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D J Kemp

Publications and source records attributed to D J Kemp.

At least 127 records · Page 7Linked to original sources
PubMed1986

Anchoring a secreted plasmodium antigen on the surface of recombinant vaccinia virus-infected cells increases its immunogenicity.

We show that the subcellular location of foreign antigens expressed in recombinant vaccinia viruses influences their effectiveness as immunogens. Live recombinant viruses induced very poor antibody responses to a secreted repetitive plasmodial antigen (the S-antigen) in rabbits and mice. The poor response accords with epidemiological data suggesting that S-antigens are poorly immunogenic. Appending the transmembrane domain of a membrane immunoglobulin (immunoglobulin G1) to its carboxy terminus produced a hybrid S-antigen that was no longer secreted but was located on the surface of virus-infected cells. This recombinant virus elicited high antibody titers to the S-antigen. This approach will facilitate the use of live virus delivery systems to immunize against a wide range of foreign nonsurface antigens.

Amino Acid Sequence↗
PubMed1986

Sorting large numbers of clones expressing Plasmodium falciparum antigens in Escherichia coli by differential antibody screening.

We describe an approach to classifying a large number of clones expressing Plasmodium falciparum antigens in Escherichia coli by virtue of their differing reactivities with 100 human anti-malarial sera. Individual sera exhibited marked differences in the patterns of reactivity with these clones. These patterns led to the identification of sets of clones, here termed "serological families", which were shown to encode distinct P. falciparum antigens. A serological family was found to be composed of non-identical clones derived from portions of the same antigen. Using this approach six new P. falciparum antigens were identified. One of these is described in detail and is a 102 X 10(3) Mr antigen, predominantly of schizonts. Sequencing studies on four cDNA clones encoding parts of this antigen revealed blocks of hydrophilic dipeptide and tripeptide repeats and so the antigen has been termed the acidic basic repeat antigen (ABRA).

Animals↗
PubMed1985

Sequence of a cDNA encoding a small polymorphic histidine- and alanine-rich protein from Plasmodium falciparum.

We describe the expression in Escherichia coli, isolation by immunological screening and complete nucleotide sequence of a cDNA clone from the malaria parasite Plasmodium falciparum. The deduced amino acid sequence contains separate blocks of repetitive hexapeptide and pentapeptide sequences and we have confirmed that these represent epitopes by reaction of the corresponding synthetic peptides with human antibodies. As the predicted size is Mr 21,000 and the overall composition is 30% His and 29% Ala, the polypeptide has been termed the small histidine-alanine rich protein (SHARP). This polypeptide is highly polymorphic in different P. falciparum isolates and cross reacts immunologically with a distinct gene product of P. falciparum. Although it is related to the Histidine Rich Protein (HRP) of P. lophurae by virtue of its high His content, it shows no obvious sequence relationship to the HRP outside the repeats.

Amino Acid Sequence↗
PubMed1985

Localization of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum in merozoites and ring-infected erythrocytes.

Immunoelectron microscopy with protein A gold has been used to determine the subcellular location of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum. RESA was associated with dense vesicles presumed to be micronemes within merozoites. RESA was not detected on the surface of merozoites but was located at the membrane of erythrocytes infected with ring-stage parasites. RESA within merozoites was largely soluble in the nonionic detergent Triton X-100, but was insoluble in this detergent when associated with the erythrocyte membrane.

Animals↗
PubMed1985

Conserved sequences flank variable tandem repeats in two S-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.

Amino Acid Sequence↗
PubMed1985

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.

Animals↗
PubMed1985

A blood stage antigen of Plasmodium falciparum shares determinants with the sporozoite coat protein.

A cDNA clone expressing a Plasmodium falciparum blood-stage antigen in Escherichia coli was identified by colony immunoassay using immune human sera. Antibodies affinity-purified on extracts of this clone reacted with both asexual blood stages and sporozoites of P. falciparum, recognizing a Mr23,000 protein in the blood stages. The nucleotide sequence of the cDNA revealed a signal peptide and an internal hydrophobic sequence typical of transmembrane anchor sequences. Located 3' to the putative anchor are two tetramers, Asn-Ala-Asn-Pro and Asn-Ala-Asp-Pro, which are closely related to the repeats of the circumsporozoite protein of P. falciparum. The blood stage protein is conserved amongst several isolates of P. falciparum, and antibodies against it are common in the sera of individuals living in the area where the parasite is endemic.

