Molecular Approaches to Epidemiology and Clinical Aspects of Malaria

Molecular Approaches to Malaria

Molecular Approaches to Epidemiology and Clinical Aspects of Malaria G.V. Brown, H-P. Beck, M. Molyneux and K. Marsh Malaria is a problem of global importance, responsible for 1–2 million deaths per year, mainly in African children, as well as considerable morbidity manifested as severe anaemia and encephalopathy in young children. Fundamental to the development of new tools for malaria control in humans is an increased understanding of key features of malaria infection, such as the diversity of outcome in different individuals, the understanding of different manifestations of the disease and of the mechanisms of immunity that allow clinical protection in the face of ongoing low-grade infection (concomitant immunity or premunition). Here, Graham Brown and colleagues review some of the ways in which molecular approaches might be used to increase our understanding of the epidemiology and clinical manifestations of malaria, as discussed at the Molecular Approaches to Malaria conference (MAM2000), Lorne, Australia, 2–5 February 2000. A high proportion of the world’s population remains at risk of malaria, which presents increasing problems because of breakdown of traditional control methods, civil disturbance, mobile populations, resistance to insecticides and resistance to cheap, safe, antimalarial drugs. An effective global strategy for malaria control demands specific approaches for its control under different endemic conditions, effective application of tools currently available, enhanced surveillance, welltrained teams and the development of new, effective disease control tools, such as drugs and vaccines. Potentially valuable advances are likely to arise from increased investments in basic molecular science, although field research is essential for proper evaluation. Field research can also generate hypotheses that can be tested at the molecular level. Clinical disease Nearly all of the deaths from malaria in African children are due to Plasmodium falciparum, usually in association with severe anaemia or cerebral malaria (CM) (symptoms referred to together as severe malaria). Many clinical indicators are associated with an increased risk of fatal outcome, but the actual causes of death, and possible contributing mechanisms, are not well established. Recent observations suggest, for example, that CM is not a homogeneous condition. Some children with CM might be in a coma as a result of subclinical status epilepticus, whereas others appear to have metabolic encephalopathy associated with severe acidosis that might be amenable to medical intervention. Among the indicators of poor prognosis in CM is Graham V. Brown is at the Department of Medicine, University of Melbourne and The Walter and Eliza Hall Institute of Medical Research, Victoria, Australia. Hans-Peter Beck is at the Swiss Tropical Institute, Basel, Switzerland. Malcolm Molyneux is at The Wellcome Trust Research Laboratories, College of Medicine, Blantyre, Malawi. Kevin Marsh is at the Centre for Geographic Medicine Research, Coast, KEMRI, Kilifi, Kenya. Tel: +61 3 8344 5490, Fax: +61 3 9347 1863, e-mail: g.brown@medicine. unimelb.edu.au 448

a very high plasma concentration of tumour necrosis factor (TNF). The role of NO production has been debated in the light of apparently contradictory results1,2, and its mode of action is not known. However, polymorphisms of the gene encoding NO synthase suggest that NO might have an important role in CM3–5. The molecular nature of these inflammatory responses, and the factors that initiate them are not yet understood sufficiently well to be able to translate these findings into improved clinical outcomes for patients6–9. Recent clinical studies suggest that reduced microvascular flow leading to tissue oxygen debt and anaerobic metabolism is common in many cases of severe malaria10–12. There is immunohistochemical evidence for inducible NO synthetase (iNOS) and nitrotyrosine, a marker for NO production, being present in cerebral capillaries of children dying of CM13. A key role was suggested for the P. falciparum toxin glycosylphosphatidylinositol (GPI)14, which appears to be a necessary and sufficient trigger for TNF release in tissues. These observations of systemic inflammatory response are clear indicators that exacerbated immune responses are associated with severe malaria and might accompany the sequestration of infected cells in target organs. A vicious cycle is established, with sequestration leading to cytokine release, increased endothelial receptor expression and, thereby, the propensity of the parasites to adhere. Current molecular techniques allowing more-detailed descriptions of events at cellular and molecular levels in fatal human malaria are of enhanced value only if clinical details preceding death have been documented. Several techniques have been applied to human tissue obtained at autopsy within hours of death in Malawian children15. Notably, there is a nonrandom distribution of parasitized red blood cells (RBC) in different parts of the brain that might have implications for the pathogenesis of CM by causing altered cerebral function despite a relatively low tissue parasite load. Parasitaemia in cerebral vessels was higher than that found in peripheral blood, and all stages of parasitized RBC were seen in brain smears, confirming that sequestration begins prior to the appearance of pigment in parasitized RBC. Subsequent rupture of mature schizonts, leading to release of toxins such as GPI, might potentially be inhibited by appropriate therapy, but this would need to be confirmed by field studies. By comparing findings from peripheral blood in fatal, severe, nonfatal and nonsevere malaria with autopsy findings from individuals who died from severe malaria, it might be possible to define the characteristics of host and parasite that associate with severe and fatal malaria. These could be useful in suggesting hypotheses that might be tested by therapeutic intervention. Clinical and autopsy studies also lead to hypotheses that can be tested in laboratory studies and vice versa, eg. laboratory studies leading to the description of chondroitin sulfate A as a receptor for P. falciparum16 were followed by field studies17 suggesting that chondroitin

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Molecular Approaches to Malaria sulfate A might be important in the pathogenesis of malaria in pregnancy. All relevant results were obtained by studying parasites taken directly from placenta and demonstrated that such parasites were far more likely to bind to chondroitin sulfate A than were isolates taken from the peripheral blood of pregnant women, from children or from nonpregnant adults18. Hyaluronic acid (HA) was later described as a receptor for placental malaria19. The demonstration of antigenic variation of P. falciparum in vitro20 and that antigenic variants had different binding properties20 were evidence on which the hypothesis that antigenic variation could allow the proliferation of parasites with propensity to bind to specific receptors in an organ-specific manner was based. Binding in the placenta would stimulate variant-specific protective immune responses to the antigens associated with this phenotype, in the same way that protection occurs following exposure to any variant antigen expressed in clinical isolates21,22. Molecular investigations of such host–parasite interactions, including studies of the interaction under physiological flow conditions23,24, will provide the background to the development of reagents for preventing or reversing cytoadherence in malaria. Vaccines: learning from experiments of nature Critical for the development of an effective vaccine against malaria is the need to define and understand antigenic diversity of malaria antigens, and to understand the fine specificity of immune responses and their value in protection against clinical disease. It is clear that not only might monoclonal antibodies (mABs) inhibit parasite growth in vitro, but that many different epitopes might also be involved. In turn, many epitopes might elicit strong immune responses but give no protection. For example, antibodies preventing the necessary processing of the P. falciparum merozoite surface protein 1 (PfMSP-1) prior to invasion could inhibit invasion and, therefore, parasite growth25. Further investigations revealed that naturally occurring antibodies against the same or a different epitope could inhibit the invasionblocking antibodies. Furthermore, those nonprotective, detrimental antibodies could also be generated by different epitopes located at the N-terminal region of the PfMSP-1 (Ref. 26; see also Blackman, this issue). Without understanding these events at a molecular level, certain contradictory results would not have been understood, which would have made it impossible to assess which antigen fragments would be most suitable for inclusion in a blood-stage vaccine. The ultimate goal is to produce a fragment that induces inhibitory antibodies without allowing the production of another family of antibodies that inhibit the first (protective) antibodies. One major impediment to the development of effective malaria vaccines for use in humans is the diversity of many of the candidate antigens. For example, PfMSP-2, a component of a vaccine currently undergoing trial in Papua New Guinea (PNG)27 is composed of a variety of conserved, dimorphic and highly polymorphic regions. It is not yet understood which region is an immunological target or whether this molecule plays a role in protection. A detailed sero-epidemiological study in children from endemic areas of PNG revealed a low responsiveness against the constant region, infrequent recognition of the dimorphic region and a highly variable response against the polymorphic region, with Parasitology Today, vol. 16, no. 10, 2000

