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New Vaccine Development: Establishing Priorities, Volume II, Diseases of Importance in Developing Countries Appendix D-10 The Prospects for Immunizing Against Plasmodium spp. DISEASE DESCRIPTION Malaria is a leading cause of death in the developing world. The pathogenic agents are four protozoan species of the genus Plasmodium. The disease is characterized by the destruction of erythrocytes and by a systemic inflammatory response resulting in chills, fevers, headache, and other manifestations. Malaria caused by Plasmodium falciparum, or “malignant malaria,” is fatal in a high proportion of cases if untreated, but responds well to appropriate chemotherapy. Although a variety of drugs are highly effective against malaria parasites, the organisms, especially P. falciparum, develop resistance to drugs in general use. For this reason, the control of malaria through chemotherapy requires continuous development of new drugs. A comprehensive review of recent research on malaria and its control has been prepared by the Special Programme for Research and Training in Tropical Diseases (SPRTTD, 1985). PATHOGEN DESCRIPTION The four protozoa that cause malaria in humans, P. falciparum, P. vivax, P. ovale, and P. malariae, have complex life cycles (Reisberg, 1980). Sporozoites are inoculated into humans by female anopheline mosquitoes during a blood meal. The sporozoites rapidly disappear from the circulation into liver parenchymal cells. There, each proliferates into thousands of individual merozoites. When mature, the merozoites rupture the hepatocytes and enter the circulation. Many are cleared from the circulation and destroyed, but the remainder attach to specific receptor sites on the red blood cell where they penetrate the red cell membrane and begin further development. The committee gratefully acknowledges the efforts of C.L.Diggs, who prepared major portions of this appendix. The committee assumes full responsibility for all judgments and assumptions.
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New Vaccine Development: Establishing Priorities, Volume II, Diseases of Importance in Developing Countries During the intraerythrocytic phase, the merozoite develops into a trophozoite (ring form), which then enlarges and begins to divide. Nuclear division initiates the schizont stage. Each schizont contains 6 to 24 merozoites, depending on the species. Proliferation of the merozoites leads to rupture of the erythrocyte, and the cycle begins again. A small number of the merozoites that enter red blood cells develop into male and female gametocytes. These do not rupture the red blood cells and require ingestion by the anopheline mosquito for further development. Fertilization of the gametocytes occurs within the stomach of the mosquito. The parasite then undergoes several additional changes that lead to the production of sporozoites. Their inoculation into a new human host starts the cycle again. In P. vivax and P. ovale, some sporozoites may remain dormant in hepatic cells for months or years; these are referred to as hypnozoites. When they do begin to proliferate, they may cause a relapse, often long after the primary infection. Such relapses do not occur with p. falciparum or P. malariae. The four species also differ in disease manifestations. HOST IMMUNE RESPONSE The immune response to malaria also is quite complex. Shortly after infection, antibodies that react with a wide variety of parasite antigens can be detected in the blood; however, this serologic response does not indicate a significant degree of immunity to subsequent infection. Reinfections usually are clinically less severe, as judged by the fever curve, although this effect is minor. individuals reexposed to infected mosquitoes have repeated episodes of malaria (Miller et al., 1984). Repeated infections eventually lead to a relative increase in immunity, however, which is made apparent by the profound difference between the clinical manifestations of malaria in young children and in adults. Young children have the most severe attacks, often resulting in death if untreated. By adulthood, most individuals who live in endemic areas have developed a degree of immunity such that reinfection results in a relatively mild disease. After repeated episodes of infection, some individuals may become almost entirely refractory to challenge with P. falciparum (Miller et al., 1984). Nevertheless, absolute immunity is difficult to establish; instead, a continuum appears to exist from high susceptibility to high resistance. Immunity to malaria is largely antibody-mediated. The classic studies by Cohen et al. (1961) in The Gambia indicated that antiparasitic activity could be transferred with IgG from donors in endemic areas, but not with control IgG from Europeans. The antiparasitic activity of immune serum also can be demonstrated in in vitro experiments (Chulay et al., 1981). The immune response to sporozoites, the stage of the parasite responsible for transmission of disease from mosquito to man, is of great interest. Antibodies to sporozoites can be found in individuals
