Antigens of Treponema pallidum Recognized by IgG and IgM Antibodies During Syphilis in Humans

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1 THE JOURNL OF INFECTIOUS DISESES VOL. 151, NO.2. FERURY by The University of Chiago. ll rights reserved /85/ $01.00 ntigens of Treponema pallidum Reognized by IgG and IgM ntibodies During Syphilis in Humans Sharon. aker-zander, Edward W. Hook III, Paul onin, H. Hunter Handsfield, and Sheila. Lukehart From the Department of Mediine, Shool of Mediine, University of Washington; Department ofmediine, Harborview Medial Center; and Seattle-King County Department of Publi Health, Seattle, Washington IgG and IgM antibody speifiities for antigens of Treponema pallidum Nihols strain were determined by using sodium dodeyl sulfate-polyarylamide gel eletrophoresis and the western blot tehnique in sera from patients with untreated syphilis, normal persons, persons with biologi false-positive tests for syphilis, and sexual ontats of persons with infetious syphilis. IgG reativities of sera from individuals with primary syphilis varied onsiderably but onsistently exhibited strong reativity to a 48-kilodalton band. Sera from patientswith seondaryandearly latent syphilis uniformlydemonstrated reativity to 22 separate polypeptide antigens; dereased reativity was seen in late latent syphilis. Normal and biologi false-positive sera showed weak IgG reativity against none to 12 polypeptides. Sera from asymptomati ontats of persons with infetious syphilis showed reativity to a varying number of treponemal antigens, inluding some reations not seen with normal sera. IgM reativity was most prominent in seondary syphilis but was demonstrable at all stages of disease. Serologial testing is an important tool for diagnosis of syphilis and the hief method for assessing the effiay oftherapy; these tests inlude the Venereal Disease Researh Laboratory (VDRL) and rapid plasma reagin (RPR; nontreponemal) tests and the fluoresent treponemal antibody-absorbed (FT-S) and hemagglutination (treponemal) tests [1]. lthough these diagnosti tests are used routinely, little is known about the speifiity of antibodies to individual Treponema pallidum antigens in various linial stages ofsyphilis or the development and persistene of these antibodies during the ourse of syphilis. haraterization of speifi treponemal antigens and a lear understanding of the host's immune response to treponemal antigens during infetion are prerequisites Reeived for publiation June 4, 1984, and in revised form ugust 7, Written informed onsent was obtained from the serum donors in this study. This work was presented in part at the Fifth nnual Meeting of the International Soiety for Sexually Transmitted Diseases Researh, held ugust 1-3, 1983, in Seattle, Washington. This study was supported by Publi Health Servie grants I and I from the National Institutes of Health. We thank Ron Roddy and June Nakata for providing serum speimens and Keith Neal for tehnial assistane. Please address requests for reprints to Sharon. aker Zander, Department of Mediine, Harborview Medial Center, 325 Ninth venue, Seattle, Washington for development of improved methods for serodiagnosis. Using sera from patients with syphilis or experimentally infeted rabbits, several investigators [2-8] have demonstrated antibody reative with 11-23treponemal antigens. The present report desribes the responses of both IgG and IgM to moleules of T. pallidum Nihols strain in patients in various stages of untreated syphilis. We also report for the first time the speifiities of antibodies from sexual ontats of persons with early syphilis as well as persons with biologi false-positive reations and normal seronegative individuals. Patients and Methods Soure oft.pallidum. T.pallidum Niholsstrain was propagated in rabbits as previously desribed [9]. dult male New Zealand white rabbits were obtained from a loal supplier (R & R Rabbitry, Stanwood, Wash). Eah rabbit was tested before use for evideneofinfetion with Treponema paraluis-uniutiby the VDRL and FT-S tests. These tests were performed as desribed by the Centers for Disease Control [10] with modifiations [11]. ll rabbits were housed individually at C and were given antibioti-free food and water. Patients. Sera were olleted from patients presenting to the Harborview-Department of Publi Health Sexually Transmitted Disease Clini (Seat- 264

