Analysis of the Humoral Immune Response to Treponema pallidum in the Different Stages of Untreated Human Syphilis

Zbl. Bakt. 271, 171-179 (1989)

Analysis of the Humoral Immune Response to Treponema pallidum in the Different Stages of Untreated Human Syphilis MATTHAus MOSKOPHIDIS Hygienisches Institut, Medizinaluntersuchungsanstalt, D-2000 Hamburg 28

With 2 Figures· Received July 12, 1988 . Accepted January 23, 1989

Abstract It has been shown that using treponema-specific and lipoidal assays for the demonstration of antibodies to Treponema pallidum subsp. pallidum in pooled sera of syphilitic patients, each stage of the untreated infection can be characterized by a typical immune response as far as specific immunoglobulin M (lgM) and G (IgG) antibody reactivity is concerned. In primary syphilis, IgM antibodies predominate over those of the IgG class. Moreover, IgM antibody reactivity with decreased titres can be detected in the later stages of untreated treponemal infection. Furthermore, it has been shown bI using the double antibody radioimmunoprecipitation assay with detergent-solubilized [ 5S] methionine-labelled Treponema pallidum as antigen followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography, that IgM and IgG antibodies in all stages of infection are directed against eight major antigenic proteins of Treponema pallidum with a Mr of 15500,33000,34500,37000,41000,44500,47000 and 71000.

Zusammenfassung Bei Nachweis von Antikorpern gegen Treponema pallidum subsp. pallidum in gepoolten Seren von Patienten mit Syphilis wird mit Hilfe treponemen-spezifischer und Lipoid-Tests gezeigt, daB jedes Stadium der unbehandelten Infektion durch eine typische Immunantwort charakterisiert werden kann, wenn die spezifische Immunglobulin M (IgM) and G (IgG)Antikorperreaktivitiit dabei beriicksichtigt wird. Bei der primiiren Syphilis dominieren die IgM-Antikorper gegeniiber solchen vom IgG-Typ. Eine IgM-Antikorperreaktivitiit mit abnehmenden Titern liiBt sich jedoch auch in den spiiteren Stadien der unbehandelten Treponema-Infektion nachweisen. Weiterhin wird unter Verwendung des Doppelantikorper-Radioimmunpriizipitationsassays mit detergentiensolubilisiertem [35S] Methioninmarkiertem Treponema pallidum als Antigen und nachfolgender Natrium DodecylsulfatPolyacrylamidgel-Elektrophorese sowie Autoradiographie gezeigt, daB die IgM- und IgGAntikorper in allen Stadien der Infektion gegen acht Hauptproteinantigene von Treponema pallidum mit den Mr 15500, 33000, 34500, 37000, 41000, 44500, 47000 und 71000 gerichtet sind.

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Introduction Serological testing in syphilis is an important tool for diagnosis and the main method to assess the efficacy of therapy. These tests include the treponemal assays, Treponema pallidum hemagglutination assay (TPHA) (16), fluorescent treponemal antibody absorption (FTA-ABS) test (7), and the non-treponemallipoidal assays like cardiolipincomplement fixation (Card.-CF) or the Venereal Disease Research Laboratory (VDRL) assay. Immunoglobulin G (IgG)-FTA-ABS, fractionated immunoblogulin M (IgM)FTA-ABS (12) or Treponema pallidum-IgM enzyme-linked immunosorbent assay (TpIgM-ELISA) (13) permit a separate demonstration of IgG or IgM antibodies to T. pallidum. Although these diagnostic tests are used routinely, little is known about the development and persistence of IgG and IgM antibodies and their specificities fiir T. pallidum protein antigens in human syphilis. A characterization of specific main treponemal protein antigens and a clear understanding of the host's immune response to T. pallidum during infection are prerequisites for the development of improved methods of serodiagnosis. The present report describes the development and persistence of both IgG and IgM antibodies to T. pallidum subsp. pallidum (Nichols strain) and the specificities of these antibodies for individual treponemal protein antigens in the different stages of untreated human syphilis. Materials and Methods

