Development of monoclonal antibodies that recognize Treponema pallidum.

We developed a panel of monoclonal antibodies to Treponema pallidum (Nichols) antigens, some of which recognize treponemal antigens on T. pallidum (Nichols), T. pallidum strain 14, and Treponema phagedenis biotype Reiter. The antibodies were detected by either an enzyme-linked immunosorbent assay or a radioimmunoassay.     

Experimental congenital syphilis: guinea pig model.

Neonates born to female guinea pigs of either a highly susceptible (C4D) or a resistant (Albany) strain, infected prior to or during pregnancy with a single dose of Treponema pallidum, showed in their sera from the first day of life immunoglobulin M (IgM) antibodies to T. pallidum, circulating immune complexes consisting of IgM antibodies and treponemal antigens, and IgM rheumatoid factor. Although the animals were asymptomatic for a 6-month observation period, several lines of evidence indicated that they were infected in utero. Molecular analysis of whole sera, purified serum IgM fraction, or dissociated immune complexes demonstrated IgM reactivity against one (47 kDa) or more of several T. pallidum peptides (15, 17, 37, 42, 45, and 87 kDa) recognized as integral membrane components. Sequential analysis of the neonates' sera by immunoblot and enzyme-linked immunosorbent assay, using alcohol-treated T. pallidum, T. phagedenis biotype Reiter, and T. vincentii, demonstrated early IgM antibodies followed 3 to 4 months later by IgG2- and IgG1-specific antibodies to T. pallidum. Moreover, an infectivity test done in five rabbits with pooled tissue extracts prepared from liveborn or stillborn animals evoked a seroconversion in two rabbits (reactive Venereal Disease Research Laboratory and fluorescent treponemal antibody tests), suggesting the presence of T. pallidum in the organs. Sera from neonates born to either T. phagedenis biotype Reiter-injected mothers or three normal pregnant females were all serologically negative. The model offers new possibilities for exploration of factors responsible for asymptomatic infection often observed in human congenital syphilis.     

Cloning and expression of the major 47-kilodalton surface immunogen of Treponema pallidum in Escherichia coli.

Monoclonal antibodies directed against the 47-kilodalton (kDa) major outer membrane surface immunogen of virulent Treponema pallidum subsp. pallidum were used to select Escherichia coli recombinant clones expressing the 47-kDa immunogen. The phenotype of the clones was dependent on the presence of recombinant plasmid in the host cell. Southern hybridization revealed that the cloned T. pallidum subsp. pallidum DNA sequence was an accurate representation of the T. pallidum subsp. pallidum genomic DNA arrangement. Purified immunoglobulin G from rabbits experimentally infected with T. pallidum subsp. pallidum and human secondary syphilitic sera specifically reacted with the clones, while normal human serum or immunoglobulin G from normal rabbit serum did not. Results of Southern hybridization indicated that a homologous 47-kDa immunogen gene was absent in at least four species of nonpathogenic treponemes tested, as well as from total rabbit genomic DNA. Rabbit anti-T. phagedenis biotype Reiter (treponemal nonpathogen) antiserum and a monoclonal antibody directed against a common treponemal determinant were unreactive with the clones. Western blotting and radioimmunoprecipitation experiments with specific monoclonal antibodies revealed that the recombinant (E. coli) and native (T. pallidum subsp. pallidum) forms of the antigen had identical electrophoretic mobilities. The availability of recombinant 47-kDa immunogen provides a new opportunity for biochemical analysis of the protein, structure-function studies, examination of its role in microbial pathogenesis, and assessment of its diagnostic and vaccinogenic potentials.     

Antigenic cross-reactivity between Treponema pallidum and other pathogenic members of the family Spirochaetaceae.

The antigenic cross-reactivity between Treponema pallidum and several pathogenic members of the family Spirochaetaceae was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. Blots of T. pallidum antigens were incubated with antiserum from rabbits infected or immunized with T. pallidum, Treponema paraluiscuniculi, Treponema hyodysenteriae (strains B204 and T22), Borrelia hermsii serotype 7, or Leptopsira interrogans serogroup Canicola. T. pallidum contained 22 antigenic molecules ranging from 85,000 to 12,000 daltons which were recognized by serum from rabbits infected with T. pallidum. Serum from rabbits infected with T. paraluiscuniculi cross-reacted with 21 of these molecules and faintly reacted with a band at 15,000 daltons which was not recognized by anti-T. pallidum serum. Antisera directed against strains B204 and T22 of T. hyodysenteriae cross-reacted with 11 and 10 antigens of T. pallidum, respectively. B. hermsii and L. interrogans serogroup Canicola antisera detected 11 and 10 treponemal antigens, respectively. Many of the T. pallidum antigens detected by antisera against T. hyodysenteriae, B. hermsii, or L. interrogans serogroup Canicola have been previously identified as containing moieties also found on the nonpathogenic Treponema phagedenis, biotype Reiter, and may therefore represent group antigens common to members of the family Spirochaetaceae.     

