Immunity in experimental syphilis. IV. Serological reactivity of antigens extracted from gamm-irradiated Treponema pallidum and Treponema reiteri

Miller, James N. (University of California School of Medicine, Los Angeles), J. H. De Bruijn, and J. H. Bekker. Immunity in experimental syphilis. IV. Serological reactivity of antigens extracted from gamma-irradiated Treponema pallidum and Treponema reiteri. J. Bacteriol. 91:583-587. 1966.-Ultrasonic lysate preparations extracted from virulent Treponema pallidum, Nichols strain, suspensions exposed to 652,800 R of gamma-irradiation exhibited a loss in the serological reactivity of their heat-labile antigens; the heat-stable components of both the lysate and residue antigens were unaffected. The activity of heat-stable, cardiolipin T. pallidum complement-fixing antigen obtained from similarly irradiated organisms was also unaltered. gamma-Irradiation of the cultivable Treponema reiteri with dosages as high as 6,500,000 R failed to alter serologically either the heat-labile or heat-stable component of its lipopolysaccharide-protein (Reiter protein) antigen. The reactivity of the lipopolysaccharide portion of the Reiter protein complex with an antiserum to T. pallidum Nichols indicates previously unsuspected antigenic differences between the rabbit-adapted Nichols strain of the organism and so-called "wild" human strains of T. pallidum in which this antigen is generally absent.     

Use of the skin protection assay in experimental syphilis to assess protective immunity against a specific Treponema pallidum surface epitope

We have recently shown that a monoclonal antibody, designated M131, that binds a surface phosphorylcholine epitope on Treponema pallidum possesses complement-dependent killing activity and confers partial protection in rabbits following passive immunization (Blanco et al., 2005, Infect. Immun. 73:3083-3095). In this study, the protective potential of M131 was further tested using the rabbit skin protection assay of Titus and Weiser. Both M131 and infection-derived immune rabbit serum resulted in significant lesion delays corresponding to at least a 90% reduction of the treponemal challenge inoculum. The skin protection assay provides a way to assess the protective potential of specific immunogens while using far less antibody than in passive immunization protocols.     

Characterization of lymphocyte responsiveness in early experimental syphilis. I. In vitro response to mitogens and Treponema pallidum antigens

Lymphoid cells from spleens and lymph nodes of rabbits infected with T. pallidum respond by proliferation to concanavalin A (Con A) and T. pallidum antigens. Spleen cell responsiveness to treponemal antigens appears 6 days after infection, is 100 to 600 fold higher than the response of uninfected control rabbits, and is maintained throughout the 31-day observation period. Specifically responding cells in the inguinal and popliteal lymph nodes of infected animals are demonstrable on day 10, and the magnitude of the response increases throughout the observation period. Specific responsiveness to T. pallidum antigens in vitro is enhanced in purified T cell populations and is abolished by treatment with goat anti-rabbit thymocyte serum and complement. The response of spleen and lymph node cells to Con A is unaffected during syphilitic infection. These results are consistent with a role for T cell-mediated specific immunity to treponemal antigens early after infection and do not support a hypothesis of depressed cellular immunity during syphilitic infection.     

Circulating immune complexes in experimental syphilis and their relation to immunological response against Treponema pallidum

It was found that circulating immune complexes (CIC) were formed in rabbits at different times after infection with Treponema pallidum. The CIC which appeared at the beginning of the disease were short-lived (2-6 weeks) but those appearing later than 20 weeks after infection remained for 10-25 weeks. CIC contained both IgM and IgG classes of immunoglobulin. The antibodies present in CIC were found to be specific and nonspecific for T. pallidum. The presence of CIC led to a marked decline of treponemal antibodies in rabbit sera. The cell-mediated immune response measured by the macrophage migration inhibition (MMI) test at the beginning of the disease (up to 12 weeks) was not decreased. However, when syphilis lasted for more than 14 weeks and when CIC were formed mainly from IgG, a distinct decrease in the ability of lymphocytes to cause MMI was observed. These findings strongly suggest that IgG-complexes suppress the immunological responsiveness of lymphocytes against T. pallidum which in turn facilitates the multiplication of treponemes in the host.     