Sera. The investigated sera had been collected by dermatologists. On the basis of clinical manifestations or a history of infection, 30 patients with reactive treponemal serological tests (TPHA and FTA-ABS) were divided into: (a) those with untreated primary syphilis (n = 10, three to six weeks after infection), (b) those with untreated secondary syphilis (n = 10, three to six months after infection) and (c) those with untreated late (tertiary) syphilis (n = 10, three to five years after infection). A diagnosis of primary syphilis was based on the presence of darkfield-positive genital lesions (chancre). All patients with secondary syphilis had a characteristic rash and/or palmar and plantar lesions, and lymphadenitis. Those with late syphilis were found to have been infected 3-5 years ago on the basis of epidemiological studies and reactive treponemal serology. To exclude individual variations in the humoral immune response to T. pallidum, the sera of five patients with the same clinical features and history of infection were pooled for serological tests and double antibody radioimmunoprecipitation assay. Pooled sera from 15 blood donors with negative TPHA and FTA test (titres < 1: 5) served as negative controls. Hemagglutination assay. The T. pallidum hemaglutination assay (TPHA) described by Rathlev (16) was carried out quantitatively according to the manufacturer's (Fujirebio, Tokyo, Japan) instructions, starting with a dilution of 1120. FTA-ABS test. The method for demonstration of T. pallidum-specific antibodies in human sera after absorption with nonpathogenic treponemes by indirect immunofluorescence has been described elsewhere (7). The Nichols strain of T. paUidum served as antigen. Cross-reacting treponemal antibodies were absorbed, using an ultrasonicate of T. phagedenis biotype Reiter (Biologische Arbeitsgemeinschaft, Lich, FRG). A goat antihuman immunoglobulin serum conjugated with fluorescein-isothiocyanate (FITC) was used in a working dilution of 11200 (Bio-Merieux, Niirtingen, FRG). JgG- and JgM-FTA-ABS test. The technique for demonstration of IgG and IgM antibodies to T. pallidum was performed quantitatively by indirect immunofluorescence on slides with air-dried T. pallidum, Nichols strain, as antigen and a FITC-conjugated goat anti-human IgG (y-chain specific) or anti-human IgM (!-I-chain specific) serum (BioMerieux) in a working dilution of 1/200 for IgG or 1150 for IgM.

