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Infect Immun. 2006 November; 74(11): 6244–6251. Published online 2006 August 21. doi: 10.1128/IAI.00827-06. | PMCID: PMC1695500 |
Copyright © 2006, American Society for Microbiology Antigenic Variation of TprK V Regions Abrogates Specific Antibody Binding in Syphilis ![[down-pointing small open triangle]](/corehtml/pmc/pmcents/x25BF.gif) † Rebecca E. LaFond,1 Barbara J. Molini,2 Wesley C. Van Voorhis,1,2 and Sheila A. Lukehart1,2* Departments of Pathobiology,1 Medicine, University of Washington, Seattle, Washington2 Received May 22, 2006; Revised June 25, 2006; Accepted August 11, 2006. The tprK gene in the syphilis spirochete, Treponema pallidum subsp. pallidum, undergoes antigenic variation in seven variable (V) regions. tprK is highly variable within T. pallidum strains, and a method has been developed to derive clones of T. pallidum that express a single, unique tprK sequence. Rabbits were infected with three different T. pallidum clones or the parent strain from which the clones were derived, and their sera were examined by immunoassay for antibody reactivity against synthetic peptides representing the TprK V regions from each clone. The parent strain expresses many different V region sequences, and infection with this strain induced antibody responses against a wide variety of V regions. In rabbits infected with the Chicago C clone, antibodies developed against all of the V regions except V1, while antibodies developed against only V5, V6, and V7 in Chicago A-infected rabbits. During Chicago B infection, antibodies developed against all of the V regions except V1 and V3. Antibodies were highly specific for the V regions of the infecting clone, and cross-reactivity was rare. The demonstration that the V regions 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 T. pallidum to establish chronic infection. Treponema pallidum subsp. pallidum causes syphilis, a multistage disease that persists in the absence of appropriate antibiotic therapy. Strains of T. pallidum isolated from infected individuals are made up of a mixed population of organisms carrying diverse tprK alleles ( 4, 8, 15). tprK belongs to the 12-member tpr gene family, and it is the only one of the tpr genes shown thus far to be heterogeneous within a single T. pallidum strain. Several other tpr genes (e.g., tprC and - D) vary in sequence among strains ( 6, 17). The sequence of tprK is heterogeneous in seven distinct variable (V) regions. These V regions are flanked by unique 4-bp repeats, and gene conversion with potential donor sequences found upstream and downstream of the tprD gene has been proposed as a mechanism by which new V region sequences are created ( 5). The 92-kDa TprK protein is predicted to have a cleavable signal sequence and two hydrophobic transmembrane domains ( 3), making it a putative outer membrane protein. Examination of the immune response to TprK determined that infection with T. pallidum induces antibodies against the V regions, while T-lymphocyte activity is focused on the constant regions ( 14). This suggests that variability in tprK may permit the organism to escape the antibody response in the infected host. Using an in vivo method of cloning T. pallidum, we have derived three T. pallidum clones from a single parent strain, and we demonstrate a high level of specificity in antibody responses against the TprK V regions. These results provide further evidence that antigenic variation of TprK abrogates binding of existing antibodies and thus may contribute to the ability of T. pallidum to evade host immunity to establish chronic infection. Isolation of T. pallidum clones. T. pallidum clones were derived from the T. pallidum Chicago parent strain, which is highly diverse in tprK, as described previously ( 4, 5). Briefly, the Chicago parent was inoculated intravenously into a rabbit whose back was kept free of hair. As disseminated lesions developed on the back, three lesions were biopsied and treponemes extracted from the biopsy tissue were propagated intratesticularly in individual naïve rabbits. Treponemes were then harvested at the peak of intratesticular infection (judged by orchitis) and subjected to another round of intravenous inoculation, lesion biopsy, intratesticular propagation in naïve rabbits with treponemes extracted from skin biopsy tissue, and harvest of treponemes from testis tissue. The resulting three T. pallidum 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 tprK gene may take place during propagation. To determine the sequence of tprK in the T. pallidum clones used in the experiments described below, DNA was extracted, the tprK gene was amplified, and the amplicon was ligated into the pCR-II TOPO vector (Invitrogen) and sequenced as previously described ( 8). Infection with the T. pallidum Chicago parent and isolated clones. Each T. pallidum clone and the Chicago parent strain were harvested from infected rabbit testes by mincing the testis tissue in 0.9% saline-10% normal rabbit serum. Treponemes were quantitated by dark-field microscopy, and the treponemal suspension was diluted to 106 T. pallidum organisms per ml. In a preliminary experiment, three rabbits were infected with Chicago C; in 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 on 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 T. pallidum clones and the Chicago parent; serum was extracted and heated for 30 min at 56°C. Immunoassays using clone-specific TprK V region synthetic peptides. Synthetic peptides used in the immunoassays represent each V region (see Fig. ) 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% pure by high-pressure liquid chromatography. Enzyme-linked immunosorbent assays (ELISAs) were performed in triplicate as previously described ( 14). Each peptide was diluted in phosphate-buffered saline (pH 7.2) to a concentration of 10 μg/ml, and 96-well plates (MaxiSorp Immunoassay; Nunc, Rochester, NY) were coated with 50 μl peptide and incubated overnight at 4°C. Plates were washed and blocked as previously described ( 14). Sera from day 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 μl serum was added to each well, and the plates were incubated for 1 hour at 37°C. 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 temperature ( 14). Plates were washed three times and then developed with 50 μl/well of 1-mg/ml para-nitrophenylphosphate (Sigma-Aldrich, St. Louis, MO) for 1 hour at room temperature. Absorbance in wells was measured at 405 nm, and the mean ± standard error for triplicate wells was calculated for synthetic peptides tested against sera from all time points from each rabbit. A negative reaction by ELISA may occur if the peptide is not adhering to the plate. For this reason, those peptides that were not recognized in ELISA by any postinfection rabbit serum sample were assayed by dot blotting. Polyvinylidene difluoride membranes were placed briefly in 100% methanol, washed with 500 ml H2O for 5 min at room temperature, and equilibrated with 0.05% Tween in Tris-buffered saline (TBST) for 10 min at room temperature. A 96-well dot blot vacuum apparatus was used to spot 5 μl of 200-μg/ml V2A, V3A, V3B, V4B, and V5A/B peptides onto membranes. As positive controls, peptides that reacted against sera by ELISA were also spotted onto membranes. Membranes were fixed with 10% formalin for 1 h at room temperature and, after being washed with TBST for 5 min, blocked with 3% NFM diluted in TBST overnight at 4°C. Sera from day 0 and day 90 postinfection for each rabbit were diluted in 1.5% NFM-TBST to a final concentration of 1/250; sera were added to peptide-spotted membranes and incubated for 2 h at room temperature. Membranes were washed three times as described above and then incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G antibody (Southern Biotech) for 1 h at room temperature. After washing with TBST four times for 10 min, positive peptide-antibody reactions were detected by enhanced chemiluminescence reagents (Amersham Biosciences). Development of clonal populations of T. pallidum. To study the immune response to TprK variable regions, three separate populations of T. pallidum with limited tprK diversity were derived from the Chicago strain as described previously ( 5). Because these derived populations each arise from a single lesion and are significantly less diverse than the Chicago parent strain ( 4, 5), we refer to the populations as “clones.” Ten tprK sequences were determined for each T. pallidum clone, and the translated amino acid sequences of the tprK V4, V6, and V7 sequences are shown in Fig. (the remaining V region sequences are shown in Fig. S1 in the supplemental material). In contrast to those of the parent Chicago strain (not shown), many of the V regions from the clones have single unique sequences (Fig. ; see Fig. S1 in the supplemental material), and all V regions have identical sequences for at least 7 of the 10 sequences per clone. The single exception is V6 in Chicago B, in which only 2 sequences of 10 are identical (Fig. ). The majority sequence for each V region defines the TprK sequence for each clone. In many cases, minority V region sequences have only a single base pair change which causes an amino acid change, such as a G-to-A change in V7 of Chicago A and an N-to-S change in V4 of Chicago C (Fig. ). These and more extensive changes can generally be explained by gene conversion events between tprK V regions and the donor cassettes found upstream and downstream of the tprD allele ( 5). The fact that these changes occurred during limited propagation in the rabbit indicates the rapid rate of sequence change in some V regions and some T. pallidum clones (e.g., V6 in Chicago B). Antibodies from Chicago C-infected rabbits do not recognize TprK peptides from other T. pallidum clones. Infection with T. pallidum elicits antibodies against the V regions of TprK but not against the constant regions ( 14). Because Chicago A, Chicago B, and Chicago C are derived from the same parent strain and are therefore perhaps more likely to have TprK V region sequences that are more related than V region sequences between two different strains, we performed an initial experiment to determine whether infection with Chicago C would elicit antibodies against the TprK V region peptides corresponding to the three Chicago clones. The sequences of the synthetic peptides used to test antibody reactivity are shown in Fig. . Sera collected at days 0, 30, 60, and 90 after intradermal infection of three rabbits with Chicago C were tested for antibody reactivity by ELISA. Antibodies from all three animals reacted with peptides specific for only V4, V6, and V7 of the Chicago C clone (Fig. ). Antibodies did not develop against the corresponding peptides in Chicago A or Chicago B. Stretches of V4 and V7 sequence are shared among the three clones (Fig. ); thus, minimal sequence change to a small portion of a V region appears to be sufficient to abrogate the ability of anti-Chicago C antibodies to bind to nonhomologous peptides. These data demonstrate exquisite specificity of the antibody response against the TprK V regions. Specificities of antibodies from rabbits infected with Chicago A, Chicago B, and Chicago C. In the experiments described above, rabbits were infected with one T. pallidum clone (Chicago C). To expand our investigation of V region-specific antibody reactivity to clones with other TprK variants, one group of three rabbits was infected with T. pallidum Chicago C, while two other groups of three rabbits each were infected with either Chicago A or Chicago B. Sera were collected at the time points described above and tested by ELISA against the V region peptides; the results for V7 are shown in Fig. . Antibodies from rabbits infected with each of the clones reacted only against the homologous V7 peptide and not against V7 peptides corresponding with the other two clones. Table 1 shows the highest mean antibody responses by ELISA against the V region peptides at any time point postinfection in rabbits infected with Chicago A, Chicago B, or Chicago C. Peptides that did not react with infected rabbit sera by ELISA were reassayed by dot blotting; these results are also shown in Table 1. In most cases, infection with a T. pallidum clone elicited antibodies against only homologous synthetic peptides (Table 1). Chicago C infection induced the development of specific antibodies against a greater number of V regions than infection with either Chicago A or Chicago B. All three Chicago C-infected rabbits developed specific antibodies against V5C, V6C, and V7C; two rabbits developed specific antibodies against V4C; and one rabbit each developed specific antibodies against V2B/C and V3C (Table 1). None of the Chicago C-infected rabbits developed an antibody response against V1A/C or, except for V5A/B, against any of the Chicago A- or Chicago B-specific peptides. | TABLE 1.Reactivity of antibodies from rabbits infected with T. pallidum clones against homologous and heterologous TprK V region peptides |
Although there was no difference in mean lesion size and time to ulceration among the groups infected with the different clones, the rabbits infected with the Chicago A clone developed antibodies against fewer V regions than those infected with Chicago C. Responses against the V5A/B and V7A peptides were elicited in all three rabbits, and antibodies against V6A were seen in one rabbit (Table 1). Similarly, all three Chicago B-infected rabbits developed specific antibodies against the homologous V5A/B and V7B peptides, and one rabbit developed specific antibodies against V6B. Additionally, Chicago B infection elicited the development of antibodies against V2B/C and V3B. While Chicago C-infected rabbits developed antibodies against V4C, infection with the Chicago A and Chicago B clones did not elicit antibodies against the homologous V4 peptides (Table 1). Nearly all of the rabbits infected with T. pallidum clones developed antibodies against both the V5A/B and V5C peptides. Infection with the T. pallidum Chicago parent elicits antibodies against a wide variety of V region peptides. The T. pallidum Chicago strain is known to be highly diverse in most tprK V regions ( 4, 5); therefore, it was not surprising that rabbits infected with this strain developed antibody reactivity against a variety of the TprK V region peptides. All three rabbits made antibody to the V7 peptide from Chicago B (V7B), and two of the rabbits produced antibodies against V7A and V7C (Fig. ). In all, 13 of the 18 V region peptides were recognized by antibodies from at least one of the Chicago-infected rabbits (Fig. ). Antibody responses elicited against the V regions due to infection with the Chicago strain were variably reactive. For example, antibodies against the V4C and V7B peptides were strongly reactive, while moderate antibody responses against V6B, V6C, V7A, and V7C were evident in all rabbits. A weak to moderate antibody response was induced against the V1 and V2 peptides in some Chicago-infected rabbits. These results mirror those observed for the T. pallidum clones, in that Chicago infection induced antibodies against all three V7 peptides and against V4C, V5A/B, and V5C. Antibodies against V2B/C, but not against V2A, V3A, or V3C, developed in Chicago-infected rabbits, similar to the pattern of antibody responses in Chicago A-, Chicago B-, and Chicago C-infected animals. Heterogeneous tprK sequences have been found in every T. pallidum strain that has been examined to date. We have shown that T. pallidum strains with little or no tprK diversity become more diverse when placed under immune pressure in rabbits ( 5, 9), and we have proposed a gene conversion method through which tprK diversity may develop ( 5). Anti-TprK antibodies elicited during infection are targeted to the V regions ( 14); therefore, generation of heterogeneity in the tprK gene results in antigenic variation in T. pallidum. To further study antibody reactivity against different TprK proteins, we used a newly developed cloning procedure ( 5) to derive three clones from a T. pallidum parent with diverse tprK sequences. The clones used in these experiments each underwent a double-cloning step. Although the clones usually carry a single tprK sequence when first obtained by skin lesion biopsy, even a single rapid propagation in testes to obtain enough treponemes for our experiments resulted in the acquisition of a small degree of sequence variability in some clones. V6, which has a greater degree of diversity than the other V regions ( 5, 8), had greater sequence variability in the Chicago B strain than in the Chicago A, and Chicago C strains (Fig. ). We speculate that some V6 sequences may undergo gene conversion more readily than others, and we are currently performing experiments to formally examine the kinetics of gene conversion in the tprK V regions. Rabbits infected with the T. pallidum clones mount specific antibody responses against many of the V region sequences expressed in the infecting strain. Antibody reactivity against V2, V3, V4, V6, and V7 was highly specific. For example, only the rabbits infected with Chicago B, Chicago C, or the Chicago parent strain developed antibodies against V2B/C (Table 1 and Fig. ), and similarly, antibodies against V4C were evident only in those rabbits infected with Chicago C or the Chicago parent. No antibodies were elicited against the V2A sequence, suggesting that this peptide is not immunogenic and that PNGNVPAGV, the N-terminal portion of the sequence unique to V2B/C (Fig. ), is immunogenic. Chicago C-infected rabbits develop cross-reactive antibodies against the V2 peptide that correspond with the Nichols TprK sequence ( 13), which contains the amino acid sequence GNVPAGV. This same sequence is found in V2B/C, implicating GNVPAGV as the reactive portion of the sequence. In contrast, V7 antibody reactivity is specific in Chicago and Nichols ( 13), and sequence differences between Nichols V7 and Chicago C V7 are found across the length of the peptide. V7 reactivity is also specific in Chicago A, Chicago B, and Chicago C infection-induced antibodies; the few differences in the V7A, V7B, and V7C sequences are found in the N-terminal half of the peptide (Fig. ). This demonstrates that V7 sequences are more similar within the clones derived from the Chicago strain than between different strains and suggests that the N-terminal portion of V7 may be the B-cell epitope. In this way, assaying antibody reactivity against a wide variety of V region peptides is useful in determining finer epitope mapping. Like V2A, other V region sequences, including V4A and V4B, did not induce the development of antibodies that were detectable with either immunoassay. Another reason that certain peptides may not react with infection-induced antibodies is because they may be part of a conformation-dependent epitope. Several bacterial proteins have been shown to contain conformation-dependent B-cell epitopes. Two monoclonal antibodies against pneumolysin, a cytolytic protein produced by Streptococcus pneumoniae, recognize 6- to 9-amino-acid-long linear epitopes on the protein, while one monoclonal antibody recognizes only those fragments of pneumolysin that are 150 amino acids in length or longer ( 16), suggesting that conformation is critical for this epitope. A neutralizing monoclonal antibody against the major outer membrane protein (MOMP) of Chlamydia pneumoniae reacts against immunoprecipitated, but not against denatured, forms of the protein ( 19). Similarly, a monoclonal antibody against MOMP in Chlamydia trachomatis loses its neutralizing activity when preabsorbed with viable elementary body forms of the organism but maintains neutralizing activity when heat-treated elementary bodies or short MOMP peptides are used for preabsorption ( 7). In the same way, certain V region sequences may be part of a conformational epitope. For example, antibody reactivity to V4A may depend upon the secondary structure of a larger section of TprK that includes V4A, while antibodies against V4C are able to recognize the linear peptide and therefore are not dependent upon the conformation of the protein. The immunoassays in our studies use linear synthetic peptides of 19 to 29 amino acids in length to test for reactivity of polyclonal antibodies against TprK. It is possible that immunoassays using longer peptides with folded structure would reveal additional anti-TprK antibodies. It is also possible that different TprK V region sequences may have an effect on the folding of all or part of the TprK protein and thus may change the portions of TprK that are exposed to antibody-producing cells. An individual T. pallidum clone expresses a unique majority TprK sequence, and therefore TprK is likely to be folded into the same structure in the majority of organisms in a given clone. In contrast, the Chicago parent strain expresses many different TprK proteins, and these different proteins may have different structures. Some V region peptides, such as V1A/C and V1B, did not react with antibodies from rabbits infected with T. pallidum clones but did react with antibodies elicited by infection with the Chicago parent strain (Table 1; Fig. ). We speculate that folding of TprK may expose a region on the surface of the protein, making it accessible to the immune response and a potential target for antibody production. In this way, a V region that did not induce an antibody response during infection with a T. pallidum clone might gain the ability to induce an antibody response during infection with a mixed population carrying different V region sequences. The greatest degree of cross-reactivity was observed among antibodies against the V5 peptides (Table 1). This cross-reactivity suggests that the shared portion of V5, ASQASNVFQGVFLT (Fig. ), is antigenic and also indicates that changes in a V region do not always abrogate antibody binding. However, peptides with changes in the shared portion of V5 have not been tested for their ability to bind. Changes in this portion of V5 have been observed in other T. pallidum strains ( 4), so the possibility that sequence changes are able to prevent the binding of antibodies to V5 cannot be ruled out. The T. pallidum Chicago parent strain is comprised of subpopulations carrying different tprK sequences ( 4, 5) and undoubtedly expresses more different TprK sequences than the Chicago A, Chicago B, and Chicago C clones. Therefore, the antigenic burden of individual V region sequences is lower in Chicago-infected rabbits than in rabbits infected with one of the clones. In spite of this, infection with the Chicago parent strain elicits strong antibody reactivity against several V regions, including V6, one of the most diverse V regions in TprK. This suggests that antibody reactivity develops even to some minority sequences, and it provides further evidence that some TprK V regions are immunodominant B-cell epitopes. Syphilis-infected humans carry populations of T. pallidum that are diverse in tprK ( 8), so antibodies against a wide variety of TprK V regions, like those seen in rabbits infected with the diverse Chicago strain, are likely to develop. Furthermore, the levels of antibodies that develop against TprK V regions may vary in different infected humans, as they did in the different outbred rabbits used in these experiments. During infection of both rabbits and humans, T. pallidum disseminates widely and rapidly from the site of entry, before organisms have proliferated to a sufficient number to induce an immune response. By the time an initial antibody response is mounted against TprK, some organisms at the site of T. pallidum entry and at distant sites may have varied their TprK V regions so that the antibodies do not bind. T. pallidum is known to survive for years in a wide variety of tissues, including “immune privileged” sites such as the central nervous system and the eye. Organisms that resisted the initial wave of immune clearance may continue to proliferate slowly, continuing to vary their tprK gene, which enables them to avoid existing and newly developed anti-TprK antibodies. T. pallidum is cleared from syphilitic lesions when infiltrating macrophages are activated by gamma interferon to phagocytose treponemes ( 10, 11, 18). Recognition of T. pallidum by macrophages is enhanced by opsonizing antibodies ( 1, 2), and we have reported that anti-TprK antibodies are opsonic ( 3). Furthermore, a few T. pallidum organisms remaining at the site of infection after the majority of organisms are able to resist phagocytosis by macrophages ( 12), suggesting that the surviving organisms possess distinct traits making them “invisible” to macrophages. Different TprK sequences may contribute to the ability of T. pallidum to avoid phagocytosis, a speculation that is supported by the finding that rabbits with preexisting antibody immunity against the Nichols TprK sequence are partially protected against Nichols infection but are less well protected against infection with T. pallidum strains expressing heterologous TprK sequences ( 13). The results presented here lend further evidence to the hypothesis that changes in TprK V regions in some organisms may render them resistant to binding by existing opsonic antibodies and therefore less likely to be recognized by activated macrophages. This ability to undergo antigenic variation may explain why syphilis is a chronic infection in the untreated host. This work was supported by National Institutes of Health grants AI63940, AI34616, and AI42143 (to S.A.L.) and AI43456 (to W.C.V.V.). R.E.L. was supported by Institutional Training Grant AI07140 from the NIH. We are grateful to Heidi Pecoraro for manuscript preparation and to Arturo Centurion-Lara for helpful discussions. 1. Baker-Zander, S. A., and S. A. Lukehart. 1992. Macrophage-mediated killing of opsonized Treponema pallidum. J. Infect. Dis. 165:69-74. [PubMed] 2. Baker-Zander, S. A., J. M. Shaffer, and S. A. Lukehart. 1993. Characterization of the serum requirement for macrophage-mediated killing of Treponema pallidum ssp. pallidum: relationship to the development of opsonizing antibodies. FEMS Immunol. Med. Microbiol. 6:273-279. [PubMed] 3. Centurion-Lara, A., C. Castro, L. Barrett, C. Cameron, M. Mostowfi, W. C. Van Voorhis, and S. A. Lukehart. 1999. 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