regard to both prevalence and titres (H-P. Beck, unpublished). Importantly, responses differed substantially against the two dimorphic regions, and antibodies against some domains were also found in nonexposed children, suggesting a role for antigenic mimicry in this molecule. It is clear, therefore, that an understanding at the epitope level is required to determine whether this is an effective immune response target and whether heterologous protection is obtained. Field studies of naturally acquired immunity can provide further evidence for the role of potential vaccine candidates, such as PfMSP-1 or PfMSP-2. In The Gambia, an immunoepidemiological approach was used to demonstrate an association between antibodies to the epidermal growth factor (EGF)-like motifs of PfMSP-1 and resistance to clinical malaria or high parasitaemia28. Using human immune serum, antibodies were affinity purified on the EGF-like region and shown to inhibit the growth of P. falciparum in vitro29. The studies in The Gambia suggested that the acquisition of IgG3 antibodies to PfMSP-2 was associated with development of clinical resistance to malaria in children 3–8 years old30. Analysis of the distribution of PfMSP-2 genotypes following vaccination is important in assessing the effect of this vaccine component, and identification of individual genotypes allows the investigation of infection dynamics. It has been shown that the persistence of multiple infections is associated with protection against subsequent infections and clinical disease (concomitant immunity or premunition)31,32. For assessment of the efficacy of bloodstage vaccines, which are not aimed at preventing infections, the rate of acquisition and loss of infections might provide a powerful tool for assessing efficacy. Most importantly for vaccine development, surrogate markers of protection are required so that many novel antigens, adjuvants or combinations can be assessed in small-scale trials in humans before moving to small numbers of large-scale human clinical trials. An important component of all efficacy trials in human and nonhuman primates is the search for immunological correlates of protection that could be used in future as markers of protective immunity. Such important information might arise from studies in a human challenge system in which rapid acquisition of immunity was demonstrated following repeated low-dose infection (presented by D.J. Pombo, Queensland Institute for Medical Research, Brisbane, Australia)33. Field studies are critical to the investigation of the generation and maintenance of genetic diversity of Plasmodium spp under conditions of varying endemicity in semi-immune human populations. For example, examination of polymorphic regions of P. vivax apical membrane antigen 1 (AMA-1) and PvMSP-1 suggest small differences between regions compared with the diversity detected within populations34. Understanding the population dynamics of malaria under conditions of drug pressure or immune pressure (eg. Ref. 35) is a requirement for successful implementation of control measures. These studies can also validate theories of population dynamics, such as density-dependent regulation of infection that transcends both species and genotype36. Looking to the genes of humans and Plasmodium Knowledge of the human genome will provide further insights to understanding factors responsible for the variable susceptibility of human populations, as 449