Induction of immunity to chemically-induced rat tumours by cellular or soluble antigens

Summary Radiation-attenuated, aminoazo dye-induced rat hepatoma cells were employed as a standard immunogen in tests evaluating the optimal conditions for the demonstration of resistance to subcutaneous challenge with viable tumour cells. These tests revealed that maximal sensitivity for the detection of the immunogenicity of -irradiated cells was obtained using multiple immunisations via the intraperitoneal route rather than the subcutaneous route. When 3 M KCl extracts of tumours (as soluble tumour antigens) were evaluated for the induction of specific immunoprotection under comparable conditions, weak resistance to tumour was demonstrable, but only within a restricted dose range of the antigen preparation. No significant effects upon tumour growth were observed in rats pretreated with fractions of tumour-bearer serum.     

Segregation of B and T cell epitopes of Treponema pallidum repeat protein K to variable and conserved regions during experimental syphilis infection

Robust immune responses clear millions of treponemes to resolve lesions of primary and secondary syphilis, but cannot clear the treponemes that lead to debilitating and sometimes fatal tertiary syphilis. It is also known that the rabbit model and humans can be reinfected with heterologous isolates. How some treponemes are able to escape the immune system is unknown. In our laboratories rabbits immunized with the Seattle Nichols strain Treponema pallidum repeat protein K (TprK) were previously shown to have attenuated lesion development following challenge. In other isolates, TprK was shown to have seven discrete variable regions, with sequence variation among and within isolates. Using overlapping synthetic 20-aa peptides, we demonstrate that during experimental infection with the Nichols strain, the T cell responses are directed to conserved regions, while the Ab responses are directed primarily to variable regions. Abs from rabbits immunized with recombinant TprK recognized conserved and variable regions, suggesting that the conserved regions are inherently as immunogenic as the variable regions. TprK variability may allow some treponemes to escape recognition from Abs. The variable region heterogeneity may help explain the lack of protection against heterologous isolates.     

The induction of cellular immunity with soluble murine histocompatibility antigens

Soluble transplantation antigens have been prepared from various lymphoid organs of the mouse strains A and C57BL. These preparations have been partially characterized by gel filtration on Sephadex G-200 and G-100. The distribution of various antigenic activities, such as precipitation with rabbit antisera, inhibition of the cytotoxic reactions of heterologous antisera and of alloantibodies, differed considerably among the chromatographic fractions. The soluble antigen preparations retained their antigenic and immunogenic properties, as demonstrated by their ability to block the cytotoxic reactions of alloantisera and to modify tumor growth in immunized recipients. Immunization of normal recipients with the immunogenic transplantation antigen preparations led to the production of sensitized lymphocytes, capable of destroying allogeneic target cells in vitro. Sensitized lymphocytes appeared in the regional lymph nodes after a single injection of 200–300 μg of the antigen preparation, reaching a peak level between 9 and 12 days. On reimmunization, the cytolytic activity of lymph node cells increased considerably and sensitized lymphocytes also appeared in the spleens of immunized animals.     

The cutaneous response in humans to Treponema pallidum lipoprotein analogues involves cellular elements of both innate and adaptive immunity

To extend prior studies implicating treponemal lipoproteins as major proinflammatory agonists of syphilitic infection, we examined the responses induced by intradermal injection of human subjects with synthetic lipoprotein analogues (lipopeptides) corresponding to the N termini of the 17- and 47-kDa lipoproteins of Treponema pallidum. Responses were assessed visually and by flow cytometric analysis of dermal leukocyte populations within fluids aspirated from suction blisters raised over the injection sites. Lipopeptides elicited dose-dependent increases in erythema/induration and cellular infiltrates. Compared with peripheral blood, blister fluids were highly enriched for monocytes/macrophages, cutaneous lymphocyte Ag-positive memory T cells, and dendritic cells. PB and blister fluids contained highly similar ratios of CD123(-)/CD11c(+) (DC1) and CD123(+)/CD11c(-) (DC2) dendritic cells. Staining for maturation/differentiation markers (CD83, CD1a) and costimulatory molecules (CD80/CD86) revealed that blister fluid DC1, but not DC2, cells were more developmentally advanced than their peripheral blood counterparts. Of particular relevance to the ability of syphilitic lesions to facilitate the transmission of M-tropic strains of HIV-1 was a marked enhancement of CCR5 positivity among mononuclear cells in the blister fluids. Treponemal lipopeptides have the capacity to induce an inflammatory milieu reminiscent of that found in early syphilis lesions. In contrast with in vitro studies, which have focused upon the ability of these agonists to stimulate isolated innate immune effector cells, in this study we show that in a complex tissue environment these molecules have the capacity to recruit cellular elements representing the adaptive as well as the innate arm of the cellular immune response.     

Immunization of rabbits with recombinant Treponema pallidum surface antigen 4D alters the course of experimental syphilis

Rabbits immunized with the protease resistant recombinant Treponema pallidum surface-associated Ag 4D showed an altered course of experimental syphilis after intradermal challenge with virulent T. pallidum. Vaccination trials using three different protocols were examined. In one experiment, a combined i.m.-i.v. immunization protocol was compared with an i.m. injection schedule. Four of five rabbits immunized by the i.m.-i.v. protocol developed morphologically atypical lesions at each of 16 sites, whereas the i.m. injected animals showed no evidence of attenuated disease. Moreover, immunization by both protocols elicited equally high ELISA titers of 4D-specific IgG antibody suggesting that cellular immune mechanisms may have been involved. In an effort to augment cell-mediated responses to 4D, seven rabbits received i.v. immunizations with an emulsion of 4D, purified BCG cell wall skeletons, and trehalose dimycolate in an oil-microdroplet form (4D-cell wall skeleton-trehalose dimycolate). Splenic lymphocytes from three representative immunized animals had a strong in vitro proliferative response to 4D as compared to controls. Further, all animals developed high anti-4D titers in response to immunization. After challenge with virulent T. pallidum, six of the seven rabbits developed an early cutaneous response (3-8 days) at the sites of inoculation consistent with a hypersensitivity reaction followed by morphologically atypical lesions. Aspirates from each of three representative atypical lesions were devoid of treponemes by darkfield examination at day 14, whereas motile T. pallidum were observed in aspirates from four of five control sites. Between days 20 and 24, lesions in the 4D-cell wall skeleton-trehalose dimycolate immunized rabbits began to enlarge and become more typical in appearance; aspirates from six of seven representative lesions were darkfield positive. We conclude that 1) immunization with the r4D Ag alters the course of experimental syphilis in a manner consistent with previously defined parameters of partial protection and 2) immunizations protocols containing an i.v. component are more effective than protocols using the i.m. route alone.     

Cloning and expression of Treponema pallidum antigens in Escherichia coli

A library of Treponema pallidum genomic DNA fragments produced by partial Sau3A digestion was established in Escherichia coli K12 using the plasmid vector pAT153. The library was screened using immune syphilitic rabbit serum and six recombinant phenotypes expressing eight treponemal polypeptides were detected. With two exceptions, all the recombinant gene products were the same size as polypeptides detected on Western immunoblots of T. pallidum. The genes encoding three novel gene products, with molecular masses in SDS-PAGE of 42, 17 and 15.5 kDa, which had not been cloned previously from T. pallidum were also identified. Monoclonal antibodies which reacted with four of the eight recombinant polypeptides were generated.     

Sequence diversity of Treponema pallidum subsp. pallidum tprK in human syphilis lesions and rabbit-propagated isolates

The tprK gene of Treponema pallidum subsp. pallidum, the causative agent of venereal syphilis, belongs to a 12-member gene family and encodes a protein with a predicted cleavable signal sequence and predicted transmembrane domains. Except for the Nichols type strain, all rabbit-propagated isolates of T. pallidum examined thus far are comprised of mixed populations of organisms with heterogeneous tprK sequences. We show that tprK sequences in treponemes obtained directly from syphilis patients are also heterogeneous. Clustering analysis demonstrates that primary chancre tprK sequences are more likely to cluster within a sample than among samples and that tighter clustering is seen within chancre samples than within rabbit-propagated isolates. Closer analysis of tprK sequences from a rabbit-propagated isolate reveals that individual variable regions have different levels of diversity, suggesting that variable regions may have different intrinsic rates of sequence change or may be under different levels of selection. Most variable regions show increased sequence diversity upon passage. We speculate that the diversification of tprK during infection allows organisms to evade the host immune response, contributing to reinfection and persistent infection.     

Restriction analysis of DNA from Treponema pallidum, the causative agent of syphilis

When purified DNA from pathogenic Treponema pallidum is digested with restriction endonucleases it results in the formation of discrete DNA fragments which range between 2.5 to 10 Kilobase pairs. No such precise fragmentation occurs with DNA isolated from nonpathogenic T. pallidum. The appearance of the discrete restriction fragments from the pathogenic T. pallidum DNA does not represent a contamination of satellite DNA from rabbit, the host in which the organism was propagated, but rather represents the presence of redundant DNA or DNA of less complexity in the pathogenic T. pallidum DNA preparation.