Received for publication 20 November 1981 and in revised form 13 April 1982. cell type. Second, erythrocytes infected with trophozoites and schizonts of P. vivax or a knobless strain of P. falciparum, both of which continue to circulate in vivo, did not bind to either target cell type. Third, transmission electron microscopy showed that infected erythrocytes attached to the amelanotic melanoma cells via knobs. Weconclude that cultured human endothelial cells and an amelanotic melanoma cell line share common determinants on their surface and that the mechanism of binding to these two different cell types is similar. The amelanotic melanoma cell line offers a useful substitute for endothelial cells in binding studies requiring large numbers of target cells.

Members of a serologically cross-reacting family of proteins including Ag332 and Pf11.1, megadalton proteins of schizont-infected red blood cells, and gametocytes, respectively, and Pf155-RESA, a 155-kDa protein of ring-infected red blood cells, have been reported to share amino acid repeat sequences. These repeats are rich in glutamic acid dipeptides postulated to be involved in generating serologic cross-reactivity. We report the identification and characterization of another member of this cross-reacting family, a 260-kDa glutamic acid-rich intraerythrocytic protein. Human antibodies affinity purified on the 260-kDa region of Western blots of trophozoite proteins of Plasmodium falciparum were used to screen a trophozoite-stage λ gt11 cDNA library. A 1.8-kb clone was identified and human antibodies were affinity purified on the expressing clone. Using this affinity-purified antibody and the 1.8-kb clone, the corresponding protein, its gene, and its chromosomal location were investigated. The 260-kDa corresponding protein serologically cross-reacts with Pf155-RESA, but is the product of a different gene. The 260-kDa protein is Triton X-100 soluble and is variable in molecular weight in different isolates. Immunoprecipitation of [35S]methionine-labeled infected red blood cells indicates that the protein is synthesized throughout the intraerythrocytic cycle but is most prominent in schizonts. The protein, as has been shown previously, is not immunoprecipitated from 125I surface-labeled infected red blood cells and is thus not PfEMP1, the antigen associated with cytoadherence. indirect fluorescent antibody studies using fixed infected red blood cells suggest that the protein is localized to the periphery of the intraerythrocytic parasite.

Cryptosporidium parvum, a zoonotic Apicomplexan pathogen, causes profound diarrhea, malnutrition, and dehydration in patients with AIDS. A less severe, self-limited disease occurs in immunocompetent individuals, particularly children, animal handlers, and residents of the developing world. Very little is known about the biology of the organism, the pathophysiology of the disease process, or the mechanism of protective immunity. There is no effective therapy for cryptosporidiosis, but hyperimmune bovine colostrum raised against Cryptosporidium oocysts and sporozoites has ameliorated infection and disease in some patients with AIDS, and a variety of monoclonal antibodies, as well as hyperimmune bovine colostrum, have significantly reduced cryptosporidial infection of mice and calves. We report here the identification and initial characterization of a > 900,000-M(r) Cryptosporodium sporozoite glycoprotein (GP900) that is a prominent antigen recognized by protective hyperimmune bovine colostral immunoglobulin. Three of six murine anticryptosporidial monoclonal antibodies reacted with GP900, indicating that the molecule is highly immunogenic in mice as well as in cows. GP900 is Triton X-100 soluble and N glycosylated. Western blotting of the N-deglycosylated protein, detected with antibodies eluted from recombinant clones expressing a partial GP900 fusion protein, suggested that the polypeptide backbone of the glycoprotein has an M(r) of < 190,000. GP900 is encoded by a single-copy gene that resides on the largest Cryptosporidium chromosome.

We identified a gametocyte-specific protein of Plasmodium falciparum called Pf11-1 and provide experimental evidence that this molecule is involved in the emergence of gametes of the infected erythrocyte (gametogenesis). A mutant parasite clone, which has deleted over 90% of the Pf11-1 gene locus, was an important control to establish the gametocyte-specific expression of the Pf11-1. Molecular analysis of the Pf11-1 deletion indicates that it is presumably due to a chromosome breakage with subsequent 'healing' by the addition of telomeric heptanucleotides. Moreover, similar DNA rearrangements are observed in most of the laboratory isolates during asexual propagation in vitro.

Cryptosporidium parvum, an Apicomplexan parasite of gastrointestinal epithelial cells, causes severe disease in persons with AIDS and is a common cause of self-limited diarrhea in children, animal handlers, and residents of developing countries. No approved therapy exists; in research studies, however, hyperimmune bovine colostrum raised to Cryptosporidium oocysts and sporozoites has eradicated disease or decreased parasite burden in some AIDS patients. Although the protective antigens recognized by bovine hyperimmune colostrum have not been defined, protective antigens of other Apicomplexan parasites frequently have been associated with two unique structures of invasive forms, the trilaminar pellicle and the apical complex. In order to identify immunogenic Cryptosporidium proteins that may be protective antigens for use as recombinant immunogens in passive and/or active immunotherapy, we screened two genomic DNA expression libraries with polyspecific anti-Cryptosporidium antibodies. We used an approach to cloning apical complex and pellicle protein antigens that succeeded despite the lack of large numbers of organisms that would be necessitated for conventional biochemical approaches requiring organelle or membrane purification. We report here the molecular cloning of five C. parvum genes and the characterization of the cognate sporozoite proteins having molecular masses of greater than 500, 68/95, 45, 23, and 15/35 kDa. The light microscopic immunofluorescence pattern of antibodies recognizing these protein antigens suggest that they are located in the pellicle or apical complex of Cryptosporidium sporozoites.

