Opsonization of Treponema pallidum is mediated by immunoglobulin G antibodies induced only by pathogenic treponemes.

Rabbit antisera to Leptospira interrogans, Borrelia hermsii, and Treponema phagedenis biotype Reiter, reactive to shared spirochetal antigens, failed to enhance phagocytosis of Treponema pallidum by macrophages, while immunoglobulin G to Treponema pallidum subsp. pertenue and Treponema paraluiscuniculi promoted phagocytosis. Opsonic antibodies are directed to pathogen-restricted, not shared spirochetal, antigens.     

Molecular basis of immunological cross-reactivity between Treponema pallidum and Treponema pertenue

Protein antigens of Treponema pallidum, Nichols strain, and Treponema pertenue, Gauthier strain, were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. Treponemal proteins were solubilized in 1% sodium dodecyl sulfate, electrophoresed on 12.5% polyacrylamide gels, and either stained with Coomassie brilliant blue or electrophoretically transferred to nitrocellulose paper. These antigen blots were incubated with sera from rabbits infected with either T. pallidum or T. pertenue and 125I-labeled staphylococcal protein A and exposed to X-ray film for visualization of antigenic molecules. Protein profiles of each organism separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue showed no distinguishable differences. Antigenic profiles as determined by Western blots were similar with two exceptions. A 39,500-dalton band was present on T. pertenue but absent from T. pallidum, and a 19,000-dalton band was present on T. pallidum but absent from T. pertenue (although two additional antigenic bands at 21,000 and 18,000 daltons were seen on T. pertenue). Because these differences were detected by using antisera raised against either T. pallidum or T. pertenue, these molecules must contain some antigenic determinants in common despite their differences in molecular weight.     

Genome scale identification of Treponema pallidum antigens

Antibody responses for 882 of the 1,039 proteins in the proteome of Treponema pallidum were examined. Sera collected from infected rabbits were used to systematically identify 106 antigenic proteins, including 22 previously identified antigens and 84 novel antigens. Additionally, sera collected from rabbits throughout the course of infection demonstrated a progression in the breadth and intensity of humoral immunoreactivity against a representative panel of T. pallidum antigens.     

Antigenic cross-reactivity between Borrelia burgdorferi, Borrelia recurrentis, Treponema pallidum, and Treponema phagedenis

The antigenic cross-reactivity between different Borrelia and Treponema species was determined by the indirect immunofluorescence antibody test and sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by immunoblotting. The protein profiles of Borrelia burgdorferi, Borrelia recurrentis, Treponema pallidum, and Treponema phagedenis revealed essential differences. Using immunoblotting, rabbit immune sera to B. burgdorferi and B. recurrentis exhibited strong cross-reactivities to heterologous borrelial antigens and, to a lesser extent, to treponemal antigens. Immune sera to T. pallidum and T. phagedenis reacted with heterologous treponemal antigens, but exhibited lesser cross-reactivities to borrelial antigens. Five B. burgdorferi and seven T. pallidum major antigens were not cross-reacting with antisera raised against T. pallidum and B. burgdorferi, respectively. However, absorption of the investigated antisera with a T. phagedenis ultrasonicate eliminated cross-reacting borrelial and treponemal antibodies.     

Conservation of the 15-kilodalton lipoprotein among Treponema pallidum subspecies and strains and other pathogenic treponemes: genetic and antigenic analyses.

The 15-kDa lipoprotein of Treponema pallidum is a major immunogen during natural syphilis infection in humans and experimental infection in other hosts. The humoral and cellular immune responses to this molecule appear late in infection as resistance to reinfection is developing. One therefore might hypothesize that this antigen is important for protective immunity. This possibility is explored by using both genetic and antigenic approaches. Limited or no cross-protection has been demonstrated between the T. pallidum subspecies and strains or between Treponema species. We therefore hypothesized that if the 15-kDa antigen was of major importance in protective immunity, it might be a likely site of antigenic diversity. To explore this possibility, the sequences of the open reading frames of the 15-kDa gene have been determined for Treponema pallidum subsp. pallidum (Nichols and Bal-3 strains), T. pallidum subsp. pertenue (Gauthier strain), T. pallidum subsp. endemicum (Bosnia strain), Treponema paraluiscuniculi (Cuniculi A, H, and K strains), and a little-characterized simian isolate of Treponema sp. (Fribourg-Blanc strain). No significant differences in DNA sequences of the genes for the coding region of the 15-kDa antigen were found among the different species and subspecies studied. In addition, all organisms showed expression of the 15-kDa antigen as determined by monoclonal antibody staining. The role of the 15-kDa antigen in protection against homologous infection with T. pallidum subsp. pallidum Nichols was examined in rabbits immunized with a purified recombinant 15-kDa fusion protein. No alteration in chancre development was observed in immunized, compared to unimmunized, rabbits, and the antisera induced by the immunization failed to enhance phagocytosis of T. pallidum subsp. pallidum by macrophages in vitro. These results do not support a major role for this antigen in protection against syphilis infection.     

Tpr homologs in Treponema paraluiscuniculi Cuniculi A strain

Treponema paraluiscuniculi, the etiologic agent of rabbit venereal syphilis, is morphologically indistinguishable from Treponema pallidum subsp. pallidum (T. pallidum), the human syphilis treponeme, and induces similar immune responses and histopathologic changes in the infected host. Because of their high degree of relatedness, comparative studies are likely to identify genetic determinants that contribute to pathogenesis or virulence in human syphilis. The tpr (Treponema pallidum repeat) genes are believed to code for potential virulence factors. In this study, we identified 10 tpr homologs in Treponema paraluiscuniculi Cuniculi A strain and determined their sequence architecture. Half of this group of paralogous genes were predicted to be nonfunctional due to the presence of frameshifts and premature stop codons. Furthermore, the immune response against the T. paraluiscuniculi Tpr homologs in long-term-infected rabbits was studied by enzyme-linked immunosorbent assay and lymphocyte proliferation assay, showing that TprK is the only target of the antibody and T-cell responses during experimental infection and emphasizing the importance of this putative virulence factor in venereal treponematosis.     

Serotypes of beta-hemolytic Treponema hyodysenteriae.

Cultures form 13 isolates of pathogenic, beta-hemolytic Treponema hyodysenteriae from 11 geographically separate outbreaks and 2 experimentally induced cases of swine dysentery were lyophilized and extracted with hot phenol-water. The resulting water phases were examined serologically with antisera produced in rabbits against whole-cell bacterins of the 13 isolates for evidence of antigenic classes within the species. Water-phase antigens gave precipitin reactions with homologous antisera. Results from cross-testing of each water phase with each antiserum showed four serologically distinct groups among the isolants examined. Based on precipitin reactions in agarose gel, four serotypes of pathogenic, beta-hemolytic T. hyodysenteriae are proposed.     

Cloning and characterization of Treponema hyodysenteriae antigens and protection in a CF-1 mouse model by immunization with a cloned endoflagellar antigen.

We cloned genes that code for Treponema hyodysenteriae antigens into Escherichia coli with the purpose of identifying protective antigens for vaccine development. Three different genomic libraries were screened with various antisera reactive with T. hyodysenteriae antigens. The cloned antigens and corresponding native T. hyodysenteriae antigens were analyzed for molecular size, serum reactivity, solubility in sarcosine, and segregation during phase partitioning with the nonionic detergent Triton X-114. The results from these analyses suggested that the gene products were components of either the cytoplasmic membrane, periplasm, or endoflagella of T. hyodysenteriae. The cloned antigens were tested as vaccine candidates in a CF-1 mouse model of T. hyodysenteriae infection and immunity. Intraperitoneal injection of crude E. coli extracts containing cloned antigens did not protect mice from challenge. However, serum from mice injected with a crude extract of an E. coli clone which expressed an endoflagellar antigen killed T. hyodysenteriae in vitro. Partially purified preparations of this cloned endoflagellar antigen protected mice against oral challenge with both the homologous serotype (B204) and a heterologous serotype (B234) of T. hyodysenteriae. These results suggest that the endoflagellar proteins could be used as an effective subunit vaccine against T. hyodysenteriae.     

Circulating immune complexes in experimental syphilis: identification of treponemal antigens and specific antibodies to treponemal antigens in isolated complexes.

As a prelude to characterization of the host and treponemal antigens present in purified immune complexes from the sera of rabbits with disseminated syphilis, autoradiographic and immunoenzymatic analyses of solubilized extracts of Treponema pallidum, Treponema phagedenis biotype Reiter, and Treponema refringens were performed on electroblots of polypeptides first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electroblots of purified immune complexes were developed with the same panel of antisera so that protein profiles could be compared. Eight treponemal antigens were consistently present in isolated complexes; four of these cross-reacted with antisera prepared against avirulent treponemes. The average molecular weights of these antigens were 87,000, 76,000, 66,000, and 45,000. Antibodies dissociated from isolated immune complexes, when used for the development of T. pallidum electroblots, reacted with four antigens of comparable molecular weight. Antibodies to those polypeptides were also present in the sera of animals immunized with immune complexes. The demonstration of treponemal antigens in purified immune complexes convincingly argues that their occurrence in experimental syphilis is not merely due to tissue destruction and responses to endogenous host antigens.     

Serologic analyses of cottontail rabbits for antibodies to Borrelia burgdorferi.

An enzyme-linked immunosorbent assay was developed to detect antibodies to Borrelia burgdorferi in cottontail rabbits captured in Millbrook, N.Y., and New York, N.Y. Five antigenically variable strains of B. burgdorferi were analyzed to determine the variability of serologic test results. In analyses of 79 serum samples, seropositivity ranged from 56% for a strain cultured from kidney tissues of a cottontail rabbit to 68% for a strain isolated from a larva of Ixodes dentatus, a tick that parasitized a cottontail rabbit. There were false-positive results when reference rabbit antisera to B. hermsii and Treponema pallidum were screened against B. burgdorferi. Cross-reactivity with antisera to Leptospira interrogans serovars was less pronounced. Western blot (immunoblot) analyses revealed reactivities of test sera to two or more surface or subsurface proteins of B. burgdorferi with approximate molecular masses of 18, 25 to 27, 34, 36, 41, and 59 kilodaltons. Cottontail rabbits respond immunologically to B. burgdorferi, but the observed variations in serologic test results should not be a limitation in field and laboratory investigations of Lyme borreliosis.     

Molecular characterization of proteins from porcine spirochetes.

Sonicated preparations of Treponema hyodysenteriae and Treponema innocens were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Treponemal proteins were electrophoresed on a 10% polyacrylamide slab gel in a discontinuous Tris-glycine system and either stained with Coomassie blue dye or transferred electrophoretically at 20 mA for 16 h and 30 mA for 3 h to nitrocellulose paper. Staining of the gels revealed at least 42 distinct T. hyodysenteriae and T. innocens proteins, with molecular sizes ranging from greater than 100 to 14 kilodaltons (kDa). Each species contained 12 to 16 major protein bands; five of the proteins were common to both species. Fourteen major antigens were identified in T. hyodysenteriae isolate B204 by using serum specimens from pigs in the acute stage of swine dysentery. Twelve additional antigens were detected in isolate B204 when convalescent-phase serum specimens were reacted to the blot. A wide band at 16 kDa was identified with convalescent-phase serum specimens in T. hyodysenteriae but not in T. innocens. This 16-kDa antigen was also identified in T. hyodysenteriae with colonic secretions from convalescent pigs.     

Monoclonal antibody analysis of specific antigenic similarities among pathogenic Treponema pallidum subspecies

Murine monoclonal antibodies directed against a 47,000-dalton immunodominant surface-exposed antigen of Treponema pallidum subsp. pallidum (Nichols) were isolated. These monoclonal antibodies cross-reacted with analogous 47,000-dalton antigens of two other virulent treponemes, T. pallidum subsp. pertenue and T. pallidum subsp. endemicum (Bosnia A), as determined by radioimmunoassay and immunoblot analyses. Immunoelectron microscopy confirmed that the 47,000-dalton antigen of T. pallidum subsp. pallidum was a surface-associated cellular component. Surface binding assays and immunoelectron microscopic studies also suggested that the analogous 47,000-dalton antigenic component of T. pallidum subsp. pertenue may not have been oriented toward the bacterial surface in the same way as the T. pallidum subsp. pallidum antigen or that the relevant antigenic determinant(s) may not have been exposed to the outer surface in the same way. The significance of this antigen relative to its apparent conservation among pathogenic treponemes and its possible diagnostic and vaccinogenic potentials are discussed.     

Identification of the major antigens of Treponema hyodysenteriae and comparison with those of Treponema innocens.

Eleven strains of Treponema hyodysenteriae isolated from pigs with swine dysentery were examined by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. T. hyodysenteriae strains formed a homogeneous group with respect to sodium dodecyl sulfate-soluble proteins. However, immunoblotting with antiserum from rabbits immunized with T. hyodysenteriae CN8368 revealed heterogeneity among the lipopolysaccharide complexes of different strains. Polypeptides of molecular weights between 30,000 and 36,000 were the predominant T. hyodysenteriae polypeptides detected by porcine immune serum. In contrast, Treponema innocens did not form a homogeneous group with respect to sodium dodecyl sulfate-soluble proteins. Adsorption studies and immunoblotting identified polypeptide antigens present on cells of T. hyodysenteriae which were not detected on cells of T. innocens. These unique antigens may play a role in the virulence of T. hyodysenteriae.     

Surface-associated antigens of Treponema pallidum concealed by an inert outer layer.

Soluble antigens of Treponema pallidum were examined by two dimensional immunoelectrophoresis against antisera from infected or artificially immunized rabbits. Concentrated suspensions of intact cells did not release antigens after storage at 4 degrees, incubation at 37 degrees, or vortex mixing. Antigens were released after disintegration of treponemes by ultrasonic vibration, or by treatment with non-ionic or anionic detergents. An antigenic component of sonicated treponemes, present in both the non-pathogenic, cultivable Reiter treponeme and T. Pallidum, was identified as axial filament. The combination of antibody with unfixed whole organisms was monitored by an indirect fluorescent antibody method, and whereas antibody did not combine with intact organisms, detergent-treated organisms were highly reactive. Immune electron microscopy showed that whereas in intact treponemes, axial filaments were unable to combine with antibody, detergent treatment allowed access to axial filaments by antibody. In intact treponemes the axial filaments are thought to be located beneath the outer membrane, which may thus comprise the postulated antigenically inert outer layer.     

Lipooligosaccharides from Treponema hyodysenteriae and Treponema innocens.

Lipooligosaccharides from Treponema hyodysenteriae serotypes 1 through 7, attenuated T. hyodysenteriae serotypes 1 and 2, and five strains of T. innocens were extracted with hot phenol water. The extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and analyzed by lipopolysaccharide selective silver staining and Western blot (immunoblot) immunodetection. Silver staining revealed the presence of two bands that ranged between 18,000 and 24,000 daltons and that were serotype specific for T. hyodysenteriae. Attenuation of pathogenic strains resulted in the loss of the higher-molecular-weight band. Four of five T. innocens strains also lacked this particular band. T. innocens 421 had six bands between 17,000 and 26,900 daltons. Western blots with hyperimmune rabbit sera and convalescent-phase swine sera revealed antigenic variation among serotypes of T. hyodysenteriae and attenuated serotypes of T. hyodysenteriae. Convalescent-phase swine sera failed to recognize lipopolysaccharides from T. innocens. Differences in results obtained by lipopolysaccharide selective silver staining versus immunoblotting of the lipopolysaccharide preparations probably indicate that these two methods identify separate characteristics of the same molecule.     

Selective in vitro response of thymus-derived lymphocytes from Treponema pallidum-infected rabbits.

The blastogenic response of nylon wool-separated peripheral-blood lymphocytes from Treponema pallidum-infected rabbits was tested in vitro with mitogens and T. pallidum antigens. The mitogenic response of the enriched T-cell population to concanavalin A and phytohemagglutinin was depressed during the first 3 to 4 weeks of infection, similar to the pattern observed with unfractionated cells. Shortly thereafter, levels of blastogenesis returned to values of uninfected cultures. Enhanced blast transformation was seen immediately when purified T-cells from infected rabbits were exposed in vitro to T. pallidum antigens. Although these relatively high levels of blastogenesis were maintained for the duration of the experiment, cultures of unfractionated lymphocytes from infected rabbits did not exhibit an increased blastogenic response to the same antigen preparation until 3 to 4 weeks after infection. Autologous serum from infected rabbits decreased the lymphocyte response to T. pallidum antigen. The stimulatory effects of anti-immunoglobulin G and lipopolysaccharide on nylon wool-fractionated or unfractionated lymphocytes from both infected and control rabbits were similar throughout the course of infection. During the first 6 weeks of experimental disease, there was a 25 to 31% increase in the number of lymphocytes circulating in the peripheral blood of T. pallidum-infected rabbits.     

Host response to treponema pallidum infection. II. Rabbit leukocyte migration inhibition in the presence of homologous organ extracts.

Peripheral leukocytes of 67 rabbits infected intratesticularly with Treponema pallidum, Nichols' strain for various lengths of time were examined by the migration inhibition test for their response to extracts of normal rabbit heart, skin, brain and T. pallidum antigen. A control group of 14 animals injected intratesticularly with extract of normal rabbit tests was similarly examined for the leukocyte response to the same antigens except the brain extract. The percentage of infected animals responding to T. pallidum antigen with significant migration inhibition varied from 13 to 31. Transitional cellular response to heart and skin but not brain was observed (12-28%). Leukocytes of all but 2 control rabbits responded to the organ extracts within the limit of 2 SD. The response of the infected animals to the homologous organ extracts may suggest that during infection, lymphocytes are activated by the host tissue antigens.     

Extensive cross reactivity between Treponema pallidum and cultivable treponemes demonstrated by sequential immunoadsorption

Extensive cross reactivity between Treponema pallidum and three nonpathogenic species of treponemes was demonstrated by the Western blot technique. Rabbit antiserum produced by adjuvant immunization with solubilized T. pallidum antigens reacted with 34 T. pallidum antigens and with approximately 30 antigens each of T. phagedenis biotype Reiter, T. noguchii and T. vincentii. Adsorption of the antiserum with T. phagedenis Reiter removed only about half of the cross-reacting antibodies. Sequential adsorption with all three nonpathogenic treponemes removed antibodies to all but three polypeptides of 36,000, 34,000 and 27,000 daltons.     

Specific immunofluorescent staining of pathogenic treponemes with a monoclonal antibody.

Two hybrid cell lines which produced mouse monoclonal antibody to the DAL-1 street strain of Treponema pallidum subsp. pallidum were established. These monoclonal antibodies strongly reacted with T. pallidum subsp. pallidum (Nichols strain, DAL-1, and two other street strains, strains MN-1 and MN-3) and T. pallidum subsp. pertenue by indirect microimmunofluorescent antibody and enzyme-linked immunosorbent assay techniques, but they did not react with normal rabbit testicular tissue. These monoclonal antibodies did not react with nonpathogenic treponemes, such as T. phagedenis Reiter, T. denticola MRB, T. refringens Noguchi, or other spirochetes, such as Borrelia burgdorferi and Leptospira interrogans serovar pomona in microimmunofluorescent antibody smear slides or in Western blots (immunoblots). While unlabeled antibodies are useful for investigating the antigenic structures of T. pallidum, we labeled these monoclonal antibodies with fluorescein isothiocyanate and used them for diagnosing syphilis by direct staining of lesion exudate or T. pallidum subsp. pallidum in formalin-fixed tissues from patients suspected of having syphilis. Both monoclonal antibodies were directed against antigens of T. pallidum subsp. pallidum with a molecular weight of 37,000 as determined by the Western blotting technique.     

Cell-mediated immunity in Treponema pallidum infected rabbits: in vitro response of splenic and lymph node lymphocytes to mitogens and specific antigens.

Peripheral blood lymphocytes from Treponema pallidum infected rabbits respond poorly to mitogen and specific antigens when cultured in the presence of autologous serum. Reactivity of lymphocytes from the spleen and popliteal lymph nodes of T. pallidum infected rabbits have therefore been examined by lymphocyte transformation using the mitogens phytohaemagglutinin (PHA) and concanavalin A (Con A) and extracts of T. pallidum. Spleen cell populations, both T cell enriched (by nylon wool elution) and non-nylon wool treated, which respond to T. pallidum as early as ten days post infection in normal serum, were suppressed in responses to T. pallidum when cultured in autologous serum. The same lymphocytes responded normally to PHA and Con A. Lymph node cells from infected rabbits responded normally to both T. pallidum antigen and mitogens in either autologous or normal rabbit serum. These data indicate that splenic lymphocytes are sensitive to regulatory factors in autologous serum during the early stages of T. pallidum infection whereas lymph node cells are not.