BSP malaria meeting, February 1990

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BSP Malaria Meeting, February 1990 C. Facer, M. Lockyer, G. Mitchell, M. Pudney and D. Walliker The second annual malaria meeting of the British Society for Parasitology was held with the background of the recent crisis of confidence in US research into the development of a malaria vaccine. The scientific reasons for this crisis were in the foreground at the UK meeting. Some sources of funding may have diminished, but this has not lessened the need or vigour of work on malaria. The meeting is developing into an important international forum, this year attended by 200 delegates from not only the UK but several European centres of malaria research also. The subject matter was wide-ranging, from basic biology to chemotherapy and vaccination. Some SO papers were presented; inevitably it is not possible to provide more than a brief and rather selective account of the meeting's main themes here.

Polymorphism and Immunogenetics A major hindrance to the development of effective malaria vaccines, especially those based on synthetic polypeptides, is the remarkable polymorphism of Plasmodium. The first session of the meeting examined the relationship of this polymorphism to the human immune response for several such candidate molecules. The circumsporozoite protein (CSP) of P. falciparum, like most malaria antigens, contains an immunodominant repetitive B-cell epitope. The interpretation of polymorphism in T-cell epitopes of the molecule with respect to human MHC is still an unresolved and contentious issue. Richard Pink (F. Hoffman-LaRoche, Switzerland)reviewed data from his own laboratory and others showing that although the main T-cell response in endemic areas is against polymorphic epitopes, human T cells can also recognize two non-variant epitopes (one within the repeats). Pink also presented new evidence that epitopes within polymorphic regions are recognized by B cells, thus immune selection by humoral responses may also contribute to the diversity of these regions. David Arnot (University of Edinburgh, UK) disputed the idea that selection pressure from human T cells maintains polymorphism in the variant CSP T-cell epitopes. Arnot rightly emphasized the first and most fundamental problem in interpreting data from molecular bio-

logical and immunological observations, namely, our ignorance of basic sporozoite biology and of the role of the CSP. He argued that the prevalence of nonsynonymous mutations in the CSP and other genes of Plasmodium is the natural consequence of an A+T-rich genome, since synonymous codons of opposite bias will be selected against. Thus, a 'quasi-neutralist' hypothesis could explain the lack of synonymous substitutions. Dimorphic regions of the precursor to the major merozoite surface antigen (MSA- I ) of P. fdciparum are the regions most strongly recognized by endemic human sera, compared to conserved and strain-specific sequences (Klaus Fruh, Zentrum fur Molekulare Biologie, Heidelberg, FRG). However, Pink claimed that, even within a conserved region of MSA-I, there is considerable MHC isotypic diversity in the T-cell epitopes recognized by different individuals. The generally poor immunogenicity of vaccine candidates is not, however, explicable solely in terms of MHCrestriction of the immune response, since Eleanor Riley (Medical Research Council, The Gambia) demonstrated very variable responses to gametocyte surface antigens in individuals with identical M H C D Q and DR alleles. MSA-2, a second candidate merozoite surface antigen, like MSA- I displays allelic polymorphism primarily due to variation in tandem repeat sequences (Brian Fenton, University of Edinburgh, UK), which must raise questions as to the likelihood of this molecule being a suitable vaccine candidate. With regard to the ring-infected cell surface antigen Pf155/RESA, vaccines based on this molecule have been used in several monkey trials, but with only partial success. There appears to be little polymorphism in this antigen, which may be due to its non-surface location. Passive transfer to Aotus monkeys of affinity-purified human IgG against the immunodominant RESA repeats allowed most of the animals to self-cure (Klaus Berzins, University of Stockholm, Sweden). Antibodies to RESA-repeats cross-react with other antigens, and, in vitro, can inhibit red cell invasion of parasites not expressing Pf155/RESA. Studies on antigenic variation in P. chabaudi (Lisa Brannan, University of Glasgow, UK) have shown that recrudescent parasites derived from a cloned line contain a mixture of clones that are

