Robert T. Reese

Plasmodium falciparum antigens associated with membrane structures in the host erythrocyte cytoplasm

Hybridomas were made from mice immunized with plasma membranes from erythrocytes infected with Plasmodium falciparum. Among the monoclonal antibodies produced, a series reacted with antigens in the host cell cytoplasm. Immunoelectron microscopy, along with indirect fluorescent antibody double labeling experiments, were used to further localize the antigens to membrane structures (presumably Maurers clefts) in the erythrocyte cytoplasm. The epitopes thus localized are found on three parasite proteins (20 kDa, 29 kDa, and 45 kDa) and one parasite glycoprotein (45 kDa). They are likely to be part of a transport system for the parasite.

Characterization of a high-molecular-weight phosphoprotein synthesized by the human malarial parasite Plasmodium falciparum

During its intra-erythrocytic cycle, Plasmodium falciparum synthesizes a protein of apparent Mr 250,000-300,000. Its precise size is dependent on the P. falciparum isolate examined. This protein contains phosphate covalently bound to one or more serine residues and hence is termed PP300. Monoclonal antibody, McAb4-1F, binds to PP300 on immunoblots of protein extracts from all parasite isolates tested, both those exhibiting and those lacking the knob phenotype. Using McAb4-1F, the polypeptide was shown to be physically associated with the plasma membrane in a membrane-isolation procedure. However, in an indirect immunofluorescence assay the McAb appeared to bind to antigen associated with the erythrocyte plasma membrane in parasitized cells. However, it reacted only to fixed, not unfixed, parasitized erythrocytes indicating that the epitope is not normally exposed to extracellular antibodies. Clone 29-2 was isolated by a McAb4-1F immunoscreen of a P. falciparum complementary DNA (cDNA) expression library created in pUC8. Rat anti-clone serum which was raised to the purified protein encoded by the lacZ-29-2 fusion in pUC8 reacted with PP300 in immunoblots of parasite antigen. In Southern-blot analyses of parasite DNA digested with EcoRI, HindIII, or EcoRV, the 29-2 DNA insert hybridized to more than one fragment even though the insert lacked internal sites for these enzymes. In addition, hybridization studies were conducted using two oligodeoxy-nucleotides which were constructed based on the sequence of a cDNA clone which encoded part of a similar high-molecular-weight P. falciparum protein [Coppel et al., Mol. Biochem. Parasitol. 20 (1986) 265-277]. Analysis of these results indicates that the two cDNA sequences are parts of the same gene or a family of related genes.

Cross-reactive asparagine-rich determinants shared between several blood-stage antigens of Plasmodium falciparum and the circumsporozoite protein.

From a Plasmodium falciparum cDNA expression library derived from mRNA of the asexual blood stages, we isolated and sequenced five different cDNA clones whose predicted protein products were unusually rich in asparagine (Asn). Two of the clones, R5 and G5, contain tandem imperfectly repeated sequences based on Asn-Asn-Thr (NNT) and Asn-Asn-Met (NNM) respectively. The other three, E4, C5 and R13, as well as G5, contain stretches of polyasparagine varying in length from 2 to 26 residues. Results of DNA blotting experiments with the individual cDNA sequences as probes suggest that each of the five clones corresponds to a different P. falciparum gene. The fragments of P. falciparum proteins expressed by the cDNA clones shared cross-reactive antigenic determinants which were present on multiple P. falciparum proteins. In immunoblotting experiments, owl monkey antibodies selected for binding to the polypeptide expressed by clone E4, C5 or G5 reacted with the expressed proteins from all 5 clones, and with at least 10 proteins from schizont infected erythrocytes. The cross-reactive epitopes could be modeled by two Asn-rich peptide structures: (1) (NNT)8, whose sequence was based on the R5 repeat; and (2) (NPNA)6, whose sequence was based on the Asn-rich repeat of the P. falciparum circumsporozoite protein (CSP). Antibodies that bound to each peptide were selected from sera of immune monkeys that had never been exposed to sporozoites. The selected antibodies bound all 5 expressed proteins in immunoblotting assays and also bound to several proteins from parasitized erythrocytes. Such cross reactivity between the CSP repeating unit and several blood-stage antigens has not been previously reported.

Comparison of the antibody produced by DNCB fed, sensitized and conventionally immunized strain 13 guinea pigs

Abstract Using a procedure which yields approx 80% of the serum antibody detectable by precipitation, anti-DNP antibody was purified from strain 13 guinea pigs immunized with highly conjugated DNP-KLH in complete Freunds adjuvant. The antibody from the two experimental groups compared was obtained from animals either sensitized with DNCB or fed DNCB in oil before immunization with DNP-KLH. Primary structural analysis of the purified IgG2 anti-DNP antibody detected definite differences in the Hv1 and Hv3 regions of the antibodies from the two different groups. Nevertheless, the only Hv-containing peptide which consistently appeared to be composed of a high proportion of distinguishing variants was that containing Hv3. From the data it is clear that there are cells present in the B-lymphocyte pool of fed animals which generally dominate in the response when normal animals are immunized with DNP-KLH. It is also apparent that these cells both can and do respond, but that there is clearly incomplete dominance to the extent that 35–50% of the antibody product isolated appears to be representative of another major group (or groups) of cells.

Antibody responses stimulated in rabbits,, guinea-pigs and mice by recombinant and synthetic portions of a 75 kDa malarial merozoite protein

The 75 kDa heat-shock-related protein (p75) of Plasmodium falciparum is an abundant, highly conserved, merozoite surface protein. A bacterial clone, C7, produces a polypeptide (C7Ag) of approximately 30 kDa representing the C-terminal 40% of p75. In several species of animals, the C7Ag stimulated high titre IgG antibodies which cross-react with p75. Two major portions of the C7Ag, theoretically predicted to have strong secondary structural preferences, were modelled with four synthetic peptides. An alpha-helical, hydrophilic region, modelled with a 28-mer, proved a poor immunogen in guinea-pigs and several strains of inbred mice, even though it had been a strong immunogen in rabbits. A disulphide-bonded region of the C7Ag was modelled with three peptides of increasing length, namely 49-, 64- and 76-amino acid residues. In general, the order of immunogenicity was 49 less than 64 less than 76-mer. Antibodies to the 76-mer and the 64-mer reacted strongly with the native parasite protein. The data also suggested that the 76-mer was a good model for the region of the molecule it was made to represent.