Alan H. Cochrane

Cloning and expression in E. coli of the malarial sporozoite surface antigen gene from Plasmodium knowlesi.

The malarial sporozoite, the infective stage found in the salivary gland of the insect vector, bears highly immunogenic surface antigen(s). Repeated exposure to irradiated sporozoites induces protection against malaria in several host species, including man1. Further, monoclonal antibodies that confer passive immunity react with the immunogenic surface determinants of different sporozoite species2–4. One approach to prevent malaria, therefore, would be to produce a vaccine that induces high titres of circulating antibodies against the sporozoite surface determinant(s). However, production of such a vaccine has not been possible since sporozoites cannot be cultivated in vitro and, therefore, only limited amounts of surface antigen may be obtained. To overcome this problem, we have prepared mRNA from Plasmodium knowlesi-infected mosquitoes to construct a cDNA library. From this library we have isolated a clone that expresses the sporozoite surface antigen as a β-lactamase fusion protein in the plasmid pBR322. This is the first potentially protective malarial antigen to be cloned by recombinant DNA technology.

The circumsporozoite gene of the plasmodium cynomolgi complex

An analysis of the circumsporozoite (CS) genes of six closely related plasmodia is presented. Like other plasmodial antigens, the CS protein contains tandem repeats flanked by conventional nonrepeated sequences. Our analysis shows that the repeats, which encode the immunodominant epitope of the CS protein, diverge more rapidly than the remainder of the gene, and that the maintenance and evolution of the repeats cannot be explained as the result of selection at the protein level. We argue that a mechanism acts directly on the DNA sequence to constrain the internal divergence of the repeats, and as a result promotes their rapid divergence between taxa.

Anophelines in the state of Acre, Brazil, infected with Plasmodium falciparum, P. vivax, the variant P. vivax VK247 and P. malariae.

Anophelines collected indoors and in the peri-domiciliary area in 3 localities in the Amazon region, state of Acre, Brazil, from August 1990 to January 1991 were examined by enzyme-linked immunosorbent assay (ELISA) using specific monoclonal antibodies directed against the repeats of the circumsporozoite proteins of Plasmodium falciparum, P. vivax, P. vivax V247, and P. malariae. Of the 3056 specimens collected, 2610 were Anopheles oswaldoi, 362 A. deaneorum, 60 A. triannulatus and 24 were A. darlingi. The infection rates of A. oswaldoi were 3.41% for P. falciparum, 2.26% for P. vivax, 1.22 for P. vivax VK247, and 0.42% for P. malariae. For A. deaneorum, the infection rates were 2.76% for P. falciparum, 0.55% for P. vivax, and 0.82% for P. vivax VK247. All samples of the other 2 species collected (A. triannulatus and A. darlingi) were negative in the ELISA. There were certain differences in the anopheline distribution and infection rates between these localities, and in one only A. oswaldoi was found to be infected. These results strongly point to A. oswaldoi as the main malaria vector in the region. No difference was found between the potential vectors of P. vivax and P. vivax VK247. The significance of these findings for malaria control is discussed.

Comparative studies on the immunogenicity of infective and attenuated sporozoites of Plasmodium berghei

Abstract Experiments to characterize the immune response induced by the inoculation of infective sporozoites were undertaken in an experimental rodent malaria system. Since earlier work had shown that the blood phase of the infection suppresses the humoral anti-sporozoite response, the development of parasitaemia was completely prevented by treating the animals with either chloroquine or primaquine shortly after sporozoite inoculation. The results obtained by immunization with infective Plasmodium berghei sporozoites were compared with those obtained using γ-irradiated sporozoites, by measuring the antibody levels of the immunized mice and determining their degree of protection against challenge. The level of protection in animals immunized with infective sporozoites and submitted to chloroquine treatment was found to be comparable to that induced with irradiated sporozoites. Under both conditions, a single intravenous inoculation of 10 4 sporozoites protected approximately 50% of the immunized mice from challenge with infective sporozoites. When two immunized doses were given, over 90% of the animals from both groups were protected. Immunization of primaquine-treated animals with infective sporozoites resulted in a somewhat lower level of protection. The antibody response to inoculation of infective sporozoites was either comparable to or somewhat greater than that induced by irradiated sporozoites. In vitro incubation of sporozoites with a high concentration of chloroquine abolished their infectivity. These chloroquine-attenuated sporozoites, however, were immunogenic and induced a protective immune response.

Susceptibility of Anopheles culicifacies species A and B to Plasmodium vivax and Plasmodium falciparum as determined by immunoradiometric assay

We have used a two-site immunoradiometric assay and species-specific antisporozoite monoclonal antibodies to determine the relative roles that sibling species A and B of the Anopheles culicifacies complex play in malaria transmission in western Uttar Pradesh, India. The results unequivocally establish species A as the primary vector of both Plasmodium vivax and P. falciparum in this area. Our results indicate active transmission of P. vivax from May to October and of P. falciparum from August to December. The identification of species A as the primary malaria vector in northern India will now allow suitable malaria control strategies to be designed.