We report the identification of the product of the Plasmodium falciparum Pf11‐1 gene and demonstrate that it is a gametocyte‐specific protein that has a potential role in the rupture of the host erythrocyte and emergence of the gametes (gametogenesis). The Pf11‐1 gene is a large locus (30 kb) whose sequence predicts a glutamic acid‐rich polypeptide. Our identification of the Pf11‐1 gene product as gametocyte specific was greatly facilitated by the isolation of a mutant parasite clone in which greater than 90

To facilitate studies of the biology of Cryptosporidium parvum, we have developed an in vitro culture system using Madin-Darby canine kidney (MDCK) cells as the host cell. Oocysts or free sporozoites were incubated 37 degrees C with monolayers of MDCK cells in supplemented RPMI 1640 medium and the cells were examined at various time intervals after initiation of the culture. High rates of infection (up to 90% of MDCK cells) were achievable. Sequential development of trophozoites, meronts, microgametocytes, and macrogametocytes was observed over a 72-h period of culture. Between 72 and 96 h we observed formation of oocyst walls, but fully sporulated oocysts were not observed. This culture system provides access to both the asexual and sexual intracellular stages of C. parvum.

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Antigenic variation of infectious organisms is a major factor in evasion of the host immune response. However, there has been no definitive demonstration of this phenomenon in the malaria parasite Plasmodium falciparum. In this study, cloned parasites were examined serologically and biochemically for the expression of erythrocyte surface antigens. A cloned line of P. falciparum gave rise to progeny that expressed antigenically distinct forms of an erythrocyte surface antigen but were otherwise identical. This demonstrates that antigenic differences on the surface of P. falciparum-infected erythrocytes can arise by antigenic variation of clonal parasite populations. The antigenic differences were shown to result from antigenic variation of the parasite-encoded protein, the P. falciparum erythrocyte membrane protein 1.

The survival of Plasmodium falciparum-infected erythrocytes is enhanced by the sequestration of mature trophozoites and schizonts from the peripheral circulation. Cytoadherence of infected erythrocytes in vivo is associated with the presence of knobs on the erythrocyte surface, but we and others have shown recently that cytoadherence to C32 melanoma cells may occur in vitro in the absence of knobs. We show here that a knobless clone of P. falciparum adheres to the leukocyte differentiation antigen, CD36, suggesting that binding to CD36 is independent of the presence of knobs on the surface of the infected erythrocyte. This clone showed little cytoadherence to immobilized thrombospondin or to endothelial cells expressing the intercellular adhesion molecule 1. Furthermore, an Mr approximately 300-kD trypsin-sensitive protein doublet was immunoprecipitated from knobless trophozoite-infected erythrocytes. Finding a P. falciparum erythrocyte membrane protein 1 (PfEMP1)-like molecule on these infected erythrocytes is consistent with a role for PfEMP1 in cytoadherence to CD36 and C32 melanoma cells.

The Plasmodium falciparum 11.1 gene locus on chromosome 10 extends over 30 kb and contains approximately 22 kb of a tandemly repeated 27-bp sequence. Biochemical and size similarities have been noted between the reported 11.1 antigen and a variable-Mr, surface-radioiodinatable protein which appears to be involved in the cytoadherence of red blood cells infected with mature intraerythrocytic parasites to venular endothelium. We attempted to determine if these proteins were identical.

Intraerythrocytic Plasmodium falciparum parasites at the trophozoite and schizont stages synthesize a > 200-kDa protein, the mature erythrocyte surface antigen (MESA), that is localized at the membrane of infected red blood cells and manifests size polymorphism and antigenic diversity among parasite isolates. Because MESA is localized in the host cell membrane, we examined parasites with differing knob and cytoadherence phenotypes to determine whether MESA expression correlated with knob formation and cytoadherence. A cloned line of P. falciparum that was cultured with repeated selection for the knobbed and cytoadherent phenotypes did not express MESA, due to at least partial deletion of the single-copy MESA gene. In contrast, parasites from the same clone that were cultured without this selection lost the knobbed and cytoadherent phenotypes, but continued to express MESA. These results indicate that MESA is apparently not required for differentiation and multiplication of erythrocyte stage P. falciparum parasites in vitro, or for knob formation and cytoadherence. We speculate that MESA may have a role in evasion of the host immune response by P. falciparum.

Trophozoites of Plasmodium falciparum obtain free amino acids for protein synthesis by degrading host erythrocyte hemoglobin in an acidic food vacuole. We previously reported that leupeptin and l-trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64), two inhibitors of the cysteine class of proteinases, blocked hemoglobin degradation in the trophozoite food vacuole, and we identified a 28-kDa trophozoite cysteine proteinase as a potential food vacuole hemoglobinase. We now report that the biochemical properties of the trophozoite cysteine proteinase closely resembled those of the lysosomal cysteine proteinases cathepsin B and cathepsin L. The trophozoite proteinase had a pH optimum of 5.5–6.0, near that of both lysosomal proteinases, and it was efficiently inhibited by highly specific diazomethylketone and fluoromethylketone inhibitors of cathepsin B and cathepsin L. The trophozoite proteinase preferred peptide substrates with arginine adjacent to hydrophobic amino acids, as does cathepsin L. Micromolar concentrations of the fluoromethylketone inhibitor Z-Phe-Ala-CH2F blocked the degradation of hemoglobin in the trophozoite food vacuole and prevented parasite multiplication. In previous studies much higher concentrations of the inhibitor were not toxic for mice. Our results provide additional evidence that the 28-kDa trophozoite proteinase is a food vacuole hemoglobinase and suggest that specific inhibitors of the enzyme may have potential as antimalarial drugs.