Origin of Nontreponemal Antibodies During Treponema pallidum Infection: Evidence From a Rabbit Model

Origin of Nontreponemal Antibodies During Treponema pallidum Infection: Evidence From a Rabbit Model Abstract The origin of nontreponemal antibodies during syphilis infection is hotly debated. Here, we analyzed the immune response in rabbits immunized with various antigens. Inactivated treponemes elicited the production of low-titer nontreponemal antibodies in some rabbits. Cardiolipin combined with bovine serum albumin also induced anticardiolipin antibody production. These findings indicate that Treponema pallidum contained a cardiolipin antigen with weak immunogenicity. However, active T. pallidum induced higher nontreponemal antibody production with strong immunogenicity at an earlier time point, and the antibody titer was consecutive, suggesting the high nontreponemal antibody titer resulted from the combined effects of both the T. pallidum cardiolipin antigen and the damaged host–cell cardiolipin antigen during syphilis infection, the latter of which plays a major role in the induction of nontreponemal antibody production. Our study provides direct animal evidence of the origin of nontreponemal antibodies during T. pallidum infection. syphilis, inactivated treponemes, nontreponemal antibody, cardiolipin antigen Syphilis is a multistage sexually transmitted infection caused by Treponema pallidum subspecies pallidum [1]. Due to the absence of in vitro culture and simple staining methods for the detection of T. pallidum [2], serological detection methods are regarded as the mainstay for the diagnosis of syphilis and for monitoring the efficacy of subsequent antibiotic treatment [1, 3]. Syphilitic infection elicits 2 distinct types of antibody responses, traditionally designated “treponemal” and “nontreponemal” [4, 5]. Treponemal antibodies are directed against the polypeptide antigens of T. pallidum, whereas nontreponemal antibodies (also called “reagin”) recognize lipoidal antigens and are detected via a defined combination of phosphatidylcholine, cholesterol, and cardiolipin, among which cardiolipin is the major antigenic component [5–7]; these antibodies may cross-react with autoimmune antigens [8]. The origin of nontreponemal antibodies during T. pallidum infection is a great mystery of syphilis immunology and is hotly debated [4, 9]. Two origin theories have been proposed. The first theory is that cardiolipin complexes are liberated from host cells damaged by the treponeme and that these complexes subsequently provoke an autoimmune reaction [4]. Another theory is that nontreponemal antibodies are induced by the lipids of T. pallidum [4, 10, 11]. To the best of our knowledge, there have been few studies and little direct animal evidence of the origins of nontreponemal antibodies during T. pallidum infection. Here, we conducted a comprehensive investigation using a cohort of infected rabbits to explore the immune responses and reveal the origins of nontreponemal antibodies during T. pallidum infection. We analyzed the immune responses based on the variation tendency of the treponemal and nontreponemal antibodies in rabbits immunized with various treponeme antigens, including active treponemes, inactivated treponemes, whole-cell lysate proteins, and cardiolipin antigens. MATERIALS AND METHODS Ethics Statement This study was approved by the Institutional Ethics Committee of Zhongshan Hospital, Medical College of Xiamen University. All the rabbit experimental protocols were approved by the Animal Experimental Ethics Committee of the Medical College of Xiamen University. Propagation and Harvesting of T. pallidum From Rabbits Adult male New Zealand white rabbits (approximately 2.5 kg) were purchased from Xiamen University Laboratory Animal Center. The animals were fed antibiotic-free food and water and housed individually at 18°C–20°C. Sera collected from all the animals were tested using the Venereal Disease Research Laboratory (VDRL) test and the T. pallidum particle agglutination assay (TPPA) to ensure that these results were negative prior to inclusion of the animals [12]. The T. pallidum Nichols strain was kindly provided by Lorenzo Giacani, PhD (University of Washington, Seattle), and was propagated intratesticularly in the New Zealand white rabbits by serial passage in our laboratory to maintain virulence as previously described [13, 14]. Then, treponemes were extracted from the infected testes using 0.9% warm saline. In brief, the testes were removed aseptically. Several lengthwise cuts were made in each testis, and the treponemes were extracted on a rotary shaker in 0.9% saline. Gross testicular debris was sedimented by centrifugation at 400g for 15 minutes, and the organisms in the supernatant were counted under a darkfield microscope and adjusted to 2 × 107 organisms/mL for use. T. pallidum Antigen Preparation and Immunization An overview of the immunization procedure with various treponemal antigens is shown in Figure 1A. Figure 1. View largeDownload slide Experimental overview of the immunization scheme. A, Immunization with the Treponema pallidum antigen. (i) In the active T. pallidum group, rabbits (n = 4) were injected with active T. pallidum once. (ii) In the formalin-treated group, rabbits (n = 4) were injected with formalin-treated T. pallidum once per week for a total of 10 injections. (iii) In the heat-treated group, rabbits (n = 4) were immunized with heat-treated T. pallidum once per week for a total of 10 injections. (iv) In the ultrasonicated group, rabbits (n = 10) were immunized with ultrasonicated T. pallidum once every 2 weeks for a total of 5 injections. B, Immunization with cardiolipin antigen. (i) In the cardiolipin group, rabbits (n = 4) were immunized with cardiolipin antigen as described for the ultrasonicated group. (ii) In the cardiolipin–bovine serum albumin (BSA) group, rabbits (n = 4) were immunized with the cardiolipin-BSA antigen. Abbreviations: BSA, bovine serum albumin; PO4–, the phosphate radical of cardiolipin; TPPA, Treponema pallidum particle agglutination assay; VDRL, Venereal Disease Research Laboratory. Figure 1. View largeDownload slide Experimental overview of the immunization scheme. A, Immunization with the Treponema pallidum antigen. (i) In the active T. pallidum group, rabbits (n = 4) were injected with active T. pallidum once. (ii) In the formalin-treated group, rabbits (n = 4) were injected with formalin-treated T. pallidum once per week for a total of 10 injections. (iii) In the heat-treated group, rabbits (n = 4) were immunized with heat-treated T. pallidum once per week for a total of 10 injections. (iv) In the ultrasonicated group, rabbits (n = 10) were immunized with ultrasonicated T. pallidum once every 2 weeks for a total of 5 injections. B, Immunization with cardiolipin antigen. (i) In the cardiolipin group, rabbits (n = 4) were immunized with cardiolipin antigen as described for the ultrasonicated group. (ii) In the cardiolipin–bovine serum albumin (BSA) group, rabbits (n = 4) were immunized with the cardiolipin-BSA antigen. Abbreviations: BSA, bovine serum albumin; PO4–, the phosphate radical of cardiolipin; TPPA, Treponema pallidum particle agglutination assay; VDRL, Venereal Disease Research Laboratory. For the active T. pallidum group, the backs of 4 normal rabbits were shaved, cleaned with 75% ethanol, and injected intradermally with 0.1 mL of a fresh, active T. pallidum extract at a density of 2.0 × 107 organisms/mL at a total of 10 areas. The rabbits in this group were intradermally injected once. For the formalin-treated group, neutral-buffered formalin (Sinopharm, Shanghai, China) was added to the treponemes for a final concentration of 0.2%, and the sample was incubated overnight at room temperature. Then, the samples were centrifuged at 12000g for 30 minutes, the precipitate was washed twice in sterile 0.9% saline, and the treponemes were resuspended in 0.9% saline to a density of 2 × 107 organisms/mL for subsequent immunization at a total of 10 areas [15]. Four rabbits’ backs were shaved and injected intradermally with formalin-treated treponemes once per week for a total of 10 injections. Prior to immunization, the treponemes in this group were confirmed to be completely inactivated (methods are described below). For the heat-treated group, the treponeme suspension at a density of 2 × 107 organisms/mL was heated at 56°C for 1 hour to inactivate the organisms [16]. Four rabbits’ backs were shaved and injected intradermally using the same method described for the formalin-treated group. Prior to immunization, the treponemes in this group were confirmed to be completely inactivated (methods are described below). For the ultrasonicated group, to obtain the whole-cell lysate of T. pallidum, the treponemal suspension was centrifuged at 12000g for 30 minutes. The wet weight of the precipitate was measured. Afterward, the organisms were suspended in a 5-fold volume of ultrasonication buffer (50 mM of Tris-Hydrochloric acid and 1 mmol/L of ethylenediaminetetraacetic acid, pH 7.4). The suspension was sonicated using a Sonics Vibra Cell Disrupter System (Sonics & Materials, Newtown, Connecticut) at 20 kHz and 60 W on ice. The ultrasonication time was 7.7 seconds and was performed at 9.9-second intervals for 10 minutes [17–19]. Then, the protein concentration was determined using the bicinchoninic acid Protein Assay Kit (Takara Bio, Dalian, China). Ten rabbits were immunized subcutaneously with the abovementioned treponemal cell lysate mixed with an equal volume of Freund adjuvant (Boson Biotech, Xiamen, China). The initial immunization was implemented with Freund complete adjuvant, and Freund incomplete adjuvant was used in additional immunizations. Each immunization delivered 200 μg of antigen per rabbit. Additional immunizations were delivered every 2 weeks following the initial treponeme injection for a total of 5 injections [12, 20, 21]. Cardiolipin and Cardiolipin–Bovine Serum Albumin Antigen Preparations and Immunization Cardiolipin from the bovine heart (Sigma-Aldrich, St Louis, Missouri) was diluted with phosphate-buffered saline to a concentration of 400 μg/mL for immunization. In addition, cardiolipin was linked to bovine serum albumin (BSA) through covalent binding, which was accomplished by the Boson Biotech Company (Xiamen, China). The final concentration of the cardiolipin-BSA antigen was 400 μg/mL after dilution with phosphate-buffered saline. The cardiolipin antigen and cardiolipin-BSA antigen were mixed with an equal volume of Freund adjuvant, and immunized