Circumsporozoite proteins of malaria parasites contain a single immunodominant region with two or more identical epitopes.

We have used panels of monoclonal antibodies to circumsporozoite (CS) proteins of Plasmodium falciparium, P. vivax, and P. knowlesi to determine the number of topographically independent epitopes of these antigens. The results of competition binding assays indicated that single regions of the CS molecules were recognized by the homologous monoclonal antibodies. Competition binding assays were also used to study the specificity of antibodies contained in the sera of humans and monkeys that had developed sterile immunity after immunization with irradiated, intact sporozoites. We found that single monoclonal antibodies inhibited 70-95% of the specific binding of the polyclonal antibodies to crude extracts of sporozoites. It appears, therefore, that CS proteins are among the most immunogenic constituents of sporozoites, and that a single region of these molecules contains most of the immunogenic activity. An additional finding was that the immunodominant region of CS molecules is multivalent with regard to the expression of a single epitope. This was demonstrated by the ability of monomers of CS proteins to bind simultaneously two or more molecules of the same monoclonal antibody.

Rationale for development of a synthetic vaccine against Plasmodium falciparum malaria.

Protective immunity against malaria can be obtained by vaccination with irradiated sporozoites. The protective antigens known as circumsporozoite (CS) proteins, are polypeptides that cover the surface membrane of the parasite. The CS proteins contain species-specific immunodominant epitopes formed by tandem repeated sequences of amino acids. Here it is shown that the dominant epitope of Plasmodium falciparum is contained in the synthetic dodecapeptide Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro-Asn-Ala-Pro or (NANP)3. Monoclonal antibodies and most or all polyclonal human antibodies to the sporozoites react with (NANP)3, and polyclonal antibodies raised against the synthetic peptide (NANP)3 react with the surface of the parasite and neutralize its infectivity. Since (NANP)3 repeats are present in CS proteins of P. falciparum from many parts of the world, this epitope is a logical target for vaccine development.

Identification of circumsporozoite proteins in individual malaria-infected mosquitoes by western blot analysis.

Circumsporozoite (CS) proteins of rodent (Plasmodium berghei), simian (P. knowlesi), and human (P. falciparum) malaria parasites extracted from dead and dried mosquitoes have been identified by the Western blot (immunoblot) technique. Dried mosquitoes which were laboratory-reared and infected with Plasmodium or freshly dissected sporozoites were triturated in sample reducing buffer and the extracts electrophoresed in a 10% SDS-polyacrylamide gel. After transferring the proteins to nitrocellulose, the molecular weights of both precursor and surface CS proteins of the plasmodial species were identified by probing with homologous monoclonal antibodies followed by 125I-labeled anti-mouse IgG. The molecular weights of the CS proteins extracted from dried mosquitoes were identical to those of CS proteins extracted from viable sporozoites. Immunoblot analysis is sensitive in that it can detect as few as 100 sporozoites. This technique is of epidemiological significance because it permits the identification of CS proteins of different plasmodial isolates extracted from individual, dried field-collected mosquitoes.

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.

Immuno-electron microscopic observations on Plasmodium knowlesi sporozoites: localization of protective antigen and its precursors.

Cryoultramicrotomy, an antisporozoite monoclonal antibody (2G3) and protein A-gold particles were used to determine the distribution of the protective surface antigen (Pk 42) of sporozoites of Plasmodium knowlesi and its precursors (Pk 52 and Pk 50). Gold particles bound uniformly to both the outer plasma and the inner pellicular membranes, indicating a uniform distribution of antigen on these membranes. Intracellular gold particles were frequently found to be associated with micronemes, but less frequently with rhoptries. Limited proteolysis of P. knowlesi sporozoites totally eliminated binding of label to the outer plasma membrane. In contrast, distribution of label on the inner pellicular membranes, rhoptries, and micronemes of trypsinized sporozoites was relatively unaltered. On the basis of the present and earlier data obtained upon metabolic labeling of these parasites, it is likely that the antigen associated with the intracellular organelles corresponds to the precursor polypeptides Pk 52 and Pk 50.

Sero-epidemiological studies of malaria in Indian tribes and monkeys of the Amazon Basin of Brazil.