A study on the pathogenesis of human cerebral malaria and cerebral babesiosis

Cerebral complications are important, but poorly understood pathological features of infections caused by some species of Plasmodium and Babesia. Patients dying from P. falciparum were classified as cerebral or non-cerebral cases according to the cerebral malaria coma scale. Light microscopy revealed that cerebral microvessels of cerebral malaria patients were filled with a mixture of parasitized and unparasitized erythrocytes, with 94% of the vessels showing parasitized red blood cell (PRBC) sequestration. Some degree of PRBC sequestration was also found in non-cerebral malaria patients, but the percentage of microvessels with sequestered PRBC was only 13%. Electron microscopy demonstrated knobs on the membrane of PRBC that formed focal junctions with the capillary endothelium. A number of host cell molecules such as CD36, thrombospondin (TSP) and intercellular adhesion molecule I (ICAM-1) may function as endothelial cell surface receptors for P. falciparum-infected erythrocytes. Affinity labeling of CD36 and TSP to the PRBC surface showed these molecules specifically bind to the knobs. Babesia bovis infected erythrocytes produce projections of the erythrocyte membrane that are similar to knobs. When brain tissue from B. bovis-infected cattle was examined, cerebral capillaries were packed with PRBC. Infected erythrocytes formed focal attachments with cerebral endothelial cells at the site of these knob-like projections. These findings indicate that cerebral pathology caused by B. bovis is similar to human cerebral malaria. A search for cytoadherence proteins in the endothelial cells of cattle may lead to a better understanding of the pathogenesis of cerebral babesiosis.

A circumsporozoite-like protein is present in micronemes of mature blood stages of malaria parasites

We demonstrate for the first time the presence of a circumsporozoite (CS)-like protein in invasive blood stages of malaria parasites. Immunogold electron microscopy using antisporozoite monoclonal antibodies localized these antigens in the micronemes of merozoites. Western immunoblot and two-dimensional gel electrophoresis of mature blood stage extracts of Plasmodium falciparum, P. berghei, P. cynomolgi, and P. brasilianum identified polypeptides having the same apparent molecular mass and isoelectric points as the corresponding sporozoite (CS) proteins. The CS-like protein of merozoites is present in relatively minor amounts, compared to the CS protein of sporozoites. Mice with long-term P. berghei blood-induced infections develop antibodies which react with sporozoites.

Prevalence and level of antibodies to the circumsporozoite proteins of human malaria parasites, including a variant of Plasmodium vivax, in the population of two epidemiologically distinct areas in the state of Acre, Brazil.

A seroepidemiological study of the prevalence of antibodies against the repeating epitopes of circumsporozoite (CS) proteins of human malaria parasites was conducted in 2 different areas in the state of Acre, Brazil in 1987 and 1990. In 1987 antibodies against the CS protein of the VK 247 variant Plasmodium vivax as well as antibodies against the CS proteins of P. falciparum and the classic P. vivax were found at relatively high rates in the 2 areas, but significant microepidemiological differences were observed. In 1990, when large scale migration in Amazonia had ceased and control measures were applied in the study areas, the malaria endemicity decreased, as determined by the declining prevalence of anti-sporozoite antibodies against all Plasmodium species, and the small number of individuals with positive blood smears. Antibodies against sporozoites of the variant P. vivax did not cross-react with the CS proteins of the classic P. vivax, nor with antibodies against sporozoites of P. falciparum and P. malariae. Sera containing antibodies against the CS protein of P. malariae were found at a very low frequency, and only in 1987. The anti-CS protein antibody response to all Plasmodium species was age-related.

Circumsporozoite protein of Plasmodium gallinaceum characterized by monoclonal antibodies

Summary Monoclonal antibodies (MoAb) were produced against both salivary gland sporozoites (SGS) and oocyst sporozoites (OS) of Plasmodium gallinaceum, an avian malaria parasite. By indirect immunofluorescence, all of the MoAbs reacted with both SGS and OS of P. gallinaceum and two of the MoAbs cross‐reacted weakly with P. berghei sporozoites. None of the MoAbs reacted with sporozoites of six additional species of mammalian plasmodia. In Western blot analysis of extracts of either SGS or OS of P. gallinaceum, these MoAbs identified two polypeptides with molecular weights of approximately 76000 and 64000 D. The results of a MoAb inhibition of binding assay and a two‐site one‐antibody immunoradiometric assay indicate that the circumsporozoite protein of P. gallinaceum, like those of mammalian malaria parasites, contains a repetitive immunodominant epitope. Two of the anti‐P. gallinaceum MoAbs were tested in a sporozoite neutralization assay and decreased, but did not abolish, the infectivity of sporozoites for chickens, indicating that the polypeptide of P. gallinaceum identified by immunoblot is probably the protective antigen.

Antigenic diversity of the circumsporozoite proteins in the Plasmodium cynomolgi complex

Antigenic diversity was observed in the circumsporozoite (CS) proteins of five of the six Plasmodium cynomolgi isolates (NIH, Mulligan, London, Gombak, Ceylon, RO) that we examined. Monoclonal antibodies were produced against salivary gland sporozoites of three of the isolates. Interaction of these monoclonal antibodies with the sporozoites was isolate specific, the exception being the anti-NIH monoclonals which also reacted with Mulligan strain sporozoites. Inhibition of binding between the different monoclonal antibodies indicated that for each of the NIH, London, and Gombak strains, the homologous monoclonals were recognizing the same or a topographically close immunodominant epitope on the respective CS protein. Also the binding of a polyvalent anti-NIH rhesus serum to the homologous antigen could only be inhibited by anti-NIH monoclonal antibody. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of sporozoite extracts demonstrated clear differences in the apparent molecular weights of the CS proteins of four of the six isolates. This is the first study which provides evidence of antigenic diversity in the CS proteins of different isolates of a primate plasmodial species.