DNA cloning of Plasmodium falciparum circumsporozoite gene: amino acid sequence of repetitive epitope.

A clone of complementary DNA encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum has been isolated by screening an Escherichia coli complementary DNA library with a monoclonal antibody to the CS protein. The DNA sequence of the complementary DNA insert encodes a four-amino acid sequence: proline-asparagine-alanine-asparagine, tandemly repeated 23 times. The CS beta-lactamase fusion protein specifically binds monoclonal antibodies to the CS protein and inhibits the binding of these antibodies to native Plasmodium falciparum CS protein. These findings provide a basis for the development of a vaccine against Plasmodium falciparum malaria.

Cloning and characterization of a novel Plasmodium falciparum sporozoite surface antigen, STARP.

A novel Plasmodium falciparum sporozoite antigen, STARP (Sporozoite Threonine and Asparagine-Rich Protein), detected consistently on the surface of sporozoites obtained from laboratory strains and field isolates, has been identified and cloned, following a systematic approach aimed at isolating novel non-CS sporozoite surface antigens. The 2.0-kb STARP gene has a 5' miniexon/large central exon structure and contains a complex repetitive region encoding multiple dispersed motifs and tandem 45- and 10-amino acid repeats. In sporozoites, transcription of the STARP gene has been conclusively demonstrated by reverse PCR and Northern blot hybridisation and the 78-kDa protein has been localized by immunofluorescence and immunoelectron microscopy to the sporozoite surface. STARP is also expressed in liver stages, as revealed by immunofluorescence assays using antisera raised either to the central repetitive region or the C-terminal non-repetitive region. Expression is also detected in early ring stages, though not in mature erythrocytic or sexual stages. Identification and elucidation of this novel antigen is a step forward in current efforts aimed at developing an effective preerythrocytic-stage malaria vaccine.

Antibodies against malaria sporozoites in patients with acute uncomplicated malaria and patients with cerebral malaria.

Serum samples from 95 patients with acute uncomplicated falciparum malaria (AM) and 95 patients with cerebral malaria (CM) were tested by the indirect immunofluorescent assay (IFA) for IgG and IgM antibodies against Plasmodium falciparum and P. vivax sporozoites. Forty-six (48%) CM patients were positive for antibodies against P. falciparum sporozoites whereas only 23 (24%) were positive for antibodies against P. vivax sporozoites (P less than 0.002). A similar result was obtained in AM patients. However, CM patients had significantly lower mean IgG anti-sporozoite titer for P. falciparum than did AM patients (P less than 0.05), especially when only anti-sporozoite antibody-positive CM and AM patients were compared (P less than 0.0005), suggesting that CM patients had relatively less exposure and were probably less immune to malaria than were AM patients. The persistence of anti-sporozoite antibodies also was investigated in paired sera taken 63 days apart from 108 patients with acute falciparum malaria. There were significant decreases in the mean antibody titers in the follow-up sera during the period of stay in the malaria-free area. It was proposed that determination of anti-sporozoite antibody be made as a substitute for, or in addition to, anti-blood stage antibody for seroepidemiological study of malaria, especially in the monitoring of the success of the malaria control program.

Anti-sporozoite antibodies induced by natural infection.

Serum samples from 120 individuals living in a malaria-endemic area, 31 patients with Plasmodium falciparum infection, and 58 healthy blood donors were tested for antibodies against P. falciparum and P. vivax sporozoites. Specific antibodies were determined by the circumsporozoite precipitation (CSP) reaction and indirect immunofluorescent (IFA) tests for IgG and IgM antibodies. It was found that a high proportion of adults living in the endemic area had IFA anti-sporozoite antibodies, usually IgG. Children and healthy donors were either negative or had low antibody titers. A positive correlation was found between IgG antibody titers against P. falciparum sporozoites and those against P. vivax sporozoites. CSP reactivity was demonstrated in 5 of 31 sera from patients with falciparum malaria, and was always associated with a high level of IFA antibodies. The anti-sporozoite antibodies were found to be stage- and species-specific.

Investigation of malaria sporozoites by immunoradiometric assay.