2 IgG and IgM Responses to Treponemal ntigens 265 tie) in various stages of untreated syphilis or with a historyof reent sexual ontat with persons with primary or seondary syphilis. diagnosis of primary syphilis was based on the presene of a darkfield-positive hanre(s); a diagnosis of seondary syphilis was based on the presene ofa typial papular or maular rash, in many ases dark-field positive, and on reative nontreponemal and treponemal serologial tests for syphilis. Earlylatentandlate latent syphilis were defined by reative serologial tests in the absene of linial manifestations and were determined on the basis of interview to be of less than or greater than one year's duration, respetively. ll serum samples from sexual ontats of persons with infetious syphilis were RPR, VDRL, and FT S nonreative when sera were obtained. Sera from biologi false-positive reators (those exhibiting a reative VDRL but a nonreative FT-Stest) were obtained from the Seattle-King County Department of Publi Health Laboratories. Normal sera were obtained from volunteers in the researh laboratoryand from individuals with no history of serologial evidene ofsyphilis who wereseen at Harborview Medial Center for other reasons. Serologial tests for Figure 1. Western blot reativity of human sera against antigens of T. pal/idum: () IgG reativity of two representative normal sera and () IgG reativity of three representative biologi false-positive sera. The approximate moleular weights of some bands are indiated. syphilis (VDRL slide test, RPR irle ard test, and FT-S test) were performed in aordane with standard proedures. ll sera were tested for rheumatoid fator with the eosin latex rheumatoid slide test (Difo, Detroit) and were found to be negative. Immunofluoresene (IF) testing. IgG and IgM titers in sera were determined by a modifiation of the standard FT-S test. Sera were diluted 1:5 in sorbent, and serial twofold dilutions were made in PS. Dilutions were tested using ommerially prepared T. pallidum antigen (ekman Diagnostis, naheim, Calif) as desribed by the Centers for Disease Control [10]. Fluoresein isothioyanate-onjugated goat antibody to human (Fe fragment) IgG (heavy hain speifi) and goat antibody to human IgM (/ hain speifi; Cappel Laboratories, West Chester, Pa) were used as the seond antibodies; the working titers were determined to be 1:1,200 and 1:100, respetively. Slides were examined with a fluoresene mirosope, and the degree of fluoresene was reorded on a sale of The end point was the highest dilution that still yielded 2 + fluoresene. Pooled syphiliti rabbit serum. Sera from four -80k -41 k --3Sk -30k

3 266 aker-zander et al. IgG IgM IgG IgM IgG IgM -80k -69k rabbits were obtained three months after intratestiular infetion with 2-6 x 10 7 T.pallidum Nihols strain. Sera were pooled, heat inativated, and stored in l-ml aliquots at - 20 C until use. The IF titer of this pool was 1:2,048. Preparation of treponemal antigens and SDS PGE. T. pallidum antigens were prepared by the method of Lukehart et al. [12]. Whole washed organisms were solubilized in 1010 SDS and eletrophoresed on 12.5% polyarylamide slab gels in the disontinuous 'Iris-glyine system desribed by Laemmli [13]. bout 2 x 10 8 organisms (1"\.125 JJg of protein) were loaded on 5-mm lanes; gels were 12em long and 1.5mm thik. The eletrophoresis was ontinued until the dye front waswithin 1em of the bottom of the gel. Protein standards ( kilodaltons [k]) for estimating moleular weights (iorad, Rihmond, Calif) were inluded on representative gels. pproximate moleular weights were determined by the method of Weber and Osborn [14]. Western blot tehnique. Eletrophoreti transfer ofproteins to nitroellulose paper (western blot) -37k Reiproal 8 IF Titer