Molecular Approaches to Malaria observed at a cellular level, eg. resistance of Duffynegative RBC to P. vivax37 or partial protection afforded by ovalocytosis38,39 Genetics of the immune responses are obviously important40,41 and details might be clarified once the human genome can be searched. Completion of the Malaria Genome Project and availability of new technologies for genome-wide comparison of genomes and expression profiling will also help identify key targets in biochemical pathways that are parasite-specific, and which might be interrupted without deleterious consequences for the host. Of particular importance to understanding the cell biology of malaria is the availability of new techniques for deleting or mutating genes or introducing new genes to determine the effect on growth, invasion or binding to RBC or to other host receptors42. Understanding the genetics of resistance to the antimalarial drugs currently available is an important goal to monitor spread of resistance, and to develop new and efficient drugs against this devastating disease. Conclusions In this Review, a variety of ways has been indicated in which molecular approaches might be used to enhance our understanding of clinical features of malaria, its pathogenesis, mechanisms of the immune response and features that correlate with poor outcome of infection. Many of the sessions at MAM2000 highlighted just how many possibilities there are for applying the latest approaches of molecular and cellular biology to the understanding of the establishment and maintenance of malaria infections, which cause so much suffering for individuals and populations of disease-endemic areas. It is hoped that this increased understanding of malaria could be used as the basis of design for new strategies, for prevention and treatment of the disease. References 1 Al-Yaman, F.M. et al. (1996) Association between serum levels of reactive nitrogen intermediates and coma in children with cerebral malaria in Papua New Guinea. Trans. R. Soc. Trop. Med. Hyg. 90, 270–273 2 Anstey, N.M. et al. (1996) Nitric oxide in Tanzanian children with malaria: inverse relationship between malaria severity and nitric oxide production/nitric oxide synthase type 2 expression. J. Exp. Med. 184, 557–567 3 Burgner, D. et al. (1998) Inducible nitric oxide synthase polymorphism and fatal cerebral malaria. Lancet 352, 1193–1194 4 Anstey, N.M. et al. (1999) Nitric oxide, malaria, and anemia: inverse relationship between nitric oxide production and hemoglobin concentration in asymptomatic, malaria-exposed children. Am. J. Trop. Med. Hyg. 61, 249–252 5 Levesque, M.C. et al. (1999) Nitric oxide synthase type 2 promoter polymorphisms, nitric oxide production, and disease severity in Tanzanian children with malaria. J. Infect. Dis. 180, 1994–2002 6 Kwiatkowski, D. et al. (1990) TNF concentration in fatal cerebral, non-fatal cerebral, and uncomplicated Plasmodium falciparum malaria. Lancet 336, 1201–1204 7 Kwiatkowski, D. et al. (1993) Anti-TNF therapy inhibits fever in cerebral malaria. Q. J. Med. 86, 91–98 8 Kwiatkowski, D. et al. (1991) Cerebral malaria. Lancet 337, 1281–1282 9 Clark, I.A. and Rockett, K.A. (1996) Nitric oxide and parasitic disease. Adv. Parasitol. 37, 1–56 10 English, M. et al. (1997) Lactic acidosis and oxygen debt in African children with severe anaemia. Q. J. Med. 90, 563–569 11 English, M. et al. (1996) Clinical overlap between malaria and severe pneumonia in hospitalized African children. Trans. R. Soc. Trop. Med. Hyg. 90, 658–662 12 English, M. et al. (1997) Acidosis in severe childhood malaria. Q. J. Mal. 90, 263–270 13 Clark, I.A. et al. (1993) Nitric oxide and cerebral malaria. Lancet 341, 632–633