A sero-epidemiological study of malaria, with special emphasis on Plasmodium brasilianum/P. malariae, was conducted on 4 Indian tribes living in the Amazon Basin of northern Brazil: the Arara, the Parakana, the Asurini, and the Metuktire. The incidence of malaria, as determined by blood films, was very low in all tribes. Parasitemia levels in most individuals were less than 0.02%; determination of the plasmodial species was not feasible. High levels of antibodies to both blood stages and sporozoites were detected for P. brasilianum/P. malariae, P. falciparum and P. vivax. The anti-sporozoite antibody response against all 3 plasmodial species was age related. All of the Metuktire adults and almost 90% of the Asurini adults had anti-sporozoite antibodies against P. brasilianum/P. malariae. The presence of P. brasilianum was confirmed in many of the indigenous monkeys by blood films and serology. This suggests that the monkeys, which are often kept as pets, serve as reservoir hosts. Anopheles darlingi mosquitoes, infected with P. brasilianum/P. malariae, were found in the study area.

Further studies on the antigenic diversity of the circumsporozoite proteins of the Plasmodium cynomolgi complex.

The circumsporozoite (CS) proteins of strains of the Plasmodium cynomolgi complex have been examined using antisporozoite monoclonal antibodies (Mab) in various immunologic assays. We found extensive antigenic diversity in the repeating immunodominant epitope of the CS proteins of the various strains. Based on the antigenicity and the electrophoretic mobility of their CS protein, the 11 strains that we examined can be placed in 7 distinct groups. Our data also indicate homology between the immunodominant repetitive epitopes of the CS proteins of the Berok strain of P. cynomolgi and the human malaria parasite P. vivax.

First field trial of an immunoradiometric assay for the detection of malaria sporozoites in mosquitoes.

An immunoradiometric assay (IRMA) using a monoclonal antibody to the major surface protein of Plasmodium falciparum sporozoites was used to assess the P. falciparum sporozoite rate in a West African population of Anopheles gambiae (s.1.). Unlike current dissection techniques, the IRMA could detect sporozoite antigen in dried as well as fresh mosquitoes. In a controlled comparison, the sensitivity of the IRMA was comparable that of the dissection technique. Additionally, the IRMA was species specific and quantitative. Sensitivity of the assay was sufficient to detect sporozoite infections resulting from the development of a single oocyst.

Potential vectors of malaria and their different susceptibility to Plasmodium falciparum and Plasmodium vivax in northern Brazil identified by immunoassay.

During the period from May 1983 to July 1985 we conducted an epidemiological study to determine potential vectors of malaria in 6 districts in the state of Pará in northern Brazil. The examination of random human blood smears, prepared at the time of mosquito capture, indicated overall human infection rates of 16.7% and 10.9% for Plasmodium falciparum and P. vivax, respectively. Two immunoassays, the immunoradiometric assay (IRMA) and the enzyme-linked immunosorbent assay (ELISA), based on the use of species-specific antisporozoite monoclonal antibodies, were used to analyze a total of 9,040 field-collected Anopheles mosquitoes for plasmodial infection. P. falciparum sporozoite antigen was detected in A. darlingi at rates varying from 2.7% to 4.2%, and in small numbers of A. oswaldoi collected in 1 of the districts. In contrast, sporozoite antigen of P. vivax was found in A. darlingi, A. triannulatus, A. nuneztovari, and A. albitarsis at rates ranging from 0.9% to 12.0%. By dissection, sporozoites were found in the salivary glands of these same 4 species at rates ranging from 0.8% to 2.2%. The latter 3 species had not previously been implicated as malaria vectors of any significance in northern Brazil.

Estimate of Plasmodium falciparum sporozoite content of Anopheles stephensi used to challenge human volunteers.

Plasmodium falciparum infected Anopheles stephensi, taken from a group of mosquitoes which had been used to challenge recipients of (NANP)3-TT vaccine, were tested for P. falciparum sporozoite content by an immunoradiometric assay. Seventy-six percent were infected with mean and median sporozoite equivalents per mosquito of 220,994 and 217,398, respectively (SD = 54,911). This sporozoite density is greater than that usually found in the field. These data suggest that this challenge for evaluating P. falciparum sporozoite vaccines is a demanding test of immunity.

Widespread reactivity of human sera with a variant repeat of the circumsporozoite protein of Plasmodium vivax.

A panel of Brazilian and Indian sera was screened for reactivity with a variant strain of Plasmodium vivax recently isolated in Thailand. This strain has been shown to have a unique repeat region which differs from the previously described P. vivax CS proteins. A total of 21/343 human sera were found to react with a synthetic peptide representing the variant P. vivax repeat. All of the sera that reacted with the variant repeat peptide, (ANGAGNQPG)4, also reacted with variant P. vivax sporozoites. Both the anti-peptide and the antisporozoite reactivity were totally abolished by adsorption with the variant peptide. Some of the human sera contained variant antibodies that were species specific and could only be adsorbed with the specific variant peptide. These findings suggest that the variant strain of P. vivax might have a worldwide distribution. We also found that some of the variant positive sera reacted with P. brasilianum sporozoites and with the P. brasilianum/P. malariae CS repeat. The adsorption of these sera with the P. brasilianum/P. malariae repeat peptide, (NAAG)4, significantly reduced the reactivity of these sera with the P. vivax variant. In addition, polyclonal and monoclonal antibodies of mice immunized with P. brasilianum sporozoites cross-reacted with the variant P. vivax CS. These findings suggest that exposure to P. brasilianum or P. malariae may give rise to sporozoite antibodies which cross-react with the P. vivax variant CS.

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.

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.

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.

Prevalence of antibodies to the repeat epitope of the circumsporozoite protein of Plasmodium vivax in San Luis Potosi, Mexico.

The prevalence of antibodies against the repeat epitope of the circumsporozoite protein (cs) of the standard (PV210) and variant (PVK247) strain of Plasmodium vivax was determined by ELISA in 1170 sera from individual residents of seven localities of the Region Huasteca of San Luis Potosi, Mexico. The capture antigens were the synthetic peptides DDAAD and (ANGAGNQPG) that correspond to the repeats of the PV210 and PVK247 cs proteins, respectively. Of the analyzed serum samples, 34.1% (400/1170) were positive with one or both of these antigens. Of the sera, 18.2% (214/1170) reacted with the DDAAD peptide and 6.6% (78/1170) were positive with the variant synthetic peptide. Additionally, 9.2% (108/1170) of the samples reacted with both peptides. A sample of 10% of positive sera for the variant cs repeat (18/78) was tested with the cs repeat peptide of P. malariae/P. brasilianum (NAAG); almost all of them (16/18, 89%) being positive. These results confirm that the transmission of the variant strain of P. vivax is a common phenomenon in endemic regions in Latin America, as well as in other tropical regions of the world. These findings may have implications for the development of aP. vivax vaccine since that based on the standard cs repeat only would not be universally protective.

Immunological relationship of Plasmodium inui with two other quartan malaria parasites, P. malariae and P. brasilianum.

A series of monoclonal antibodies was produced against sporozoites of the OS strain of Plasmodium inui, a simian quartan malaria parasite, and used to characterize the circumsporozoite protein of this parasite. The results confirm that the immunodominant epitope of the circumsporozoite protein of P. inui is immunologically distinct from those of 2 other quartan parasites, the human P. malariae and simian P. brasilianum, which are identical.

Characterization of cross-reactive blood-stage antigens of the Plasmodium cynomolgi complex using anti-Plasmodium vivax monoclonal antibodies.

Five out of 18 monoclonal antibodies (moAB's) produced against blood stages of a Brazilian (Belem) strain of Plasmodium vivax were shown to cross-react with all of the 11 strains of the P. cynomolgi complex that were assayed. The 5 moAB's produced 3 different patterns of immunofluorescence, identical for both P. vivax and P. cynomolgi. Three of these moAB's appeared to react with antigens associated with the cytoplasm or membranes of infected erythrocytes. By Western blot analysis, 2 of these 3 moAB's identified an antigen with an apparent molecular weight of 31 kDa in extracts of parasitized erythrocytes of both species; the third of these moAB's reacted with an antigen with an apparent molecular weight of 95 kDa. By immunofluorescence, the 2 other moAB's reacted only with parasites at all developmental stages. The target antigen of these 2 moAB's was not identified. Immunoradiometric assays indicated that the moAB's are directed against 3 or possibly 4 distinct nonrepetitive epitopes. None of the moAB's inhibited merozoite invasion or growth of the parasites in an in vitro culture system of the Berok strain of P. cynomolgi.

Inhibition of phagosome-lysosome fusion in macrophages by soluble extracts of virulent Brucella abortus.

Water extracts of virulent Brucella abortus were able to inhibit phagosome-lysosome fusion in unelicited murine peritoneal macrophages following ingestion of yeast. Extracts from an avirulent strain were unable to produce a similar effect. Lipopolysaccharide from B abortus did not appear to be involved with the ability of the extracts to inhibit fusion.

Role of Anopheles culicifacies s.l. and An. pulcherrimus in malaria transmission in Ghassreghand (Baluchistan), Iran.

A 2-site immunoradiometric assay (IRMA) was performed on the head and thorax of Anopheles culicifacies s.l. and An. pulcherrimus females, the 2 most common anopheline species in the District of Ghassreghand (Baluchistan, Iran), collected during the 2 peak malaria transmission seasons (May and September-October 1991). Positive IRMA results revealed the 2 species as potential vectors of malaria in this highly endemic district. This finding serves as the first report on natural infection of An. pulcherrimus in Iran and is the second on natural infection of An. culicifacies since the previous report of 1959.