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New Vaccine Development: Establishing Priorities, Volume II, Diseases of Importance in Developing Countries living in areas endemic for malaria (Nardin et al., 1979). It is possible that these antibodies may convey immunity to challenge, but this has not been well studied. The recent review by Miller et al. (1984) provides further detailed information on the immune response to malaria, as well as on prospects for vaccination. DISTRIBUTION OF DISEASE Geographic Distribution Malaria can exist in any climate suitable for the anopheles mosquito. Although the highest incidence of malaria is in the tropics, temperate zones are not immune. In the past, such areas as the southern United States had very high incidences of the disease. At present, the bulk of the malaria disease burden is in sub-Saharan Africa, South Asia, Oceania, and South America (Stürchler, 1984). Disease Burden Estimates It is difficult to obtain reliable information on the worldwide incidence of malaria. The World Health Organization is the major source of such information. Estimates over the past decade have been in the range of 100 to 300 million cases of malaria and 1 to 2 million malaria-related deaths annually (Lancet, 1975; Wyler, 1983). Sub-Saharan Africa is the largest endemic focus. Asia and Central and South America also have large areas where malaria is highly prevalent. It is estimated that 365 million people live in areas where malaria is highly endemic and where no specific antimalaria measures are used. Those living in areas where malaria is endemic but where some measures are used number an additional 2.217 billion. Thus, nearly half of the world’s population is at some risk (SPRTTD, 1985). Estimates of disease rates provided by Walsh (personal communication, 1985) have been used as a starting point for disease burden calculations. In the most highly endemic areas (without control), it is presumed that all infants (birth cohort about 11 million, presuming a crude birth rate of 32 per 1,000; see Chapter 4, Table 4.1) become infected in the first year of life. In other endemic areas with some control it is assumed that 20 percent of the birth cohort (about 90 million) becomes infected in the first year of life, that is, about 14 million, for a total of 25 million infant infections each year.* An * It is possible that less than one-fifth of children in moderately endemic areas contract the disease in the first year of life because the risk in some of these areas may be relatively low. It is also likely that the estimated birth cohort for heavily endemic areas may be low since the crude birth rate of 32 per 1,000 reflects the developing world average. Many of the heavily endemic areas are in
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New Vaccine Development: Establishing Priorities, Volume II, Diseases of Importance in Developing Countries additional 25 million cases are assumed to occur during the annual seasonal malaria epidemics that are common in India, Pakistan, the Middle East, southern China, and Central America. In addition, about 100 million reinfections are estimated to occur annually in heavily endemic regions, as immunity to the parasite wanes, for a total annual incidence of about 150 million cases. Seasonal epidemic and reinfection cases are divided into the four age groups used in the disease burden analysis according to the relative populations of these age groups (see Chapter 4 and Population Reference Bureau, 1984). Annual deaths in the under 5 years age group are assumed to be about 1 million, in line with an estimate by Gilles (1981). This yields a case fatality rate of 3.5 percent for this group. Although no data exist on which to base case fatality rates in older age groups, they have been estimated in such a manner as to reflect a decline in disease severity through middle age (5 to 14 years, 0.75 percent; 15 to 59 years, 0.25 percent), followed by an increase in older populations (60 years and over, 0.47 percent). These computations yield a total of 1.5 million deaths, or an overall case fatality rate of 1 percent. The assignment of cases to morbidity categories used in this analysis reflects the general pattern of increased severity in younger age groups (under 5 years, 10 percent in morbidity category A, 40 percent in category B, 50 percent in category C; 5 to 14 years, 25 percent in category A, 50 percent in category B, 25 percent in category C; 15 to 59 years, 50 percent in category A, 40 percent in category B, 10 percent in category C; 60 years and older, 50 percent in category A, 40 percent in category B, 10 percent in category C). Table D-10.1 shows the distribution of morbidity and mortality estimated to arise from malaria infections using the assumptions outlined above. PROBABLE VACCINE TARGET POPULATION The total population at risk of malaria is estimated to be 2.6 billion (SPRTTD, 1985). This includes 365 million persons in highly endemic regions and an additional 2.217 billion in areas where control measures have reduced the endemic level. Table D-10.2 shows the size of the birth cohort at risk of malaria based on the most recent available data for specific regions. For calculation of the potential benefits of each vaccine candidate, it is necessary to decide the likely target population. For the envisaged second generation vaccine, anticipated to be based on the circumsporozoite proteins of P. falciparum, P. vivax, P. ovale, and P. malariae, it is assumed that it eventually would routinely be delivered to the whole birth cohort at risk, that is, 78 million infants. sub-Saharan Africa, where the birth rate is much higher—45 per 1,000 in some areas. These two considerations would counterbalance each Other.