rabbits were treated in the same manner as the rabbits in the ultrasonicated treponeme group (Figure 1B). Serological Analysis Serum samples were collected from the ear arteries of all the rabbits every week starting 1 week after the initial antigen injection, and syphilis serological tests were performed in a blinded fashion using VDRL (Becton Dickinson and Company, Franklin Lakes, New Jersey) and TPPA kits (Fujirebio, Tokyo, Japan) following the manufacturers’ instructions [22, 23]. The serum anticardiolipin antibody titer was monitored with cardiolipin enzyme-linked immunosorbent assay (ELISA; Boson Biotech, Xiamen, China) as previously described [24]; the cutoff value was 2.1 times the mean value of the negative sample. Live/Dead T. pallidum Fluorescence Assay The LIVE/DEAD BacLight Viability Kit (Molecular Probes, Leiden, the Netherlands) was used according to the manufacturer’s instructions to determine whether the treponemes exhibited activity after inactivation [25]. The ratio between the 2 dyes was 1:1. The active treponemes, which were stained with SYTO 9, exhibited green fluorescence, and the dead treponemes, which were stained with propidium iodide, exhibited red fluorescence. Images were acquired on a Nikon ECLIPSE 80i fluorescence microscope (Nikon Instruments, Shanghai, China) equipped with a Nikon DS-Ri2 CCD camera. One hundred fields were counted to determine the proportion of the T. pallidum that exhibited red fluorescence. Statistical Analysis Serum VDRL and serum TPPA, which are quantitative measures of serial 2-fold dilutions, are presented as geometric means for the correlation analysis. Statistical analyses were conducted using SPSS version 17.0 (SPSS, Chicago, Illinois) for Windows with repeated-measures analysis of variance and general estimating equations. A 2-sided P value <.05 was considered statistically significant. RESULTS Effect of Inactivation on T. pallidum Treponemal activity analysis showed that the treponemes in the formalin- and heat-treated groups all exhibited red fluorescence (Supplementary Figure 1A and 1B), whereas the treponemes in the active T. pallidum group exhibited green fluorescence (Supplementary Figure 1C). Thus, the treponemes used for immunization were completely inactivated in the formalin- and heat-treated groups. Moreover, the treponemes had a mostly intact cell structure after inactivation. Reactivity of Serum TPPA and VDRL in the Inactivated Treponeme Groups First, we explored the variation tendency of treponemal antibodies in rabbits immunized with inactivated treponemes, including the formalin- and heat-treated treponemes. All the rabbits had positive serum TPPA, indicating that the inactivated treponemes had potent immunogenicity and could elicit the production of treponemal antibodies. The TPPA antibodies began to appear 3 weeks and 4 weeks after the initial injection of treponemes in the heat- and formalin-treated groups, respectively (Table 1). The TPPA test titers in these 2 inactivated treponeme groups increased over time. The highest TPPA titer in the heat-treated group was 1:2560, which was similar to the titer achieved in the formalin-treated group. No significant difference was found in the trend of TPPA titer change between the 2 inactivated treponeme groups (P > .05; Figure 2). In contrast, the TPPA test titer in the active treponemal group showed positive results and a rapid increase at an earlier time point than that in the formalin- and heat-treated groups (Table 1). All the rabbits in this group showed a positive TPPA result 1 week after the initial treponeme injection. At the same time, the highest titer in the active treponemal group was 1:10240, which was much higher than the titers achieved in the inactivated treponeme groups (P < .001; Figure 2). One rabbit that died 32 days after the initial treponeme injection in the heat-treated group was excluded from the experiment. Table 1. Reactivity of Serum Treponema pallidum Particle Agglutination Assay Postimmunization in the Formalin-Treated Group, Heated-Treated Group, and Active Group Group  Animal  TPPA Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  Formalin-treated group  1  –  –  –  –  –  1:320  1:640  1:320  1:320  1:160  1:320  1:160  1:80  1:80    2  –  –  –  1:80  1:80  1:640  1:640  1:640  1:640  1:640  1:640  1:640  1:640  1:640    3  –  –  –  1:80  1:80  1:320  1:640  1:640  1:1280  1:640  1:320  1:320  1:320  1:320    4  –  –  –  –  1:80  1:640  1:2560  1:1280  1:2560  1:1280  1:1280  1:640  1:640  1:640  Heat-treated group  1  –  –  1:80  1:80  1:160  1:160  1:320  1:320  1:320  1:320  1:160  1:160  1:160  1:160    2  –  –  –  –  Deceased    3  –  –  1:80  1:160  1:1280  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:1280  1:1280  1:1280    4  –  –  1:160  1:320  1:640  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:1280  Active Treponema pallidum group  1  –  1:2560  1:2560  1:1280  1:1280  1:2560  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120    2  –  1:320  1:640  1:640  1:1280  1:5120  1:5120  1:5120  1:10240  1:5120  1:5120  1:5120  1:5120  1:5120    3  –  1:640  1:320  1:640  1:1280  1:2560  1:2560  1:5120  1:5120  1:10240  1:10240  1:5120  1:5120  1:5120    4  –  1:640  1:1280  1:1280  1:2560  1:2560  1:5120  1:5120  1:5120  1:10240  1:5120  1:5120  1:5120  1:5120  Group  Animal  TPPA Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  Formalin-treated group  1  –  –  –  –  –  1:320  1:640  1:320  1:320  1:160  1:320  1:160  1:80  1:80    2  –  –  –  1:80  1:80  1:640  1:640  1:640  1:640  1:640  1:640  1:640  1:640  1:640    3  –  –  –  1:80  1:80  1:320  1:640  1:640  1:1280  1:640  1:320  1:320  1:320  1:320    4  –  –  –  –  1:80  1:640  1:2560  1:1280  1:2560  1:1280  1:1280  1:640  1:640  1:640  Heat-treated group  1  –  –  1:80  1:80  1:160  1:160  1:320  1:320  1:320  1:320  1:160  1:160  1:160  1:160    2  –  –  –  –  Deceased    3  –  –  1:80  1:160  1:1280  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:1280  1:1280  1:1280    4  –  –  1:160  1:320  1:640  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:1280  Active Treponema pallidum group  1  –  1:2560  1:2560  1:1280  1:1280  1:2560  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120    2  –  1:320  1:640  1:640  1:1280  1:5120  1:5120  1:5120  1:10240  1:5120  1:5120  1:5120  1:5120  1:5120    3  –  1:640  1:320  1:640  1:1280  1:2560  1:2560  1:5120  1:5120  1:10240  1:10240  1:5120  1:5120  1:5120    4  –  1:640  1:1280  1:1280  1:2560  1:2560  1:5120  1:5120  1:5120  1:10240  1:5120  1:5120  1:5120  1:5120  Abbreviations: –, negative; TPPA, Treponema pallidum particle agglutination assay. View Large Table 1. Reactivity of Serum Treponema pallidum Particle Agglutination Assay Postimmunization in the Formalin-Treated Group, Heated-Treated Group, and Active Group Group  Animal  TPPA Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  Formalin-treated group  1  –  –  –  –  –  1:320  1:640  1:320  1:320  1:160  1:320  1:160  1:80  1:80    2  –  –  –  1:80  1:80  1:640  1:640  1:640  1:640  1:640  1:640  1:640  1:640  1:640    3  –  –  –  1:80  1:80  1:320  1:640  1:640  1:1280  1:640  1:320  1:320  1:320  1:320    4  –  –  –  –  1:80  1:640  1:2560  1:1280  1:2560  1:1280  1:1280  1:640  1:640  1:640  Heat-treated group  1  –  –  1:80  1:80  1:160  1:160  1:320  1:320  1:320  1:320  1:160  1:160  1:160  1:160    2  –  –  –  –  Deceased    3  –  –  1:80  1:160  1:1280  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:1280  1:1280  1:1280    4  –  –  1:160  1:320  1:640  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:1280  Active Treponema pallidum group  1  –  1:2560  1:2560  1:1280  1:1280  1:2560  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120    2  –  1:320  1:640  1:640  1:1280  1:5120  1:5120  1:5120  1:10240  1:5120  1:5120  1:5120  1:5120  1:5120    3  –  1:640  1:320  1:640  1:1280  1:2560  1:2560  1:5120  1:5120  1:10240  1:10240  1:5120  1:5120  1:5120    4  –  1:640  1:1280  1:1280  1:2560  1:2560  1:5120  1:5120  1:5120  1:10240  1:5120  1:5120  1:5120  1:5120  Group  Animal  TPPA Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  Formalin-treated group  1  –  –  –  –  –  1:320  1:640  1:320  1:320  1:160  1:320  1:160  1:80  1:80    2  –  –  –  1:80  1:80  1:640  1:640  1:640  1:640  1:640  1:640  1:640  1:640  1:640    3  –  –  –  1:80  1:80  1:320  1:640  1:640  1:1280  1:640  1:320  1:320  1:320  1:320    4  –  –  –  –  1:80  1:640  1:2560  1:1280  1:2560  1:1280  1:1280  1:640  1:640  1:640  Heat-treated group  1  –  –  1:80  1:80  1:160  1:160  1:320  1:320  1:320  1:320  1:160  1:160  1:160  1:160    2  –  –  –  –  Deceased    3  –  –  1:80  1:160  1:1280  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:1280  1:1280  1:1280    4  –  –  1:160  1:320  1:640  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:1280  Active Treponema pallidum group  1  –  1:2560  1:2560  1:1280  1:1280  1:2560  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120    2  –  1:320  1:640  1:640  1:1280  1:5120  1:5120  1:5120  1:10240  1:5120  1:5120  1:5120  1:5120  1:5120    3  –  1:640  1:320  1:640  1:1280  1:2560  1:2560  1:5120  1:5120  1:10240  1:10240  1:5120  1:5120  1:5120    4  –  1:640  1:1280  1:1280  1:2560  1:2560  1:5120  1:5120  1:5120  1:10240  1:5120  1:5120  1:5120  1:5120  Abbreviations: –, negative; TPPA, Treponema pallidum particle agglutination assay. View Large Figure 2. View largeDownload slide Variation tendency in the postimmunization Treponema pallidum particle agglutination assay (TPPA) titer in the groups immunized with various treponemal antigens. The test titers are presented as the geometric means and were assessed using generalized estimating equations (***P < .001). Figure 2. View largeDownload slide Variation tendency in the postimmunization Treponema pallidum particle agglutination assay (TPPA) titer in the groups immunized with various treponemal antigens. The test titers are presented as the geometric means and were assessed using generalized estimating equations (***P < .001). Second, we analyzed the variation tendency of nontreponemal antibodies throughout the experimental phase. One rabbit in the heat-treated group (number 1) appeared to have a persistent VDRL titer of 1:1 9 weeks after the initial treponeme injection, and another rabbit in the formalin-treated group (number 4) had a persistent titer at 2 weeks after the initial treponeme injection (Table 2). Their titers did not increase over time. In addition, several rabbits appeared to have weakly discontinuous VDRL reactivity. In contrast, positive