Recombinant proteins and synthetic peptides as malaria vaccine candidates.

Introduction Malaria is a severe, very widespread disease, particularly in third-world countries. Approximately 200 million people develop this infection yearly, with a high toll of morbidity and considerable mortality, particularly in young children. In my discussion of recombinant and peptide vaccines for malaria, I will raise some general issues, which I believe apply to the development of molecular vaccines in general. ~ i r s i , however, I would like to review the rationale for the development of a vaccine as a control measure for malaria. The progression of malaria in Brazil, from 50 000 diagnosed cases in 1971 to more than 500 000 cases in 1985, just in the state of Amazonas, reflects the situation of malaria in many developing areas of the world, namely, that the incidence of malaria is increasing greatly, in recent years, in spite of ongoing control measures, such a the use of insecticides and drug treatment. On the basis of the particular situation in Brazil, I will discuss the reasons for this recent increase in malaria also in other tropical areas. Malaria has been eliminated from most areas of Brazil, except for the Amazon Region, where more than 95% of the current cases originate. The difficulty of controlling the disease in this area is due to the enormous extent of this region, and also to the fact that a large proportion of individuals who live in that area live in huts or tents. not in houses. Since their habitations frequently have no walls, there is no possibility of spraying insecticides, even though the mosquitoes in this area are still susceptible to dichlorodiphenyltrichloroethane (DDT) and other residual insecticides. A similar situation also occurs in many areas of Africa and Asia. The second circumstance that also frustrates the attempts to control this disease is that in many of these regions there is an increasing level of resistance of the parasites to chloroquine, the best and least expensive antimalarial drug currently available. Frequently, in fact, the parasites in this area display multiple drug resistance, so that the physicians dispose of only few, very expensive alternatives for the treatment of their malaria-infected paiients. Based on the rather complex life cycle of malaria parasites, there are many potential targets for vaccine development. We chose to use sporozoites, the infective stage of the malaria parasite, as the target for our research. Since malaria is transmitted by the bite of infected mosquitoes, which during their blood meal inject sporozoites, we considered it important to induce sporozoite-neutralizing antibodies. These antibodies would abolish the infectivity of the sporozoites and prevent their development in the liver, where the parasites differentiate and reproduce very rapidly. This would also prevent the appearance of blood stages, responsible for the symptomatology of this disease. The remainder of my presentation will be focused exclusively on antisporozoite immunity, the main target of our laboratories' research effort. Initially we immunized experimental animals with attenuated parasites, obtained from the salivary glands of infected mosquitoes. For this purpose we used laboratory-bred mosquitoes fed on animals infected with various malarial species. Approximately 2 weeks after the infective blood meal, the sporozoites, i. e., needle-shaped, elongated unicellular parasites, migrate to the mosquito salivary glands. When such an infected mosquito bites, it takes a blood meal, and simultaneously introduces the parasites into the host, transmitting the disease. We first used a classical approach to immunization, namely, radiation attenuated parasites. Irradiated sporozoites are no longer able to reproduce, nor to cause the disease, although they are still alive, motile, and capable of invading liver cells. Mice were immunized by the repeated intravenous injection with irradiated sporozoites, and we found that this provides very effective protection. In fact, the sporozoites contained in the salivary glands of a single mosquito are sufficient to immunize and totally protect a mouse against an otherwise lethal malaria infection (Nussenzweig et al. 1967). This immunity is stage specific, which indicates that the protective antigen(s) vary from one stage of development of the parasite to the next. In fact when we bypassed the mosquito stage and challenged the sporozoite-immunized protected mice with blood stages, these mice developed a normal course of infection, i.e., they were not protected against blood-induced infection (Nussenzweig et al. 1969). The immunity is also specific for each of the malarial species. However, and this is a very important property for a vaccine, the immunity is not limited to the parasite strain or isolate used for the immunization. This was clearly shown in experiments done by Dr. David Clyde and collaborators (1975), at the University of Maryland, in which he immunized and protected a small number of human volunteers by the bite of malaria-infected irradiated mosquitoes. This unquestionably is the most original mode of immunization ever employed, namely, to use infected irradiated mosquitoes to deliver a vaccine. Under these conditions, it was found that those individuals who were protected against Plasmodium falciparum were not only protected against sporozoites of the isolate used for the immunization but were also protected against challenge with P . falciparum from various other geographic regions. The same was also true for the immunization with Plasmodium v i v a , an equally prevalent human malaria parasite (Clyde 1975). Possibly the most important finding in all these early experiments was that the antibodies, present in the serum of the protected animals and in man, were directed against the surface of the sporozoites. This indicated that the important, protective antigen(s) of this parasite stage was localized on the sporozoite surface. In fact when sporozoites were incubated with immune serum or purified antibody, we detected by electron microscopy a deposition of immunoglobulin all around the parasite. This indicated that the corresponding antigen is distributed over the entire sporozoite surface. We called it the circumsporozoite (CS) antigen (Cochrane et al. 1976).