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New Vaccine Development: Establishing Priorities, Volume II, Diseases of Importance in Developing Countries TABLE D-10.1 Disease Burden: Malaria—All Under 5 Years 5–14 Years 15–59 Years 60 Years and Over Morbidity Category Description Condition Number of Cases Duration Number of Cases Duration Number of Cases Duration Number of Cases Duration A Moderate localized pain and/or mild systemic reaction, or impairment requiring minor change in normal activities, and associated with some restriction of work activity Chills, mild fever 2,850,000 3 8,812,500 2 38,250,000 2 4,750,000 3 B Moderate pain and/or moderate impairment requiring moderate change in normal activities, e.g., housebound or in bed, and associated with temporary loss of ability to work Fever 11,400,000 4 17,625,000 2 30,600,000 2 3,800,000 4 C Severe pain, severe short-term impairment, or hospitalization Severe fever, complications of malaria 14,250,000 6 8,812,500 4 7,650,000 3 950,000 5 D Mild chronic disability (not requiring hospitalization. institutionalization, or other major limitation of normal activity, and resulting in minor limitation of ability to work) n.a. n.a. n.a. n.a. E Moderate to severe chronic disability (requiring hospitalization, special care, or other major limitation of normal activity, and seriously restricting ability to work) n.a. n.a. n.a. n.a. F Total impairment n.a. n.a. n.a. n.a. G Reproductive impairment resulting in infertility n.a. n.a. n.a. n.a. H Death 1,000,000 n.a. 264,375 n.a. 191,250 n.a. 44,375 n.a.
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New Vaccine Development: Establishing Priorities, Volume II, Diseases of Importance in Developing Countries TABLE D-10.2 Derivation of Birth Cohort at Risk of Malaria, 1982 Region Population in Malarious Areas (millions) Birth Rate (per 100,000) Births/Year (millions) Africa (sub-Saharan) 350 45 16 Central America 53 31 2 South America 72 31 2 Asia (west of India) 154 30 5 Asia (mid-south) 794 30 24 East Asia and Oceania 973 30 29 Total 2,396 78 NOTE: Figures are the most recently available data for specific regions. SOURCE: World Health Organization (1984). For the envisaged first generation vaccine, anticipated to provide protection only against P. falciparum, there are reasons to believe that the target population would be smaller. Although it is estimated that 80 percent of malaria worldwide is caused by P. falciparum, there are regions where other strains are equally or more prevalent (World Health Organization, 1984). In these areas it is possible that a vaccine likely to protect against only one strain would not be used, especially if expensive. However, if a vaccine gave long-lasting protection (as assumed in the predictions on vaccine development), then it might be used despite these considerations since it would provide protection against the most serious form of the disease caused by P. falciparum It is therefore assumed that a P. falciparum vaccine would eventually be used routinely in the entire birth cohort of the at-risk population. This assumption may overestimate its potential health benefits since malaria may occur in areas where it may not be used, but it also overestimates the costs of vaccine where it may not be purchased. Immediately after licensure, both first and second generation vaccines are likely to be used in most of the other at-risk age groups. Both vaccines probably will be used by travelers and the military. However, these groups are small in number relative to the local population at risk. Assuming that a vaccine can be developed that induces immunity at an early age, it appears that a malaria vaccine could be introduced into the World Health Organization Expanded Program on Immunization. Such a vaccine would probably be readily adopted in endemic areas.
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New Vaccine Development: Establishing Priorities, Volume II, Diseases of Importance in Developing Countries Vaccine Preventable Illness* Because of its high burden of morbidity and mortality, P. falciparum malaria is now the focus of vaccine development. The first generation vaccine is likely to be limited to this strain; hence, it may only prevent that portion of the disease burden caused by P. falciparum. The SPRTTD (1985) estimates that 80 percent of all malaria cases worldwide, including most of the serious disease, are caused by P. falciparum. Estimates for the proportion of deaths due to P. falciparum are not available, but it is assumed in estimating the disease burden for P. falciparum (Table D-10.3) that it is responsible for nearly all mortality (99 percent) in all age groups. The relatively greater severity of P. falciparum compared to the other Plasmodium species also is reflected in the distribution of cases into each severity category; for each age group, 70 percent of cases in group A are assumed to be due to P. falciparum, 80 percent of cases in group B, and 95 percent of cases in group C. All of the disease burden represented in Table D-10.3 is potentially preventable with the first generation P. falciparum vaccine. These estimates yield a disease burden value of 2,082,083, which represents 0.9859 of the total burden of malaria as potentially preventable with the first generation P. falciparum vaccine. A second generation vaccine against P. falciparum, P. vivax, P. malariae, and P. ovale could potentially prevent all malaria in humans. SUITABILITY FOR VACCINE CONTROL Control of malaria is badly needed, and vaccination offers the most hopeful approach. Vaccination could be achieved before the peak of disease because it appears that immunity can be induced. Immunization has the potential for preventing disease without manipulation of the environment and, therefore, is simpler than mosquito control. In addition, it is hoped that vaccine-induced immunity will last for months or years; protection by drugs and/or vector control is measured in days after cessation of administration. These theoretical advantages make vaccination highly suitable for malaria control. Alternative Control Measures and Treatments Various measures have been shown to be effective against malaria, and all should be exploited fully for the foreseeable future, whether or not a vaccine becomes available (Bruce-Chwatt, In press). The * Vaccine preventable illness is defined as that portion of the disease burden that could be prevented by immunization of the entire target population (at the anticipated age of administration) with a hypothetical vaccine that is 100 percent effective (see Chapter 7).
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New Vaccine Development: Establishing Priorities, Volume II, Diseases of Importance in Developing Countries TABLE D-10.3 Disease Burden: P. falciparum Under 5 Years 5–14 Years 15–59 Years 60 Years and Over Morbidity Category Description Number of Cases Duration Number of Cases Duration Number of Cases Duration Number of Cases Duration A Moderate localized pain and/or mild systemic reaction, or impairment requiring minor change in normal activities, and associated with some restriction of work activity 1,995,000 3 6,168,750 2 26,775,000 2 3,325,000 3 B Moderate pain and/or moderate impairment requiring moderate change in normal activities, e.g., housebound or in bed, and associated with temporary loss of ability to work 9,120,000 4 14,100,000 2 24,480,000 2 3,040,000 4 C Severe pain, severe short-term impairment, or hospitalization 13,537,500 6 8,371,875 4 7,267,500 3 902,500 5 D Mild chronic disability (not requiring hospitalization, institutionalization, or other major limitation of normal activity, and resulting in minor limitation of ability to work) n.a. n.a. n.a. n.a. E Moderate to severe chronic disability (requiring hospitalization, special care, or other major limitation of normal activity, and seriously restricting ability to work) n.a. n.a. n.a. n.a. F Total impairment n.a. n.a. n.a. n.a. G Reproductive impairment resulting in infertility n.a. n.a. n.a. n.a. H Death 990,000 n.a. 261,731 n.a. 189,338 n.a. 43,931 n.a.
