1995. in antibody reactivity to rTRAP-C1 occur after recent exposure to infects the intestinal mucosas of various mammals, including humans. This apicomplexan parasite can cause asymptomatic infection or result in self-limited diarrhea, sometimes accompanied by nausea, vomiting, abdominal cramping, and fever in healthy hosts (4). However, infection with species can also result in persistent, sometimes fatal, diarrheal disease and malnutrition, especially in those with an underlying immunodeficiency (1, 4, 22). In other species of the phylum (26), (9, 23), and (15, 27), respectively, are characterized by similar structures and functions (12, 25). TRAP proteins are important in parasite attachment and invasion of host cells (3, 10, 14, 20, 25). Thrombospondin-1 stimulates focal adhesion disassembly through a sequence known as the Hep 1 peptide, which then mediates signaling through a receptor co-complex involving calreticulin and a low-density lipoprotein receptor-related protein (20, 21). TRAP epitopes are recognized by both the humoral (2, 11) and cellular (13) immune O4I2 systems and serve as potential candidates for vaccines (5, 7). Recently, the gene encoding the thrombospondin-related adhesive protein of 1 1 (TRAP-C1) has been cloned and sequenced, and a fragment of the encoded polypeptide has been produced in sporozoites by immunolocalization, raising the possibility that this protein has adhesive properties similar to those described for other parasites. The objective of this study was to characterize the antibody response to recombinant TRAP-C1 (rTRAP-C1) in healthy volunteers exposed to and their association with clinical illness. MATERIALS AND METHODS Human subjects, evaluation of stools, and definition of terms. Informed consent was obtained from all participating volunteers. This study was approved by The University of Texas-Houston Health Science Center Committee for the Protection of Human Subjects. Blood was collected on days 0 to 5, 30, and 45 postchallenge from healthy volunteers who participated in studies of infectivity as previously described (18). All volunteers were seronegative with respect to whole-oocyst antigens, as determined by enzyme-linked immunosorbent assays (ELISA) prior to challenge. Seven volunteers received the TAMU isolate (inocula containing from 10 to 300 oocysts), and 24 volunteers received the UCP isolate (inocula containing from 500 to 105 oocysts). Sera were separated and stored at ?80C until use. Clinical information available included symptoms and characteristics of all stools passed for the first 2 weeks of the study and of two 24-h collections thereafter for a total of 6 weeks after challenge. Stools were examined for the presence of species by direct immunofluorescence assay (DFA) (6). Clinical and parasitologic data were categorized into the following groups. Volunteers with diarrhea included subjects who passed three unformed stools within an 8-h period, four or more unformed stools within a 24-h period, or stools with a total unformed weight of more than 200 g per 24-h period accompanied by at least two of the following gastrointestinal symptoms: abdominal pain and/or cramping, fecal urgency, excessive gas, tenesmus, nausea, or vomiting on at least one day during the duration of the study. Subjects were presumed infected when oocysts were detected in their stools by DFA or when they met the definition for diarrhea described above. Volunteers were presumed uninfected when they did not develop diarrhea as defined and stools were DFA negative. Purification of rTRAP-C1. Competent XL1-Blue MRF cells were transformed with a polyhistidine-tagged plasmid construct coding for the gene that expresses a fragment of TRAP-C1 from amino acid 295 to 491, as O4I2 previously described (24). Protein expression was induced with the addition of 2 mM isopropyl-d-thiogalactopyranoside (IPTG) (Sigma, St. Louis, Mo.). Protein purification of rTRAP-C1 from the supernatant was performed by nickel affinity chromatography, as previously described (24). Protein concentration was determined by a bicinchoninic acid protein assay (Pierce, Rockford, Ill.) O4I2 per the manufacturer’s instructions. ELISA for anti-rTRAP C1 antibodies. Optimal concentrations of antigen and serum dilutions were determined by checkerboard analysis. Individual wells of Nunc flat 96-well immuno plates (Maxisorp Immuno Plates, Rochester, N.Y.) were coated with 0.2 g of rTRAP-C1 in carbonate buffer (pH 9.5) overnight at 5C. The plates were Rabbit Polyclonal to TBC1D3 then washed six times with 0.2 M phosphate-buffered.