Sensitivity and specificity of monoclonal antibodies directed against antigenic determinants of Treponema pallidum Nichols in the diagnosis of syphilis.

Murine anti-Treponema pallidum monoclonal antibodies were employed in studies on sensitivity and specificity of binding to examine their potential for use in the detection of low numbers of pathogenic treponemes present in various body fluids. Monoclonal antibodies were used as a primary antibody source in a solid-phase immunoblot assay system. All monoclonal antibodies assayed were capable of detecting ca. 1.0 X 10(3) to 2.5 X 10(3) treponemes. Of 13 monoclonal antibodies examined, 3 were able to detect 10(3) virulent treponemes, and 1 of these antibodies was able to reveal the presence of as few as 500 organisms. Western blot analyses showed that all anti-T. pallidum monoclonal antibodies exhibiting high sensitivities for the detection of T. pallidum cells were directed against an abundant, 47,000-dalton surface-exposed antigen of the organism (S. A. Jones, K. S. Marchitto, J. N. Miller, and M. V. Norgard, Abstr. Annu. Meet. Am. Soc. Microbiol. 1984, B173, p. 46; K. S. Marchitto, S. A. Jones, and M. V. Norgard, Abstr. Annu. Meet. Am. Soc. Microbiol. 1984, B182, p. 48). Differences in binding properties of the various monoclonal antibodies were most likely a reflection of differential binding affinities or their specificities for different epitopes on the 47,000-dalton surface antigen. With two possible exceptions, the monoclonal antibodies tested reacted specifically with T. pallidum, either purified or found within a high-contaminating tissue background, and not with Treponema phagedenis biotype Reiter, Haemophilus ducreyi, Neisseria gonorrhoeae, herpes simplex virus type 2, or normal rabbit testicular tissue. The high sensitivity and specificity exhibited by these anti-T. pallidum monoclonal antibodies make them excellent candidates for employment in new syphilis or other treponemal diagnostic tests designed to detect very low numbers of pathogenic treponemes in lesion exudates or other body fluids.     

Immunogenicity of three recombinant Treponema pallidum antigens examined in guinea pigs

The immunogenicity of recombinant treponemal antigens TmpA, TmpB and TmpC incorporated in RIBI adjuvant and injected into inbred strain 2 guinea pigs has been examined. The immune status of these animals has been challenged by infection with Treponema pallidum, Nichols. The immune response evaluated by the fluorescent-antibody test, microhemagglutination test and ELISA demonstrated high titers of antibodies to the T. pallidum antigens. The immunoblot analysis proved that the antibodies were directed to the 43-(Tmp A) 34- (Tmp B) and 35-kdalton (Tmp C) polypeptides. Antibodies cross-reacting with Treponema phagedenis biotype Reiter were, however, also detected. In spite of high titers of antibodies the animals were not protected against challenging infection with 10(8) organisms of T. pallidum.     

Antigenic interrelationship between endoflagella of Treponema phagedenis biotype Reiter and Treponema pallidum (Nichols): molecular characterization of endoflagellar proteins.

Purified endoflagella from nonpathogenic Treponema phagedenis biotype Reiter were characterized biochemically and compared antigenically with the endoflagellar proteins of Treponema pallidum. T. phagedenis biotype Reiter endoflagella were dissociated into constituent polypeptides by incubation under conditions which disrupt noncovalent bonds. Chymotrypsin peptide maps of T. phagedenis biotype Reiter endoflagellar proteins revealed that the 37- and 33-kilodalton (kDa) major components shared significant homology with the 27- and 30-kDa minor components, respectively. The peptide maps also suggested that the major components shared a lesser degree of structural similarity with each other. These relationships were confirmed by Western blots of T. phagedenis biotype Reiter endoflagellar proteins employing antibodies that were purified against the individual endoflagellar polypeptides. Western blots of T. pallidum with the purified antibodies also demonstrated strong cross-reactivity between the T. phagedenis biotype Reiter endoflagellar proteins and T. pallidum proteins of identical or similar molecular weights. A unique Western blotting technique that we called epitope bridging was used to determine that the 37-kDa subunit contains most of the external epitopes on T. phagedenis biotype Reiter endoflagella. Immunoelectron microscopy with human syphilitic serum and rabbit T. phagedenis biotype Reiter endoflagellar antiserum confirmed the presence of cross-reactive epitopes on the surface of intact T. phagedenis and T. pallidum endoflagella.     