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New Vaccine Development: Establishing Priorities, Volume II, Diseases of Importance in Developing Countries first general category is vector control, which involves control of breeding sites and the use of insecticides to suppress vector populations. Efforts of this type have been successful in freeing limited areas from malaria, but the magnitude of the continuing effort required on a global scale would be prohibitive. The development of vector resistance to insecticides has forced vector control programs to use new and more expensive insecticides, which further complicates the effort. The second major alternative, which has been highly successful in some instances, is chemoprophylaxis and chemotherapy. The development of parasite resistance to drugs, however, renders this approach less than optimal (SPRTTD, 1985). Currently, for example, there is no adequate prophylactic drug for prevention of P. falciparum malaria in Southeast Asia. Resistance to chloroquine and Fansidar are widespread. Mefloquine is highly effective as a prophylactic agent, but because resistance to this drug already has occurred in Southeast Asia (SPRTTD, 1985), its use as a prophylactic agent would certainly accelerate widespread resistance to it. PROSPECTS FOR VACCINE DEVELOPMENT Interest in vaccination against malaria is strong, and knowledge is accumulating rapidly on various aspects of the associated problems (Miller, 1985; Norrby, 1985; SPRTTD, 1985). A comprehensive review of strategies for the development of antimalarial vaccines has recently been published (Ravetch et al., 1985). Therefore, this section will be restricted to an outline of approaches and problems. The prospects for vaccination against malaria were greatly aided by the development in the mid-1970s of methods for malaria culture in vitro (Traeger and Jensen, 1976). Modern immunologic and genetic engineering techniques have subsequently made possible various approaches to the problem of vaccination. Several vaccination strategies are being investigated. Vaccines are being targeted against the sporozoite stage, with the objective of blocking infection and eliminating subsequent disease (and hence transmission). Vaccines targeted at merozoites (blood stage) or their interaction with erythrocytes could result in reduced morbidity and transmission. Additionally, blocking the development of the sexual stages of the parasite could interrupt transmission but would not affect any individual’s illness. Work is in progress on all these possibilities (Ravetch et al., 1985). Work on immunization against sporozoites is the most advanced. Possibly, a combination of these approaches will be necessary to provide the most effective protection. Immunization with irradiated sporozoites provides protection against malaria in rodents (Nussenszweig et al., 1969). The surface of a sporozoite is largely composed of a single protein termed the circumsporozoite protein (Ravetch et al., 1985). The gene coding for the circumsporozoite protein of P. falciparum has been cloned, and the antigen has been expressed in E. coli (Dame et al., 1984). Knowing the
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New Vaccine Development: Establishing Priorities, Volume II, Diseases of Importance in Developing Countries exact structure of the protein has made possible the design of a wide variety of antigens that can be tested for their ability to induce protective immunity (Ravetch et al., 1985). These antigens are being used in three different ways: Synthetic peptides representing the relevant portions of the antigen molecule can be used in combination with a carrier of suitable molecular size in a semisynthetic vaccine (Miller, 1985; Nussenszweig et al., 1985). Recombinant DNA products (fusion proteins), including the relevant antigenic sequences, can be used in a vaccine (Young et al., 1985). Genes can be inserted into a microorganism, which then serves as the vehicle for immunization. An example of the third approach is the experimental use of vaccinia virus as a carrier for Plasmodium knowlesi circumsporozoite protein genes in the immunization of experimental animals (Smith et al., 1984). Phase one clinical trials of candidate vaccines using the first two approaches with P. falciparum are expected to begin in the near future. Unfortunately, the circumsporozoite protein may not be an optimal approach to vaccination against malaria. Sporozoites disappear rapidly from the circulation into hepatic cells, leaving little or no time for an anamnestic