antigenically distinct from one another and from the original parent clone. These recrudescent parasites vary in their immunogenicity and in the level of parasitaemia they attain. Molecular probes for these antigenswould allowthe genetic mechanisms responsible for their variable expression to be elucidated. It is clear that the potential for antigenic .polymorphism in malaria is far greater than initially anticipated. This, together with the poor immunogenicity of candidate vaccines, suggests that new strategies for vaccine design will be needed. One such strategy, an 'antidisease' vaccine, was described by Chris Bate (Middlesex Hospital, UK). In vitro, the parasite secretes heat stable, water soluble, non-polymorphic and protease insensitive antigens (ie. toxins) that, like bacterial lipopolysaccharide (LPS), induce tumour necrosis factor (TNF) secretion. Antibodies to the 'toxin' can block TNF production, but do not prevent LPS from triggering TNF release. The antibodies that recognize the toxin are of IgG3 and IgM type, however, since the toxins are T-cell independent antigens, no memory is induced. An antidisease vaccine, therefore, may need to make use of anti-idiotypes to promote a suitable anamnestic response.

GametocyteAntigens and Development Vaccines based on gametocyte antigens are still an active area of interest. The object of such vaccines is to block development of the parasite in the vector, thus preventing its transmission to a new host. Richard Carter (University of Edinburgh, UK) reviewed previous work, pointing out that three groups of antigens, particularly those of 250 and 48/45 kDa, associated with the surface of gametocytes, and one of 25 kDa, associated with the ookinete surface, have been identified as possible vaccine candidates. Although antibodies to these antigens have been found to be effective in blocking development in the mosquito, work in Sri Lanka and elsewhere has shown that natural antibodies to these antigens can actually enhance oocyst production when present in low concentrations. Such enhancement has been shown with some 14% of sera from patients in malaria-endemic regions; interestingly, a much lower proportion (approximately 4%) of sera from non~) 1990,ElsevierSciencePublishersLtd, (UK) 0169 4707190/$02.00

Parasitology Today, voL 6, no. 7, 1990 immune migrants showed such enhancement. Corinne Ong and her colleagues (London School of Hygiene and Tropical Medicine, UK) found that immune individuals generally showed a lower humoral response to a 48/45 kDa antigen than patients who had experienced only a few episodes of malaria. Five distinct epitopes on this molecule have been identified by competitive ELISA tests with a panel of monoclonal antibodies. These epitopes were recognized by antibodies in sera from patients in The Gambia and Papua New Guinea, although to varying degrees. The variability seen in the response of different patients to gametocyte antigens could be due to either MHC-restriction or to antigen-specific immune suppression. Carter discussed work on nonresponders that suggested active suppression may occur by CD8 ÷ cells blocking T-cell help. Evidence against the MHC-restriction hypothesis was presented by Riley, who showed that individuals in The Gambia with identical haplotypes for HLA-DQ ancl -DR alleles exhibited variable lympho-proliferative responses to the 48/45 kDa gametocyte antigens. Genes involved in gametocyte differentiation are now being identified in several laboratories. Earlier work by Andrew Waters and Thomas McCutchan (National Institutes of Health, USA) showed that for ribosomal RNA, a switch in expression from the rRNA-A gene to the rRNA-C gene occurred during gametogenesis. Pietro Alano and colleagues (University of Edinburgh, UK) have identified an internal protein (Pfg 27/25) specific to gametocytes and present very early in their development. Chris Delves and colleagues (University of Edinburgh, UK) and Steven Holloway and John Hyde (University of Manchester, Institute of Science and Technology, UK)in studies on tubulins, have shown that the two a-tubulin genes present in P. falciparum are expressed differentially in the life cycle, the ~-I gene being active in both asexual forms and gametocytes, and a-II only in gametocytes. The I]-tubulin gene appeared to be expressed as three transcripts in gametocytes, but only as one in asexual stages. None of these studies has answered the critical question of what commits a given parasite to develop as an asexual form or as a gametocyte, but the answer may emerge from studies of the expression of gametoo/te-specific genes. Tubulins are ubiquitous proteins, one of their primary functions being in the formation of spindle microtubules in dividing cells. Very little is known about

203 the genes involved in mitosis of malaria parasites, but they are of obvious importance to our understanding of the mechanisms of cell division, and their products may be potential targets for chemotherapy. Don Williamson and Petra Ross-Macdonald (National Institute of Medical Research, UK) have isolated a gene, designated pfc2, which shows homology to the p34 c~c2 protein kinase gene of the yeast Schizosaccharomyces pombe; the product of this gene is important in the initiation of mitosis in S, pombe and other eukaryotes.

Parasite- Host Cell Relations This session, with the exception of one paper, concentrated on induced change and its consequences in P. falciparum-infected red cells. Russell Howard (DNAX, San Francisco, USA) distinguished between the phenomena of rosetting of uninfected erythrocytes around parasitized cells, and of the cytoadherence of parasitized cells to capillary endothelial cells. Rosetting precludes cytoadherence, and if rosettes are disrupted mechanically, these cells are then able to attach to endothelial cells. Studies on the flow of parasitized cells in rat capillaries showed that rosetting is just as effective as cytoadherence in causing obstruction. Rosetting has only been found in some isolates of P. falciparum. When wild isolates in patients' blood were examined, cytoadherence via thrombospondin and CD36 binding was closely related, perhaps indicating a single co-expressed receptor, while rosetting was a quite distinct phenomenon. Mats Wahlgren (University of Stockholm, Sweden) showed that knobs on the infected cell surface, which at one time were thought to mediate in cytoadherence, are not required for rosetting in vitro, and that some human sera can inhibit the formation of rosettes. He suggested that rosetting is a way of facilitating merozoite invasion of new erythrocytes by avoiding exposure to the host's immune cells and molecules. Graham Brown (Walter and Eliza Hall Institute, Australia) has shown that clones from an established laboratory clone differ widely in their possession of knobs, their ability to bind to melanoma or endothelial cells, and in their ability to agglutinate; sub-clones lacking knobs could bind to CD36, and express a molecule having the characteristics of PfEMPI. Similarly, Tony Berendt (John Radcliffe Hospital, Oxford, UK) has used transfected COS cells and human umbilical vein endothelial cells (HUVEC) that express another cell surface molecule

ICAM-I, but not CD36, and that, like CD36-negative melanomacytes, permit adhesion of parasitized erythrocytes. The expression oflCAM- I can be stimulated by tumour necrosis factor, serum levels of which are often elevated in malaria patients. This may have implications for the development of severe cerebral malaria. Gerard Nash (University of Birmingham, UK) described the marked mechanical changes in P. falciparum-infected red cells, but emphasized the need for comparative studies to clarify the contribution of these changes to pathology. He also placed cytoadherence in a physiological setting by showing that the binding was sufficiently strong to resist the shear forces generated in venules. In one paper addressing sporozoiteliver cell interactions, J. van Pelt (University Hospital of Nijmegen, The Netherlands) showed that P. folciparum sporozoites, incubated with solubilized hepatocyte membranes, bound 65 kDa and 20 kDa parasite proteins. The technique depended on reversibly crosslinking these ligands to their recognition sites. When, in turn, the 20 kDa protein was used to probe sporozoites, a 16 kDa protein was bound, and this is therefore a candidate for the sporozoite's liver cell membrane recognition molecule.

Immunopathology The characteristic immunodominant repeat regions of P. falciparurn surface proteins are thought to trigger redundant non-protective polyclonal B-cell responses. Interestingly, there is a geographic co-distribution between Epstein-Barr virus (EBV), malaria and endemic Burkitt's lymphoma, the B-cell monoclonal tumour of children. Christine Facer (London Hospital Medical College, UK) asked what new laboratory evidence there was for malaria as the malignant cofactor in triggering this tumour. Although EBV is able to transform B cells, it lacks oncogenic capacity per se. From observations derived from experiments involving the interaction between lymphocytes, malaria antigens and EBV, Facer made three suggestions as to how malaria may play a role: (I) malaria antigens stimulate DNA synthesis in EBV infected B cells, (2) the immune suppressive effect of malaria infections impairs cytotoxic T-cell control of EBV infected lymphocytes, and (3) the germinal centre hyper-activity and polyclonal B-cell activation triggered by the repeated epitopes of malaria antigens increases the probability of a cytogenetic event. However, puzzles still

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remain. For example, why is EBV not detected in all cases of sporadic Burkitt's lymphoma? Also, why is the tumour so rare ( I - 2 cases per 10 000) in a population of child ren who carry both the virus and appreciable levels of P. falciparum? Double infections of EBV and a quartan malaria species, Plasmodium brasilianum, in marmosets are associated with the development of glomerulonephritis (David Davies, St Thomas' Hospital, London, UK), which is not seen when the monkeys have single infections. It was notable that neither the severity of the disease (oedema, proteinuria) nor the histopathological changes correlated with immune complex deposition in the glomerular mesangium. In murine malaria, a chronic progressive and irreversible nephritis develop. DNA-binding antibodies have been implicated in the initiation of renal pathology in the autoimmune disease, systemic lupus erythematosus (SLE). By ELISA, Clare Lloyd (King's College, London, UK) found that DNA-binding antibody titres increased in mice infected with Plasmodium vinckei, P. yoelii and P. berghei after changes in renal function had been initiated by the parasite. The auto-antibodies may contribute to the nephritis by the formation of immune complexes in the glomeruli. Using murine monoclonal DNA-binding antibody, deposition of DNA was shown in late-stage infected kidneys.

Chemotherapy The most alarming practical problem with malaria is the rapid worldwide spread of drug resistance. David Warrell (John Radcliffe Hospital, Oxford, UK) explained that the useful life of the last line of defence against resistant malaria, quinine, is now also severely limited by the appearance of resistance in Southeast Asia. An understanding of the genetic and molecular basis of drugresistance should enable us to find ways of restoring the usefulness of existing drugs, as well as of circumventing the development of resistance to new drugs. In those areas where quinine still remains effective, Warrell stressed the need for a form of the drug that can be administered intramuscularly. in the last two years, our understanding of the mechanisms of resistance to the antifolate drug, pyrimethamine, have progressed greatly. There is good evidence from several laboratories that point mutations in the gene for dihydrofolate reductase-thymidylate synthase (DHFP~I-S) cause resistance, probably by affecting the ability of the drug to bind to the enzyme. John Hyde (University of

Manchester Institute of Science and Technology, UK) reported expression of the P. falciparum DHFR/TS gene in E. coil, grown in a medium containing trimethoprim (which binds to the E. coil DHFR). Thus, the Plasmodium DHFR complemented the disabled gene of the bacterium. In a similar type of experiment, Mohammed Shahabuddin and colleagues (University of Edinburgh, UK) obtained expression of the P. fdciparum hypoxanthine phosphoribosyl transferase (HPRT) gene in a Salmonella typhimurium mutant that lacked the equivalent gene. This type of approach will, hopefully, lead to the development of new drugs targeted on these enzymes, as well as to a clearer understanding of the mechanisms of resistance to them. A different approach to the inhibition of Plasmodium DHFR/TS was described by Richard Franklin (University of Basel, Switzerland), who has attempted to block production of the enzyme by inhibiting mRNA translation with antisense nucleotides. Some success was achieved with one 49 nucleotide molecule that markedly reduced synthesis of a 71 kDa protein exhibiting TS activity. Apart from pyrimethamine, little is known of the mode of action and metabolism of the diverse range of antimalarial drugs in use. Nuala Helsby (Liverpool School of Tropical Medicine, UK) presented data on her work investigating variations in the process by which proguanil is changed to the active metabolite, cycloguanil. In humans, the frequency distribution of the ratio of circulating proguanil to cycloguanil is skewed from normal, with some individuals being very poor at metabolizing proguanil. There also seems to be distinct inter-ethnic variation. In vitro, the metabolism of proguanil in human liver microsomes is competitively inhibited by mephenytoin. The latter is metabolized in a genetically polymorphic manner, and Helsby suggested a role for mephenytoin hydroxylase in the conversion of proguanil to cycloguanil. Individuals who are poor metabolizers of proguanil may not only suffer treatment failure but, by exposing parasites to sub-lethal levels of the active drug, may also provide an origin of drug resistant parasites. A major contributory factor to the development of drug resistance is the inappropriate use of the drugs already available. A very clear indication of the effect that the disease can have on the pharmacokinetics of compounds was given by Sharif Mansour (University of Liverpool, UK), using quinine. He was able to show that during the malaria infection, changes in alpha I-acid glycoprotein and fever can individually affect

the disposition of quinine in animal models. The role of calcium in the uptake of chloroquine is now the subject of research in several laboratories, following the findings two years ago that calcium channel blockers such as verapamil can reverse chloroquine resistance. Studies of calcium transport in erythrocytes infected with P. berghei or P. falciparum have shown that the uptake of calcium is markedly elevated compared to unparasitized red cells (Patrick Bray, Liverpool School of Tropical Medicine, UK). However, the picture is still far from clear, since verapamil has been found to be unable to reverse the resistance of the P. berghei RC strain. Despite gaps in our knowledge of the precise molecular mechanism underlying chloroquine resistance, Geoff Kirby (London School of Hygiene and Tropical Medicine, UK) was able to show that resistance in P. falciparum is not accompanied by alterations in the characteristics of protein synthesis by the parasite. Thus a resistant (KI) and sensitive (T9-96) parasite were equally sensitive to the in vitro antimalarial activity of quassinoid compounds despite a 16-fold difference in sensitivity to chloroquine. Derived from plants and used pantropically to treat malaria, these compounds are known for their ability to inhibit protein synthesis in Plasmodium. There is also a need for new methods to detect and determine antimalarial drugs in body fluids for use, in vitro and in vivo, on drug susceptibility, chemoprophylaxis compliance and drug use. T. Eggelte (Royal Tropical Institute, Amsterdam, The Netherlands) elaborated on the development of ELISA tests using monoclonal antibodies to a variety of antimalarial drugs, some of which also react with metabolites of the drugs in question. In studies carried out in rural African hospitals, a high percentage of malaria patients had high concentrations of chloroquine in the urine or blood prior to treatment, indicating widespread abuse of antimalarials, a situation of considerable concern with regard to the spread of drug-resistant strains of P. falciparum. When a new antimalarial drug becomes available, one of the first questions asked is when will resistance arise? Mary Pudney (Wellcome Research Laboratory, UK) described how resistance to Wellcome's newly developed hydroxynaphthoquinone antimalarial can be induced, in vitro, by progressively selecting parasites from relapses arising from sub-optimal drug regimens. The strain selected by this technique has, however, retained sensitivity to other

Parasitology Today, vol. 6, no. 7, 1990 antimalarials, and resistance does not seem to relate to transport processes or to the pyrimidine pathway, but seems to be concerned with mitochondria. The ease with which P. falciparum becomes drug-resistant highlights the urgent need to discover drugs with novel modes of action. As John Horton (Smith Kline Beecham, UK) pointed out, the depressing fact remains that the less developed world is an unpromising marketplace to gain a return from the long and expensive processes of drug development and marketing. This is the main reason why so few new compounds are currently being,, investigated as anti malaria& Future Directions

In addition to the subjecls mentioned above, the meeting included talks on

205 numerous other topics as diverse as mitochondrial RNA polymerase (Malcolm Gardner, National Institute for Medical Research, UK), a novel fluorescent technique for malaria diagnosis (Michael Makler, Veterans Administration Medical Center, Portland, USA), the possible role of tumour necrosis factor and interleukin- I in pathogenesis (Dominic Kwiatkowski, Medical Research Council, The Gambia), and exchange transfusion as a treatment for severe P. falciparum malaria (Peter Chiodini, Hospital for Tropical Diseases, London, UK). Although the 1980s produced remarkable advances in our knowledge of the molecular biology of the parasite, most of this effort has been directed towards candidate antigens for an eventual vaccine. The disappointments of the first vaccine trials have emphasized the need for research into all aspects of the biology of the parasite if novel methods

of control are to be produced. The 1990 BSP malaria meeting provided ample food for thought on new ways of looking at the capricious agent that causes this disease. Acknowledgements

The British Society for Parasitology(BSP)2nd Malaria Meeting was held at the Zoological Society of London on 8-9 February, 1990. The BSPand the Malaria Meeting Committee gratefully acknowledge generous sponsorship from the Wellcome Foundation and Smith Kline and Beecham. The 3rd BSP Malaria Meeting is scheduledto take place at the Royal College of Physicians,Edinburgh, on February 14-15~ 199 I. This account of the 1990 meeting has been compiled by Caroline Ash, Christine Facer (London Hospital Medical College, UK), Michael Lockyer (Wellcome Biotech, UK), Graham Mitchell (Guy's Hospital Medical School, London, UK), Mary Pudney (Wellcome Research Laboratories, UK) and David Walliker (University of Edinburgh, UK).

Biological Control of Pests and Vectors: Pros and Cons I, Popiel and W. Olkowski Biological control is 'the utilization of parasites, predators and pathogens for the regulation of host population densities '~. This definition encompasses both preventative and curative pest control practices that suppress pest populations and keep them below intolerable injury levels. Ideally, methods of biological control are non-toxic to the operators, leave no residues to contaminate food chains, produce no secondary pest outbreaks, do not harm non-target organisms and rarely lead to resistant pest populations. In addition, reg,Jlatory controls are expected to remain minimal compared to existing regulation of modern synthetic pesticides. Biological control has traditionally bee.n incorporated within integrated pest management (IPM) programmes with conservation, augmentation and importation of natural enemies as available tactics. Despite the overwhelming benefits that biological control offers, however, complex technical, regulatory, and philosophical issues impede its widespread implementation. Production, Storage and Application

Production. The efficacy of any biological control agent is irrelevant if economiC) 1990,ElsevierSciencePublishersLtd~(UK) 0169~707/90/$02.00

cally acceptable production methods cannot be developed and, in industrialized countries, affordable production generally requires automated mass production methods. This requirement is a severe limitation for the commercialization of many biological agents because, with only a few exceptions, such methods either do not exist or are currently too costly. In countries where the cost of labor is low compared to the cost of sophisticated production equipment, labor intensive methods are appropriate. For example, in China and the USSR, parasitoid insects are used on a large scale, most commonly egg parasitoids in the hymenopteran genus Trichogramma. Although production has been mechanized, in most areas hand labor is still used. Seasonally released insects, particularly Trichogramma sp., have been released on the scale of over 20 and 25 million acres in China and the USSR, respectively2. Mermithid nematodes and insect-pathogenic bacteria, fungi and viruses are also produced en masse in China. In the west, the labor costs of these methods are too high, and the lack of economically efficient methods may delay the commercialization of these organisms for many years to come. Notable exceptions to the problem of available mass production tech-

nology are the fermentation processes developed for the bacterium, Bacillus thuringiensis, and insect pathogenic steinernematid nematodes. B. thuringiensis has been commercially produced in fermenters for decades and is widely used for the control of a number of agriculturally important insect pests as well as the larvae of medically important mosquitoes and black flies. More recently, Biosys (Palo Alto, CA, USA) has developed a fermentation process to produce steinernematid nematodes and is using a 15 000 I reactor for commercial production. Storage. In an industry with such seasonal markets, a long shelf life is required for economic efficiency, and the poor storage properties of biological agents compared with chemicals are a major problem for all producers. For example, insectaries producing entomophages need to maintain colonies throughout the year even though there is only demand for the product during brief periods. Poor storage properties can be partially offset by the use of artificial diets, cold storage methods (including induced diapause) and overnight shipments by air. The shelf life at room temperature of baculoviruses and the infective stages of steinernematid nematodes is limited to