Two-site immunoradiometric assay (IRMA) (Zavala et al., 1982) using monoclonal antibodies to P. falciparum and P. vivax was applied to detect sporozoites in laboratory-maintained An. dirus and also mosquitoes collected from endemic areas of malaria in Thailand. Study in P. falciparum infected mosquitoes revealed that the circumsporozoite (CS) antigen was first found in the abdominal portion on day 10 post-infection, while it could be observed in the salivary glands from day 15 onwards. The head-thorax portion of wild-caught mosquitoes were investigated by IRMA compared with the dissection technique. The results showed that none of the mosquitoes collected from Phrae was positive for malaria. The mosquitoes collected from Chantaburi showed 4 out of 1243 An. dirus that were positive for P. falciparum by IRMA, with sporozoites ranging from 207 to 3875. Among 3123 An. minimus collected from Kanchanaburi, 3 were positive by IRMA, 2 for P. falciparum and one P. vivax with sporozoites found in head-thorax portion were 1880, 2380 and 1026 respectively. Not a single sporozoite was found in the mosquitoes collected from these areas by the dissection technique. However 7 out of 1219 An. minimus from Kanchanaburi were found to possess undeveloped oocysts in the stomach wall. It is evident that the IRMA is efficient, convenient and suitable for the investigation of sporozoites in this region. The application of this technique in further epidemiological study of malaria is in progress.

Occurrence of a common epitope in circumsporozoite proteins of Plasmodium falciparum isolated from different areas in Thailand.

Fifteen isolates of P. falciparum sporozoites obtained from patients with acute falciparum malaria from various malaria endemic areas in Thailand were tested for the presence of a common antigenic determinant in their CS protein molecules. SDS-PAGE and Western blot analysis using MAB or human serum antibodies specific to the CS proteins of the parasites revealed a common epitope shared in the CS proteins of all strains of P. falciparum tested. However, the CS proteins exhibited M.W. variation when different strains of the parasites were compared. A similar result was obtained when the human serum antibodies were used. The present study clearly indicated the occurrence of the common epitope in phenotypically different CS proteins among isolates of P. falciparum sporozoites and supported the notion that antigens containing these repetitive epitopes could be used as the candidates for the sporozoite vaccine against P. falciparum infection.

Antibodies against circumsporozoite proteins of Plasmodium falciparum induced by natural infection.

Sera from 10 individuals who lived in a malaria endemic area, 10 patients with acute uncomplicated falciparum malaria and 10 patients with cerebral malaria and hyperimmune mouse serum were tested for their reactivities against Plasmodium falciparum sporozoite antigens by Western blot analysis using 125I-labeled staphylococcal protein A as the detecting reagent. These sera were shown by indirect immunofluorescence and/or circumsporozoite precipitation test to have antibodies reacting against the parasites. It was found that all serum antibodies from the three groups of individuals and the mouse serum reacted in a similar pattern with circumsporozoite (CS) proteins of P. falciparum. Ten sera from normal individuals were negative in all reactions. Monoclonal antibody (MAB) specific against CS proteins of the parasites showed that the proteins exhibited as four different molecular weight (MW) polypeptides, i.e., 67,000, 65,000, 60,000, and 58,000 daltons. These CS proteins of P. falciparum were found to be species and stage specific. Radioimmunoprecipitation using 35S-methionine-labeled parasites and sera of individuals from the various categories or MABs gave a similar result. Another protein antigen of P. falciparum sporozoites had a MW of 80,000 daltons. This antigen was not species specific, probably not membrane associated and was present in a minute quantity in the parasite's extract.

Evidence for diversity of Plasmodium falciparum sporozoite surface antigens derived from analysis of antibodies elicited in humans.

We have compared the reactivities of antibodies developed by individuals frequently exposed to Plasmodium falciparum infections with the epitopes contained within the repeats of the circumsporozoite (CS) protein and their reactivities with the epitopes of a native molecule(s) accessible on the sporozoite surface. Results of direct-binding assays and competition assays between artificial and native molecules or between human antibodies and anti-CS monoclonal antibodies suggest that humans respond preferentially to epitopes not contained within the repeats of the CS protein and probably not contained in the whole CS protein. Human monoclonal antibodies reactive with P. falciparum sporozoite surface antigens were produced by Epstein-Barr virus transformation of human lymphocytes. Their pattern of reactivity with sporozoites from a number of different isolates indicates the existence of several distinct epitopes on the parasite surface. Differences between isolates and between sporozoites within a given sample were observed. No single human monoclonal antibody capable of detecting an epitope expressed in all the parasites studied was found.