Amino Acid Sequence↗
PubMed1985

Interspersed blocks of repetitive and charged amino acids in a dominant immunogen of Plasmodium falciparum.

We describe an antigen of Plasmodium falciparum that is a dominant immunogen in man. The corresponding cDNA clone, Ag231, expressing this antigen in Escherichia coli reacted in an in situ colony assay with sera from up to approximately equal to 93% of 65 people living in an area in which P. falciparum is endemic. Human antibodies affinity purified on immobilized Ag231 lysates identified the corresponding parasite antigen as a polypeptide of Mr approximately equal to 300,000. It was present in schizonts and also in ring-stage trophozoites, where a speckled immunofluorescence pattern suggested an association with the erythrocyte. Its mRNA was enriched in merozoites relative to other blood stages, a distinctive property shared by a recently described antigen located on the surface of ring-infected erythrocytes, and it is encoded by a single gene having a number of allelic variants. The complete nucleotide sequence of Ag231 revealed a structural unit composed of 13 hexapeptide repeats flanked by a highly charged region containing both acidic and basic amino acids. This structural unit is itself repeated, so that blocks of repeats and charged units are interspersed along the molecule. The sequences within the repeats vary much more extensively than those in the charged units.

Amino Acid Sequence↗
PubMed1985

Potential vaccine antigens of the asexual blood-stages of Plasmodium falciparum.

We have constructed a cDNA clone library that contains many natural immunogens of the asexual blood-stages of Plasmodium falciparum. The corresponding parasite antigens have been identified with antisera raised against the antigens expressed in Escherichia coli or with monospecific human antibodies purified on adsorbents prepared from various clones. Sequencing studies on the clones have revealed that many malaria antigens contain extensive sequence repeats. These repeats encode antigenic epitopes that in several proteins have been shown to be immunodominant. One candidate vaccine molecule, the Ring-infected Erythrocyte Surface Antigen (RESA), which is transferred from inside merozoites to the erythrocyte surface at about the time of merozoite invasion, contains two blocks of antigenically cross-reactive repeats. The structures of other antigens containing extensive repeats are described and their possible significance to the host-parasite relationship is discussed.

Animals↗
PubMed1984

The expression of Plasmodium falciparum bloodstage antigens in Escherichia coli.

A library of cDNA clones expressing proteins of the asexual blood stages of a Papua New Guinean isolate of Plasmodium falciparum (isolate FCQ27/PNG (FC27] was constructed in the bacteriophage vector lambda gt11-Amp3. In an in situ colony immunoassay, human serum was used to identify colonies producing natural immunogens. Sera from donors of defined clinical status, or reactive to a defined subset of natural immunogens were used to identify clones of particular interest (for example, clones reacting with convalescent but not with acute serum or clones expressing the isolate specific S-antigen of FC27). Antisera raised by immunizing mice and rabbits with cloned antigens were used to characterize the P. falciparum proteins corresponding to the antigen-positive clones. Nucleotide sequence analysis of an antigen found on the surface of cells infected with ring stage parasites revealed an unusual sequence coding for eight, four and three amino acid repeats rich in acidic amino acids. The discussion centres on the use of cloned antigens as tools for the analysis of the host-protective immune response and selection of candidate vaccine molecules.

Animals↗
PubMed1984

Identification of a cDNA clone encoding a mature blood stage antigen of Plasmodium falciparum by immunization of mice with bacterial lysates.

A cDNA library was constructed in pBR322 using mRNA from blood stages of a Papua New Guinean isolate of Plasmodium falciparum. Expression of parasite antigens was not directly detectable by conventional immunological assays. To circumvent this, mice were immunized with lysates of cDNA clones, and the antisera raised were assayed for anti-parasite reactivity. One cDNA clone was identified which reliably elicited antibodies to P. falciparum. The mouse antisera were used to characterize the native P. falciparum protein as a 120-kd protein, which is antigenic during natural infection. The protein occurs in late trophozoite and schizont stages and is found in isolates of the parasite from widely separated geographical areas. The genomic context of the antigen gene is conserved in the different isolates.

