitric oxide; PR: Pattern recognition receptors; T H : T helper.
Vascular occlusionNOToxic radicals
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This ‘roadmap’ for malaria vaccine development is widely accepted by the research community [101] but implementing it will depend heavily on having a better understanding of how immunity to the parasite is induced, what the best effector mechanisms are and which antigens to target.
3. Naturally acquired immunity to malaria
Naturally acquired immunity to malaria reduces but does not prevent the establishment of blood-stage infection, consequently ‘immune’ individuals residing in malaria endemic areas often carry parasites at low density but without symptoms. Immunity to severe clinical symptoms is acquired relatively rapidly, possibly after only one or two infections [12] , whereas immunity to milder disease – which is associated with the ability to limit parasite replication – seems to develop rather more slowly. Susceptibility to disease thus declines in a predictable fashion, with the risk of severe disease and death declining first, followed somewhat later in life by a decline in the risk of mild disease. In areas of high malaria transmission intensity, where infectious mosquito bites are common, this can happen very quickly. On the contrary, in areas of low transmission, clinical immunity may not be acquired until late childhood or adolescence. In areas of infrequent transmission, immunity may not be achieved at any age.
Although superficially consistent with much of the epidemiological data, the notion that immunity to malaria develops simply as a function of the number of infectious bites received has been challenged. Studies in migrant populations exposed to malaria for the first time revealed that adults become immune much more quickly than children [13,14] . Subsequently, studies in a region of highly diverse malaria transmission revealed that age-related patterns in disease presentation were similar at very different levels of transmission [15] . Together, these studies suggested that age per se rather than cumulative exposure to malaria might be the crucial determinant of developing antimalarial immunity. There has also been considerable debate about which type of immune response, anti-disease or anti-parasite [10,16] , is most effective. Recently, using an age-structured mathematical model of malaria transmission, the authors found that typical age-prevalence patterns of malaria infection and disease are best explained by two types of immune response: one that reduces susceptibility to clini-cal disease and develops with age and exposure (with a half-life of ∼ 5 years) and a second that results in more rapid clearance of parasitaemia, is acquired later in life and is longer lasting (half-life of > 20 years) (Filipe et al. , manuscript in publication). Importantly, the outputs of this model suggest that the development of this second type of immune response (anti-parasite immunity) is dominated by age-dependent physiological processes rather than the magnitude of exposure (provided some exposure occurs). The nature of these age-dependent physiological processes is
not entirely clear but maturation of dendritic cell function [17] , recognition of polysaccharide antigens [18] or ability of B lymphocytes to undergo class-switching to highly cytophilic IgG3 subclasses [19] have all been shown to be age dependent. Acceptance that young children may be inherently less able than older children to make antiparasitic immune responses to malaria is important because, although most malaria vaccine development programmes are focussed on the need to protect neonates and infants from severe malaria (which our model, and others [12] , suggest is feasible), these vaccines are typically evaluated by looking for a reduction in mild disease or reduced parasite densities in the blood, which are markers of antiparasitic immunity. Lack of efficacy by these criteria may simply reflect the age of the vaccinees rather than any lack of efficacy of the vaccine. Furthermore, the models suggest that a vaccine which has minimal impact on infection or mild disease could have a major effect on severe disease.