response after challenge. The escape of even a few sporozoites from the immune surveillance system could result in disease. For this reason, work continues on the development of vaccines based on the complex array of blood-stage and gametocyte antigens. Progress and problems associated with the development of vaccines against the asexual erythrocyte stages of the parasite have been described by Ravetch et al. (1985). Such efforts currently have two major thrusts: identification of merozoite antigens for testing as potential immunogens, and investigation of the possibility of interfering with the interaction of merozoites with erythrocytes. Although these approaches appear to have considerable promise, more basic research is needed to identify a particular approach that is most likely to yield success. Antigametocyte antibodies would prevent fertilization and development of the zygote in the stomach of the mosquito after a blood meal. A vaccine based on this approach would block transmission of the parasite but would not affect the clinical course of the disease in immunized persons; hence, it has been termed “altruistic.” Such vaccines might be used in combination and might slow the evolution of mutant parasites that escape destruction by developing new sporozoite/ merozoite antigens. Ravetch et al. (1985) also reviewed progress in this approach. In summary, three potentially viable approaches for the production of a malaria vaccine are currently being studied. The sporozoite approach, which theoretically applies to all four Plasmodium species, is most advanced. Probably, a vaccine based on these antigens will be ready to be fielded within the next 5 years.
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New Vaccine Development: Establishing Priorities, Volume II, Diseases of Importance in Developing Countries Problems Although various observations suggest that vaccination against malaria may be possible, a number of factors suggest that development of a vaccine conferring effective long-lasting immunity against all or most strains and species will be difficult. Recrudesent infections may last as long as 2 years for P. falciparum to 30 years for P. malariae. Natural immunity builds slowly, and sterile immunity is rarely achieved (Miller, 1985; Perrin et al., 1984). It may be possible, however, to raise the level of immunity (e.g., to sporozoite antigens) to a greater extent by vaccination since natural exposure to sporozoite before sequestration of the parasite in the liver is brief. In the case of antigenic fragments (e.g., from sporozoites), it may be possible to develop vaccines conferring long-lasting immunity by coupling fragments to carriers (e.g., toxoids), or by incorporating fragments into immunogenic bacterial fusion proteins. That protracted exposure to the natural disease is required for the development of immunity suggests that the parasite has evolved mechanisms for evading the human immune response. This may be particularly problematic in selecting merozoite antigens as candidate immunogens. Knowledge of parasite immune variation is incomplete, as is knowledge of the roles of humoral and cell-mediated responses in combatting the disease. These and other potential problems are discussed more fully by Ravetch et al. (1985). REFERENCES Bruce-Chwatt, L.J. In press. Malaria and selective primary health care. Rev. Infect. Dis. Chulay, J.D., M.Aikawa, C.Diggs, and J.D.Haynes. 1981. Inhibitory effects of immune monkey serum on synchronized Plasmodium falciparum cultures. Am. J. Trop. Med. Hyg. 30:12–19. Cohen, S., I.A.McGregor, and S.Carrington. 1961. Gamma-globulin and acquired immunity to human malaria. Nature 192:733–737. Dame, J.B., J.L.Williams, T.F.McCutchan, J.L.Weber, R.A.Wirtz, W.T.Hockmeyer, W.L.Maloy, J.D.Haynes, I.Schneider, D.Roberts, G.S.Sanders, E.P.Reddy, C.L.Diggs, and L.H.Miller. 1984. Structure of the gene in encoding the immunodominant surface antigen on the sporozoite of the human malaria parasite Plasmodium falciparum. Science 225:593–599. Gilles, H.M. 1981. Malaria. Brit. Med. J. 283:1382–1385. Lancet. 1975. Epitaph for global malaria eradication. Lancet II: 662. Miller, L.H. 1985. Research toward a malaria vaccine: A critical review. Pp. 1–11 in Vaccines 85. Molecular and Chemical Basis of Resistance to Parasitic, Bacterial, and Viral Diseases, R.A. Lerner, R.M.Chanock, and F.Brown, eds. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratories. Miller, L.H., P.H.David, and T.J.Hadley. 1984. Perspectives for malaria vaccination. Phil. Roy. Royal Soc. London 307:99–108.