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14 Schofield, L. et al. (1996) Glycosylphosphatidylinositol toxin of Plasmodium upregulates intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin expression in vascular endothelial cells and increases leukocyte and parasite cytoadherence via tyrosine kinase-dependent signal transduction. J. Immunol. 156, 1886–1896 15 Brown, H. et al. (1999) Cytokine expression in the brain in human cerebral malaria. J. Infect. Dis. 180, 1742–1746 16 Rogerson, S.J. et al. (1995) Chondroitin sulfate A is a cell surface receptor for Plasmodium falciparum-infected erythrocytes. J. Exp. Med. 182, 15–20 17 Fried, M. and Duffy, P.E. (1998) Maternal malaria and parasite adhesion. J. Mol. Med. 76, 162–171 18 Rogerson, S.J. and Brown, G.V. (1997) Chondroitin sulfate A as an adherence receptor for Plasmodium falciparum-infected erythrocytes. Parasitol. Today 13, 70–75 19 Beeson, J.G. et al. (2000) Adhesion of Plasmodium falciparuminfected erythrocytes to hyaluronic acid in placental malaria. Nat. Med. 6, 86–90 20 Biggs, B.A. et al. (1991) Antigenic variation in Plasmodium falciparum. Proc. Natl. Acad. Sci. U. S. A. 88, 9171–9174 21 Marsh, K. and Howard, R.J. (1986) Antigens induced on erythrocytes by P. falciparum: expression of diverse and conserved determinants. Science 231, 150–153 22 Bull, P.C. et al. (1998) Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria. Nature Med. 4, 358–360 23 Cooke, B.M. et al. (1994) Rolling and stationary cytoadhesion of red blood cells parasitized by Plasmodium falciparum: separate roles for ICAM-1, CD36 and thrombospondin. Br. J. Haematol. 87, 162–170 24 Cooke, B.M. et al. (1996) Adhesion of malaria-infected red blood cells to chondroitin sulfate A under flow conditions. Blood 88, 4040–4044 25 Blackman, M.J. et al. (1991) Processing of the Plasmodium falciparum major merozoite surface protein 1: identification of a 33kilodalton secondary processing product which is shed prior to erythrocyte invasion. Mol. Biochem. Parasitol. 49, 35–44 26 Holder, A.A. et al. (1999) Merozoite surface protein 1, immune evasion, and vaccines against asexual blood stage malaria. Parassitologia 41, 409–414 27 Genton, B. et al. (2000) Safety and immunogenicity of a threecomponent blood-stage malaria vaccine in adults living in an endemic area of Papua New Guinea. Vaccine 18, 2504–2511 28 Egan, A.F. et al. (1996) Clinical immunity to Plasmodium falciparum malaria is associated with serum antibodies to the 19 kDa Cterminal fragment of the merozoite surface antigen, PfMSP-1. J. Infect. Dis. 173, 765–769 29 Egan, A.F. et al. (1999) Human antibodies to the 19 kDa C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 inhibit parasite growth in vitro. Parasite Immunol. 21, 133–139 30 Taylor, R.R. et al. (1998) IgG3 antibodies to Plasmodium falciparum merozoite surface protein 2 (MSP2): increasing prevalence with age and association with clinical immunity to malaria. Am. J. Trop. Med. Hyg. 58, 406–413 31 Al-Yaman, F. et al. (1997) Reduced risk of clinical malaria in children infected with multiple clones of Plasmodium falciparum in a highly endemic area: a prospective community study. Trans. R. Soc. Trop. Med. Hyg. 91, 602–605 32 Beck, H.P. et al. (1997) Analysis of multiple Plasmodium falciparum infections in Tanzanian children during the phase III trial of the malaria vaccine SPf66. J. Infect. Dis. 175, 921–926 33 Cheng, Q. et al. (1997) Measurement of Plasmodium falciparum growth rates in vivo: A test of malaria vaccines. Am. J. Trop. Med. Hyg. 57, 495–500 34 Figtree, M. et al. (2000) Plasmodium vivax synonymous substitution frequencies, evolution and population structure deduced from diversity in AMA1 and MSP1 genes. Mol. Biochem. Parasitol. 108, 53–66 35 Paul, R.E. et al. (1999) Genetic analysis of Plasmodium falciparum infections on the north-western border of Thailand. Trans. R. Soc. Trop. Med. Hyg. 93, 587–593 36 Bruce, M.C. et al. (2000) Cross-species interactions between malaria parasites in humans. Science 287, 845–848 37 Miller, L.H. et al. (1976) The resistance factor to Plasmodium vivax in blacks. The Duffy-blood-group genotype, Fy/Fy. New Engl. J. Med. 295, 302–304 38 Kidson, C. et al. (1981) Ovalocytic erythrocytes from Melanesians are resistant to invasion by malaria parasites in culture. Proc. Natl. Acad. Sci. U. S. A. 78, 5829–5832

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Molecular Approaches to Malaria 39 Genton, B. et al. (1995) Ovalocytosis and cerebral malaria. Lancet 378, 564–565 40 Hill, A.V.S. et al. (1991) Common West African HLA antigens are associated with protection from severe malaria. Nature 352, 595–600 41 Kun, J-F. et al. (1998) Polymorphism in promoter region of

inducible nitric oxide synthase gene and protection against malaria. Lancet 351, 265–266 42 O’Donnell, R.A. et al. (2000) Functional conservation of the malaria vaccine antigen MSP-119 across distantly related Plasmodium species. Nat. Med. 6, 91–95

Pathogenesis of Malaria I.A. Clark and L. Schofield As the mortality rate of 20–30% for severe falciparum malaria under even the best clinical conditions testifies, access to antimalarial drugs is not sufficient to prevent an appreciable mortality from this disease. Understanding the cause of death at a cellular level is essential if additional rational treatments are to be developed. Here, Ian Clark and Louis Schofield discuss recent work presented at the Molecular Approaches to Malaria conference, Lorne, Australia, 2–5 February 2000, that updates the cytokine-based concept of malarial disease. The syndrome caused by Plasmodium falciparum in African children typically consists of fever, metabolic acidosis, hypoglycaemia, seizures, coma and cerebral oedema1,2. For all its dramatic manifestations, the disease seen in severe falciparum malaria is remarkably similar to many other conditions, including some, such as heatstroke, that are not caused by infectious agents. The coma exhibited by severe cases of falciparum malaria has traditionally been explained as resulting from mechanical blockage of blood vessels by sequestered parasitized red blood cells (PRBC), causing local cerebral hypoxia. Yet the condition is mimicked exactly by chronic salicylate poisoning3, a condition accepted to be a complex metabolic dysfunction4, devoid of PRBC, sequestered or otherwise, and not remarkable for hypoxia. Salicylate has been shown to cause a systemic inflammatory state5,6, precisely the terminology used by Brian Maegraith7 in 1948 to describe his then revolutionary view of severe falciparum malaria, which he likened functionally to systemic bacterial infections. Over 30 years later, it was proposed that Maegraith’s observations could be explained by host cells, under the influence of a long-proposed malaria toxin, releasing large amounts of the soluble mediators now called proinflammatory cytokines8, which then triggered the onset of pathology. Evidence supporting this concept now includes the ability of experimental malaria to prime tumour necrosis factor (TNF) production8, the close similarity to malaria of the side effects and biochemical changes observed when TNF is administered to tumour patients9, and clinical studies showing a correlation between disease severity and the circulating levels of TNF in African10,11 and Melanesian12 children and European adults13. In vitro studies14 on the interaction between the pro-inflammatory cytokines and interleukin-10 (IL-10) Ian Clark is at the Australian National University, Canberra, ACT 0200, Australia. Louis Schofield is at the Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC 3052, Australia. Tel: +61 2 6249 4363, Fax: +61 2 6249 0313, e-mail: [email protected] Parasitology Today, vol. 16, no. 10, 2000

have shown that IL-10 counter-regulates the proinflammatory response to P. falciparum. The studies also produced evidence consistent with severe falciparum malaria being associated with an inadequate negative feedback response by IL-10. This argument has recently been reinforced by the in vivo evidence15 of plasma IL-10 levels being significantly lower in fatal cases of malaria than in survivors. Recent work in monkey malaria has shown that FAS ligand, another member of the TNF family, might also be elevated in malaria16. Furthermore, a recent pilot study has shown a tendency for the injection of a polyclonal-specific Fab fragment directed against human TNF to inhibit a range of clinical manifestations of falciparum malaria17. In recent years, work on this cytokine-based concept of disease has focussed on the pathophysiological implications of the ability of these cytokines to generate the inducible form of nitric oxide synthase (iNOS), and thus produce a continuous, potentially large supply of nitric oxide (NO) in tissues that normally experience only low, tightly controlled levels of this ubiquitous cellular messenger. In critical locations, this iNOS could be functionally important, accounting for some of the reversible cerebral symptoms18,19 and also immunosuppression20 and weight loss21 seen in experimental malaria. As recently reviewed, a parallel argument has been developing for sepsis22. It has, therefore, been reasoned that the coma in some patients given a clinical diagnosis of cerebral malaria (CM) is an integral part of a wider syndrome caused by systemic cytokine –iNOS excess rather than a local entity caused by mechanical blockage of blood vessels, and that it is part of a systemic change that has more in common with the metabolic encephalopathies than with simple hypoxia23. Nevertheless, hypoxia has an important role in this view of malaria as systemic inflammation, in that, as recently reviewed in a malaria context24, it synergizes with inflammatory cytokines to enhance iNOS induction. Sequestration of PRBC could, therefore, play a vital role in exacerbating the inflammatory role of this class of cytokines, thus making a given concentration of these mediators more potent in falciparum than in vivax malaria. At the Molecular Approaches to Malaria conference, the stage was set for an update of the role of cytokines in the pathogenesis of malaria. The range of invited speakers allowed a rare and successful opportunity for practical experience to blend with basic science. A clinical basis for cellular studies Kevin Marsh (Kenya Medical Research Institute, Kilifi, Kenya) summarized his view that there is no one

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