Antibodies were highly specific for the V regions of the infecting clone, and cross-reactivity was rare. illness, antibodies formulated against all the V areas except V1 and V3. Antibodies were highly specific for the V regions of the infecting clone, and cross-reactivity was rare. The demonstration the V areas elicit a variant-specific antibody response supports the hypothesis that TprK variants may help organisms to avoid the developing immune response in infected individuals, contributing to the ability of to establish chronic illness. subsp. causes syphilis, a multistage disease Dauricine that persists in the absence of appropriate antibiotic therapy. Strains of isolated from infected individuals are made up of a combined population of organisms carrying varied alleles (4, 8, 15). belongs to the 12-member gene family, and it is the only one of the genes demonstrated thus far to be heterogeneous within a single strain. Several other genes (e.g., and -is definitely heterogeneous in seven unique variable (V) areas. These V areas are flanked by unique 4-bp repeats, and gene conversion with potential donor sequences found upstream and downstream of the gene has been proposed like a mechanism by which new V region sequences are created (5). The 92-kDa TprK protein is predicted to have a cleavable transmission sequence and two hydrophobic transmembrane domains (3), making it a putative outer membrane protein. Examination of the immune response to TprK identified that illness with induces antibodies against the V areas, while T-lymphocyte activity is focused within the constant areas (14). This suggests that variability in may permit the organism to escape the antibody response in the infected sponsor. Using an in vivo method of cloning clones from a single parent strain, and we demonstrate a high level of specificity in antibody reactions against the TprK V areas. These results provide further Dauricine evidence that antigenic variance of TprK abrogates binding of existing antibodies and thus may contribute to the ability of to evade sponsor immunity to establish chronic infection. MATERIALS AND METHODS Isolation of clones. clones were derived from the Chicago parent strain, which is definitely highly varied in clones are called Chicago A, Chicago B, and Chicago C. After the cloning process, the Chicago A clone was propagated intratesticularly two times, and Chicago B and Chicago C were propagated intratesticularly once. Changes to the V regions of the gene may take place during propagation. To determine the sequence of in the clones used in the experiments explained below, DNA was extracted, the gene was amplified, and the amplicon was ligated into the pCR-II TOPO vector (Invitrogen) and sequenced as previously explained (8). Infection with the Chicago parent and isolated clones. Each clone and the Chicago parent strain were harvested from infected rabbit testes by mincing the testis cells in 0.9% saline-10% normal rabbit serum. Treponemes were quantitated by dark-field microscopy, and the treponemal suspension was diluted to 106 organisms per ml. In a preliminary experiment, three rabbits were infected with Chicago C; inside a subsequent experiment, four groups of three rabbits were infected with the Chicago parent, Chicago A, Chicago B, and Chicago C. Each group of three rabbits was injected intradermally at 12 sites within the clipped back with 0.1 ml of treponemal suspension. After all intradermal injections were completed, remaining treponemes were judged to be viable by active motility observed by dark-field microscopy. At 30, 60, and 90 days postinfection, blood was collected from rabbits infected with the clones and the Chicago parent; serum was extracted and heated for Dauricine 30 min at 56C. Immunoassays using clone-specific Rabbit Polyclonal to BRP16 TprK V region synthetic peptides. Synthetic peptides used in the immunoassays represent each V region (observe Fig. ?Fig.2)2) and were made on a Rainin-PTI Symphony instrument (Fred Hutchinson Cancer Research Center, Seattle, WA). Peptides were subjected to a desalting step and found to be at least 70% genuine by high-pressure liquid chromatography. Enzyme-linked immunosorbent assays (ELISAs) were Dauricine performed in triplicate as previously explained (14). Each peptide was diluted in phosphate-buffered saline (pH 7.2) to a concentration of 10 g/ml, and 96-well plates (MaxiSorp Immunoassay; Nunc, Rochester, NY) were coated with 50 l peptide and incubated over night at 4C. Plates were washed and clogged as previously explained (14). Sera from day time 0 and the various time points postinfection were each diluted in 10% nonfat milk (NFM) in phosphate-buffered saline to a final concentration of 1/20; 100 l serum was added to each well, and the plates were incubated for 1 hour at 37C. Plates were washed three times and then incubated with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G antibody (Sigma-Aldrich, St. Louis, MO) for 1 hour at room temp (14). Plates were.