Animals↗
PubMed1984

Genes of the protozoan parasite Babesia bovis that rearrange to produce RNA species with different sequences.

We describe a highly polymorphic gene family from the protozoan parasite Babesia bovis that encodes a complex pattern of transcripts. Gene rearrangements at this locus (designated BabR ) have generated RNA species that differ in sequence. The naturally occurring virulent field isolate Kv expresses two different sized transcripts that are identical in sequence at their 5' ends but different at the 3' ends. An avirulent derivative of this isolate, Ka, expresses not only these two transcripts but a third, of intermediate size. Analysis of cloned genomic fragments from Ka identified genes encoding all three transcripts. Two fragments contained tandemly repeated copies of the BabR genes. The gene encoding the intermediate sized transcript has undergone a rearrangement, substituting a 5' region different to that shared by the other transcripts. These results suggest that sequence diversity may play an important role in the biological function of the BabR locus.

Amino Acid Sequence↗
PubMed1984

Accurate transcription of a cloned gene from Babesia bovis in Saccharomyces cerevisiae.

Two cloned chromosomal segments of the hemoprotozoan Babesa bovis bearing genes from the polymorphic locus designated BabR were inserted into plasmid vector YCp19, cloned in Escherichia coli and then introduced into Saccharomyces cerevisiae. Both genes were transcribed producing poly(A+) RNAs. In at least one case, the transcript was very similar if not identical to that produced in B. bovis. It is therefore possible that S. cerevisiae can recognize at least some hemoprotozoan promoter and poly(A+)-addition signals.

Animals↗
PubMed1984

DNA polymorphisms and subpopulations in Babesia bovis.

Independent isolates of Babesia bovis differ by only a limited number of polypeptides, some of which may be important as host protective antigens. Avirulent derivatives of these parasites also differ from their virulent counterparts in only a few polypeptides. To identify genes encoding such polypeptides we have isolated cDNA clones corresponding to poly(A)+ RNAs that are expressed only in certain isolates. For this purpose a cDNA clone library was constructed from poly(A)+ RNA of the K-avirulent isolate (KA). These clones were screened by colony hybridization using [32P]cDNA complementary to poly(A)+ RNA from KA and from virulent isolates, in order to identify clones that selectively hybridize to one cDNA probe. Hybridization of DNA from three clones, designated pK4, pK5 and pK6 to poly(A)+ RNA from various isolates revealed different and complex patterns. The gene represented by clone pK5 appeared to be transcribed predominantly in avirulent parasites. Analysis of genomic DNA by the Southern procedure enabled each isolate to be distinguished and suggested that most isolates are comprised of a heterogeneous mixture of subpopulations. Analysis of genomic DNA from parasites obtained after passage of KA through the tick vector (Boophilus microplus) suggested that a subpopulation was being selected that more closely resembled KV than KA.

Animals↗
PubMed1984

Differential antibody screening of cloned Plasmodium falciparum sequences expressed in Escherichia coli: procedure for isolation of defined antigens and analysis of human antisera.

We describe a procedure that uses polyspecific human sera for screening Escherichia coli colonies expressing cloned Plasmodium falciparum cDNA sequences in order to detect colonies that react differentially with different sera. This procedure can be used for two distinct purposes. First, it enables the isolation of clones encoding specified antigenic sequences present in the complex mixture, without purification of either antigens or antibodies by conventional procedures. This requires that the antigen can be expressed in E. coli and that antisera are available that differ substantially in their reactivities to the component of interest. To develop the procedure, we used two polyspecific sera that shared many anti-P. falciparum specificities but differed in that only one was reactive to the isolate-specific S antigen of P. falciparum strain FCQ27/PNG (called FC27). Differential screening with the two sera identified 30 cDNA clones, and colony hybridization confirmed that 25 of these express S-antigen sequences. Second, the procedure identifies defined antibody specificities within polyspecific human sera by virtue of their ability to react with any given cDNA clone. The procedure has been used here to identify antibody specificities that increase dramatically in titer between the acute and convalescent phases of malaria in certain individuals and, hence, to isolate clones encoding the corresponding antigens.

Animals↗