cDNA sequence encoding a Plasmodium falciparum protein associated with knobs and localization of the protein to electron-dense regions in membranes of infected erythrocytes.

Plasmodium falciparum modifies the host erythrocyte's plasma membrane by the formation of electron-dense structures called knobs. We have produced monoclonal antibodies (McAbs) which specifically bind to the knobs in immunoelectron microscopic experiments with thin sections of parasitized erythrocytes. However, the McAbs fail to bind to the surface of live parasitized erythrocytes. Immunoblotting experiments with these McAbs show the antigen is localized to the erythrocyte plasma membrane. The antigen with which the McAbs react varies in mol. wt from 80 to 95 kd in different knob-producing isolates of P. falciparum and is absent in knobless variants. The McAbs react with the expressed product of a P. falciparum cDNA clone, thus demonstrating that the clone encodes part of this knob-associated protein. The sequence of the cDNA fragment partially overlaps a published cDNA sequence reported to encode the amino-terminal portion of the knob protein, and extends the predicted open reading frame by 190 amino acids. The carboxyl-terminal portion of the predicted amino acid sequence contains a highly charged stretch of approximately 100 amino acid residues. We suggest that this unusual, highly charged region participates in intermolecular salt bridging leading to dense packing of these molecules. This would create the electron-dense regions observed by electron microscopy and might also explain the insolubility of the knob-associated protein in the absence of strong ionic detergents or chaotropic agents.

A 75 kd merozoite surface protein of Plasmodium falciparum which is related to the 70 kd heat-shock proteins.

Proteins on the merozoite surface of the human malarial parasite Plasmodium falciparum are targets of the host's immune response. The merozoite surface location of p75, a 75 kd P. falciparum protein, was established by immunoelectron microscopy using antisera raised to the expressed product of a cDNA clone. Immunoprecipitation from protein extracts biosynthetically labeled during different periods of the asexual cycle showed that p75 is made continuously, although ring-stage parasites appear to synthesize larger quantities. p75 is conserved and invariant in size in eight isolates of P. falciparum. The 880 bp cDNA sequence encoding part of p75 reveals one open reading frame containing a repetitive sequence unit of four amino acids. The predicted reading frame is correct since antisera to a synthetic peptide corresponding to the repetitive region recognize p75 in immunoblots. The sequence of p75 is homologous with the sequences of proteins from the ubiquitous, highly conserved family of 70 kd heat-shock proteins, suggesting an important physiological function for p75. The cDNA fragment encoding part of p75 hybridizes with multiple genomic fragments, whose sizes are identical in DNA from nine P. falciparum strains, suggesting that the gene for p75 is well conserved and may be part of a gene family.

Expression of Plasmodium falciparum surface antigens in Escherichia coli.

The asexual blood stages of the human malarial parasite Plasmodium falciparum produce many antigens, only some of which are important for protective immunity. Most of the putative protective antigens are believed to be expressed in schizonts and merozoites, the late stages of the asexual cycle. With the aim of cloning and characterizing genes for important parasite antigens, we used late-stage P. falciparum mRNA to construct a library of cDNA sequences inserted in the Escherichia coli expression vector pUC8. Nine thousand clones from the expression library were immunologically screened in situ with serum from Aotus monkeys immune to P. falciparum, and 95 clones expressing parasite antigens were identified. Mice were immunized with lysates from 49 of the bacterial clones that reacted with Aotus sera, and the mouse sera were tested for their reactivity with parasite antigens by indirect immunofluorescence, immunoprecipitation, and immunoblotting assays. Several different P. falciparum antigens were identified by these assays. Indirect immunofluorescence studies of extracellular merozoites showed that three of these antigens appear to be located on the merozoite surface. Thus, we have identified cDNA clones to three different P. falciparum antigens that may be important in protective immunity.

Zymodeme and species specificities of monoclonal antibodies raised against Trypanosoma cruzi.

A series of hybrid cell lines was generated by the fusion of Sp2/0 myeloma cells with spleen cells from BALB/c mice that had been immunized or infected with Trypanosoma cruzi zymodemes (Z1, Z2, Z3 ). Four immunoglobulin isotypes, IgM, IgG1, IgG2a and IgG3 were represented amongst the monoclonal antibodies secreted by 22 hybridoma clones. On indirect immunofluorescence (IFAT) antibodies bound to flagellum, cytoplasm, cell membrane or stained the whole organism. Two antibodies were epimastigote-specific. Enzyme-linked immunosorbent assays (ELISA) and a dot immunobinding test were used to evaluate the zymodeme and species specificities of 13 antibodies: four reacted with all T. cruzi zymodemes tested, two reacted strongly with all except Z1, two predominantly with Z1, two predominantly with Z2, and three predominantly with Z3 . Two IgM antibodies cross reacted with Trypanosoma rangeli, T. brucei, Leishmania mexicana, L. braziliensis and L. donovani. Five antibodies were used in a preliminary immunobinding test, performed blindly, to compare monoclonal reactivities and zymodeme groups. The results suggested a correlation between the two methods of characterization. Anti-T. cruzi monoclonal antibodies are considered to have important applications to epidemiological studies and the improved diagnosis and prognosis of Chagas' disease.

A monoclonal antibody with specificity for Trypanosoma cruzi, central and peripheral neurones and glia.

Of 26 hybridomas secreting anti-Trypanosoma cruzi antibodies, two reacted with murine brain and spinal cord extracts but not other tissues. Both antibodies (5H7 and 3H3) had identical isotypes (IgM) and specificities as judged by western blotting. Antigens of molecular weight 58,000 and 37,000 in mouse brain and spinal cord, and 58,000 and 35,000 in T. cruzi were detected. Different tissue antigens were recognized by a previously reported T. cruzi mammalian neural tissue cross-reacting monoclonal antibody CE5; on immunofluorescence 5H7 stained some glia, and some central and peripheral neurones in rat tissue sections. Pooled sera from mice chronically infected with T. cruzi competed with binding of 5H7 to T. cruzi antigens.

Purified Trypanosoma cruzi specific glycoprotein for discriminative serological diagnosis of South American trypanosomiasis (Chagas' disease).

Chagas' disease, leishmaniasis, and Trypanosoma rangeli infection are endemic and their distributions overlap in vast regions of South and Central America. Serological cross-reactivities can confuse epidemiological studies of these infections, and their differential diagnosis has been assigned a high priority by the World Health Organisation. A lectin-affinity-purified, 90,000 molecular weight glycoprotein (GP90) is present in the known principal strains (zymodemes) of Trypanosoma cruzi and absent from Leishmania and T rangeli. Patients with T cruzi infection have antibody to GP90, whereas patients with leishmaniasis do not and the two infections can be distinguished in an ELISA system using this antigen. In a mouse model, the same test can differentiate between T cruzi and T rangeli infections. Antigens purified by affinity chromatography clearly provide a practical basis for very precise, even strain-specific, diagnostic tests.