VDRL results were observed in all the rabbits as early as 2 weeks after the initial injection, and the VDRL titer was notably increased in the active treponemal group. Consecutive and higher VDRL titers were observed in all the rabbits in the active treponemal group. At 3 weeks following the initial treponeme injection, the VDRL test titer reached a peak at 1:64 and then decreased. Table 2. Reactivity of Serum Venereal Disease Research Laboratory Postimmunization in the Formalin-Treated Group, Heat-Treated Group, and Active Group Group  Animal  VDRL Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  Formalin-treated group  1  –  –  –  –  –  –  –  –  ±  1:1  1:1  1:1  1:1  1:1    2  –  –  –  –  –  –  –  –  –  ±  –  –  ±  ±    3  –  –  –  –  –  –  –  –  –  –  –  –  –  ±    4  –  –  –  –  –  –  –  –  –  ±  ±  –  –  ±  Heat-treated group  1  –  ±  –  –  –  –  ±  ±  ±  –  –  –  –  –    2  –  –  –  –  Deceased    3  –  –  –  –  –  –  –  –  ±  –  –  –  –  –    4  –  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  Active Treponema pallidum group  1  –  1:16  1:16  1:16  1:16  1:8  1:4  1:4  1:4  1:2  1:1  1:1  1:1  1:1    2  –  1:8  1:16  1:16  1:16  1:16  1:8  1:8  1:4  1:4  1:2  1:1  1:1  1:1    3  –  1:4  1:8  1:8  1:4  1:2  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1    4  –  1:16  1:64  1:32  1:8  1:4  1:4  1:4  1:4  1:2  1:2  1:1  1:1  1:1  Group  Animal  VDRL Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  Formalin-treated group  1  –  –  –  –  –  –  –  –  ±  1:1  1:1  1:1  1:1  1:1    2  –  –  –  –  –  –  –  –  –  ±  –  –  ±  ±    3  –  –  –  –  –  –  –  –  –  –  –  –  –  ±    4  –  –  –  –  –  –  –  –  –  ±  ±  –  –  ±  Heat-treated group  1  –  ±  –  –  –  –  ±  ±  ±  –  –  –  –  –    2  –  –  –  –  Deceased    3  –  –  –  –  –  –  –  –  ±  –  –  –  –  –    4  –  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  Active Treponema pallidum group  1  –  1:16  1:16  1:16  1:16  1:8  1:4  1:4  1:4  1:2  1:1  1:1  1:1  1:1    2  –  1:8  1:16  1:16  1:16  1:16  1:8  1:8  1:4  1:4  1:2  1:1  1:1  1:1    3  –  1:4  1:8  1:8  1:4  1:2  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1    4  –  1:16  1:64  1:32  1:8  1:4  1:4  1:4  1:4  1:2  1:2  1:1  1:1  1:1  Abbreviations: –, negative; –, nonreactive; ±, weakly reactive; VDRL, Venereal Disease Research Laboratory. View Large Table 2. Reactivity of Serum Venereal Disease Research Laboratory Postimmunization in the Formalin-Treated Group, Heat-Treated Group, and Active Group Group  Animal  VDRL Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  Formalin-treated group  1  –  –  –  –  –  –  –  –  ±  1:1  1:1  1:1  1:1  1:1    2  –  –  –  –  –  –  –  –  –  ±  –  –  ±  ±    3  –  –  –  –  –  –  –  –  –  –  –  –  –  ±    4  –  –  –  –  –  –  –  –  –  ±  ±  –  –  ±  Heat-treated group  1  –  ±  –  –  –  –  ±  ±  ±  –  –  –  –  –    2  –  –  –  –  Deceased    3  –  –  –  –  –  –  –  –  ±  –  –  –  –  –    4  –  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  Active Treponema pallidum group  1  –  1:16  1:16  1:16  1:16  1:8  1:4  1:4  1:4  1:2  1:1  1:1  1:1  1:1    2  –  1:8  1:16  1:16  1:16  1:16  1:8  1:8  1:4  1:4  1:2  1:1  1:1  1:1    3  –  1:4  1:8  1:8  1:4  1:2  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1    4  –  1:16  1:64  1:32  1:8  1:4  1:4  1:4  1:4  1:2  1:2  1:1  1:1  1:1  Group  Animal  VDRL Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  Formalin-treated group  1  –  –  –  –  –  –  –  –  ±  1:1  1:1  1:1  1:1  1:1    2  –  –  –  –  –  –  –  –  –  ±  –  –  ±  ±    3  –  –  –  –  –  –  –  –  –  –  –  –  –  ±    4  –  –  –  –  –  –  –  –  –  ±  ±  –  –  ±  Heat-treated group  1  –  ±  –  –  –  –  ±  ±  ±  –  –  –  –  –    2  –  –  –  –  Deceased    3  –  –  –  –  –  –  –  –  ±  –  –  –  –  –    4  –  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  Active Treponema pallidum group  1  –  1:16  1:16  1:16  1:16  1:8  1:4  1:4  1:4  1:2  1:1  1:1  1:1  1:1    2  –  1:8  1:16  1:16  1:16  1:16  1:8  1:8  1:4  1:4  1:2  1:1  1:1  1:1    3  –  1:4  1:8  1:8  1:4  1:2  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1    4  –  1:16  1:64  1:32  1:8  1:4  1:4  1:4  1:4  1:2  1:2  1:1  1:1  1:1  Abbreviations: –, negative; –, nonreactive; ±, weakly reactive; VDRL, Venereal Disease Research Laboratory. View Large Reactivity of Serum TPPA and VDRL in the Ultrasonicated Treponeme Group The results from the 2 inactivated groups indicated that T. pallidum might contain a cardiolipin antigen that could elicit the production of nontreponemal antibodies. Therefore, we immunized 10 rabbits with whole-treponemal-cell lysates. TPPA positivity first appeared 3 weeks after the initial injection (Figure 2 and Table 3). The highest TPPA titer in this group was 1:2560, which was similar to the titers of the 2 inactivated groups and did not show any significant differences (P > .05). However, TPPA positivity in the ultrasonicated treponeme group was significantly different (P < .001) from that in the active treponemal group. Regarding the VDRL test, 3 rabbits (numbers 1, 2, and 7) appeared to have a persistent VDRL titer of 1:1 (Table 4), whereas the other rabbits (numbers 4, 6, 8, and 10) had discontinuous positive VDRL results. The VDRL titers of all the rabbits were <1:1 and were similar to those of the inactivated treponeme groups. Table 3. Reactivity of Serum Treponema pallidum Particle Agglutination Assay Postimmunization in the Ultrasonicated Group Animal  TPPA Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  1  –  –  –  –  1:160  1:80  1:160  1:320  1:320  1:320  1:640  1:640  1:640  1:640  2  –  –  –  –  1:160  1:640  1:640  1:640  1:320  1:640  1:640  1:1280  1:640  1:640  3  –  –  –  –  –  –  –  1:160  1:80  1:160  1:80  1:80  –  –  4  –  –  –  –  1:160  1:160  1:2560  1:2560  1:640  1:640  1:640  1:1280  1:640  1:640  5  –  –  1:80  1:80  1:160  1:160  1:1280  1:1280  1:1280  1:640  1:640  1:640  1:640  1:640  6  –  –  –  1:80  1:80  1:160  1:640  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  1:640  7  –  –  –  1:160  1:640  1:1280  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  8  –  –  –  1:80  1:160  1:320  1:1280  1:2560  1:2560  1:1280  1:1280  1:1280  1:1280  1:1280  9  –  –  –  1:160  1:320  1:640  1:640  1:1280  1:1280  1:640  1:640  1:640  1:640  1:640  10  –  –  –  1:80  1:160  1:160  1:320  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  Animal  TPPA Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  1  –  –  –  –  1:160  1:80  1:160  1:320  1:320  1:320  1:640  1:640  1:640  1:640  2  –  –  –  –  1:160  1:640  1:640  1:640  1:320  1:640  1:640  1:1280  1:640  1:640  3  –  –  –  –  –  –  –  1:160  1:80  1:160  1:80  1:80  –  –  4  –  –  –  –  1:160  1:160  1:2560  1:2560  1:640  1:640  1:640  1:1280  1:640  1:640  5  –  –  1:80  1:80  1:160  1:160  1:1280  1:1280  1:1280  1:640  1:640  1:640  1:640  1:640  6  –  –  –  1:80  1:80  1:160  1:640  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  1:640  7  –  –  –  1:160  1:640  1:1280  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  8  –  –  –  1:80  1:160  1:320  1:1280  1:2560  1:2560  1:1280  1:1280  1:1280  1:1280  1:1280  9  –  –  –  1:160  1:320  1:640  1:640  1:1280  1:1280  1:640  1:640  1:640  1:640  1:640  10  –  –  –  1:80  1:160  1:160  1:320  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  Abbreviations: –, negative; TPPA, Treponema pallidum particle agglutination assay. View Large Table 3. Reactivity of Serum Treponema pallidum Particle Agglutination Assay Postimmunization in the Ultrasonicated Group Animal  TPPA Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  1  –  –  –  –  1:160  1:80  1:160  1:320  1:320  1:320  1:640  1:640  1:640  1:640  2  –  –  –  –  1:160  1:640  1:640  1:640  1:320  1:640  1:640  1:1280  1:640  1:640  3  –  –  –  –  –  –  –  1:160  1:80  1:160  1:80  1:80  –  –  4  –  –  –  –  1:160  1:160  1:2560  1:2560  1:640  1:640  1:640  1:1280  1:640  1:640  5  –  –  1:80  1:80  1:160  1:160  1:1280  1:1280  1:1280  1:640  1:640  1:640  1:640  1:640  6  –  –  –  1:80  1:80  1:160  1:640  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  1:640  7  –  –  –  1:160  1:640  1:1280  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  8  –  –  –  1:80  1:160  1:320  1:1280  1:2560  1:2560  1:1280  1:1280  1:1280  1:1280  1:1280  9  –  –  –  1:160  1:320  1:640  1:640  1:1280  1:1280  1:640  1:640  1:640  1:640  1:640  10  –  –  –  1:80  1:160  1:160  1:320  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  Animal  TPPA Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  1  –  –  –  –  1:160  1:80  1:160  1:320  1:320  1:320  1:640  1:640  1:640  1:640  2  –  –  –  –  1:160  1:640  1:640  1:640  1:320  1:640  1:640  1:1280  1:640  1:640  3  –  –  –  –  –  –  –  1:160  1:80  1:160  1:80  1:80  –  –  4  –  –  –  –  1:160  1:160  1:2560  1:2560  1:640  1:640  1:640  1:1280  1:640  1:640  5  –  –  1:80  1:80  1:160  1:160  1:1280  1:1280  1:1280  1:640  1:640  1:640  1:640  1:640  6  –  –  –  1:80  1:80  1:160  1:640  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  1:640  7  –  –  –  1:160  1:640  1:1280  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  8  –  –  –  1:80  1:160  1:320  1:1280  1:2560  1:2560  1:1280  1:1280  1:1280  1:1280  1:1280  9  –  –  –  1:160  1:320  1:640  1:640  1:1280  1:1280  1:640  1:640  1:640  1:640  1:640  10  –  –  –  1:80  1:160  1:160  1:320  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  Abbreviations: –, negative; TPPA, Treponema pallidum particle agglutination assay. View Large Table 4. Reactivity of Serum Venereal Disease Research Laboratory Postimmunization in the Ultrasonicated Group Animal  VDRL Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  1  –  1:1  1:1  1:1  ±  –  –  –  –  –  –  –  –  –  2  –  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  –  3  –  –  –  –  –  –  –  –  –  –  –  –  –  –  4  –  –  –  –  –  –  –  –  ±  ±  –  –  ±  –  5  –  –  –  –  –  –  –  –  –  –  –  –  –  –  6  –  ±  –  –  –  –  1:1  ±  ±  –  –  –  –  –  7  –  1:1  ±  ±  1:1  1:1  1:1  1:1  ±  ±  –  –  –  –  8  –  1:1  –  –  ±  –  –  –  1:1  –  –  –  –  –  9  –  –  –  –  –  –  –  –  –  –  –  –  –  –  10  –  –  –  –  –  ±  –  –  –  –  –  –  –  –  Animal  VDRL Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  1  –  1:1  1:1  1:1  ±  –  –  –  –  –  –  –  –  –  2  –  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  –  3  –  –  –  –  –  –  –  –  –  –  –  –  –  –  4  –  –  –  –  –  –  –  –  ±  ±  –  –  ±  –  5  –  –  –  –  –  –  –  –  –  –  –  –  –  –  6  –  ±  –  –  –  –  1:1  ±  ±  –  –  –  –  –  7  –  1:1  ±  ±  1:1  1:1  1:1  1:1  ±  ±  –  –  –  –  8  –  1:1  –  –  ±  –  –  –  1:1  –  –  –  –  –  9  –  –  –  –  –  –  –  –  –  –  –  –  –  –  10  –  –  –  –  –  ±  –  –  –  –  –  –  –  –  Abbreviations: –, negative; ±, weakly reactive; VDRL, Venereal Disease Research Laboratory. View Large Table 4. Reactivity of Serum Venereal Disease Research Laboratory Postimmunization in the Ultrasonicated Group Animal  VDRL Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  1  –  1:1  1:1  1:1  ±  –  –  –  –  –  –  –  –  –  2  –  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  –  3  –  –  –  –  –  –  –  –  –  –  –  –  –  –  4  –  –  –  –  –  –  –  –  ±  ±  –  –  ±  –  5  –  –  –  –  –  –  –  –  –  –  –  –  –  –  6  –  ±  –  –  –  –  1:1  ±  ±  –  –  –  –  –  7  –  1:1  ±  ±  1:1  1:1  1:1  1:1  ±  ±  –  –  –  –  8  –  1:1  –  –  ±  –  –  –  1:1  –  –  –  –  –  9  –  –  –  –  –  –  –  –  –  –  –  –  –  –  10  –  –  –  –  –  ±  –  –  –  –  –  –  –  –  Animal  VDRL Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  1  –  1:1  1:1  1:1  ±  –  –  –  –  –  –  –  –  –  2  –  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  –  3  –  –  –  –  –  –  –  –  –  –  –  –  –  –  4  –  –  –  –  –  –  –  –  ±  ±  –  –  ±  –  5  –  –  –  –  –  –  –  –  –  –  –  –  –  –  6  –  ±  –  –  –  –  1:1  ±  ±  –  –  –  –  –  7  –  1:1  ±  ±  1:1  1:1  1:1  1:1  ±  ±  –  –  –  –  8  –  1:1  –  –  ±  –  –  –  1:1  –  –  –  –  –  9  –  –  –  –  –  –  –  –  –  –  –  –  –  –  10  –  –  –  –  –  ±  –  –  –  –  –  –  –  –  Abbreviations: –, negative; ±, weakly reactive; VDRL, Venereal Disease Research Laboratory. View Large Immune Response Induced by the Cardiolipin and Cardiolipin-BSA Antigens Generally, cardiolipin is considered the most important antigenic substance in the nontreponemal test. We immunized 4 rabbits per group with the cardiolipin antigen and the cardiolipin-BSA antigen. The optical density of the anticardiolipin antibody was measured weekly (Figure 3). The ELISA showed that rabbits immunized with cardiolipin did not produce an anticardiolipin antibody. However, the anticardiolipin antibody test was positive at 4 weeks after the initial cardiolipin-BSA immunization and slowly increased over time. The optical density value of the anticardiolipin antibody was significantly greater in the BSA-cardiolipin group than in the cardiolipin group (P < .01), with the antibody titer in the BSA-cardiolipin group reaching 1:16000–1:64000 seven weeks after the first immunization. Figure 3. View largeDownload slide Variation tendency in the anticardiolipin antibody titer postimmunization with the cardiolipin and cardiolipin–bovine serum albumin (BSA) antigens. The differences in the optical density of the anticardiolipin antibody were assessed between 2 groups using repeated measurements (***P < .001). The variation in anticardiolipin antibody titers among 4 rabbits in the cardiolipin-BSA group is shown in the dashed box. Figure 3. View largeDownload slide Variation tendency in the anticardiolipin antibody titer postimmunization with the cardiolipin and cardiolipin–bovine serum albumin (BSA) antigens. The differences in the optical density of the anticardiolipin antibody were assessed between 2 groups using repeated measurements (***P < .001). The variation in anticardiolipin antibody titers among 4 rabbits in the cardiolipin-BSA group is shown in the dashed box. DISCUSSION The nontreponemal antibody was the first recognized serological response to infection with T. pallidum [26] and is still used for diagnosing syphilis and monitoring treatment efficacy [27]. In the last century, there has been a heated debate concerning the production of nontreponemal antibodies during T. pallidum infection [4]. Some studies have shown that nontreponemal antibodies are actually elicited by T. pallidum [28]. A previous article supporting this finding showed that T. pallidum contained 1.3 times more total lipids than proteins [29] and that 67.8% of the lipids in T. pallidum were phospholipids (including 13% cardiolipin), while the remaining lipids were neutral lipids (including 26.5% cholesterol). Therefore, T. pallidum contains a large amount of the cardiolipin antigen and can elicit nontreponemal antibodies in the host [28]. In this study, we showed direct evidence of the origin of nontreponemal antibodies during syphilis infection using an animal model. First, we applied different methods to inactivate the treponemes, including formalin, heat, and ultrasonication treatments, prior to the immunization of rabbits. The results from these immunizations were similar to those of previous studies [30, 31]. The results showed that inactivation of the treponemes stimulated the rabbits to produce nontreponemal antibodies. Because the inactivated treponemes are unable to damage host tissue cells and thus may not induce the release of cardiolipin from host tissue cells, the rabbits immunized with the rabbit cellar debris did not show a positive nontreponemal antibody result (data not shown). The antigen that causes nontreponemal antibody reactions may originate from T. pallidum. Our results indirectly demonstrate that T. pallidum has a cardiolipin antigen that can elicit nontreponemal antibody production. Why did all the rabbits show a positive VDRL test, and why were the nontreponemal antibody titers low (1:1 or weakly reactive) in our inactivated treponemal experiment? One reason may be that the cardiolipin antigen in T. pallidum has very weak immunogenicity. Cardiolipin is the only phospholipid with antigenicity [28] and theoretically can elicit antibody production [24]. In our study, we immunized rabbits with cardiolipin from bovine hearts or cardiolipin-BSA and evaluated the immunogenicity of cardiolipin in the treponemes. The results indicated that pure cardiolipin could not elicit anticardiolipin antibody production in rabbits. However, cardiolipin elicited anticardiolipin antibody production with strong immunogenicity when it was combined with BSA. Therefore, the weak immunogenicity of cardiolipin in T. pallidum may be because cardiolipin was not combined with a suitable T. pallidum protein. In addition, a previous study showed that the organisms could be labeled with VDRL antibodies when the T. pallidum outer membrane was disrupted [5], suggesting that the cardiolipin antigen might be located on the T. pallidum inner membrane [5, 32]. This possibility may be another reason for the weak immunogenicity of the cardiolipin antigen in T. pallidum. In addition to the treponemal origin theory, which explains the origins of nontreponemal antibodies, another possibility is that cardiolipin complexes are liberated from damaged cells at the site of infection and that these complexes provoke an autoimmune reaction [10, 11, 33]. In our study, active T. pallidum had significantly different immune effects when the treponemes were inactivated (by formalin, heat, or ultrasonication). Nontreponemal antibody production occurred earlier and reached higher titers in the active T. pallidum group than those in the inactivated T. pallidum groups, and the antibody titer was consecutive. The treponemal antibody titer in the active treponemal group also increased more rapidly and was higher than that in the inactivated groups. Unlike inactivated T. pallidum, active T. pallidum can proliferate during infection and attack and disrupt host cells. Consequently, the high titer of nontreponemal antibodies is a result of the combined effect of both the T. pallidum cardiolipin antigen and the damaged host–cell cardiolipin antigen during syphilis infection. We can assume that the host–cell cardiolipin antigen plays a major role in this process. T. pallidum can incorporate host fatty acids into lipids. Some articles reported that the fatty acid composition of T. pallidum was similar to that of infected testis, so the host-derived cardiolipin and other lipids are believed to be incorporated into T. pallidum, and the unique epitopes may induce nontreponemal antibody production during syphilis [4, 33]. Our study showed the origin of nontreponemal antibodies in an animal model. However, our study has some limitations. The statistical power in this study was small due to the limited sample size and the outbred nature of the rabbit animal model. In addition, this study conducted experiments using antibody tests and lacked direct pathological evidence. For this reason, additional studies are required to show that T. pallidum causes host–cell damage and that the release of the cardiolipin antigen induces the production of nontreponemal antibodies. Which specific T. pallidum protein is associated with the cardiolipin antigen and whether it can enhance immunogenicity of the cardiolipin antigen remain unknown. Furthermore, the biological function of nontreponemal antibodies is unclear—they may be protective, harmful, or simply a reflection of an immunological response in the host. These questions need to be answered in subsequent studies. In conclusion, our results show that T. pallidum contained a cardiolipin antigen with weak immunogenicity and that this antigen elicited the production of nontreponemal antibody at a low titer. High titers of nontreponemal antibody are a result of the combined effect of both the T. pallidum cardiolipin antigen and the damaged host–cell cardiolipin antigen during syphilis infection. Host–cell cardiolipin antigens play a major role in inducing nontreponemal antibody production. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Disclaimer. The funders played no role in the study design, data collection or analysis, the decision to publish, or manuscript preparation. Financial support. This work was supported by the National Natural Science Foundation of China (grant numbers 81772260, 81771312, 81672094, 81471967, 81471231, 81401749, 81201360, 81271335, 81101324, and 81171625); the Key Projects for Province Science and Technology Program of Fujian (grant number 2018D0014); the National Science Foundation for Distinguished Young Scholars of Fujian (grant number 2014D001); the Major Special Projects for Serious Illness of Xiamen (grant numbers 3502Z20159016 and 3502Z20179045); the Natural Science Foundation of Fujian Province (grant numbers 2016J01628 and 2015J01529); the Youth Foundation Project of Fujian Provincial Health Department (grant number 2017-2-105); and the Medical Innovation Project of Fujian Health Development Planning Commission (grant number 2012-CXB-33). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

Origin of Nontreponemal Antibodies During Treponema pallidum Infection: Evidence From a Rabbit Model