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New Vaccine Development: Establishing Priorities, Volume II, Diseases of Importance in Developing Countries Nardin, E.H., R.S.Nussenzweig, I.A.McGregor, and J.H.Bryan. 1979. Antibodies to sporozoites: their frequent occurrence in individuals living in an area of hyperendemic malaria. Science 206:597–599. Norrby, E. 1985. Summary. Pp. 387–394 in Vaccines 85. Molecular and Chemical Basis of Resistance to Parasitic, Bacterial, and Viral Diseases, R.A.Lerner, R.M.Chanock, and F.Brown, eds. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratories. Nussenszweig, R.S., J.Vanderberg, and H.Most. 1969. Protective immunity produced by the injection of X-irradiated sporozoites of Plasmodium, berghei IV. Dose response, specificity and humoral antibody. Mil. Med. 134(suppl.):1176–1182. Nussenszweig, R.S., F.Zavala, and V.Nussensweig. 1985. The tandem repeats of the circumsporozoite (CS) protein as a basis for malaria vaccine development. Paper presented at the conference on Modern Approaches to Vaccines, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., Sept. 11–15, 1985. Perrin, L., A.Perez, and C.Chizzolini. 1984. Malaria, immunity, vaccination and immunodiagnosis. Experimentia 40:1343–1350. Population Reference Bureau. 1984. 1984 World Population Data Sheet. Washington, D.C.: Population Reference Bureau. Ravetch, J.V., J.Young, and G.Poste. 1985. Molecular genetic strategies for the development of anti-malarial vaccines. Biotechnology 3:729–740. Reisberg, B. 1980. Malaria. Pp. 707–716 in The Biologic and Clinical Basis of Infectious Diseases, G.P.Youmans, P.Y.Paterson, and H.M. Sommers, eds. Philadelphia: W.B.Saunders. Smith, G.L., G.N.Godson, V.Nussenszweig, R.S.Nussenszweig, J. Barnwell, and B.Moss. 1984. Expression of antigenic and iminunogenic Plasmodium knowlesi circumsporozoite protein by an infectious vaccinia virus recombinant. Science 224:397–399. SPRTTD (Special Programme for Research and Training in Tropical Diseases). 1985. Tropical Disease Research. Seventh Programme Report, 1 January 1983–31 December 1984. Geneva: World Health Organization. Stürchler, D. 1984. Malaria prophylaxis in travellers: The current position. Experientia 40:1357–1362. Traeger, W., and T.B.Jensen. 1976. Human malaria parasites in continuous culture. Science 193:673–675. Walsh, J. 1985. Personal communication, Harvard Medical School, Boston, Mass. World Health Organization. 1984. World malaria situation, 1982. Rapp. Trimest. Statist. Sanit. Mond. 37:130–161. Wyler, D.J. 1983. Malaria: Resurgence, resistance and research. N. Engl. J. Med. 308:875–878. Young, J.F., W.T.Hockmeyer, M.Gross, W.R.Ballou, R.A.Wirtz, J.H.Trosper, R.L.Beaudoin, M.R.Hollingdale, L.H.Miller, C.L. Diggs, and M.Rosenberg. 1985. Expression of Plasmodium falciparum circumsporozoite proteins in Escherichia coli for potential use in a human malaria vaccine. Science 228:958–962.
Representative terms from entire chapter: