389

Malaria vaccines- progress and problems Francis E. G. Cox Developing a vaccine against malaria is a major priority of the WHO. A decade of research exploiting the techniques of molecular biology has yielded a series of potentially protective vaccines. However, progress has been frustrated by the complexity of the parasite's life cycle and the antigenic diversity exhibited by each stage. Although candidate vaccines are now entering human trials, questions still arise concerningthe nature of a successful malaria vaccine and who will benefit from it. Throughout the tropics and subtropics, over 2000 million people are at risk from infection with malaria. O f these, between 200 and 400 million are infected and one to two million, mainly children, die each year. Traditional methods of control involve the use of insecticides against the mosquito vector and drugs for the prevention and cure of infection but these have merely contained the disease and have had little impact on its eradication. Even these control measures are now becoming less effective with the worldwide spread of insecticide-resistant mosquitoes and malaria parasites that are resistant to all the-~vailable antimalarial drugs. Most malariologists agree that a vaccine will have to be an essential component of future control-and-eradication schemes and consequently great effort has been put into developing one. However, the task has been much more difficult than had been envisaged and there are those who doubt whether there will ever be a really effective vaccine against malaria.

The parasite and its life cycle Human malaria is caused by four species of protozoan parasites belonging to the genus Piasmodium of which P. falciparum, is the most important (Box 1). These parasites are very host-specific and, although they can be cultured in vitro, they cannot be maintained in laboratory animals. An understanding of the mechanisms of host-parasite interaction is important for vaccine design, and several other species of Plasmodium that naturally infect monkeys and rodents have been used with some success as experimental models. The problem with such model host-parasite systems is that they can provide only indirect informarion about what might happen in a human infected with malaria, though several important leads have come from studies on owl monkeys (Aotus trivirgatus) F. E. G. Cox is at the Division of Biomolecular Sciences, King's College London, Campden Hill Road, London W8 7AH, UK. © 1991, Elsevier Science Publishers Ltd (UK) 0167 - 9430/91/$2.00

and squirrel monkeys (Saimiri sciureus), both of which can be experimentally infected with P. falcipamm. However, the number ofthe~e monkeys available for experimental purposes is limited and they are now used to a significant extent only in South America where they are native. It is therefore essential to study human immunity to malaria in the human hosts themselves and there is no doubt that immunity to malaria does occur in humans: people do recover from the disease, and children born to immune mothers acquire an effective level of protection. In general, immunity is slow to develop, incomplete (most individuals harbour low level infections for many years) and soon diminishes. The parasites that cause malaria are single-celled eukaryotic organisms ~;ith a complex life cycle comprising several stages, each of which is structurally, biochemicaUy and antigenically distinct (Fig. 1). Each stage has been studied in detail and ma,y of the antigens recognized by the human immune system have been identified and their genes cloned and sequenced t. The immunological responses involved in protection have been much harder to determine.

Box 1. Basic facts about malaria Malaria, caused by a protozoan parasite transmitted by female mosquitoes belonging to the genus Anopheles, is now largely confined to the tropics but can occur anywhere between 64°N and 32°S. Malaria is caused by four species of the genus Plasmodium: • P. falciparum causing malignant tertian malaria is the commonest speciesand,the most dangerous to humans; • P. vivax causing benign tertian malaria has the widest geographical range (it used to occur in Europe and North America) and is the second most dangerous; • P. ovale causes ovale tertian malaria in West Africa; • P. malariae causing quartan malaria has a patchy distribution and is not regarded as being particularly dangerous.

~BTECHNOVEMBER1991 (VOL9)

390

reviews

Figure 1 The life cycle of a malaria parasite. The infection begins when sporozoites (A) injected by a mosquito enter liver cells where multiplication occurs (B), resulting in the production of a number of merozoites (C}. These invade red blood cells (D) where they multiply (E) to produce more merozoites (F), which either repeat the cycle in the blood or develop into sexual forms (G) which are taken up by a mosquito. Fertilization occurs in the gut of the mosquito and this is followed by a further phase of multiplication and the production of sporozoites.

However, it is known that immunity is predominantly stage-specific and involves several mechanisms including antibodies, cell-mediated responses, cytokines and oxygen and nitrogen radicals, operating either sequentially or simultaneously 2. Unfortunately, these responses also contribute to the pathology of the disease 3. Vaccines will therefore have to be devised to generate a response against each individual stage, and an effective vaccine is likely to be a 'cocktail' of antigens, each designed to have a specific protective role while avoiding precipitating the disease.

Anti-sporozoite vaccines Malaria infection begins when sporozoites are injected by a mosquito directly into a blood vessel. The sporozoites circulate in the blood for a few minutes before entering tile liver where the first phase of multiplication occurs. Sporozoites are therefore/deal targets for immune attack, and irradiated sporozoites were the first vaccines tested in humans and laboratory animals with some success 4. Sporozoites are completely enveloped by a thick surface coat consisting of the circumsporozoite (CS) protein. The structure of the CS protein is basically similar in all species of Pla~modium, consisting of an ilmnunodominant central region, of several tandem repeats of a small number of amino acids, flanked by non-repeat regions 5. In P.faiciparum, the CS molecule consists of about IC' 10 7 molecules and the irmnunodominant TIBTECHNOVEMBER1~.?~ (VOL.%

region is composed of about 40 tandem repeats of a sequence of four amino acids (Asn-Ala-Asn-Pro; NANP); several vaccines based on these repeats have been developed. Two such vaccines have been used in experimental trials. In each case, the vaccinated volunteers who developed the highest antibody responses were subsequently infected. In one trial, a recombinant vaccine, R.32tet32 (Table 1), expressed in Escherichia coli, was given to six volunteers: one was protected, three experienced slightly delayed infection and two were not protecteff'. In an alternative approach, a synthetic molecule coupled to tetanus toxoid, (NANP)3-TT (Table 1), was used to vaccinate three volunteers, one of whom was protected and two experienced slightly delayed infectionT; in a second trial all four volunteers had delayed infections s. Overall, these results were disappointing; although CS protein elicited good antibody responses, there was only limited protection, a situation echoed by field studies 9. Subsequent vaccine-design studies have concentrated on incorporating non-repetitive regions of the CS molecule, or using various combinations of synthetic peptides based on the repetitive and the nonrepetitive regions 1°. Such vaccines are currently undergoing trials for safety and efficacy (as determined by the production of antibodies) in different parts of Africa and Asia but no immediate long-term vaccination trials are envisaged. Few malariologists now believe that a simple anti-sporozoite vaccine will be really effective and attention has turned to the next stage in the life cycle.

Vaccines against the liver stages It requires only a single sporozoite to evade the immune response to establish an infection: once it has reached the cells of the liver, a massive phase of multiplication begins resulting in the production of approximately 30 000 daughter individuals (merozoites) over the subsequent seven days. Until recently, it was believed that there was no immunity to the liver stages and that they were antigenically distinct from the other stages in the life cycle. It is now clear that although they possess antigens that are unique, they also possess some antigens that are shared with the sporozoite It and it is likely that some of the stagespecific antigens are actually derived from the CS molecule lz. The early stages in the liver are known to be susceptible to immune attack. In mice, an antibody-independent cell-mediated immune response is involvedt3: this utilizes cytotoxic lymphocytes that recognize malaria antigens on the surface of the infected liver cell and so destroy the cell. In humans, similar cytotoxic lymphocytes are found either as a result of long exposure to mosquito bites or after experimental immunization with irradiated sporozoites or components of the CS molecule 12. All the evidence points to a cytotoxic immune response which is spedfic, genetically restricted an mediated by CD8 + lymphocytes - the characteristics of immune responses more often associated with viral

391

reviews Table 1. Molecular structure of vaccines used in human malaria trials Vaccine

Descriptiona

Ref.

R32tet32

A recombinant antigen based on the circumsporite (CS) protein molecule MDP(NANPhsNVDP(NANP)is coupled to 32 amino acids encoded by the tetracycline-resistance gene expressed in E. coli

6

(NANP)3-TT

A synthetic molecule (based on the CS protein); (NANP)3 coupled to tetanus toxoid (TT)

7

SPf66

A mixture of asexual blood-stage antigens, largely composed of the polymerb CGDELEAETQNVYAAPNANPYSLFQKEKMVLPNANPPANKKNAGC

25

The one-letteraminoacid code is used. bThe sequencescommonto R32tet3aand (NANPh-TTare underlined.

a

and tumour immunology than with immunity to para- blood stage antigens, particularly MSA-1 (Ref. 17) sitic infections. It is possible that the limited success of and RESA 19, have been tested against P.faiciparum in the CS vaccines might be, in part, due to the inhi- owl monkeys, and have achieved encouraging bition of sporozoite invasion of liver cells but, as degrees of protection. A purified parasite-derived antibodies from individuals immunized with MSA-1 antigen gave good protection in owl mon(NANP)3-TT or Ik32tet32 do not block the invasion keys2t but results obtained with a recombinant form of human liver cells TM, it is likely that this kind of were less convincing 17. In contrast, initial trials with response is also cell-mediated. The World Health recombinant RESA antigens have produced high Organization (WHO) is currently investing a consid- levels of immunity in owl monkeys t9 but these have erable amount of effort investigating the possibility of not been repeated. Another group of antigens is secreted by the merodeveloping a vaccine against the liver stagesIs, though zoite as it invades the red blood cell. These are senone is yet being developed. creted from organelles called rhoptries and the antigem are collectively known as the rhoptry antigens 1. Vaccines against the blood stages Merozoites released from the liver immediately Their main function is to facilitate entry into the invade red blood cells within which they divide and blood cell: the aim of any immunization procedure multiply. In the case of P. falciparum, each merozoite based on this stage of the life cycle must be to block yields between 8 and 16 further merozoites every 48 this process. There is little evidence in natural infections that hours. These merozoites are released and invade further cells, and the cycle is repeated indefinitely. This immunity correlates with the production of specific results in all the symptoms of malaria, ranging from antibodies 22. This suggests that the immunodominant the fevers that coincide with the release of mero- repeat regions are not involved in protection but zoites, to anaemia and blockage of the brain capil- divert the immune response away from more relevant laries by infected cells. The blood stages, which are targetsz3. Research is therefore being directed towards very easy to obtain and can be grown in culture 16, non-repeat regions of antigens such as RESA. have been intensively studied and many of their anti- Currently, MSA-1, which does not have an extensive gens are well characterized. Potentially protective repeat region, is regarded as the best candidate peptide antigens have been identified on the surface vaccine for human trials. MSA-2 has not yet been of the merozoite, on the surface of the infected red thoroughly studied, but is thought to be an antigen cell and secreted by the parasites. Two merozoite sur- with considerable potential for use as a vaccine. Parallel with this systematic approach to a bloodface antigens, /v~_SA-1 (Ref. 17) and MSA-2 (Ref. 18), and an antigen that appears on the surface of stage vaccine, a Colombian biochemist, Manuel newly invaded red blood cells, the ring-infected ery- Patarroyo, has adopted a more pragmatic approach. throcyte surface antigen (mESA) 19, have been exten- He has developed a polymer (SPf66), consisting of sively investigated. As with the spor~zoites, many three synthetic peptides corresponding to parts of blood-stage antigens, particularly tLESA, are charac- three relatively uncharacterized native molecules terized by the possession of immunodominant from blood stages of P. faldparum together with the regions containing a number of tandem repeats of CS protein repeat NANP (Table 1). SPf66 induces short runs of amino acids. MSA-1, RESA and a num- strong antibody responses in owl monkeys and prober of other antigens have been extensively used (in tects some of them against P. falciparum24. In a pretheir native form or as synthetic or recombinant mol- liminary human trial, SPf66 was administered to five ecules) in experimental-animal immunization studies. volunteers: four developed only low level infection All have produced similar results: the production of on challenge with the parasite, and three of these subspecific antibodies and limited, but incomplete pro- sequently cleared their infection completely2s. This tection in some, but not all, test animals 2°. Several vaccine is now undergoing major field trials in South TIBTECHNOVEMBER1991 (VOL9)

392

reviews America involving up to 30 000 individuals. Few details are available but some scientists are cautious about adopting this vaccine: in a further series of experiments it was not possible to obtain the same results as those originally reported in owl monkeys26 and the quality controls used in the production of the vaccine did not initially satisfy the FDA. Batches of the vaccine are now being prepared under more rigorous conditions and it is expected that they will soon be tested for safety and efficacy in controlled trials in Asia and Africa as well as in South America.

blocks adhesion and this would be a major contribution towards combatting one of the more dangerous aspects of falciparum malaria.

Diversity and variation

One of the most interesting features of malaria parasites is their diversity, not only between stages but also between different isolates and within isolates32. Most parasites exhibit some degree ofpolymorphism, in the co-existence of different forms of the same antigen. For example, both MSP-1 and MSP-2 exist in two allelic forn~ 33,34 and some degree of polymorphism has been identified in most of the major antigens Vaccines against the sexual stages During the blood phase of infection, some mero- including the CS protein 3s. Diversity can arise in varizoites enter erythrocytes where they differentiate into ous ways including point mutations occurring during sexual stages that are taken up by a mosquito when it the blood stages, and unequal crossing over and feeds (Fig. 1). These sexual stages, initially found recombination occurring within the mosquito host32. inside red blood ceils, have to escape in order for fer- Much of what we know about diversity has been tilization to occur. Once they are liberated into the derived from observations on drug action. The paramosquito gut they are susceptible to attack from anti- site's ability to exhibit spontaneous changes in both its bodies taken up in the blood meal and fertilization is molecular structure and function has already created inhibited. In laboratory models, animals irmrmnized problems of drug resistance as parasites have adapted with well-characterized sexual-stage antigens mount to, or spontaneously mutated to forms that are able immune responses that effectively block fertilization to tolerate nearly all the standard anti-malarial drugs 36. in mosquitoes after subsequent blood feeds27. Since This problem has been compounded by recombiantibodies against sexual stages occur in human infec- nation occurring between resistant and susceptible tions, some investigators have advocated the use of a forms. There are obvious parallels with the molecules 'transmission blocking' vaccine 2s. There is no doubt involved in immunity. Immunity to malaria is that such a vaccine could reduce transmission, but largely species- and strain-specific and this can now be any vaccine that relies on the immunized person partially explained in terms of this diversi:~. Such becoming infected is unlikely to be acceptable unless diversity and adaptability could easily result in selecit forms part of another vaccine, for example one tion, by immune pressure, of forms capable of survivdesigned to prevent disease rather than infection. ing in immune or immunized hosts, thus frustrating any attempts at immunization. In the case of the CS Vaccines against disease protein, there is already some evidence that this can It is well recognized that many people with ma- happen3L In this context it must be assumed that the laria are not seriously ill, so a distinction can be made simpler the vacci~le, the more likely it will be that between infection and disease. As the mechanisms resistance will arise. involved in malaria disease and pathology become clear, a vaccine that permits some degree of infection What will the vaccine be like? but prevents disease may be envisaged. Two possible To be effective in the control and possible eradicandidate vaccines are currently being investigated. cation of the disease, as well as for the protection of One is directed against 'exoantigens' released from individuals, the vaccine win have to contain several infected red cells29 and the other against tumour components. For example, such a vaccine would necrosis factor (TNF), an effector molecule which, in need to contain two sporozoite antigens (one evokexcess, is associated with cerebral malaria 30. Both ing a cell invasion blocking antibody response and approaches have produced encouraging results in one capable of inducing a cell-mediated immune experimental animals but only anti-TNF has been response against the early stages of the parasite in the tried in humans with equivocal results (unpublished). liver), several antigens associated with the blood stages (including Pf66), a toxin-inactivating molecule and Cerebral malaria one or more sexual stage antigens. Such a 'cocktail An alternative approach is to try to prevent the vaccine' directed against several discrete target antisequestration of P. falciparum in the capillaries of the gens that are encoded by different genes might be able brain. This is a major complication of falciparum to overcome some of the problems of diversity and malaria and leads to cerebral malaria which is often variation. It is difficult to envisage a synthetic fatal. Considerable efforts are being made to identify molecule that has all these components but it might the molecules involved in this adhesion, and anti- be possible to design a recombinant one. However, in bodies specific for some of the endothelial receptors this context, it is important to note that, in geninvolved do inhibit cytoadherence 3t. When the para- eral, experimental recombinant vaccines against site-derived molecules or their receptors have been malaria have been less successful than those based on identified it should be possible to design a vaccine that native or synthetic peptides. TIBTECHNOVEMBER1991 (VOL9)

393

reviews Ideally, the vaccine should be given by mouth. One possibility is the use of attenuated Salmonella spp. 3s as vector for the genes encoding the various malarial components inserted, together with genes capable of stimulating appropriate immune regulatory mechanisms (see also Charles, I. and Dougan, G. TIBTECH 8, 117-121, 1990). Possible methods for producing large amounts of the vaccines have been well reviewed a9 and indicate that although there are problems still to be overcome, large-scale production is a feasible prospect. Is a malaria vaccine realistic? Progress towards developing a malaria vaccine is being made by several approaches. However, it is easy to forget that the science of subunit and recombinant vaccines is still in its infancy. The few acceptable vaccines available for other diseases are costly and have been developed only for relatively simple pathogens such as the Hepatitis B vires 4°, with none of the problems of antigenic complexity, diversity and random variation that is characteristic of malaria. The big question is whether a malaria vaccine will fulfil the criteria for vaccines in general, namely safety, efficacy, stability, ease of administration and cost. It could be argued that a partially effective vaccine would be acceptable, but the present cholera epidemic in South America should serve as some kind of warning agaimt this approach since a vaccine against cholera exists but it only confers protection in about 50% of cases and is effective for only 3-6 months. Naturally acquired immunity to malaria quiddy wanes, and it is likely that any vaccine will be of very restricted use because of the variation that exists between different geographical isohtes, and the almost inevitable spread of new variants. Various mathematical models have been produced to predict the possible efficacy of a malaria vaccine. These suggest that it would be necessary to vaccinate virtually everyone in a population with a vaccine that was completely effective for over half the life span of the population at risk in order to achieve eradication 41. Even with such an effective vaccine, cost is a major problem, particularly in those parts of the world where malaria is endemic. There are specific problems associated with vaccination programmes in the tropics. In particular, there is the need for a reliable 'cold chain' of refi'igeration facilities to ensure that the vaccines reach their target populations in good condition, in addition to the difficulties of assembling children for vaccination, and the universal problem of failure of individuals to return for booster treatments. Other practical aspects of this topic are discussed elsewhere 42. In the long term, countries in the tropics will probably have to produce their own vaccines as major international companies become increasingly reluctant to produce products with low profit margins and with a high probability of being sued for legal damages if anything goes wrong. This will invoh, e a major shi~ of technology to the developing world and will put additional

strains on already inadequate health budgets. In the short term, an expensive and short-lasting vaccine for limited use in particular situations could soon become • available, but its use would probably be restricted to tourists and military personnel. In the longer term conventional anti-malaria measures should not be abandoned in the hope that a perfect vaccine is on its w a y 43.

References 1 Anders, R. F., Smythe, J. A., Barzaga, N. G., Forsyth, K. P., Brown, H.J., Crewther, P. E., Thomas, L. M., Coppd, R. L., Culvenor, J. G. and Brown, G. V. (1989) in Neu, Strategies in Parasitology (McAdam, K. P. W. J., ed.), pp. 19-36, Churchill Livingstone 2 Good, M. F. (1990) Immunol. Left. 25, 1-10 3 Clark, 1. A. and Chaudhri, G. (1989) in Malaria: Host Responses to lnfeaion (Stevenson, M. M., ed.), pp. 127-146, CRC Press 4 Clyde, D. F. (1990) Bull. W. H. O. 68 (Suppl.), 9-12 5 Nussenzweig, V. and Nussenzweig, R. S. (1985) Cell 42, 401-403 6 Ballou, W. R., Sherwood, J. A., Neva, F. A., Gordon, D. M., Wirtz, R. A., Wasserman, G. F., Diggs, C. L., Hoffman, S. L., Hollingdale, M. K., Hockmeyer, W. T., Schneider, 1., Young, J. F., Reeve, P. and Chuhy, j. D. (1987) Lancet i, 1277-1281 7 Herrington, D. A.,,Clyde, D. F., Lasonsky, G., Cortesia, M., Murphy, J. R., Davis, J./Baqar, S., Felix, A. M., Heimer, E. P., Gillessen, D., Nardin, E., Nussenzweig, R. S., Nussenzweig, V., Hollingdale, M. R. and Levine, M. M. (1987) Nature 328, 257-259 8 Herrington, D. A., Clyde, D. F., Davis,J. R., Baqar, S., Murphy, J. R., Cortese,J. F., Bank, R. S., Nardin, E., DiJohn, D., Nussenzweig, R. S., Nussenzweig, V., Tortes, J. R., Murillo, J., Cortesia, M., SturcMer, D., Hollingdale, M. R. and Levine, M. M. (1990) BuU. IV. H. O. 68 (Suppl.), 33-37 9 Greenwood, B. M. (1990) Bull. W. H. O. 68 (Suppl.), 184-190 10 Sinigaglia, F. and Pink, J. R. L. (1990) Parasitol. Today 6, 17-19 11 Suhrbier, A. (1991) Parasitol. Today 7, 160-163 12 Malik, A., Egan, J. E., Houghten, K. A., Sadoff, J. C. and Hoffman, S. L. (1991) Doc. Nail Acad. Sd. USA 88, 3300-3304 13 Schofield, L. (1989) Exp. Parasitol. 68, 357-364 14 Hollingdale, M. R,, Appiah, A., Leland, P., Rosario, V. E., Mailer, P., Pied, S., Herrington, D. A., Chulay, J. D., Bailou, W. R., Derks, T., Yap, S. H., Beaudoin, R. L. and Verhave, J. P. (1990) Trans. R. Soc. Trop. Med. Hyg. 84, 325-329 15 World Health Organization (1990) Malaria Vacdne Developme, t: Pre-erglhrocytic Stages. Bulletin of the World Health Organization, 68 (Suppl.), WHO, Geneva 16 Trager, W. (1982) Brit. Med. B,dl. 38, 129-148 1"7 Holder, A. A., Freeman, R. B. and Nicholls, S. C. (1988) Parasite lmmunol. 10, 607-618 18 Marshall, V. M., Coppd, R. L., Martin, R. K., Oduola, A. M.J., Anders, R. F. and Kemp, D.J. (1991) Mol. Biochem. i~arasitol. 45, 349-352 19 Collim, W. E., Anders, R. F., Pappaioanou, M., Campbell, G. H., Brown, G. V., Kemp, D.J., Coppel, R. L., Skinner, J. C., Andrysiak, P. M., Favaloro, J. M., Corcoran, L. M., Broderson, J. R., Mitchell, G. F. and Campbell, C. C. (1986) Nature 333, 259-262 20 Lockyer, M. J. and Holder, A. A. (1989) in Varxination Strategies of Tropical Diseases (Liew, F. Y., ed.), pp. 123-148, CRC Press 21 Siddiqui, W. A., Tam, L. Q., Kramer, K.J., Hui, J. S. N., Case, S. E., Yamage, K. M., Chang, S. P., Chan, E. B. T. and Kan, S. (1987) Proc. Natl Acad. Sd. USA 84, 3014-3018 22 Marsh, K., Hayes, R. H., Carson, D. C., Otoo, L., Shenton, F., Byass, P., Zavala, F. and Greenwood, B. M. (1988) Tra~u. R. Soc. Trop. Med. Hyg. 82, 532-537 23 Schofield, L. (1991) Parasitol. Today 7, 99-105 TIBTECHNOVEMBER1991 (VOL9)

394

reviews 24 Kodriguez, P., Moreno, A., Guzman, F., Calvo, M. and Patarroyo, M. E. (1990) Am.J. Trop. Med. Hyg, 43, 339-354 25 Patarroyo, M. E., Amador, R., Clavijo, P., Moreno, A., Guzman, F., Romero, P., Tascon, P,., Franco, A., Murillo, L. A., Ponton, G. and Tmjillo, G. (1988) Nature 332, 158-161 26 Ruebush, T. K., Campbell, G. H., Moreno, A., Patarroyo, M. E. and Collins, W. E. (1990) Am. J. Trop. Med. Hyg. 43, 355--366 27 Mendis, K. N., David, P. H. and Carter, R. (1991) lnt.J, parasitoi. 20, 497-502 28 Kaslow, D. C. (1990) Immunol. Lett. 25, 83-86 29 Playfair, J. H. L., Taveme, J., Bate, C. A. W. and de Souza, J. B. (1990) lmmunol. Today 11, 25-27 30 Clark, I. A., Kockett, K. A. and Cowden, W. B. (1991) Parasitol. Today 7, 205-207 31 Udeinya, I.J., Miller, L. H., McGregor, I. A. and Jensen, J. B. (1983) Nature 303, 429-431 32 Kemp, D.J., Cowman, A, F. and Walliker, D. (1990) in Advances in Parasitology, Vol. 29 (Baker, J. R.. and Muller, K., eds), pp. 75-149, Academic Press 33 Tanabe, K., Mackay, M., Goman, M. and Scaife, J. G. (1987)

j. Mol. Biol. 195, 273-287 34 Fenton, B., Clark, J. T., Khan, C. M. A., Robinson, J. V., Walliker, D., llddley, R., Scaife, J. G. and McBride, J. S. (1991) Mol. Biochem. Parasitol. 11,963-971 35 McCutchan, T. F., Good, M. F. and Miller, L. H. (1989) Parasitol. Today 5, 143-146 36 Cruz, V. F. de la, Matoy, W. L., Miller, L. H., Lal, A. A., Good, M. F. and McCutchan, T. F. (1988) in Technological Advances in VaccineDevelopment (Lasky, L,, ed.), pp. 615-624, Alan R. Liss 37 Wellems, T. E. (1991) Parasitol. Today7, 110-112 38 Dougan, G. and Maskell, D. (1989) in Vaccination Strategies of Tropical Diseases (Liew, F. Y., ed.), pp. 47-64, C R C Press 39 Lasky, L. (ed.) (1988) TechnologicalAdvances in Vaccine Development, Alan R. Liss 40 Carrier, M. (1989) in Vaccination Strategies of Tropical Diseases (Liew, F. Y , ed.), pp. 11-29, CKC Press 41 Koella, J. C. (1991) Acta Tropica 49, 1-25 42 Lussow, A. K., Aguado, M. T., Del Guidice, G. and Lambert, P-H. (1990) Immunol. Lett. 25, 255-264 43 Targett, G. A. T. (ed.) (1991) Malaria- Waitingfor the Vaccine, John Wiley

Tailoring the microenvironment of enzymes in water-poor systems Bo Mattiasson and Patrick Adlercreutz Biocatalytic systems using enzymes in organic solvents open up the possibility of performing a whole range of reactions which would not normally occur under physiological conditions. The ability to perform reverse hydrolysis, or to convert substances relatively insoluble in aqueous environments on a scale of practical value in commercial applications are among those reactions for which water-poor systems are appropriate.

The majority of traditional studies of enzyme activity optimal biocatalysis. To a large extent, such efforts have involved enzymes in aqueous environments: have used an empirical approach, and only recently such studies have established that many environmen- has there been a greater attempt to establish general tal factors (including pH, ionic strength, water activ- rules for defining operating conditions. This is an esity and temperature) control enzyme activity. It sential step in facilitating design of new process conmight be expected that the same factors will also ditions, while avoiding time-consuming and costly contribute to determining enzyme behaviour in trial-and-error methods. organic solvents, thus necessitating close control of This review summarizes our current knowledge of these parameters in the enzyme's microenviron- ways to measure the microenvironment parameters, ment. to select suitable conditions and, moreover, to Much recent research in bioorganic synthesis has manipulate the microenvironment. Despite water been devoted to developing practical conditions for being present in only minute amounts, it plays an extremely important role (see below). Other B. Mattiasson and P. Adlercreutz are at the Department of Biotech- parameters which need to be considered in detail nology, Chemical Center, Lund University, PO Box 124, S-221 O0 are partition of substrate/product, temperature and Lined, Sweden. TIBTECHNOVEMBER1991 (VOL9)

pIq.

© 1991, Elsevier Science Publishers Ltd (UK) 0167 - 9430/91/$2.00