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Abstract

Abstract The origin of nontreponemal antibodies during syphilis infection is hotly debated. Here, we analyzed the immune response in rabbits immunized with various antigens. Inactivated treponemes elicited the production of low-titer nontreponemal antibodies in some rabbits. Cardiolipin combined with bovine serum albumin also induced anticardiolipin antibody production. These findings indicate that Treponema pallidum contained a cardiolipin antigen with weak immunogenicity. However, active T. pallidum induced higher nontreponemal antibody production with strong immunogenicity at an earlier time point, and the antibody titer was consecutive, suggesting the high nontreponemal antibody titer resulted from the combined effects of both the T. pallidum cardiolipin antigen and the damaged host–cell cardiolipin antigen during syphilis infection, the latter of which plays a major role in the induction of nontreponemal antibody production. Our study provides direct animal evidence of the origin of nontreponemal antibodies during T. pallidum infection. syphilis, inactivated treponemes, nontreponemal antibody, cardiolipin antigen Syphilis is a multistage sexually transmitted infection caused by Treponema pallidum subspecies pallidum [1]. Due to the absence of in vitro culture and simple staining methods for the detection of T. pallidum [2], serological detection methods are regarded as the mainstay for the diagnosis of syphilis and for monitoring the efficacy of subsequent antibiotic treatment [1, 3]. Syphilitic infection elicits 2 distinct types of antibody responses, traditionally designated “treponemal” and “nontreponemal” [4, 5]. Treponemal antibodies are directed against the polypeptide antigens of T. pallidum, whereas nontreponemal antibodies (also called “reagin”) recognize lipoidal antigens and are detected via a defined combination of phosphatidylcholine, cholesterol, and cardiolipin, among which cardiolipin is the major antigenic component [5–7]; these antibodies may cross-react with autoimmune antigens [8]. The origin of nontreponemal antibodies during T. pallidum infection is a great mystery of syphilis immunology and is hotly debated [4, 9]. Two origin theories have been proposed. The first theory is that cardiolipin complexes are liberated from host cells damaged by the treponeme and that these complexes subsequently provoke an autoimmune reaction [4]. Another theory is that nontreponemal antibodies are induced by the lipids of T. pallidum [4, 10, 11]. To the best of our knowledge, there have been few studies and little direct animal evidence of the origins of nontreponemal antibodies during T. pallidum infection. Here, we conducted a comprehensive investigation using a cohort of infected rabbits to explore the immune responses and reveal the origins of nontreponemal antibodies during T. pallidum infection. We analyzed the immune responses based on the variation tendency of the treponemal and nontreponemal antibodies in rabbits immunized with various treponeme antigens, including active treponemes, inactivated treponemes, whole-cell lysate proteins, and cardiolipin antigens. MATERIALS AND METHODS Ethics Statement This study was approved by the Institutional Ethics Committee of Zhongshan Hospital, Medical College of Xiamen University. All the rabbit experimental protocols were approved by the Animal Experimental Ethics Committee of the Medical College of Xiamen University. Propagation and Harvesting of T. pallidum From Rabbits Adult male New Zealand white rabbits (approximately 2.5 kg) were purchased from Xiamen University Laboratory Animal Center. The animals were fed antibiotic-free food and water and housed individually at 18°C–20°C. Sera collected from all the animals were tested using the Venereal Disease Research Laboratory (VDRL) test and the T. pallidum particle agglutination assay (TPPA) to ensure that these results were negative prior to inclusion of the animals [12]. The T. pallidum Nichols strain was kindly provided by Lorenzo Giacani, PhD (University of Washington, Seattle), and was propagated intratesticularly in the New Zealand white rabbits by serial passage in our laboratory to maintain virulence as previously described [13, 14]. Then, treponemes were extracted from the infected testes using 0.9% warm saline. In brief, the testes were removed aseptically. Several lengthwise cuts were made in each testis, and the treponemes were extracted on a rotary shaker in 0.9% saline. Gross testicular debris was sedimented by centrifugation at 400g for 15 minutes, and the organisms in the supernatant were counted under a darkfield microscope and adjusted to 2 × 107 organisms/mL for use. T. pallidum Antigen Preparation and Immunization An overview of the immunization procedure with various treponemal antigens is shown in Figure 1A. Figure 1. View largeDownload slide Experimental overview of the immunization scheme. A, Immunization with the Treponema pallidum antigen. (i) In the active T. pallidum group, rabbits (n = 4) were injected with active T. pallidum once. (ii) In the formalin-treated group, rabbits (n = 4) were injected with formalin-treated T. pallidum once per week for a total of 10 injections. (iii) In the heat-treated group, rabbits (n = 4) were immunized with heat-treated T. pallidum once per week for a total of 10 injections. (iv) In the ultrasonicated group, rabbits (n = 10) were immunized with ultrasonicated T. pallidum once every 2 weeks for a total of 5 injections. B, Immunization with cardiolipin antigen. (i) In the cardiolipin group, rabbits (n = 4) were immunized with cardiolipin antigen as described for the ultrasonicated group. (ii) In the cardiolipin–bovine serum albumin (BSA) group, rabbits (n = 4) were immunized with the cardiolipin-BSA antigen. Abbreviations: BSA, bovine serum albumin; PO4–, the phosphate radical of cardiolipin; TPPA, Treponema pallidum particle agglutination assay; VDRL, Venereal Disease Research Laboratory. Figure 1. View largeDownload slide Experimental overview of the immunization scheme. A, Immunization with the Treponema pallidum antigen. (i) In the active T. pallidum group, rabbits (n = 4) were injected with active T. pallidum once. (ii) In the formalin-treated group, rabbits (n = 4) were injected with formalin-treated T. pallidum once per week for a total of 10 injections. (iii) In the heat-treated group, rabbits (n = 4) were immunized with heat-treated T. pallidum once per week for a total of 10 injections. (iv) In the ultrasonicated group, rabbits (n = 10) were immunized with ultrasonicated T. pallidum once every 2 weeks for a total of 5 injections. B, Immunization with cardiolipin antigen. (i) In the cardiolipin group, rabbits (n = 4) were immunized with cardiolipin antigen as described for the ultrasonicated group. (ii) In the cardiolipin–bovine serum albumin (BSA) group, rabbits (n = 4) were immunized with the cardiolipin-BSA antigen. Abbreviations: BSA, bovine serum albumin; PO4–, the phosphate radical of cardiolipin; TPPA, Treponema pallidum particle agglutination assay; VDRL, Venereal Disease Research Laboratory. For the active T. pallidum group, the backs of 4 normal rabbits were shaved, cleaned with 75% ethanol, and injected intradermally with 0.1 mL of a fresh, active T. pallidum extract at a density of 2.0 × 107 organisms/mL at a total of 10 areas. The rabbits in this group were intradermally injected once. For the formalin-treated group, neutral-buffered formalin (Sinopharm, Shanghai, China) was added to the treponemes for a final concentration of 0.2%, and the sample was incubated overnight at room temperature. Then, the samples were centrifuged at 12000g for 30 minutes, the precipitate was washed twice in sterile 0.9% saline, and the treponemes were resuspended in 0.9% saline to a density of 2 × 107 organisms/mL for subsequent immunization at a total of 10 areas [15]. Four rabbits’ backs were shaved and injected intradermally with formalin-treated treponemes once per week for a total of 10 injections. Prior to immunization, the treponemes in this group were confirmed to be completely inactivated (methods are described below). For the heat-treated group, the treponeme suspension at a density of 2 × 107 organisms/mL was heated at 56°C for 1 hour to inactivate the organisms [16]. Four rabbits’ backs were shaved and injected intradermally using the same method described for the formalin-treated group. Prior to immunization, the treponemes in this group were confirmed to be completely inactivated (methods are described below). For the ultrasonicated group, to obtain the whole-cell lysate of T. pallidum, the treponemal suspension was centrifuged at 12000g for 30 minutes. The wet weight of the precipitate was measured. Afterward, the organisms were suspended in a 5-fold volume of ultrasonication buffer (50 mM of Tris-Hydrochloric acid and 1 mmol/L of ethylenediaminetetraacetic acid, pH 7.4). The suspension was sonicated using a Sonics Vibra Cell Disrupter System (Sonics & Materials, Newtown, Connecticut) at 20 kHz and 60 W on ice. The ultrasonication time was 7.7 seconds and was performed at 9.9-second intervals for 10 minutes [17–19]. Then, the protein concentration was determined using the bicinchoninic acid Protein Assay Kit (Takara Bio, Dalian, China). Ten rabbits were immunized subcutaneously with the abovementioned treponemal cell lysate mixed with an equal volume of Freund adjuvant (Boson Biotech, Xiamen, China). The initial immunization was implemented with Freund complete adjuvant, and Freund incomplete adjuvant was used in additional immunizations. Each immunization delivered 200 μg of antigen per rabbit. Additional immunizations were delivered every 2 weeks following the initial treponeme injection for a total of 5 injections [12, 20, 21]. Cardiolipin and Cardiolipin–Bovine Serum Albumin Antigen Preparations and Immunization Cardiolipin from the bovine heart (Sigma-Aldrich, St Louis, Missouri) was diluted with phosphate-buffered saline to a concentration of 400 μg/mL for immunization. In addition, cardiolipin was linked to bovine serum albumin (BSA) through covalent binding, which was accomplished by the Boson Biotech Company (Xiamen, China). The final concentration of the cardiolipin-BSA antigen was 400 μg/mL after dilution with phosphate-buffered saline. The cardiolipin antigen and cardiolipin-BSA antigen were mixed with an equal volume of Freund adjuvant, and immunized rabbits were treated in the same manner as the rabbits in the ultrasonicated treponeme group (Figure 1B). Serological Analysis Serum samples were collected from the ear arteries of all the rabbits every week starting 1 week after the initial antigen injection, and syphilis serological tests were performed in a blinded fashion using VDRL (Becton Dickinson and Company, Franklin Lakes, New Jersey) and TPPA kits (Fujirebio, Tokyo, Japan) following the manufacturers’ instructions [22, 23]. The serum anticardiolipin antibody titer was monitored with cardiolipin enzyme-linked immunosorbent assay (ELISA; Boson Biotech, Xiamen, China) as previously described [24]; the cutoff value was 2.1 times the mean value of the negative sample. Live/Dead T. pallidum Fluorescence Assay The LIVE/DEAD BacLight Viability Kit (Molecular Probes, Leiden, the Netherlands) was used according to the manufacturer’s instructions to determine whether the treponemes exhibited activity after inactivation [25]. The ratio between the 2 dyes was 1:1. The active treponemes, which were stained with SYTO 9, exhibited green fluorescence, and the dead treponemes, which were stained with propidium iodide, exhibited red fluorescence. Images were acquired on a Nikon ECLIPSE 80i fluorescence microscope (Nikon Instruments, Shanghai, China) equipped with a Nikon DS-Ri2 CCD camera. One hundred fields were counted to determine the proportion of the T. pallidum that exhibited red fluorescence. Statistical Analysis Serum VDRL and serum TPPA, which are quantitative measures of serial 2-fold dilutions, are presented as geometric means for the correlation analysis. Statistical analyses were conducted using SPSS version 17.0 (SPSS, Chicago, Illinois) for Windows with repeated-measures analysis of variance and general estimating equations. A 2-sided P value <.05 was considered statistically significant. RESULTS Effect of Inactivation on T. pallidum Treponemal activity analysis showed that the treponemes in the formalin- and heat-treated groups all exhibited red fluorescence (Supplementary Figure 1A and 1B), whereas the treponemes in the active T. pallidum group exhibited green fluorescence (Supplementary Figure 1C). Thus, the treponemes used for immunization were completely inactivated in the formalin- and heat-treated groups. Moreover, the treponemes had a mostly intact cell structure after inactivation. Reactivity of Serum TPPA and VDRL in the Inactivated Treponeme Groups First, we explored the variation tendency of treponemal antibodies in rabbits immunized with inactivated treponemes, including the formalin- and heat-treated treponemes. All the rabbits had positive serum TPPA, indicating that the inactivated treponemes had potent immunogenicity and could elicit the production of treponemal antibodies. The TPPA antibodies began to appear 3 weeks and 4 weeks after the initial injection of treponemes in the heat- and formalin-treated groups, respectively (Table 1). The TPPA test titers in these 2 inactivated treponeme groups increased over time. The highest TPPA titer in the heat-treated group was 1:2560, which was similar to the titer achieved in the formalin-treated group. No significant difference was found in the trend of TPPA titer change between the 2 inactivated treponeme groups (P > .05; Figure 2). In contrast, the TPPA test titer in the active treponemal group showed positive results and a rapid increase at an earlier time point than that in the formalin- and heat-treated groups (Table 1). All the rabbits in this group showed a positive TPPA result 1 week after the initial treponeme injection. At the same time, the highest titer in the active treponemal group was 1:10240, which was much higher than the titers achieved in the inactivated treponeme groups (P < .001; Figure 2). One rabbit that died 32 days after the initial treponeme injection in the heat-treated group was excluded from the experiment. Table 1. Reactivity of Serum Treponema pallidum Particle Agglutination Assay Postimmunization in the Formalin-Treated Group, Heated-Treated Group, and Active Group Group  Animal  TPPA Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  Formalin-treated group  1  –  –  –  –  –  1:320  1:640  1:320  1:320  1:160  1:320  1:160  1:80  1:80    2  –  –  –  1:80  1:80  1:640  1:640  1:640  1:640  1:640  1:640  1:640  1:640  1:640    3  –  –  –  1:80  1:80  1:320  1:640  1:640  1:1280  1:640  1:320  1:320  1:320  1:320    4  –  –  –  –  1:80  1:640  1:2560  1:1280  1:2560  1:1280  1:1280  1:640  1:640  1:640  Heat-treated group  1  –  –  1:80  1:80  1:160  1:160  1:320  1:320  1:320  1:320  1:160  1:160  1:160  1:160    2  –  –  –  –  Deceased    3  –  –  1:80  1:160  1:1280  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:1280  1:1280  1:1280    4  –  –  1:160  1:320  1:640  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:1280  Active Treponema pallidum group  1  –  1:2560  1:2560  1:1280  1:1280  1:2560  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120    2  –  1:320  1:640  1:640  1:1280  1:5120  1:5120  1:5120  1:10240  1:5120  1:5120  1:5120  1:5120  1:5120    3  –  1:640  1:320  1:640  1:1280  1:2560  1:2560  1:5120  1:5120  1:10240  1:10240  1:5120  1:5120  1:5120    4  –  1:640  1:1280  1:1280  1:2560  1:2560  1:5120  1:5120  1:5120  1:10240  1:5120  1:5120  1:5120  1:5120  Group  Animal  TPPA Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  Formalin-treated group  1  –  –  –  –  –  1:320  1:640  1:320  1:320  1:160  1:320  1:160  1:80  1:80    2  –  –  –  1:80  1:80  1:640  1:640  1:640  1:640  1:640  1:640  1:640  1:640  1:640    3  –  –  –  1:80  1:80  1:320  1:640  1:640  1:1280  1:640  1:320  1:320  1:320  1:320    4  –  –  –  –  1:80  1:640  1:2560  1:1280  1:2560  1:1280  1:1280  1:640  1:640  1:640  Heat-treated group  1  –  –  1:80  1:80  1:160  1:160  1:320  1:320  1:320  1:320  1:160  1:160  1:160  1:160    2  –  –  –  –  Deceased    3  –  –  1:80  1:160  1:1280  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:1280  1:1280  1:1280    4  –  –  1:160  1:320  1:640  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:1280  Active Treponema pallidum group  1  –  1:2560  1:2560  1:1280  1:1280  1:2560  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120    2  –  1:320  1:640  1:640  1:1280  1:5120  1:5120  1:5120  1:10240  1:5120  1:5120  1:5120  1:5120  1:5120    3  –  1:640  1:320  1:640  1:1280  1:2560  1:2560  1:5120  1:5120  1:10240  1:10240  1:5120  1:5120  1:5120    4  –  1:640  1:1280  1:1280  1:2560  1:2560  1:5120  1:5120  1:5120  1:10240  1:5120  1:5120  1:5120  1:5120  Abbreviations: –, negative; TPPA, Treponema pallidum particle agglutination assay. View Large Table 1. Reactivity of Serum Treponema pallidum Particle Agglutination Assay Postimmunization in the Formalin-Treated Group, Heated-Treated Group, and Active Group Group  Animal  TPPA Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  Formalin-treated group  1  –  –  –  –  –  1:320  1:640  1:320  1:320  1:160  1:320  1:160  1:80  1:80    2  –  –  –  1:80  1:80  1:640  1:640  1:640  1:640  1:640  1:640  1:640  1:640  1:640    3  –  –  –  1:80  1:80  1:320  1:640  1:640  1:1280  1:640  1:320  1:320  1:320  1:320    4  –  –  –  –  1:80  1:640  1:2560  1:1280  1:2560  1:1280  1:1280  1:640  1:640  1:640  Heat-treated group  1  –  –  1:80  1:80  1:160  1:160  1:320  1:320  1:320  1:320  1:160  1:160  1:160  1:160    2  –  –  –  –  Deceased    3  –  –  1:80  1:160  1:1280  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:1280  1:1280  1:1280    4  –  –  1:160  1:320  1:640  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:1280  Active Treponema pallidum group  1  –  1:2560  1:2560  1:1280  1:1280  1:2560  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120    2  –  1:320  1:640  1:640  1:1280  1:5120  1:5120  1:5120  1:10240  1:5120  1:5120  1:5120  1:5120  1:5120    3  –  1:640  1:320  1:640  1:1280  1:2560  1:2560  1:5120  1:5120  1:10240  1:10240  1:5120  1:5120  1:5120    4  –  1:640  1:1280  1:1280  1:2560  1:2560  1:5120  1:5120  1:5120  1:10240  1:5120  1:5120  1:5120  1:5120  Group  Animal  TPPA Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  Formalin-treated group  1  –  –  –  –  –  1:320  1:640  1:320  1:320  1:160  1:320  1:160  1:80  1:80    2  –  –  –  1:80  1:80  1:640  1:640  1:640  1:640  1:640  1:640  1:640  1:640  1:640    3  –  –  –  1:80  1:80  1:320  1:640  1:640  1:1280  1:640  1:320  1:320  1:320  1:320    4  –  –  –  –  1:80  1:640  1:2560  1:1280  1:2560  1:1280  1:1280  1:640  1:640  1:640  Heat-treated group  1  –  –  1:80  1:80  1:160  1:160  1:320  1:320  1:320  1:320  1:160  1:160  1:160  1:160    2  –  –  –  –  Deceased    3  –  –  1:80  1:160  1:1280  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:1280  1:1280  1:1280    4  –  –  1:160  1:320  1:640  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:1280  Active Treponema pallidum group  1  –  1:2560  1:2560  1:1280  1:1280  1:2560  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120  1:5120    2  –  1:320  1:640  1:640  1:1280  1:5120  1:5120  1:5120  1:10240  1:5120  1:5120  1:5120  1:5120  1:5120    3  –  1:640  1:320  1:640  1:1280  1:2560  1:2560  1:5120  1:5120  1:10240  1:10240  1:5120  1:5120  1:5120    4  –  1:640  1:1280  1:1280  1:2560  1:2560  1:5120  1:5120  1:5120  1:10240  1:5120  1:5120  1:5120  1:5120  Abbreviations: –, negative; TPPA, Treponema pallidum particle agglutination assay. View Large Figure 2. View largeDownload slide Variation tendency in the postimmunization Treponema pallidum particle agglutination assay (TPPA) titer in the groups immunized with various treponemal antigens. The test titers are presented as the geometric means and were assessed using generalized estimating equations (***P < .001). Figure 2. View largeDownload slide Variation tendency in the postimmunization Treponema pallidum particle agglutination assay (TPPA) titer in the groups immunized with various treponemal antigens. The test titers are presented as the geometric means and were assessed using generalized estimating equations (***P < .001). Second, we analyzed the variation tendency of nontreponemal antibodies throughout the experimental phase. One rabbit in the heat-treated group (number 1) appeared to have a persistent VDRL titer of 1:1 9 weeks after the initial treponeme injection, and another rabbit in the formalin-treated group (number 4) had a persistent titer at 2 weeks after the initial treponeme injection (Table 2). Their titers did not increase over time. In addition, several rabbits appeared to have weakly discontinuous VDRL reactivity. In contrast, positive VDRL results were observed in all the rabbits as early as 2 weeks after the initial injection, and the VDRL titer was notably increased in