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Fractionated IgM-FTA-ABS test. Separation of IgM from IgG antibodies in sera was carried out by Sephacryl S-300 (Pharmacia, Uppsala, Sweden) filtration (12). Briefly, 0.7 ml of unheated serum was filtered through a gel column (1.5 x 14 cm) using phosphatebuffered saline (PBS), pH 7.3. Fractions of 1.3 ml were collected, and the absorbance was measured at 280 nm. The demonstration for IgM antibodies to T. pallidum in the fractions was performed quantitatively as described for the IgM-FTA-ABS test. Treponema pallidum IgM-ELISA. The technique for detection of IgM antibodies to T. pallidum in sera after IgG immunoprecipitation by an indirect enzyme-linked immunosorbent assay (ELISA) has been described previously (13). Cardiolipin complement fixation (Card.-CF) test. For the complement fixation test with cardiolipin (Behringwerke, Marburg, FRG) as antigen, a quantitative microassay according to the standardized diagnostic complement fixation method (18) was used, starting with a serum dilution of 114. Venereal Disease Research Laboratory (VDRL) test. The assay was performed quantitatively according to the manufacturer's (Behringwerke) instructions, starting with a serum dilution of 114. Intrinsic radiolabelling of T. pallidum. A suspension of sterile extracted Treponema pallidum (Nichols strain) from infected rabbit testes (5 x 10 7 treponemes/ml) in a modified basal medium (10) was incubated with 25 !lCi/ml of [35 S] methionine (specific activity 1,320 Ci/mmol; Amersham-Buchler, Braunschweig, FRG) for 22 h at 35°C under atmospheric condition (10, 11). The motility of treponemes after incubation for radiolabelling was greater than 95%. The treponemes were then pelleted by centrifugation at 15 000 x g for 1 h, washed three times in PBS, pH 7.3, pelleted again and stored at - 70°C. Radioimmunoprecipitation. Solubilization of [35 S] methionine-labelled T. pallidum in 0.1 % SDS - 1 % Triton X-IOO buffer and double antibody radioimmunoprecipitation assay were carried out as previously described (10, 11). Briefly, samples of detergent-solubilized radiolabelled treponemes were incubated with samples of human syphilitic or human normal serum for 18 h at 4°C in a total volume of 350 !ll. The second reaction with rabbit antihuman IgM or anti-human IgG serum (!l-chain or y-chain specific; Miles Laboratories, Elkhart, USA) was incubated for 30 min at 37°C and then for 2 h at 4°C. Precipitates formed were washed three times in TNE (50 mM trishydrochloride, 150 mM sodium chloride, 5 mM EDTA . Naz, pH 7.4) buffer containing 0.05% Triton X-I00. The final pellets were dried and dissolved in 50 !ll solubilizing buffer (8) followed by boiling for 3 min. Sodium dodecyl sulphate-polyacrylmide gel electrophoresis (SDS-PAGE). Radiolabelled treponemal proteins were electrophoresed on 13 % polyacrylamide slab gels in the discontinuous trisglycine system (8). After electrophoresis, slab gels were fixed and processed for autoradiography at -70°C (10). The 14C-methylated protein mixture (Amersham) was used as the relative molecular masses (Mr) marker. Approximate Mr values were estimated by the method of Weber and Osborn (19). Results

Reactivity of sera in the treponemal and lipoidal tests for syphilis Reciprocal antibody titres of the investigated sera in the serological tests for syphilis are summarized in the Table 1. All sera were reactive in the TPHA and FTA-ABS tests just as in the IgG-FTA-ABS, fractionated IgM-FTA-ABS test and Tp-IgM-ELISA. Sera of patients with primary syphilis (3 weeks after infection) were non-reactive in the lipoidal tests, Card.-CF and VDRL (titre < 1: 4). All other sera were reactive in these tests. However, Card.-CF titres were 2 steps higher than those of the VDRL test. Sera of patients with secondary (6 months after infection) and late syphilis were nonreactive in the unfractionated IgM-FTA-ABS test (titre < 1: 5).

174M. Moskophidis Table 1. Reciprocal antibody titres of treponemal and lipoidal tests for syphilis in pooled sera of patients with untreated primary, secondary and late syphilis Lipoidal tests

Treponema-specific tests

TPHA

FfAABS

IgGFfAABS

Primary (3 weeks)*

80

40

Primary (6 weeks)

Stage of syphilis

IgMFfAABS

Fract. IgMFfAABS

TpIgMELISA

Card.CF

10

40

40

400

<4

<4

VDRL

320

80

20

80

80

800

16

8

Secondary (3 months)

2,560

1,280

1,280

40

320

3,200

512

64

Secondary (6 months)

10,240

2,560

2,560

<5

640

6,400

1,024

128

Late (3 years)

10,240

2,560

2,560

<5

80

800

128

32

Late (5 years)

20,480

5,120

5,120

<5

80

800

128

32

* Time after infection

IgM and IgG reactivity to T. pallidum in sera For demonstration of IgM and IgG antibodies to T. pallidum, sera were investigated quantitatively in the IgG-FTA-ABS test, fractionated IgM-FTA-ABS test and Tp-IgMELISA (Table 1). In sera of patients with primary syphilis, treponemal IgM antibodies with high titres and IgG antibodies in small amounts were detected. Sera from patients with secondary syphilis contained high titres of IgM and IgG antibodies to T. pallidum. High IgG and lower IgM treponemal antibodies were demonstrated in sera of patients with late syphilis. In all sera, the Tp-IgM-ELISA antibody titres were 10 times higher than those demonstrated by the fractionated IgM-FTA-ABS test.