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.     

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.     

Molecular specificities of monoclonal antibodies directed against virulent Treponema pallidum.

Radioimmunoprecipitation (RIP) and Western blot analyses with specific anti-Treponema pallidum subsp. pallidum monoclonal antibodies were used to identify antigens with apparent masses of 102, 84, 54, 53, 52, 47, 32, 29, and 24 kilodaltons (kDa). Cross-reactivity of these antibodies with T. pallidum subsp. pertenue antigens and lack of cross-reactivity with T. phagedenis biotype Reiter, T. vincentii, T. refringens, T. scoliodontum, and T. denticola were also demonstrated by RIP and Western blot analyses. Reactivities in the T. pallidum immobilization test, along with the RIP of lactoperoxidase-catalyzed iodination products, suggested that the identified antigens were surface associated. The abundance and surface association of the 47- and 84-kDa antigens were supported by reactivity in the microhemagglutination test for T. pallidum and by strong reactivity of monoclonal antibodies upon indirect immunofluorescence assays with rabbit-cultivated T. pallidum subsp. pallidum, respectively, but not with T. phagedenis biotype Reiter. Anti-47-kDa and anti-84-kDa monoclonal antibodies were also reactive in indirect immunofluorescence assays using treponemes found in dark-field-positive smears of human genital ulcers.     

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.     

Natural antibodies to treponemal antigens in four strains of guinea-pigs.

A total of 185 serum samples obtained from healthy male and female guinea-pigs of inbred strains 2 and 13 and outbred strains C4D and Hartley A were examined for natural antibodies to treponemal antigens by ELISA using Treponema pallidum (TP), T. phagedenis biotype Reiter (TR) and T. vincentii (TV) antigens and by the FTA test. The prevalence and titres of natural antibodies depended on the age and strain of guinea-pig and the treponemal antigen used. One- and 7-day-old guinea-pigs contained significantly (P less than 0.001) higher levels of natural antibodies than did animals 1 or 3-6 months old. The similar high levels of natural antibodies in newborn guinea-pigs and their mothers (12-30 months old) and the sharp drop observed at the age of 1 month suggested maternal transfer as the mechanism of acquisition. In young adults 3-6 months old, the age group most susceptible to TP infection, antibodies to TP and TR were at their lowest levels, but antibodies reacting to TV had already begun to rise. Natural antibodies were of the IgG1 and IgG2 but not of the IgM class. The highest levels of natural antibodies were in the C4D guinea-pigs; the lowest were in the Hartley A strain. Natural antibody activity was inhibited or adsorbed by TR 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.     

Changes in the cell surface properties of Treponema pallidum that occur during in vitro incubation of freshly extracted organisms

We previously reported that a number of Treponema pallidum membrane proteins appear to reside on the cell surface, since intact treponemes radiolabeled by overnight incubation in medium containing [35S]methionine bind immunoglobulin G (IgG) antibodies directed against these proteins. In the present study, it was found that freshly extracted organisms radiolabeled in vitro for only 2 h inefficiently bound IgG antibodies directed against just two proteins of molecular weights 40,000 and 34,000. An in vitro incubation period of greater than 8 h was required before IgG antibodies present in rabbit syphilitic serum could recognize additional protein antigens on the cell surface. Treatment of aged treponemes, but not freshly extracted organisms, with 0.04% sodium dodecyl sulfate selectively removed a membranous layer from the treponemal surface. Only three treponemal proteins were found associated with this structure, including the same 40,000- and 34,000-molecular-weight proteins mentioned above. These two proteins most likely represent endoflagellar subunits, since they were precipitated with rabbit antisera prepared against purified endoflagellar subunits of the cultivable treponemal strain Treponema phagedenis. Further evidence also was obtained that cells of T. pallidum actively secrete into their extracellular environment a unique class of low-molecular-weight proteins.     

Capacity of virulent Treponema pallidum (Nichols) for deoxyribonucleic acid synthesis.

Treponema pallidum (Nichols) was extracted from infected rabbit tissue, and cell lysates were prepared for monitoring thymidine kinase and deoxyribonucleic acid polymerase activities. No thymidine kinase could be demonstrated in preparations of T. pallidum or the cultivable T. phagedenis biotype Reiter. Significant levels of deoxyribonucleic acid polymerase were detected in both treponemal samples. Interestingly, comparisons of polymerase activity among a spectrum of bacterial genera revealed a direct correlation between enzyme concentrations and estimated generation time. Incorporation of [3H]uridine and [3H]thymidine into macromolecules by intact T. pallidum and the Reiter treponeme was examined. Selective ribonuclease-deoxyribonuclease digestion and cesium chloride gradient banding demonstrated that T. pallidum, independent of the host, and T. phagedenis were capable of synthesizing deoxyribonucleic acid only from the [3H]-uridine precursor.     

Molecular characterization of common treponemal antigens.

A molecular characterization of cross-reactive antigens of Treponema pallidum Nichols and Treponema phagedenis biotype Reiter that are reactive with normal and syphilitic human sera is described. At least 8 common polypeptides, 14 T. pallidum-specific antigens, and 2 T. phagedenis biotype Reiter-specific antigens were identified.     

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 cloning and expression of Treponema pallidum DNA in Escherichia coli K-12.

A gene bank of Treponema pallidum DNA in Escherichia coli K-12 was constructed by cloning SauI-cleaved T. pallidum DNA into the cosmid pHC79. Sixteen of 800 clones investigated produced one or more antigens that reacted with antibodies from syphilitic patients. According to the separation pattern of the antigens produced on sodium dodecyl sulfate-polyacrylamide gels, six different phenotypes were distinguished among these 16 clones. These antigens reacted also with anti-T. pallidum rabbit serum. No antibodies against the cloned antigens were found in normal rabbit serum and in nonsyphilitic human serum. The antigens produced by the E. coli K-12 recombinant DNA clones comigrated in sodium dodecyl sulfate-polyacrylamide gels with antigens extracted from T. pallidum bacteria, suggesting that the treponemal DNA is well expressed in E. coli K-12. Several of the cosmid recombinant plasmids have been subcloned, resulting in smaller T. pallidum recombinant plasmids which are more stably maintained in the cell and produce more treponemal antigen. Monoclonal antibodies were raised against T. pallidum, and one hybridoma produced antibodies that reacted not only with an antigen from T. pallidum but also with the antigen produced by one of the E. coli clones.     

Immunodiagnostic test for detection of serum antibody to Treponema pallidum (syphilis): fibronectin as a capture vehicle for treponemal adhesins

Knowledge that Treponema pallidum adhesin proteins bind to host fibronectin (Fn) via ligand-receptor interactions has resulted in development of an ELISA for measuring specific antitreponemal antibodies in sera of syphilitic patients and infected experimental animals. As little as 50 ng of T. pallidum total protein extract added to Fn-coated wells permitted half-maximal levels of ELISA reactivity. Detection of serum antibody from intratesticularly infected rabbits occurred at dilutions greater than 1/100,000. Antibody titers in serum from patients with primary and latent syphilis were positive at 1/1 000 dilutions while serum samples from patients with secondary syphilis were reactive at 1/10,000. Furthermore, the ELISA proved useful for evaluating serum samples from individuals with other treponemal infections. Antibodies raised against the non-pathogenic spirochete, T. phagedenis biotype Reiter, were non-reactive with Fn-T. pallidum complexes. Also, Reiter treponemal proteins did not bind to Fn-coated wells. This ELISA using Fn as a capture vehicle for treponemal adhesin proteins was superior to 3 other routinely used tests for syphilis diagnosis.     

Genetics of Treponema: characterization of Treponema hyodysenteriae and its relationship to Treponema pallidum.

Saturation reassociation assays with 125I-labeled treponemal DNAs show that Treponema hyodysenteriae is genetically unrelated to T. pallidum (Nichols), T. phagedenis biotype Reiter, and T. refringens biotype Noguchi. Pathogenic and nonpathogenic isolates of T. hyodysenteriae exhibited 28% sequence homology and had an extremely low guanine-plus-cytosine content (25.8%).