4. The cellular and molecular basis of antimalarial immunity
Studies using mice experimentally infected with rodent malaria parasites have revealed a complex series of events leading to protective immunity ( Figure 2 ). Blood stage schizonts, the soluble fraction from schizont lysate [20] and the malaria pigment [21,22] , known as hemozoin, all activate dendritic cells (DCs) through the toll-like receptor (TLR) 9 family of pattern recognition receptors (PRRs) [21] . It is unlikely that hemozoin itself is a ligand for TLR-9, more probably it functions as a carrier for the true ligand, which may be malarial DNA [22] . GPI, a glycolipid which anchors proteins to the merozoite surface, is believed to be a ligand for TLR-2 through which it activates macrophages [23] to produce mediators such as TNF- α and reactive oyxgen and nitrogen species, which potentiate the phagocytic capacity of monocytes [24] . Malaria sporozoites and sporozoite-derived antigens also activate DCs, leading to priming of specific CD4 and CD8 T cells and initiation of protective immune responses [25-27] but the molecules involved in sporozoite recognition have not been identified. Interaction between PRRs and their ligands on the parasites may lead to an actin-dependent phagocytosis of iRBCs by DCs which then mature, upregulate co-stimulatory molecules on their surfaces and produce cytokines [28] , including IL-12. Murine DCs pulsed with iRBCs trigger IL-12-dependent IFN- γ production by CD4 + T cells (and also, in the case of liver-stage parasites, CD8 + T cells) promoting development of a T helper (T H )1 immune response and macrophage-mediated clearance of infected cells [26,28] . DCs can also produce IL-4 [29] and initiate T H 2 responses, which, in cooperation with antigen-specific CD4 + T cells [30] , promote develop-ment of the robust antibody response which is required for eradication of chronic blood-stage infection [31] . All the evidence points to a similar sequence of events during
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human malaria. In uncomplicated P. falciparum malaria, IL-12 concentration is inversely correlated with parasitaemia and disease severity [32-34] , serum IFN- γ and IL-12 predominate over IL-4 and IL-10 [35] , IFN- γ production is inhibited by anti-IL-12 antibodies [36] and correlates with clearance of parasites [37] and resistance to reinfection [38] .
Typically, IL-10 is co-produced with IFN- γ [39] , suggesting existence of a homeostatic feedback mechanism to limit potentially dangerous inflammatory responses. Indeed, these regulatory mechanisms seem to be conserved between mice and humans. For example, T H -1 CD4 + T cells specific for P. berghei , a rodent malaria, suppress parasite growth in immunodeficient recipients but at the same time promote anaemia and weight loss [40] ; these effects are exacerbated in IL-10-deficient hosts [41] , (Couper et al. , submitted for publication). Similarly, a high ratio of TGF- β or IL-10 to TNF- α IL-12 or IFN- γ is associated with higher density P. falciparum parasitaemia but reduced pathology and less severe symptoms [42,43] and patients with severe malaria frequently have higher levels of TNF- α than those with uncomplicated malaria [44-47] . TGF- β can be among the earliest cytokines produced during malaria infection in humans [39] and mice [48] where it is associated with suppressed type-1 responses and faster parasite growth [48,49] . By contrast, IL-10 is upregulated later in infection and in its absence, parasite clearance occurs more rapidly but at the cost of more severe pathology [41] , (Couper et al. , submitted for publication). At present, the data are consistent with a scenario by which TGF- β (from innate sources such as monocytes and macrophages) regulates the early innate inflammatory response of, for example, NK cells [50] whereas IL-10 (from adaptive Tr1 populations of regulatory cells) regulates the adaptive type 1 T-cell response. Clearly, a critical balance of pro-inflammatory to anti-inflammatory cytokine responses is essential to establish effective immunity without predisposing to pathology. Although this is challenging in terms of vaccine development, induction of a balanced T-cell response, encompassing type 1 (IFN- γ -producing) and regulatory (IL-10-producing) cells, may well be necessary.
5. Development of malaria vaccines: a case history
As of December 2006, at least 35 malaria vaccine candidates were in various stages of clinical development, mostly in Phase I or Phase IIa trials; none of the vaccines is close to licensure [102] . One vaccine, however, is much further along the clinical development pathway than the others and this vaccine (RTS,S) is an excellent example of some of the problems of malaria vaccine development, providing lessons in how these difficulties might be overcome and offering an intriguing example of how vaccine research can throw a light on underlying immunological mechanisms, which might then be exploited to enhance future vaccine research.
RTS,S is designed to interrupt the pre-erythrocytic stage of infection by targeting sporozoites and/or schizont-infected liver cells. If it were 100% effective, sterile immunity could be achieved as the vaccine would prevent the release of primary merozoites from infected hepatocytes. However, even if it induced only partial protection, the vaccine may reduce the magnitude of the first wave of merozoites emerging from the liver cell, extending the time for clinically relevant parasite densities to develop and thus allowing more time for antiblood-stage immune responses to take effect. RTS,S consists of a recombinant protein representing 19 consecutive repeats (R) of the four amino acid motif NANP and the major T-cell epitopes (T) encoded within the carboxyl terminus of the circumsporozoite protein (CSP); this is fused to the N terminus of the surface (S) antigen of hepatitis B virus and co-expressed in yeast as a particle with free S antigen [51] . When RTS,S formulated with AS02 adjuvant was given as three doses to adult Gambian men, the vaccine was found to be safe and well tolerated [52] . The concentration of antibodies against CSP correlated with the degree of vaccine-induced protection, defined as time to reinfection (i.e., parasitaemia) during a period of follow up. Over 9 weeks of follow up, the vaccine significantly reduced reinfection rates (by parasites carrying either the vaccine sequence or other variant CSP sequences) with an efficacy of 71%; however, efficacy over the next 6 weeks was 0%, bringing the vaccine efficacy to 34% over the 15-week follow-up period. A double-blind, randomised, controlled trial in Mozambican children aged 1 – 4 years old also showed promising results, protecting children from mild and severe clinical malaria over a 6-month surveillance period with efficacies of 29.9 and 57.7%, respectively [53] . The vaccine induced high levels of specific antibodies to both CSP and antigen of hepatitis B virus but these were not correlated with protection and decayed by ∼ 75% over the 6-month follow up. Moreover, careful examination of the disease-free survival curves revealed that all of the protection occurred in the first 3 months of follow up with no substantial difference in disease incidence in the second 3 months [54] . Although there has been some criticism that interpretation of the results of the trials has been overly optimistic, and in particular that the vaccine has not been tested against a homologous adjuvant-only control group and that the trials were not designed to test protection against severe disease, the conclusions drawn from these trials were that RTS,S is able to induce short-term, heterologous protection against infection and clinical disease.
The immune mechanisms that provide this protection are unclear. A role for anti-CS antibodies cannot be ruled out as correlations between antibody titres and protection have been observed in some trials but not others [52,53] . The demonstration, using intra-vital imaging, showing that antisporozoite antibodies impede sporozoite migration in the skin and hinder invasion of dermal blood vessels [55] casts a
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new light on the potential protective role of such antibodies. It is likely, however, that T-cell-derived IFN- γ [56-58] or possibly cytotoxic CD8 + T cells [59] are also required but, despite clear evidence from experimental animals that cell-mediated immune effector mechanisms (in most cases, CD8 + T-cell-derived IFN- γ ) are essential for pre-erythrocytic immunity, there is no direct evidence that these mechanism are required in humans and correlations between T-cell responses and protection are highly inconsistent between trials (reviewed in [60] ).
Intriguingly, a further 12 months of follow-up of the vaccinated Mozambican children revealed that although antibodies against CSP continued to wane, and showed no evidence of boosting, children vaccinated with RTS,S/AS02A remained significantly protected against P. falciparum (vaccine efficacy of 35.5 and 48.6% for protection against infection and severe malaria, respectively) [61] . These very promising results have provided the impetus for a series of Phase IIb optimisation trials leading to a multi-centre Phase III trial which could ultimately lead to registration of RTS,S as the first licensed malaria vaccine. Nevertheless, the explanation for the extended protection observed in RTS,S vaccinated children is very unclear and deserves careful consideration. Is it simply an indication of long-term efficacy of anti-CS immunity or is something much more interesting going on?
6. Do low dose malaria infections prime protective cellular immune responses?
Although possible, in the authors’ opinions it is unlikely that the prolonged protection of RTS,S vaccinated children [61] is due to persisting immune responses to CS antigens. First, if this were the case, it is not obvious why protection would wane to non-protective levels between 3 and 6 months postvaccination [54] and then apparently recover. Furthermore, there was no evidence that immune responses to CS protein were boosted in the intervening period; indeed, anti-CS antibody titres declined inexorably over the follow-up period. Nevertheless, postvaccination follow up of cell-mediated immune responses is required to determine whether other anti-CS responses might be providing long-term protection.
An alternative explanation, however, is that RTS,S reduces but does not completely prevent, emergence of merozoites from the liver, such that vaccinated children receive an attenuated or low-dose blood-stage infection (Sutherland et al. , submitted) which allows more effective immunity to blood-stage parasites to develop. In support of this suggestion, mathematical models based on quantitative polymerase chain reaction determination of parasitaemia in vaccinated and experimentally infected subjects have demonstrated that pre-erythrocytic malaria vaccines can significantly reduce the numbers of merozoites emerging from the liver [62] and there is considerable evidence to
suggest that such low-dose infections might induce better immunity to blood stages, leading to enhanced protection against reinfection. If true, this may explain the apparently paradoxical observation that RTS,S, which targets only liver-stage parasites, appears to confer significant protection against severe anaemia and cerebral malaria, which are mediated entirely by blood stage parasites.
The experimental evidence that low-dose, blood-stage infections induce more effective antimalarial immunity comes from intervention studies in which infants who received intermittent antimalarial treatment with sulphadoxine-pyrimethamine during the first 9 months of life were significantly less likely to develop clinical malaria over the next year than infants who received placebo [63] . As the half-lifes of these drugs are very short (< 7 days), they should not have had any prolonged protective effect. Rather, it is suggested that the drugs may have attenuated infections occurring during the first 9 months of life, providing the immune system sufficient time to generate protective responses. This suggestion is supported by a study showing that children who received malarial chemoprophylaxis sub-sequently had higher T-cell proliferative and IFN- γ responses to malaria antigen production, and were better protected against infection, than those who received placebo [64] . A study in which children were protected from mosquito bites by provision of insecticide-treated curtains in their homes showed that these children were able to clear drug-resistant parasites more effectively than children who were not protected in this way [65] . The most direct evidence in support of this hypothesis, however, comes from a study in which naive human volunteers deliberately ‘immunised’ by repeated, ultra low dose, P. falciparum iRBC infection were shown to be protected from subsequent high-dose infection; immunised individuals did not make antimalarial antibodies but protection was correlated with the presence of antigen-specific effector T cells capable of producing IFN- γ [66] .
These data from studies in humans are consistent with equivalent studies of murine malaria. For example, mice repeatedly exposed to subpatent P. chabaudi chabaudi infections developed a crossreactive T H 1 immune response as well as antibodies to conserved merozoite surface antigens and were protected against both homologous and heterologous parasite challenge [67] . Also, mice infected with fully viable Plasmodium yoelii sporozoites but concurrently dosed with chloroquine to minimise the development of blood-stage parasitaemia were highly protected against sub-sequent sporozoite-induced and iRBC-induced infections [68] . Again, protective immunity was antibody independent, T-cell dependent and mediated by IFN- γ and nitric oxide. In the latter study, immunity was abrogated by removal of liver stages with primaquine, suggesting that the target antigens may be expressed in liver stages.
Taken together, both the human and the murine studies suggest that low-dose malaria infection may effectively prime
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T-cell mediated effector mechanisms such that subsequent infections can be very rapidly cleared before reaching either parasite densities that lead to clinical disease or levels of T-cell activation that are associated with pathology. The target antigens may be expressed in the liver or in infected erythrocytes, or both, and in some instances at least, conserved antigens seem to be effectively recognised. The challenge now is to convert this knowledge into a practical vaccine strategy as immunisation with low doses of viable parasites is not thought feasible for large populations. As a first step, the acquisition of immunity to blood-stage parasites is being compared among vaccinees and controls within the ongoing Phase IIb trials of RTS,S to directly test the hypothesis that this vaccine may function in part by potentiating blood-stage immunity.
7. Problems with humoral immunity to malaria?
One surprising outcome of the RTS,S vaccination trials was the rather modest titres of antisporozoite antibodies induced by an otherwise very potent vaccine and the lack of boosting of antibody titres following natural exposure to sporozoite infection. Direct comparisons are hindered by the use of very different methods for reporting concentrations of anti-CS and anti-S antigen antibodies in many of the clinical trials but where comparison can be made, postvaccination end point titres of IgG antibodies to the S antigen appear to be 80 – 100-fold higher than those to the CS epitopes [53] . On the other hand, antibodies to both antigens appear to decline at a comparable rate, declining by 74 and 82%, respectively, 6 months postvaccination in one study [53] and by 75 and 85%, respectively, 11 months postvaccination in another [69] . Although it is possible that, with such low starting titres, levels of anti-CS antibodies may decline to undetectable levels (i.e., vaccinated individuals may revert to seronegative) sooner than levels of anti-S antibodies, it does not seem that the primary problem with the antibody response to the CS protein is its short half-life. Rather the problem seems to be lack of immunogenicity. As the vaccine itself is not inherently poorly immunogenic (as evidenced by the very high titres of anti-S antibodies and the much higher titres of anti-CS antibodies compared to previous vaccine formulations) the problem seems to lie with the immunogenicity of the CS protein. Even in its native form (i.e., displayed in multimeric form on the surface of sporozoites) the CS protein is poorly immunogenic with only rather low antibody titres in adult individuals who have been regularly bitten by sporozoite-infected mosquitoes for their entire lives [70,71] . This has traditionally been ascribed to the rather low number of sporozoites (and thus the low antigen dose) present in any single bite but the data from the vaccine trials (where much larger amounts of CS protein are present) suggest that this may not be the primary problem. Could it be that there is something inherent to the
structure of the ‘immunodominant’ NANP repeat sequence that makes it a poor B-cell immunogen?
As far as we are aware, and in stark contrast to detailed analysis of the T-cell response to pre-erythrocytic malaria vaccines (reviewed in [60] ), there are no published studies of the frequency of CS-specific memory B cells or plasma cells in humans, so it is very difficult to determine where the primary problem lies. A much more detailed analysis of the B-cell response to malaria vaccination in humans and in animal models is urgently required. One possibility worth investigating is that the tertiary structure of the NANP polymer prevents formation of high avidity complexes with the B-cell receptor, resulting in suboptimal B cell priming and memory or plasma cell development. Of interest, a study published almost 20 years ago found that different carrier proteins markedly influenced the immunogenicity of a 32-mer recombinant NANP protein (R32) but that the immunogenicity of R32 within the complex was not related to either the inherent immunogenicity of the carrier or the amount of R32 administered [72] . The authors concluded that ‘physiochemical properties of the conjugate, as related to the physical presentation of the R32 determi-nants, are of great importance in determining the magnitude of the anti-R32 antibody response, the best conjugate would position (R32) in such a manner as to ensure optimal recognition by the immune system’. Whether this ‘optimal recognition’ represents proximity of T-helper epitopes, avidity of R32 binding to B cell receptors or some other property, is an unresolved question that perhaps merits investigation.
Although the problem with existing pre-erythrocytic vaccination strategies may be to do with inducing high levels of anti-CS antibodies rather than sustaining these antibodies, maintaining high circulating levels of antimalarial antibodies is likely to be critical for protection, especially if the target is the invasive sporozoite where there is no time to boost antibody levels after re-exposure to antigen. High titre antibodies need to be present at the time of infection if they are to play a role as sporozoites are only exposed to the extracellular milieu of the host for brief periods (typically much less than an hour) before entering host cells and shedding their coat proteins. In view of this, it is worrying that there does not seem to be any effective natural boosting of anti-CS antibody titres in RTS,S vaccinated children who are exposed to sporozoite-infected mosquitoes [61] . If the protective efficacy of this vaccine is to be enhanced, one urgently needs to understand why this is the case.
Potential explanations for poor maintenance and/or boosting of anti-CS antibody responses include some problems with generation or maintenance of either long-lived plasma cells or memory B cells. On exposure to antigen, germinal centre reactions give rise to short-lived antibody-secreting cells that differentiate into plasmablasts capable of producing a large amount of antibodies, or into memory B cells responsible for the rapid antibody response that occurs after
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re-exposure to antigen [73] . A proportion of plasma cells migrate to bone marrow where they appear to persist for many years secreting significant quantities of high-affinity antibodies [74] . Highly effective vaccines, such as that against smallpox, induce memory-B cells that remain detectable (by enzyme-linked immunosorbent spot [ELISPOT] after in vitro restimulation with antigen, for example) for at least 60 years postimmunisation [75] . Moreover, the presence of serum antibodies more than 50 years after most recent exposure to virus implies either that smallpox-specific plasma cells are retained for decades or that memory B cells are somehow induced to differentiate into plasma cells in the absence of antigen. This does not appear to happen after vaccination with the existing crop of prototype malaria vaccines but we do not know why.
Survival of plasma cells requires their migration to survival niches in the bone marrow where signals are provided that prevent apoptosis and promote sustained Ig secretion [76] . Migration to bone marrow is controlled by chemokine receptors – such as CXCR4 – expressed on plasma cells which enable the cell to home via a concentration gradient of chemokines, such as CXCL12 [73] . Conversely, plasma cells generated as a result of prolonged antigenic stimulation or sustained inflammation tend to express CXCR3 and migrate (in response to gradients of chemokines such as CXCL9, 10 or 11) to inflamed tissues where they undergo apoptosis as the inflammation resolves [73,76] . It is possible that inappropriate migration or survival signals are induced after some forms of immunisation and we suggest that more research is needed into the effects of different adjuvants
Box 1. Expert opinion.
Why have past attempts to make a malaria vaccine failed?
If we knew the full answer to this question we would be much closer to having a vaccine! Nevertheless, commonly cited reasons include:
• Only a very small proportion (< 1%) of the malaria genome has been exploited for vaccine development so far; maybe there are key antigenic targets yet to be identifi ed?
• Many of the antigens studied are inherently poorly immunogenic, for reasons that are not fully understood
• Many of the antigens have complex tertiary structures that are diffi cult to fully recreate in vitro
• We do not know precisely which effector mechanisms are most effective at each stage of the life cycle. Data from animal models and in vitro experiments is almost certainly incomplete and may be misleading
• Very little attention has been paid to questions of immunological memory and the kinetics of the effector response either in the context of natural infections or after vaccination
• As we do not know the key effector mechanisms, we do not have any reliable correlates of protection by which to assess the potency of vaccines prior to clinical trials
• Vaccine effi cacy in animals, mainly mice and non-human primates, correlates poorly with effi cacy in humans
• Phase II trials are time consuming and expensive (and diffi cult to justify ethically without extensive preclinical data) and thus many prototype vaccines are abandoned before their clinical effi cacy can be assessed
Future directions?
• We need a two-pronged approach in which classical, essentially empirical, approaches to vaccine development go hand-in-hand with rational vaccine design based on a thorough understanding of the parasite and the immune response to it
• Parasitologists, immunologists and vaccinologists need to communicate better so that basic scientifi c advances feed through much more effectively and effi ciently into vaccine development
• We need to invest in a more thorough understanding of correlates of protection, making use of new high-throughput technologies to map humoral and cellular responses to the entire proteome
• We need to consider issues of timing and location of effector responses. An effector response of the right type and magnitude (e.g., 1 – 5% of circulating CD8 + T cells making IFN- γ in response to liver stage antigens), which occurs too slowly or in the wrong place (e.g., effector T cells against liver-stage antigens that cannot traffi c to the liver or arrive there after the parasite has emerged into the bloodstream) cannot protect
• We need a better understanding of our target population. A vaccine that is immunogenic in a Caucasian population may not be immunogenic in an African population with different genetic makeup and different immunological experiences
• We need to identify better clinical end points for vaccine trials that more accurately refl ect the ability of the vaccine to limit parasite growth or prevent pathology. Mathematical modelling of immunological and parasitological data offers the potential to identify proxy outcomes of vaccine effi cacy
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and the mode of antigen administration on induction of long-lived memory B cells and on plasma cell migration and survival. This kind of information cannot be gained from the simple assays of antibody concentration that are typically used to monitor the outcome of vaccine trials.
An explanation that is frequently but rather unsatisfactorily used to explain lack of persistence of antibodies after malaria vaccination is that antibody responses to malaria antigens are unusually short-lived and, by implication, that exposure to blood-stage malaria infection leads to aberrant B-cell responses (reviewed in [77] ). However, a rapid decline of serum antibody titres (to as little as 1 – 5% of their peak value) in the period immediately after clearance of the antigen is typical of responses to many infections and is not, in itself, an indication that anything is amiss [74] . The more relevant parameters are the numbers of memory B cells and plasma cells surviving but these parameters are rarely measured. In humans, this is understandable given the inaccessible locations (lymph nodes, spleen and bone marrow) in which these cells persist, but perhaps more effort should be made to measure these very important parameters in experimental animals and to consider proxy measures that might be suitable for clinical use. One option that could be explored further is the use of mathematical models based, for example, on antibody seroprevalence rates at intervals after vaccination [78] or the time taken for antibodies to reach protective concentrations following re-exposure to antigen, to infer the longevity of plasma cells or the time over which memory B cells can continue to reseed secondary lymphoid organs with antibody-producing cells.
8. Expert opinion
There is widespread agreement among public health professionals in tropical countries that deployment of an effective malaria vaccine would greatly reduce human suffering and enhance economic growth and prosperity in affected countries ( Box 1 ). There is also a broad consensus among scientists regarding the ‘roadmap’ for developing such a vaccine, but there are significant gaps in our knowledge that need to be filled before this roadmap can be fully implemented.
Phase I and II clinical trials are evaluated using proxy markers of vaccine efficacy that may not fully reflect the ability of the vaccine to prevent severe morbidity and death; consequently, some potentially valuable vaccines may be being discarded prematurely. We need to identify proxy
outcomes that more closely predict the likelihood of deve-loping severe disease and to consider the potential value of mathematical modelling approaches to do this. There is no real consensus regarding the immune effector mechanisms that are most effective in individuals who have acquired immunity by natural exposure nor on which effector mecha-nisms are responsible for protection in the most recent clinical trials. The techniques we use to evaluate vaccine-induced and naturally acquired immunity need to be reconsidered, applying approaches such as ELISPOT and flow cytometry, which are routinely used to evaluate T-cell responses to look more carefully at the B-cell response; at present, poorly understood issues include induction and retention of antibody-producing cells and memory B cells and timely reactivation of memory B cells during reinfection.
Long-term protection after immunisation with partially effective pre-erythrocytic vaccines may be explained by vaccine-induced attenuation of blood-stage infections allow-ing the development of more effective antimerozoite or anti-iRBC responses. These responses seem to be primarily T-cell rather than antibody-mediated raising concerns that, unless carefully regulated, they might predispose to immunopathology. Immunological follow-up of future trials should include assays for regulatory as well as effector responses. If ultra low-dose blood-stage infections are confirmed to efficiently induce protective immunity, converting this knowledge into practicable vaccine strategies will be extremely challenging.
Acknowledgements
J Wipasa is the recipient of a Wellcome Trust International Research Development Award (grant reference 072182). Work in our laboratories is supported by the Wellcome Trust (grant references 074538; 078925) and the UK Medical Research Council (G0400225). This article was developed from a presentation by EM Riley at the British Society for Immunology Congress, Glasgow, UK in February 2007.
Declaration of interest
J Wipasa received support from the Wellcome Trust and World Health Organization grants. EM Riley received grants form the MRC, Wellcome Trust, Royal Society, Malaria Vaccine Initiative, the European Commission and the BMGF.
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Affi liation Jiraprapa Wipasa1 & Eleanor M Riley†2 †Author for correspondence 1 Chiang Mai University, Research Institute for Health Sciences, PO Box 80 CMU, Chiang Mai 50202, Thailand 2 London School of Hygiene and Tropical Medicine, Immunology Unit, Department of Infectious and Tropical Diseases, Keppel Street, WC1E 7HT, London, UK Tel: +44 20 7927 2706 ; Fax: +44 20 7927 2807 ; E-mail: [email protected]