Protein antigens of T. pallidum precipitated by IgM antibodies The IgM reactivities in pooled sera from patients with primary syphilis (3 and 6 weeks after infection) to 35S-labelled T. pallidum proteins are shown in Fig. 1 (a and b). The serum pool (3 weeks after infection) contained IgM antibodies mainly to proteins with Mr values of 33000,37000,41000,44500,47000 and 71000. Additionally, patients with primary syphilis (6 weeks after infection) had IgM antibodies with a strong reactivity to the protein with a Mr of 15 500. The IgM reactions in pooled sera from patients with secondary syphilis (3 and 6 months after infection) to 35S-labelled proteins of T. pallidum are shown in Fig. 1 (c and d). The serum pools contained IgM antibodies to a wide spectrum of proteins with a Mr between 15500 and 115000 with strong reactivities to proteins with a Mr of '15500,33000,34500,37000,41000,44500,47000 and 71000.

Humoral Immune Response to T.pallidum

175

Fig. 1. SDS-PAGE of 35S-labelled proteins of T. pallidum precipitated by IgM antibodies followed by autoradiography. Primary (a and b), secondary (c and d), late syphilis (e and f) and (g) IgM in normal human serum. (h) total protein profile of 35S-labelled T. pallidum, (i) marker proteins, Mr x 10 3.

The serum pools from patients with late syphilis (3 and 5 years after infection) contained IgM antibodies to 35S-labelled proteins of T. pallidum (Fig. 1, e and f) with the same specificities but weaker reactivities than those from patients with secondary syphilis. Three weak precipitated bands with a Mr of 33000, 41000 and 71 000 were detected when pooled normal human serum was used (Fig. 19).

Protein antigens of T. pallidum precipitated by JgG antibodies Reactions of IgG in pooled sera from patients with primary syphilis (3 and 6 weeks after infection) to 35S-labelled treponemal proteins are shwon in Fig. 2 (a and b). A weak reaction to proteins with a Mr of 33 000,37000,41000 and 71 000 was detected with IgG antibodies in serum from patients with primary syphilis (3 weeks after infection). Patients with primary syphilis (6 weeks after infection) had additional IgG antibodies with specificities for proteins with a Mr of 15500,44500 and 47000. The serum pool of patients with secondary syphilis contained IgG antibodies to a wide spectrum of 35S-labelled treponemal proteins with a Mr between 15500 and 115000 with strong reactivities to proteins with Mr values of 15500,33000,34500, 37000,41000,44500,47000 and 71000 (Fig. 2, c and d). Pooled sera of patients with late syphilis contained IgG antibodies to treponemal proteins with the same molecular specificities as those of patients with secondary syphilis (Fig. 2, e and f).

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M. Moskophidis

Two weak precipitated bands with a Mr of 33000 and 71 000, respectively, were detected when pooled normal human serum was used (Fig. 2g).

Fig. 2. SDS-PAGE of 35S-1abelled proteins of T. pallidum precipitated by IgG antibodies followed by autoradiography. Primary (a and b), secondary (c and d), late syphilis (e and f) and (g) IgG in normal human serum. (h) total protein profile of 35S-labelled T. pallidum, (i) marker proteins, Mr x 103.

Discussion Specific serological assays for T. pallMum infection (IgG-FTA-ABS, fractionated IgM-FTA-ABS or Tp-IgM-ELISA) allow the demonstration of IgG and IgM antibodies in the sera of syphilitic patients (11, 12, 13). Both IgM and IgG antibodies to T. pallidum were demonstrated in the sera of patients with untreated primary, secondary and late syphilis (Table 1). In primary syphilis, IgM antibodies were found to predominate over those of the IgG class. However, IgM antibodies to T. pallidum with decreased titers can persist in the serum of patients with latent syphilis for several years or even decades. After antibiotic treatment, specific IgM antibodies disappear (14). In unfractionated sera of patients with secondary (6 months after infection) and late syphilis, IgM antibody reactivity cannot be demonstrated by the IgM-FTA-ABS test because of competitive inhibition of IgM binding to treponemal antigens by high IgG antibody concentration (4). In addition, IgM rheumatoid factor in syphilitic sera may cause false-positive results in the unfractionated IgM-FTA-ABS test (9). For these

Humoral Immune Response to T. pallidum

177

reasons, the un fractionated IgM-FTA-ABS test is not suitable for demonstration of IgM antibodies in the sera of patients suffering from syphilis. Lipoidal antibodies appear late in the course of human syphilis and are not demonstrable through the early period of primary syphilis even three weeks after infection (2) (Table 1). Therefore, the nontreponemal lipoidal tests for syphilis, Card.-CF and VDRL, are not suitable for the diagnosis of early primary syphilis. Recently, using the highly sensitive labelling of T. pallidum with [35 S] methionine, 32-35 proteins were differentiated by SDS-PAGE (10, 17). All these proteins (except the weak bands) were precipitated by IgG antibodies in human syphilitic sera (10) and identified as antigens of T. pallidum. Our investigations with sera of patients in various clinical stages of untreated syphilis have lead us to the conclusion that the humoral immune response to T. pallidum starts with synthesis of IgM antibodies to treponemal proteins with Mr values of 33 000, 37000,41000,47000 and 71 000 (primary syphilis 3 weeks after infection). In the next stages of infection (secondary and late syphilis), IgM and IgG antibodies to all proteins of T. pallidum are synthesized. Antibodies to treponemal proteins with Mr values of 15500,33000,34500,37000,41000,44500, 47000 and 71 000 predominate. These antigens seem to be the main immunogenic proteins of T. pallidum. Utilizing sera from syphilitic patients and the Western blot technique, Hanff et al. (5) characterized six major antigenic proteins of T. pallidum with Mr values of 15500, 16500,30000,33000,42000 and 45000. By the same technique, Hensel et al. (6) have shown that in various stages of syphilis IgG and IgM antibodies against treponemal antigens with a Mr of 14000 and especially 17000 predominate. In immunoblot studies of syphilitic sera in various stages of infection, Baker-Zander et al. (2) demonstrated a strong IgG antibody reactivity to at least 12 treponemal antigens with a Mr between 12 000 and 80000 in most syphilitic sera and a strong IgM antibody reactivity to treponemal antigens with an Mr of 14000, 37000, 48000 and 80000 in some syphilitic sera. Our investigations indicate that during syphilis in humans, the IgM antibody response persists with a strong reactivity to at least eight main antigenic proteins of T. pallidum. The weak IgM and IgG reaction of normal human serum with T. pallidum protein antigens in the highly sensitive double antibody radioimmunoprecipitation assay (10) and Western blot (5) may be explained by antibodies against nonpathogenic hostindigenous treponemes (3) not being detected by TPHA and FTA test. Slight differences in the Mr values of major antigenic proteins of T. pallidum may be caused by different gel concentrations (8-20%) and varying protein markers. In a cooperative effort of 16 laboratories, it was possible to correlate the position of 16 of the major protein bands of T. pallidum in SDS-PAGE, but identification of many proteins has remained ambiguous, particularly in the low Mr range (15). Our understanding of the role of T. pallidum in the pathogenesis of syphilis is limited by the inability to cultivate this bacterium in vitro. Most of the main protein antigens of T. pallidum are localized on the surface of treponemes (1) and expressed by recombinant DNA in Escherichia coli (15). We anticipate that the availability of cloned T. pallidum protein antigens will provide new tools for investigating the pathogenesis and immunobiology of syphilis. Moreover, a convenient, inexpensive source of specific main T. pallidum protein antigens could lead to the development of more specific serodiagnostic tests for syphilis, possibly even tests that will accurately assess the stage of infection.

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