Robert B. Saint

Conserved sequences flank variable tandem repeats in two α-antigen genes of Plasmodium falciparum

We describe the isolation of two chromosomal DNA fragments from Plasmodium falciparum. These fragments encode the antigenically distinct S antigens of two different P. falciparum isolates, namely FC27 from Papua New Guinea and NF7 from Ghana. The complete nucleotide sequences of both fragments are presented. The fragments are homologous over most of their lengths, including the entire regions flanking the protein coding sequences. Whereas the N- and C-terminal portions of sequences encoding the S antigens are homologous, major portions of the coding sequences are not. The nonhomologous regions are comprised of tandemly repeated sequences, of 33 bp in FC27 and predominantly of 24 bp in NF7. The 33 bp tandem repeats encoded by the FC27 S-antigen gene could not be detected in the NF7 genome. Conversely, the 24 bp tandem repeats encoded by the NF7 S-antigen gene could not be detected in the FC27 genome. The pattern of sequence variation within the repeats of both genes suggests a mechanism for the generation of S-antigen diversity.

Plasmodium falciparum: Identification and localization of a knob protein antigen expressed by a cDNA clone

Differential screening of cDNA libraries constructed from knobby and predominantly knobless Plasmodium falciparum isolates, identified the sequence SD17. Chromosome blotting experiments have shown that this sequence, which is located on chromosome 2 of most isolates, was deleted in the cloned parasite line E12 of the FCQ27/PNG isolate. Here we show that erythrocytes infected with the SD17-containing cloned line D10 have typical knob structures on their surfaces, whereas those infected with the line E12 lack knobs. An expression clone was constructed from SD17 and used to affinity purify antibodies from the sera of individuals living in areas of Papua New Guinea where malaria is endemic. The antibodies reacted in immunoblotting experiments with a single polypeptide that varied in Mr from 85,000 to 105,000 among different isolates. The antigen was not expressed in the knobless clone E12. Postembedding immunoelectron microscopy showed localization of the antigen over the knobs of FC27 and two other isolates, largely on the cytoplasmic side. We conclude that the parasite antigen corresponding to clone SD17 is a knob protein.

Expression of Taenia taeniaeformis antigens in Escherichia coli.

Two important features of infection of mice with larvae of Taenia taeniaeformis are the ready demonstration of host protective antibodies and the ability to immunize susceptible strains of mice against first infection using crude parasite preparations. Candidate immunogens in established larvae and the invasive oncosphere have been identified by immunoprecipitation of radiolabeled parasite proteins with host-protective antibodies. To overcome the difficulties associated with purification of these antigens from parasite material, the alternative strategy of expressing parasite proteins in Escherichia coli has been adopted. Double stranded DNA complementary to mRNA from 28 day old liver larvae was inserted into the beta-galactosidase gene of the bacteriophage lambda Amp 3. Some recombinants express a fusion protein with additional parasite-encoded epitopes located at the C-terminal end of the beta-galactosidase protein. Four clones that reacted with antibodies in an E. coli colony immunoassay were selected for detailed characterization. Analysis of lysates of the selected clones by SDS-PAGE and Western blotting revealed that each clone produced an abundant fusion protein that reacted specifically with a hyperimmune anti-oncosphere serum. Sibling analysis revealed that the four antiserum-positive clones encoded three immunologically-distinct parasite antigens. The identity of the native protein of larvae encoded by one clone (designated TA10) was an abundant antigen of Mr 70,000. This approach allows the assessment of antigens expressed in E. coli as vaccines in susceptible strains of mice by direct immunization and challenge and thus the development of a model defined-antigen vaccine against a larval cestode parasite.

Plasmodium falciparum: Differentiation of isolates with DNA hybridization using antigen gene probes

Chromosomal DNA was prepared from seven Plasmodium falciparum isolates that had been cultured in vitro and from a cloned P. falciparum line. The DNA was cleaved with restriction endonucleases, fractionated by agarose gel electrophoresis, blotted to nitrocellulose, and hybridized with a series of radioactively labeled DNA probes. The probes had been derived from cDNA clones encoding portions of P. falciparum antigens. Simple, reproducible band patterns that differed for many of the isolates were obtained. Parasite isolates collected from different continents could be readily distinguished, as could some but not all isolates collected from one restricted region of Papua New Guinea. Application of this technique for the identification and differentiation of parasite strains was explored. The patterns of hybridization observed were consistent with the proposition that blood stages of P. falciparum have a haploid genome.

Changes in repeat number, sequence, and reading frame in S-antigen genes of Plasmodium falciparum.

The S antigens from different isolates of Plasmodium falciparum exhibit extensive size, charge, and serological diversity. We show here that the S-antigen genes behave as multiple alleles of a single locus. The size heterogeneity results from different numbers, lengths, and/or sequences of tandem repeat units encoded within the S-antigen genes. Two genes studied here encode antigenically different S antigens but nevertheless have closely related tandem repeat sequences. We show that antigenic differences can arise because repeats are translated in different reading frames.

Expression of Schistosoma japonicum antigens in Escherichia coli

A cloned library of DNA complementary to the mRNA of adult Schistosoma japonicum has been prepared and expressed as fusion proteins with Escherichia coli beta-galactosidase. Colonies expressing the S. japonicum cDNA clones were screened both with antibodies from individuals with a history of schistosomiasis and with antibodies obtained from a rabbit immunized with whole adult worms. In both cases colonies were detected which bound antibody, although the frequency of antigen-positive clones was much higher with the rabbit antiserum than with human sera. In both cases the proportion of colonies reacting with antibodies was markedly lower than that published for equivalent screens of Plasmodium falciparum cDNA with sera from individuals with a history of falciparum malaria. Several major S. japonicum antigens were identified by the affinity purification of antibodies using immobilised fusion proteins produced during lytic growth of the recombinant bacteriophage.

Immunochemical analysis of Taenia taeniaeformis antigens expressed in Escherichia coli

Previously we reported the isolation of several Escherichia coli clones expressing fragments of Taenia taeniaeformis antigens as beta-galactosidase fused proteins (Bowtell, Saint, Rickard & Mitchell, 1984). Here we describe the isolation of additional antigen-expressing clones from a larval cDNA library and the assignment of these clones to 7 antigen families. These were isolated with a polyspecific rabbit antiserum raised to the oncosphere. Since this serum was capable of reacting with a large number of antigens, it was important to develop techniques for rapidly determining the identity of the native T. taeniaeformis molecule corresponding to a cloned antigen gene. These included active immunization of rabbits with fused proteins and several techniques involving affinity purification on immobilized fused proteins. The reactivity of the antigen-positive clones with sera from humans infected with related parasites was also assessed. Finally, immunization of mice with several fused proteins failed to protect against subsequent infection, although antigens previously identified as candidate host-protective antigens (Bowtell, Mitchell, Anders, Lightowlers & Rickard, 1983) have yet to be identified in the expression library.