the active treponemal group. Consecutive and higher VDRL titers were observed in all the rabbits in the active treponemal group. At 3 weeks following the initial treponeme injection, the VDRL test titer reached a peak at 1:64 and then decreased. Table 2. Reactivity of Serum Venereal Disease Research Laboratory Postimmunization in the Formalin-Treated Group, Heat-Treated Group, and Active Group Group  Animal  VDRL Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  Formalin-treated group  1  –  –  –  –  –  –  –  –  ±  1:1  1:1  1:1  1:1  1:1    2  –  –  –  –  –  –  –  –  –  ±  –  –  ±  ±    3  –  –  –  –  –  –  –  –  –  –  –  –  –  ±    4  –  –  –  –  –  –  –  –  –  ±  ±  –  –  ±  Heat-treated group  1  –  ±  –  –  –  –  ±  ±  ±  –  –  –  –  –    2  –  –  –  –  Deceased    3  –  –  –  –  –  –  –  –  ±  –  –  –  –  –    4  –  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  Active Treponema pallidum group  1  –  1:16  1:16  1:16  1:16  1:8  1:4  1:4  1:4  1:2  1:1  1:1  1:1  1:1    2  –  1:8  1:16  1:16  1:16  1:16  1:8  1:8  1:4  1:4  1:2  1:1  1:1  1:1    3  –  1:4  1:8  1:8  1:4  1:2  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1    4  –  1:16  1:64  1:32  1:8  1:4  1:4  1:4  1:4  1:2  1:2  1:1  1:1  1:1  Group  Animal  VDRL Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  Formalin-treated group  1  –  –  –  –  –  –  –  –  ±  1:1  1:1  1:1  1:1  1:1    2  –  –  –  –  –  –  –  –  –  ±  –  –  ±  ±    3  –  –  –  –  –  –  –  –  –  –  –  –  –  ±    4  –  –  –  –  –  –  –  –  –  ±  ±  –  –  ±  Heat-treated group  1  –  ±  –  –  –  –  ±  ±  ±  –  –  –  –  –    2  –  –  –  –  Deceased    3  –  –  –  –  –  –  –  –  ±  –  –  –  –  –    4  –  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  Active Treponema pallidum group  1  –  1:16  1:16  1:16  1:16  1:8  1:4  1:4  1:4  1:2  1:1  1:1  1:1  1:1    2  –  1:8  1:16  1:16  1:16  1:16  1:8  1:8  1:4  1:4  1:2  1:1  1:1  1:1    3  –  1:4  1:8  1:8  1:4  1:2  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1    4  –  1:16  1:64  1:32  1:8  1:4  1:4  1:4  1:4  1:2  1:2  1:1  1:1  1:1  Abbreviations: –, negative; –, nonreactive; ±, weakly reactive; VDRL, Venereal Disease Research Laboratory. View Large Table 2. Reactivity of Serum Venereal Disease Research Laboratory Postimmunization in the Formalin-Treated Group, Heat-Treated Group, and Active Group Group  Animal  VDRL Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  Formalin-treated group  1  –  –  –  –  –  –  –  –  ±  1:1  1:1  1:1  1:1  1:1    2  –  –  –  –  –  –  –  –  –  ±  –  –  ±  ±    3  –  –  –  –  –  –  –  –  –  –  –  –  –  ±    4  –  –  –  –  –  –  –  –  –  ±  ±  –  –  ±  Heat-treated group  1  –  ±  –  –  –  –  ±  ±  ±  –  –  –  –  –    2  –  –  –  –  Deceased    3  –  –  –  –  –  –  –  –  ±  –  –  –  –  –    4  –  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  Active Treponema pallidum group  1  –  1:16  1:16  1:16  1:16  1:8  1:4  1:4  1:4  1:2  1:1  1:1  1:1  1:1    2  –  1:8  1:16  1:16  1:16  1:16  1:8  1:8  1:4  1:4  1:2  1:1  1:1  1:1    3  –  1:4  1:8  1:8  1:4  1:2  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1    4  –  1:16  1:64  1:32  1:8  1:4  1:4  1:4  1:4  1:2  1:2  1:1  1:1  1:1  Group  Animal  VDRL Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  Formalin-treated group  1  –  –  –  –  –  –  –  –  ±  1:1  1:1  1:1  1:1  1:1    2  –  –  –  –  –  –  –  –  –  ±  –  –  ±  ±    3  –  –  –  –  –  –  –  –  –  –  –  –  –  ±    4  –  –  –  –  –  –  –  –  –  ±  ±  –  –  ±  Heat-treated group  1  –  ±  –  –  –  –  ±  ±  ±  –  –  –  –  –    2  –  –  –  –  Deceased    3  –  –  –  –  –  –  –  –  ±  –  –  –  –  –    4  –  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  Active Treponema pallidum group  1  –  1:16  1:16  1:16  1:16  1:8  1:4  1:4  1:4  1:2  1:1  1:1  1:1  1:1    2  –  1:8  1:16  1:16  1:16  1:16  1:8  1:8  1:4  1:4  1:2  1:1  1:1  1:1    3  –  1:4  1:8  1:8  1:4  1:2  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1    4  –  1:16  1:64  1:32  1:8  1:4  1:4  1:4  1:4  1:2  1:2  1:1  1:1  1:1  Abbreviations: –, negative; –, nonreactive; ±, weakly reactive; VDRL, Venereal Disease Research Laboratory. View Large Reactivity of Serum TPPA and VDRL in the Ultrasonicated Treponeme Group The results from the 2 inactivated groups indicated that T. pallidum might contain a cardiolipin antigen that could elicit the production of nontreponemal antibodies. Therefore, we immunized 10 rabbits with whole-treponemal-cell lysates. TPPA positivity first appeared 3 weeks after the initial injection (Figure 2 and Table 3). The highest TPPA titer in this group was 1:2560, which was similar to the titers of the 2 inactivated groups and did not show any significant differences (P > .05). However, TPPA positivity in the ultrasonicated treponeme group was significantly different (P < .001) from that in the active treponemal group. Regarding the VDRL test, 3 rabbits (numbers 1, 2, and 7) appeared to have a persistent VDRL titer of 1:1 (Table 4), whereas the other rabbits (numbers 4, 6, 8, and 10) had discontinuous positive VDRL results. The VDRL titers of all the rabbits were <1:1 and were similar to those of the inactivated treponeme groups. Table 3. Reactivity of Serum Treponema pallidum Particle Agglutination Assay Postimmunization in the Ultrasonicated Group Animal  TPPA Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  1  –  –  –  –  1:160  1:80  1:160  1:320  1:320  1:320  1:640  1:640  1:640  1:640  2  –  –  –  –  1:160  1:640  1:640  1:640  1:320  1:640  1:640  1:1280  1:640  1:640  3  –  –  –  –  –  –  –  1:160  1:80  1:160  1:80  1:80  –  –  4  –  –  –  –  1:160  1:160  1:2560  1:2560  1:640  1:640  1:640  1:1280  1:640  1:640  5  –  –  1:80  1:80  1:160  1:160  1:1280  1:1280  1:1280  1:640  1:640  1:640  1:640  1:640  6  –  –  –  1:80  1:80  1:160  1:640  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  1:640  7  –  –  –  1:160  1:640  1:1280  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  8  –  –  –  1:80  1:160  1:320  1:1280  1:2560  1:2560  1:1280  1:1280  1:1280  1:1280  1:1280  9  –  –  –  1:160  1:320  1:640  1:640  1:1280  1:1280  1:640  1:640  1:640  1:640  1:640  10  –  –  –  1:80  1:160  1:160  1:320  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  Animal  TPPA Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  1  –  –  –  –  1:160  1:80  1:160  1:320  1:320  1:320  1:640  1:640  1:640  1:640  2  –  –  –  –  1:160  1:640  1:640  1:640  1:320  1:640  1:640  1:1280  1:640  1:640  3  –  –  –  –  –  –  –  1:160  1:80  1:160  1:80  1:80  –  –  4  –  –  –  –  1:160  1:160  1:2560  1:2560  1:640  1:640  1:640  1:1280  1:640  1:640  5  –  –  1:80  1:80  1:160  1:160  1:1280  1:1280  1:1280  1:640  1:640  1:640  1:640  1:640  6  –  –  –  1:80  1:80  1:160  1:640  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  1:640  7  –  –  –  1:160  1:640  1:1280  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  8  –  –  –  1:80  1:160  1:320  1:1280  1:2560  1:2560  1:1280  1:1280  1:1280  1:1280  1:1280  9  –  –  –  1:160  1:320  1:640  1:640  1:1280  1:1280  1:640  1:640  1:640  1:640  1:640  10  –  –  –  1:80  1:160  1:160  1:320  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  Abbreviations: –, negative; TPPA, Treponema pallidum particle agglutination assay. View Large Table 3. Reactivity of Serum Treponema pallidum Particle Agglutination Assay Postimmunization in the Ultrasonicated Group Animal  TPPA Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  1  –  –  –  –  1:160  1:80  1:160  1:320  1:320  1:320  1:640  1:640  1:640  1:640  2  –  –  –  –  1:160  1:640  1:640  1:640  1:320  1:640  1:640  1:1280  1:640  1:640  3  –  –  –  –  –  –  –  1:160  1:80  1:160  1:80  1:80  –  –  4  –  –  –  –  1:160  1:160  1:2560  1:2560  1:640  1:640  1:640  1:1280  1:640  1:640  5  –  –  1:80  1:80  1:160  1:160  1:1280  1:1280  1:1280  1:640  1:640  1:640  1:640  1:640  6  –  –  –  1:80  1:80  1:160  1:640  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  1:640  7  –  –  –  1:160  1:640  1:1280  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  8  –  –  –  1:80  1:160  1:320  1:1280  1:2560  1:2560  1:1280  1:1280  1:1280  1:1280  1:1280  9  –  –  –  1:160  1:320  1:640  1:640  1:1280  1:1280  1:640  1:640  1:640  1:640  1:640  10  –  –  –  1:80  1:160  1:160  1:320  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  Animal  TPPA Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  1  –  –  –  –  1:160  1:80  1:160  1:320  1:320  1:320  1:640  1:640  1:640  1:640  2  –  –  –  –  1:160  1:640  1:640  1:640  1:320  1:640  1:640  1:1280  1:640  1:640  3  –  –  –  –  –  –  –  1:160  1:80  1:160  1:80  1:80  –  –  4  –  –  –  –  1:160  1:160  1:2560  1:2560  1:640  1:640  1:640  1:1280  1:640  1:640  5  –  –  1:80  1:80  1:160  1:160  1:1280  1:1280  1:1280  1:640  1:640  1:640  1:640  1:640  6  –  –  –  1:80  1:80  1:160  1:640  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  1:640  7  –  –  –  1:160  1:640  1:1280  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  1:2560  8  –  –  –  1:80  1:160  1:320  1:1280  1:2560  1:2560  1:1280  1:1280  1:1280  1:1280  1:1280  9  –  –  –  1:160  1:320  1:640  1:640  1:1280  1:1280  1:640  1:640  1:640  1:640  1:640  10  –  –  –  1:80  1:160  1:160  1:320  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  1:1280  Abbreviations: –, negative; TPPA, Treponema pallidum particle agglutination assay. View Large Table 4. Reactivity of Serum Venereal Disease Research Laboratory Postimmunization in the Ultrasonicated Group Animal  VDRL Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  1  –  1:1  1:1  1:1  ±  –  –  –  –  –  –  –  –  –  2  –  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  –  3  –  –  –  –  –  –  –  –  –  –  –  –  –  –  4  –  –  –  –  –  –  –  –  ±  ±  –  –  ±  –  5  –  –  –  –  –  –  –  –  –  –  –  –  –  –  6  –  ±  –  –  –  –  1:1  ±  ±  –  –  –  –  –  7  –  1:1  ±  ±  1:1  1:1  1:1  1:1  ±  ±  –  –  –  –  8  –  1:1  –  –  ±  –  –  –  1:1  –  –  –  –  –  9  –  –  –  –  –  –  –  –  –  –  –  –  –  –  10  –  –  –  –  –  ±  –  –  –  –  –  –  –  –  Animal  VDRL Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  1  –  1:1  1:1  1:1  ±  –  –  –  –  –  –  –  –  –  2  –  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  –  3  –  –  –  –  –  –  –  –  –  –  –  –  –  –  4  –  –  –  –  –  –  –  –  ±  ±  –  –  ±  –  5  –  –  –  –  –  –  –  –  –  –  –  –  –  –  6  –  ±  –  –  –  –  1:1  ±  ±  –  –  –  –  –  7  –  1:1  ±  ±  1:1  1:1  1:1  1:1  ±  ±  –  –  –  –  8  –  1:1  –  –  ±  –  –  –  1:1  –  –  –  –  –  9  –  –  –  –  –  –  –  –  –  –  –  –  –  –  10  –  –  –  –  –  ±  –  –  –  –  –  –  –  –  Abbreviations: –, negative; ±, weakly reactive; VDRL, Venereal Disease Research Laboratory. View Large Table 4. Reactivity of Serum Venereal Disease Research Laboratory Postimmunization in the Ultrasonicated Group Animal  VDRL Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  1  –  1:1  1:1  1:1  ±  –  –  –  –  –  –  –  –  –  2  –  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  –  3  –  –  –  –  –  –  –  –  –  –  –  –  –  –  4  –  –  –  –  –  –  –  –  ±  ±  –  –  ±  –  5  –  –  –  –  –  –  –  –  –  –  –  –  –  –  6  –  ±  –  –  –  –  1:1  ±  ±  –  –  –  –  –  7  –  1:1  ±  ±  1:1  1:1  1:1  1:1  ±  ±  –  –  –  –  8  –  1:1  –  –  ±  –  –  –  1:1  –  –  –  –  –  9  –  –  –  –  –  –  –  –  –  –  –  –  –  –  10  –  –  –  –  –  ±  –  –  –  –  –  –  –  –  Animal  VDRL Titer Postimmunization  Week 1  Week 2  Week 3  Week 4  Week 5  Week 6  Week 7  Week 8  Week 9  Week 10  Week 11  Week 12  Week 13  Week 14  1  –  1:1  1:1  1:1  ±  –  –  –  –  –  –  –  –  –  2  –  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  1:1  –  3  –  –  –  –  –  –  –  –  –  –  –  –  –  –  4  –  –  –  –  –  –  –  –  ±  ±  –  –  ±  –  5  –  –  –  –  –  –  –  –  –  –  –  –  –  –  6  –  ±  –  –  –  –  1:1  ±  ±  –  –  –  –  –  7  –  1:1  ±  ±  1:1  1:1  1:1  1:1  ±  ±  –  –  –  –  8  –  1:1  –  –  ±  –  –  –  1:1  –  –  –  –  –  9  –  –  –  –  –  –  –  –  –  –  –  –  –  –  10  –  –  –  –  –  ±  –  –  –  –  –  –  –  –  Abbreviations: –, negative; ±, weakly reactive; VDRL, Venereal Disease Research Laboratory. View Large Immune Response Induced by the Cardiolipin and Cardiolipin-BSA Antigens Generally, cardiolipin is considered the most important antigenic substance in the nontreponemal test. We immunized 4 rabbits per group with the cardiolipin antigen and the cardiolipin-BSA antigen. The optical density of the anticardiolipin antibody was measured weekly (Figure 3). The ELISA showed that rabbits immunized with cardiolipin did not produce an anticardiolipin antibody. However, the anticardiolipin antibody test was positive at 4 weeks after the initial cardiolipin-BSA immunization and slowly increased over time. The optical density value of the anticardiolipin antibody was significantly greater in the BSA-cardiolipin group than in the cardiolipin group (P < .01), with the antibody titer in the BSA-cardiolipin group reaching 1:16000–1:64000 seven weeks after the first immunization. Figure 3. View largeDownload slide Variation tendency in the anticardiolipin antibody titer postimmunization with the cardiolipin and cardiolipin–bovine serum albumin (BSA) antigens. The differences in the optical density of the anticardiolipin antibody were assessed between 2 groups using repeated measurements (***P < .001). The variation in anticardiolipin antibody titers among 4 rabbits in the cardiolipin-BSA group is shown in the dashed box. Figure 3. View largeDownload slide Variation tendency in the anticardiolipin antibody titer postimmunization with the cardiolipin and cardiolipin–bovine serum albumin (BSA) antigens. The differences in the optical density of the anticardiolipin antibody were assessed between 2 groups using repeated measurements (***P < .001). The variation in anticardiolipin antibody titers among 4 rabbits in the cardiolipin-BSA group is shown in the dashed box. DISCUSSION The nontreponemal antibody was the first recognized serological response to infection with T. pallidum [26] and is still used for diagnosing syphilis and monitoring treatment efficacy [27]. In the last century, there has been a heated debate concerning the production of nontreponemal antibodies during T. pallidum infection [4]. Some studies have shown that nontreponemal antibodies are actually elicited by T. pallidum [28]. A previous article supporting this finding showed that T. pallidum contained 1.3 times more total lipids than proteins [29] and that 67.8% of the lipids in T. pallidum were phospholipids (including 13% cardiolipin), while the remaining lipids were neutral lipids (including 26.5% cholesterol). Therefore, T. pallidum contains a large amount of the cardiolipin antigen and can elicit nontreponemal antibodies in the host [28]. In this study, we showed direct evidence of the origin of nontreponemal antibodies during syphilis infection using an animal model. First, we applied different methods to inactivate the treponemes, including formalin, heat, and ultrasonication treatments, prior to the immunization of rabbits. The results from these immunizations were similar to those of previous studies [30, 31]. The results showed that inactivation of the treponemes stimulated the rabbits to produce nontreponemal antibodies. Because the inactivated treponemes are unable to damage host tissue cells and thus may not induce the release of cardiolipin from host tissue cells, the rabbits immunized with the rabbit cellar debris did not show a positive nontreponemal antibody result (data not shown). The antigen that causes nontreponemal antibody reactions may originate from T. pallidum. Our results indirectly demonstrate that T. pallidum has a cardiolipin antigen that can elicit nontreponemal antibody production. Why did all the rabbits show a positive VDRL test, and why were the nontreponemal antibody titers low (1:1 or weakly reactive) in our inactivated treponemal experiment? One reason may be that the cardiolipin antigen in T. pallidum has very weak immunogenicity. Cardiolipin is the only phospholipid with antigenicity [28] and theoretically can elicit antibody production [24]. In our study, we immunized rabbits with cardiolipin from bovine hearts or cardiolipin-BSA and evaluated the immunogenicity of cardiolipin in the treponemes. The results indicated that pure cardiolipin could not elicit anticardiolipin antibody production in rabbits. However, cardiolipin elicited anticardiolipin antibody production with strong immunogenicity when it was combined with BSA. Therefore, the weak immunogenicity of cardiolipin in T. pallidum may be because cardiolipin was not combined with a suitable T. pallidum protein. In addition, a previous study showed that the organisms could be labeled with VDRL antibodies when the T. pallidum outer membrane was disrupted [5], suggesting that the cardiolipin antigen might be located on the T. pallidum inner membrane [5, 32]. This possibility may be another reason for the weak immunogenicity of the cardiolipin antigen in T. pallidum. In addition to the treponemal origin theory, which explains the origins of nontreponemal antibodies, another possibility is that cardiolipin complexes are liberated from damaged cells at the site of infection and that these complexes provoke an autoimmune reaction [10, 11, 33]. In our study, active T. pallidum had significantly different immune effects when the treponemes were inactivated (by formalin, heat, or ultrasonication). Nontreponemal antibody production occurred earlier and reached higher titers in the active T. pallidum group than those in the inactivated T. pallidum groups, and the antibody titer was consecutive. The treponemal antibody titer in the active treponemal group also increased more rapidly and was higher than that in the inactivated groups. Unlike inactivated T. pallidum, active T. pallidum can proliferate during infection and attack and disrupt host cells. Consequently, the high titer of nontreponemal antibodies is a result of the combined effect of both the T. pallidum cardiolipin antigen and the damaged host–cell cardiolipin antigen during syphilis infection. We can assume that the host–cell cardiolipin antigen plays a major role in this process. T. pallidum can incorporate host fatty acids into lipids. Some articles reported that the fatty acid composition of T. pallidum was similar to that of infected testis, so the host-derived cardiolipin and other lipids are believed to be incorporated into T. pallidum, and the unique epitopes may induce nontreponemal antibody production during syphilis [4, 33]. Our study showed the origin of nontreponemal antibodies in an animal model. However, our study has some limitations. The statistical power in this study was small due to the limited sample size and the outbred nature of the rabbit animal model. In addition, this study conducted experiments using antibody tests and lacked direct pathological evidence. For this reason, additional studies are required to show that T. pallidum causes host–cell damage and that the release of the cardiolipin antigen induces the production of nontreponemal antibodies. Which specific T. pallidum protein is associated with the cardiolipin antigen and whether it can enhance immunogenicity of the cardiolipin antigen remain unknown. Furthermore, the biological function of nontreponemal antibodies is unclear—they may be protective, harmful, or simply a reflection of an immunological response in the host. These questions need to be answered in subsequent studies. In conclusion, our results show that T. pallidum contained a cardiolipin antigen with weak immunogenicity and that this antigen elicited the production of nontreponemal antibody at a low titer. High titers of nontreponemal antibody are a result of the combined effect of both the T. pallidum cardiolipin antigen and the damaged host–cell cardiolipin antigen during syphilis infection. Host–cell cardiolipin antigens play a major role in inducing nontreponemal antibody production. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Disclaimer. The funders played no role in the study design, data collection or analysis, the decision to publish, or manuscript preparation. Financial support. This work was supported by the National Natural Science Foundation of China (grant numbers 81772260, 81771312, 81672094, 81471967, 81471231, 81401749, 81201360, 81271335, 81101324, and 81171625); the Key Projects for Province Science and Technology Program of Fujian (grant number 2018D0014); the National Science Foundation for Distinguished Young Scholars of Fujian (grant number 2014D001); the Major Special Projects for Serious Illness of Xiamen (grant numbers 3502Z20159016 and 3502Z20179045); the Natural Science Foundation of Fujian Province (grant numbers 2016J01628 and 2015J01529); the Youth Foundation Project of Fujian Provincial Health Department (grant number 2017-2-105); and the Medical Innovation Project of Fujian Health Development Planning Commission (grant number 2012-CXB-33). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

The Journal of Infectious DiseasesOxford University Press

Published: Apr 21, 2018

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