Figure 2. Western blot reativity of representative sera from patients with primary syphilis. Of eah pair, the left lane shows IgG reativity and the right lane shows IgM reativity. The duration of lesions in individual patients was () one week, () two weeks, and (C) three to four weeks. The approximate moleular weights of some bands are indiated. The numbers beneath eah lane are the reiproals of the IgG and IgM IF titers, respetively. was performed by the method of Towbin et al. [15] using a Trans lot Cell (iorad). Proteins weretransferred at 5-7 V for 18 hr at room temperature (1"\.123 C)~ Effiieny of transfer was determined by staining the proteins (inluding moleular weight markers) on nitroellulose paper with amido blak [15]. Inubation of nitroellulose paper with antiserum was performed as previouslydesribed [12] exept that antiserum (l:100 for testing for IgG reativity or 1:50 for testing for IgM reativity) and 12sI-labeled staphylooal protein (New England Nulear, oston) were diluted in 10 mm Tris and 0.09% NaCI (ph 7.4) ontaining 0.5% bovine serum albumin and Triton X-I00. 12sI-Labeled staphylooal protein was used at a final onentration of x 105 pm/ml. The IgG fration of goat antibody to human IgM {JJ hain speifi; Cappel) was radioatively labeled with arrier-free (mersham Corp., rlington Heights, Ill) by using Iodogen" (Piere iohemials, Rokford, Ill) and diluted as above to x 10 6 pm/ml. fter inubation western blots wereexposed to Cronex'"

4 IgG and IgM Responses to Treponemal ntigens 267 MRF 32 film (DuPont, Wilmington, Del) at - 70 C with enhaning sreens. Neither 1251-labeled staphylooal protein nor 1251-labeled antibody to IgM bound treponemal antigens in the absene of antibody to treponemal antigen. Results Reativity ofnormal and biologi false-positive serum. Twenty serum speimens from individuals with nonreative serologial tests and no history of syphilis were reated with T. pallidum antigens. Individual sera exhibited very weak IgG reativity most frequently with six treponemal antigens: 80,48,41, 35, 33, and 30 k. Four sera reated weakly with seven to 12 antigens (the six above plus 85, 75, 69, 54, 43, and 37 k), and only two sera failed to reat with any treponemal antigens. Two representative blots are IgG IgM IgG IgM IgG IgM IgG IgM shown in figure 1. Four sera were tested for IgM reativity; no reativity was demonstrated in one, and three reated weakly with the 80-, 33-, and 30-k bands (data not shown). Fifteen sera from individuals with biologi falsepositive tests for syphilis were tested using the western blot tehnique. Individual IgG reativity varied but was indistinguishable from that seen in normal sera; one serum sample did not reat with treponemal antigens. No additional bands were demonstrated. Three representative blots are shown in figure 1. Four sera were tested for IgM reativity; all reated to varying degrees with five treponemal antigens (80, 41, 35, 33, and 30 k; data not shown). Reativity in primary syphilis. Sera from eight patients with primary syphilis were reated with T. pallidum antigens using the western blot tehnique with 1251-labeled staphylooal protein and 125 1_ -8Dk -69k - 37k Reiproal IF Titer Figure 3. Western blot reativity of representative sera from patients with seondary syphilis. Of eah pair, the left lane shows IgG reativity and the right lane shows IgM reativity. The duration of seondary symptoms in individual patients was () 1 week, () 2 weeks, (C) 4 weeks, and (D) 10 weeks. The approximate moleular weights of some bands are indiated. the numbers beneath eah lane are the reiproals of the IgG and IgM IF titers, respetively. o

5 268 aker-zander et at. IgG IgM IgG IgM IgG IgM IgG IgM -80k -69k k Reiproal IF Titer Figure 4. Representative blots of sera from patients with untreated early latent syphilis. Of eah pair, the left lane shows IgG reativity and the right lane shows IgM reativity. The approximate moleular weights of some bands are indiated. The numbers beneath eah lane are the reiproals of the IgG and IgM IF titers, respetively. labeled antibody to IgM. utoradiographs of three representative sera are shown in figure 2. Figure 2 shows the reativity of serum from a patient with a dark-field-positive primary lesion of one week in duration; IgO reativity ourred with six bands and IgM reativity with eight bands. lthough the VDRL and FT-S tests were nonreative in this patient, the IgM IF titer was 1:128. Strong IgO reativitywas seen in the blot with bands only weakly seen with normal human sera; no T. pallidum- or pathogenspeifi moleules were identified. Figure 2 shows the reativity of serum from a patient with multiple dark-field-positive lesions of two weeks in duration, a VDRL titer of 1:2, and a reative FT-S test. Strong IgO reativity against the 48-k band is seen, as well as weak reativity against six other bands, inluding the 12-k moleule. Very weak IgM reativity ourred against the 80-,69-,48-,41-,37-, and 33-k moleules. The IF titers for IgO and IgM were o 1:1 and 1:64, respetively. Figure2C shows the reativity ofserum from a patientwhose dark-field-positive lesion had appeared three to four weeks earlier; although this serum was VDRL nonreative, it was FT-S reative. Strong IgO reativity is noted against at least 12 bands; the IgO IF titer was 1:2. Veryweak IgM reativity ourred against all ofthese bands exept the 12-k moleule; the IgM IF titer was 1:8. Reativity in seondary syphilis. Sera from 13 patients with seondary syphilis were tested for IgO and IgM reativity against T. pallidum antigens. The results from four representative sera are shown in figure 3. These patients had the rash of seondary syphilis for 1,2,4, and 10weeks and all had reative nontreponemal and treponemal serologial tests. Sera from all four individuals showed substantial IgO reativity against treponemal antigens and produed profiles similar to that seen with the referene sera

6 [go and IgM Responses to Treponemal ntigens 269 from infeted rabbits. Stronger IgM reativity was seen in seondarythanin primarysyphilis and may reflet the higher antibody titers observed during this stage. This reativity appeared to wane with inreasing duration of symptoms. Reativity in early latent syphilis. The results with representative sera from sevenpatients with untreated early latent syphilis are shown in figure 4. s in seondary syphilis, IgG reativity was strong in both the western blot tehnique and by IF. IgM reativity, however, varied onsiderably among individuals; IgM IF titers were low in all ases, ranging from 1:2 to 1:32. Reativity in late latent syphilis. Sera from eight individuals with untreated late latent syphilis were tested against T. pallidum antigens. Representative results are shown in figure 5. Some of these sera showed dereased IgG reativity (, C, and D) ompared with those from seondary or early latent IgG IgM IgG IgM IgG IgM IgG IgM syphilis; the lower IgG IF titers reflet these differenes. Most IgM reativity, as well,was lost, although very weak IgM reativity did ontinue to persist in some patients. IgM antibody ould not be deteted by IF in these sera. Reativity in ontats of persons with early syphilis. Sera from seven seronegative persons who were sexual ontats of individuals with infetious syphilis were tested with the western blot proedure. Contat had ourred within 30 days of diagnosis and treatment ofthe index ases; four were ontats ofpersons with primary syphilis, and three wereontats ofpersons with seondarysyphilis. ll ontats denied a previous history of syphilis. ll seven sera showed IgG reativity; figure 6 shows sera from ontats of persons with primary ( and ) and seondary (C and D) syphilis. broad range of reativity, similar to thatfound with sera from patientswith symptomati primary syphilis, is seen. Strong rea- - 80k -69k -37k D Reiproal IF Titer Figure 5. Representative blots of sera from patients with untreated late latent syphilis. Of eah pair, the left lane shows IgO reativity and the right lane shows IgM reativity, pproximate moleular weights of some bands are indiated. The numbers beneath eah lane are the reiproals of the IgO and IgM IF titers, respetively.

7 270 aker-zander et al. o - 80k - 69k - 48k - 37k Figure 6. IgG reativity of sera from sexual ontats of persons with infetious syphilis: ( and ) sera from two ontats of persons with primary syphilis and (C and D) sera from two ontats of persons with seondary syphilis. Contat had ourred within 30 days of diagnosis of the index ase. The approximate moleular weights of some bands are indiated. ll sera were nonreative in the RPR, VDRL, and FT-S tests. tivity to the 80- and/or 48-k protein was found in all ases. Two patients demonstrated a weak response to the 12- and lor 14-k moleule not seen in normal or biologi false-positive sera. Two of seven sera showed no IgM reativity, whereas five had demonstrable IgM reativity against the 80-, 35-, 33-, and 30-k bands (data not shown). IgG or IgM antibody to treponemal antigens was not deteted by IF. Disussion We examined the development of the humoral immune response to T. pallidum antigens during the natural ourse of syphilis in humans. The IgG reativities in sera from patients with primary syphilis varied between patients but appeared to reflet the duration of linial symptoms. Even in persons who had not yet developed VDRL or FT-S reativ- ity, moreintense, well-defined bands were seen ompared with the faint reativity displayed by normal or biologi false-positive sera. In addition, reativity against a 12-k protein shown previously to ontain pathogen-speifi determinants [12] appeared during primary syphilis but was never seen in normal or biologi false-positive sera. Sera from individuals with seondary syphilis uniformly displayed broad IgG reativity, direted against as many as 22 treponemal antigens. This strong humoral response ontinued in early lateny but waned, as demonstrated by diminished reativity, in late latentsyphilis. However, reativity against the 48-k moleule persisted. These results are onsistent with those desribed previously [6]. Moskophidis and Muller [5] did not examine sera from patients with early and late latent syphilis. IgM reativity developed early in the ourse of syphilis and was demonstrable through the early latent period; IgM was not, however, deteted against all treponemal antigens, as reported by Moskophidis and Muller [5]. IgM antibody to treponemal antigens was not demonstrable by IF in late latent syphilis, although weak reativity ould be deteted by the western blot tehnique. These results are onsistent with those of Julian et ai. [16] in whih only 33% of primary and of seondary syphiliti sera tested displayed speifi IgM reativity to treponemal antigens by IF, whereas and , respetively, were reative in the FT S test [17-19]. These observations indiate that by the time linial signs develop most patients have both IgG and IgM antibody. The results of testing sera from experimental infetion in himpanzees [20] and rabbits (authors' unpublished observations) show development of IgM and IgG antibodies onurrent with linial symptoms. Our results suggest this sequene may also be the ase in human infetion. IgM reativity may be underestimated by IF or the western blot tehnique using unfrationated serum beause of potential ompetitive inhibition of IgM binding to treponemal antigens by IgG, thereby masking IgM reativity [21]. IgG antibodies generally have higher avidity and maintain a more stable ombination with gram-negative baterial antigens than do IgM antibodies [22]. The ontinued presene of IgM in latent and even late syphilis has been demonstrated previously by serologial testing [23-25]. Muller and Oelerih [26] desribed the existene of 8S IgM antibody in some individuals,

8 IgG and IgM Responses to Treponemal ntigens 271 predominantly in late latent syphilis. lthough we were able to demonstrate speifi IgM reativity in late latent syphilis, Van Eijk and van Embden [8] ould not. Moskophidis and Muller [5] and Hanff et al. [6] did not investigate IgM reativity beyond the seondary stage. ll sera in our study were tested and found negative for rheumatoid fator, a finding exluding the possibility of false-positive IgM reativity. Previous desriptions of reativity to treponemal antigens in normal human serum have used both radioimmunopreipitation [4, 5] and western blot [6] tehniques. We were able to identify antibody to as many as 12 T. pallidum antigens in normal human serum. Five of these antigens orrespond to those determined by Lukehart et al. [12] to be ommon to both T.pallidum and the nonpathogen Treponema phagedenis biotype Reiter; they also orrespond to the four to 15 ross-reative antigens desribed by others [5,27,28]. Differenes in reported moleular weights and numbers of antigens may be due to differenes in methodology. Comparisons of reativity to T. pallidum in normal and biologi false-positivesera with those of sera from persons in various stages of syphilis suggest that several moleules may be promising andidates for improved diagnosti tehniques. We have previously shown that the 48-, 14-, and 12-k moleules ontain pathogen-speifi determinants [12]. The 12 and 14-k moleules were not identified with normal or biologi false-positive sera but did reat with some sera from primary and inubating syphilis as well as with most sera from seondaryand latent syphilis. lthough weak reativity to the 48-k moleule was seen in normal and biologi false-positive sera, muh stronger reativity was seen in all stages of syphilis, inluding inubating infetion. In serum adsorption studies [12] and using pathogen-speifimonolonal antibodies to T. pallidum [29], we have been able to show that this band appears either to share both ommon and pathogen-speifi moieties or to onsist of omigrating polypeptides. The presumptive pathogen-speifi determinants on these moleules may provide the basis for more speifi, sensitive diagnosti tests and suggest promising antigens for vaine development. Referenes 1. Sparling PF. Diagnosis and treatment of syphilis. N Engl J Med 1971;284: lderete JF, aseman J. Surfae-assoiated host proteins on virulent Treponema pallidum. Infet Immun 1979;26: lderete JF, aseman J. Surfae haraterization of virulent Treponema pallidum, Infet Immun 1980;30: aseman J, Hayes EC. Moleular haraterization of reeptor binding proteins and immunogens of virulent Treponema pallidum. J Exp Med 1980;151: Moskophidis M, Muller F. Moleular analysis of immunoglobulins M and G immune response to protein antigens of Treponema pallidum in human syphilis. Infet Immun 1984;43: Hanff P, Fehniger TE, Miller IN, Lovett M. Humoral immune response in human syphilis to polypeptides of Treponema pallidum. J Immunol 1982;129: HanffP, ishop NH, Miller IN, Lovett M. Humoral immune response in experimental syphilis to polypeptides of Treponema pallidum. J Immunol 1983;131: Van Eijk RVW, van Embden JD. Moleular haraterization of Treponema pallidum proteins responsible for the human immune responseto syphilis.ntonie VanLeeuwenhoek 1982;48: Lukehart S, aker-zander S, Sell S. Charaterization of lymphoyte responsiveness in early experimental syphilis. I. In vitro response to mitogens and Treponema pallidum antigens. J Immunol 1980;124: U.S. Department of Health, Eduation, and Welfare, Publi Health Servie. Manual of tests for syphilis. tlanta: Centers for Disease Control, Lukehart S, aker-zander S, Lloyd RMC, Sell S. 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9 272 aker-zander et al. 21. Cohen IR,Norins LC, Julian J. Competition between and effetiveness of IgG and IgM antibodies in indiret fluoresent antibody and other tests. J ImmunoI1967;98: Robbins J, Kenny K, Suter E. The isolation and biologial ativitiesof rabbit y M- and y G- anti-salmonella typhimudum antibodies. J Exp Med 1965;122: Shannon R, ooth SD. The pattern of immunologial responses at various stages of syphilis. r J Vener Dis 1977; 53: O'Neill P, Niol CS. IgM lass antitreponemal antibody in treated and untreated syphilis.r J VenerDis 1972;48: Muller F, Lindenshmidt E-G. Demonstration of speifi 19S (IgM) antibodies in untreated and treated syphilis. r J Vener Dis 1982;58: Muller F, Oelerih S. Identifiation of a low moleular weight IgM antibody with Treponema pallidum speifiity in sera of patients with hroni syphilis. Klin Wohenshr 1979; 57: aughn RE, dams C, Musher DM. Cirulating immune omplexes in experimental syphilis: identifiation of treponemal antigens and speifi antibodies to treponemal antigens in isolated omplexes. Infet Immun 1983;42: Hanff P, Miller IN, Lovett M. Moleular haraterization of ommon treponemal antigens. Infet Immun 1983; 40: Lukehart S, Tam MR, Hom J, aker-zander S, Holmes KK, Nowinski RC. Charaterization of monolonal antibodies to Treponema pallidum. J Immunol 1985;134: