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Sep 3, 1982 - of syphilis is described. Polypeptides of motile, virulent f. pallidurn, purified from rabbit testes, were separated on. SDS polyacrylamide gels and ...
0022-1767/82/1293-1287$0200/0 THE JOURNAL OF lMMUNOLOGY Copyright 0 1982 by The American Association of lmrnunologlsts

Vol 129. No 3.September 1982 Pnnted m U . S A.

HUMORAL IMMUNE RESPONSE IN HUMAN SYPHILIS TO POLYPEPTIDES OF TREPONEMA PALLIDUM ' PHILIP A. HANFF,2THOMASE.FEHNIGER,

JAMES N.MILLER,

AND

MICHAEL A. LOVETT3

From the Departmentof Medicine, Division o f lnfectious Diseases; the Molecular Biology Institute;a n d the Department of Microbiology and Immunology, U. C. L. A. School of Medicine, i o s Angeles, CA 90024

A molecular characterization of the polypeptide antiThe development of a method to purify motile and virulent T. gensof Treponema pallidurn reactive withserafrom pallidurn (Hanff, Norris, Lovett, and Miller, manuscript in prepof syphilis is described. aration) and the recent availability of techniques to transfer patientswithdifferentstages f . pallidurn, purifiedfrom polypeptides separated by SDS-polyacrylamide gelelectroPolypeptides of motile, virulent rabbittestes,wereseparatedon SDS polyacrylamide phoresis (SDS-PAGE) to solid-phase matrices for antigenic gels and electrophoretically transferred to nitrocellulose analysis (8, 9) have enabled us to study the humoral immune IgG response in human syphilis to individual proteins forantigenicanalysis("Westernblotting").Serum of T. pallidurn. from uninfected individuals reacts weakly with three polyClassically, untreated disease progresses throughthe primary, peptides of 45,000, 33,000,and 30,000 m.w.Inthis secondary, and earlyand late latentstages, and may ultimately study patients with primary syphilis have IgM antibody, end in the destructive late stage (1 0).Thus, we have attempted and allpatients with syphilis have IgG antibodyattoleast todefine and correlate the principal antigens recognized in four polypeptides of45,000,33,000,30,000, and 15,500 each stage with the humoral immune response to T. pailidurn m.w.Antibody topolypeptidesof 42,000 and 16,500 infection. m.w. appear to be markers for nonprimary syphilis. These six polypeptides have been termed the major antigenic MATERIALS AND METHODS T. pallidurn. Those patientsstudied proteins(MAP)of of T. Source pallidurn. T. pallidurn, Nichols strain, was maintained by with secondary and earlylatent syphilis acquire antibody rabbit testicular passage twice weekly as described by Lukehart and Miller to asetof 16 additionalpolypeptideantigens.Those (1 1). Testicles harvested 8 to 10 days post-infection were washed once in patients studiedwith late latent or late syphilishave PES. pH 7.2. Organisms were then extractedin PES. followed by two antibody to a much smaller set of five or four antigens, differential centrifugations at 450 x G for 7 min. Supernatants containing respectively, in addition to MAP. ?ne results suggestthat 2 to 9 X I O 8 T. pallidum/ml were subjected to Percoll (Pharmacia Fine Chemicals) density gradient centrifugation at 30.000 x G for 30 min at 4°C a correlation exists between acquisition of antibody and to remove host tissue contaminants. The bottom band was harvested and thedevelopment of "chancreimmunity."Additionally, examined for purity and motility by darkfield microscopy. SDS-PAGE analthelossofantibody that characterizes late latent and ysis of purified and original treponemal suspensions, as well as normal late syphilis may be associated with the potential devel- rabbit testicular tissue and serum components, has confirmed the purity of the T. pallidurn suspensions. Virulence and long-term motility studies indiopment of destructive late syphilis.

cated that the organisms were unaffected by the Percoll procedure(Hanff, Norris, Lovett. and Miller. manuscript in preparation). After removal of the Percoll at 100,000 X G for 1 hr at 4 T . , T. pallidurn was resuspended in Although it is well established that immunity to reinfection electrophoresis sample buffer (FSB)4 (0.0625 M Tris, pH6.8, 2% NaDodSO,, 5% 2-mercaptoethanol), and stored at -2OOC. Normal rabbit, develops during the course ofhuman syphilitic infection (1, 2), testicular material and normal rabbit serum were prepared in FSB for use the mechanisms involved remain poorly understood. In part, as controls. this is due tothe fact that the etiologic agent, Treponema Sera. Sera from patients with primary, secondary, and latent syphilis pallidurn, has not until recently (3) been grown in vitro. Addiwere collected from patients attending Los Angeles County Venereal Distionally, studies related to the immunobiology of the organism ease Clinics. Diagnosis of primary syphiliswas based on the presence of a darkfield-positive lesion. All patients withsecondary syphilis hada charachave been limited by the fact that T. pallidurn must be transteristic rash and/or palmar and plantar lesions and reactive nontreponemal and previous ferred in vivo by rabbittesticularinoculation, and treponemal tests. Those with latent syphilis were determined, on the attempts to purify the organism from host tissue have resulted basis of epidemiologic studies and reactive serology, to have been infected in the loss of its characteristic motility and virulence (4-7). As for less or greater than 4 yr duration for early or late latent disease, respectively. Sera from patients with late syphilis were generously supplied a result, pure T. pallidurn antigens have not been available to by the Center for Disease Control. Atlanta, GA. and false positive (FP) sera study the host immune response to infection. were kindly provided by Dr. Hunter Handsfield (University of Washington School of Medicine,Seattle. WA). A diagnosis of FP was based on reactive nontreponemal and nonreactiveFTA-ABS tests onserum from patients with Received for publication March 9, 1982. no history or epidemiologic evidence of syphilis. Normal human sera (NHS) Accepted for publication June 14, 1982. exhibiting nonreactive VDRL tests were obtained from medical students and The Costs Of publication of this article were defrayed in part by the payment laboratory personnelwho denied a previous history of syphilis. Of Page charges. This article must therefore be hereby marked advertisement in '251-labeledproteins. Protein A from Staphylococcus aureus (Pharmacia accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Fine Chemicals). marker proteins (Pharmacia Fine Chemicals) for m.w. ' This work was presented in part atthe 1st Sexually Transmitted Disease determination, and rabbit anti-human IgM (heavy chain specific) antibody, 15-21. 1981. This work was World Congress. San Juan. Puerto Rico. November supported by a giff from the Cetus Corporationto M. A. L., and by United States a gift from Dr. Ronald H. Stevens, Department of Microbiology and ImmuPublic Health Service GrantA l l 2601 and World Health Organization Agreement nology, U. C . L. A. School of Medicine, were labeled with '251 (Amersham) by the lactoperoxidase method described by Marchalonis (1 2). RadioimV3-181-26 to J. N. M. munoassay analysis of the class-specific antiserum by Dr. Stevens indicated Current and correspondence address: Clinical Microblology Laboratories; North Carolina Memorial Hospital; University of North Carolina; Chapel Hill, North Carolina 2751 4. Abbreviations usedin this paper: MAP. major antigenic proteins; TSA. buffer Reprint requests should be addressed to Michael A. Lovett, M.D., ph.D., containing 50 mM Tris. 0.9% saline, and0.2% sodium azide; OA. 5% ovalbumin; Department of Medicine. Division of Infectious Diseases, u. c . L. A. School of NHS.normalhumanserum;FP.falsepositive; FSB, electrophoresissample Medicine. Los Angeles.CA 90024. buffer. 1287

1288

POLYPEPTIDES OF TREPONEMA PALLIDUM

that the anti-human IgM contained no y- or a-chain binding activity. Gel electrophoresis. Gradient NaDodSOa-polyacrylamideslab gels of 8 to 2090 or 10 to 2090 were used with the discontinuous buffer system of Laemmli (1 3). Electrophoresis was performed at 5 mA overnight and continued at 20 mA until 1 hr after the dye front left the gel. Before electrophoresis, samples containing approximately 2 to 7 x 10" T. pallfdurn as well as marker proteins were boiled at 100°C for 5 min in the presence of FSB. Samples were loaded into individual5-mm wells or applied to a single 11 8mm well. '"'I-labeled marker protelns were included with each electrophoresls In a separate well. Protein transfer to nitrocellulose and antigenfc analysfs. Electrophoretic transfer of proteins from the gel to nitrocellulose paper was carried out using a modification of the method described by Towbin et a/. (9). The gel and 0.45 pm nitrocellulose sheet (Sartorius) were sandwichedbetween Scotch-Brite pads (3M Company) and a plastic grid. With the paper facing the anode, proteins were transferred electrophoretically at 195 mA for 3 hr in 2.5 mM Tris/192 mM glycme/20% methanol. Efficacy of transfer was assessed by staining the paper "blot" for 3 min with amido schwarz (9) followed by destaln In H:,O. Depending upon the purpose of the protein separation and transfer. the blots either were cut into 8- to IO-mm strips or remamed Intact. Strips or whole blots were placed in a buffer containing 50 mM Tris. pH 7.5. 0.9O6 NaCI. 0.2% sodium azide (TSA) containing 5% ovalbumin (OA) for 1 hr at roomtemperature. All subsequent steps were also at room temperature. Strtps and/or blots were probed with serum diluted 1 :10 or 1 : 100 inTSA-OA for 16 to 18 hr. The blots were then washed for 1 hr with five changes of TSA, and incubated for 2 hr with '?protein A in TSA-OA. Blots reactedwlth normal and prlmarysera were also probed with '%rabbit anti-human IgM diluted similarly. Unbound label was removed by washing with several changes of TSA over a 4-hr perlod. Blots were rinsed in HTO, air-dried,andautoradiographedusing Dupont MRF-32clear base x-ray film. RESULTS

T. pallidum polypeptides. The total protein profile of T. pallidum, Nichols strain, is shown in Figure l . The efficiency of transfer to nitrocellulose paper varied as a function of the m.w. Utilizingradioactivemarkers,transferefficiencyreacheda maximum at 3 hr, and was determined to be approximately 90% for a-lactalbumin and trypsin inhibitor(1 4,400 and 20,100 m.w.), 70% for carbonic anhydrase, ovalbumin, and albumin (30,000, 43,000, and 67,000 m.w.), and 34% for phosphorylase b (94,000 m.w.1. Normal human and FP samples. The reaction of sera from uninfectedindividuals with blots of T. pallidum is shown in Figure 2. Serum from 16 normal individuals and three FP were tested utilizing the ""I-protein A probe for IgG. Although only three representative reactions with NHS are shown, 14 of 16 showed IgG reactivity with a 45,000 m.w. antigen. Further, 1 1 of the 16 sera detected weak treponemal antigens of 33.000 and 30.000 daltons. Identical results were obtained with serum diluted 1 : l o . Normal sera that were probed for IgM antibody to T. pallidum polypeptides only detected the33,000 and 30,000 m.w. antigens. Serum from patients diagnosed as FP reactors also reacted weakly to the 45,000, 33.000. and 30.000 m.w. proteins at the 1 :10 dilution. Primary syphilis. Representative reactions of sera from patients with primary syphilis with T. pallidum blots are shown in Figure 3. All the patients with primary syphilis had IgM and IgG antibody to polypeptides with m.w. of 45,000, 33.000, 30,000, and 15.500. In all four primary sera, there was IgM antibody to one or more antigens not detectedwith "51-protein A, the probe for IgG. Further, in one of the four sera, additional proteins between 33,000 and 45,000 daltons were detected. Faint but detectable IgM antibody to the 45.000, 33.000, and 15,500 m.w. proteins was found in the serum from a patient with a nonreactive VDRL and a darkfield-positive chancre (lane D), whereas IgG antibody was not detectable (lane C). Identical results were obtained with these sera at a 1:lO dilution (not shown). Secondary and earlylatentsyphilis. Representative reac-

Figure I . Separation ofTreponema pallidurn polypeptides. Intact. Percollcentrifuged T. pallidurn. Nichols strain, were separated on an 8 to 20% gradient NaDodSO. polyacrylamide/Bisacrylamide gel.andthegelwasstainedwith Coomassle Blue. A, T. pallfdurn polypeptides: B. marker protein. The numbers m.w. ( x of the proteins. refer to the

-

-94 -67 -

Figure 2. Detection of T. pallfdum polypeptide antigens with sera from nonsyphilitic individuals. A bot of T. pallidurn polypeptides was prepared using an 8 to 2090 gradient NaDodSO, polyacrylamide/Bis gel as described. The blot was cut into 10-mm-wide strips, and these were incubated for 16 hr with sera diluted 1 :10 or 1 : l o 0 in TSA buffer containing 5% OA. Antigenic proteins were detected with '"1-protein A and autoradiographed with Dupont MRF-32 x-ray film. A-C. normal human seradiluted 1:loo; D-E. FP seradiluted 1 :10. The numbers refer to them.w. ( x of theproteins. Identical results were obtained withNHS diluted 1:lO.

tions of sera from five patients with secondary andfive patients with early latent syphilis are shown in Figure 4. All had antibody to the 45.000, 33,000, 30,000. and 15.500 m.w. proteins just as in primary syphilis. In addition,secondaryseradetected strong antigens of 115,000, 94,000. 79,000, 76,000, 62,000, 54,000.47.000.35.500,29,000, and 16,500 daltons. The broad highly antigenic band in the m.w. range of 40.000 to

1 289

POLYPEPTIDES OF PALLIDUM TREPONEMA

of 47,000 and 40,000 m.w. and to five treponemal antigens in the m.w. range of 17,400 to26,800 that were detected by sera from patients with secondary and early latent syphilis. DISCUSSION

Figure 3. Detection of T. pallidurn protein antigens with sera from four patients with primary syphilis. A blot of T. pallidurnpolypeptides was prepared using a 10 to 20% gradient NaDodSO. polyacrylamide/Bis gel as described in Materials and Methods. Strips were incubated for16 hr with sera diluted1 : 100. Antigenic proteins were detected byeither '*%protein A ( A . C. E. G) or Iz5l-rabbit anti-human IgM (heavy chain specific) (8.D. F, H) and autoradiographed. The numbers refer to the m.w. ( x 1 0-3)of theproteins. Identical results were obtained with sera diluted 1 :lo.

The data presented in this report show that patients with syphilis have either IgM in the case of primary syphilis and/or IgG antibody to at least four polypeptides from T. pallidurn, Nichols strain, of m.w. 45,000, 33,000, 30,000, and 15,500. Additionally, all the patients studied with secondary, early latent, late latent, and late syphilis have IgG antibody to polypeptides of 42,000 and 16,500 daltons. We have termed these six antigens the major antigenic proteins (MAP) of T. pallidurn, Nichols strain. The weak but consistent reaction of NHS with T. pallidurn antigens may be significant, because there are data showing that NHS contains imnlobilizing and treponemicidal activity against T. pallidurn (14-1 7 ;and Hanff and Miller, manuscript submitted for publication). Inasmuch as FP and normal human

I-30x 14. Fi,rure 4 . Detection of T. pallidurn protein antigens bysera from five patients with ! econdary and five patients with early latent syphilis. Blots of T. pallidurn protens were prepared using 8 to 20% gradient polyacrylamide gels as described. and strips were incubated for 16 hr with sera diluted l :loo.A-€, secondary sera; F-J. early latent sera. The numbers refer to the m.w. ( x of the proteins.

45,000 on underexposure of the autoradiogram revealed three distinct proteins of 40.000, 42,000, and 45,000 daltons. Weaker reactions can be seen with treponemal proteins of 28.500,26.800.24,500.22,000, 19,500, and 1 7,400 daltons. To test the specificity of the reaction of syphilitic sera with treponemal antigens, blots of normal rabbit testicular tissue and normal rabbit serum were probed with the five highly reactive secondary syphilitic sera tested above; no bands were detected (data not shown). The treponemal antigens detected with sera from patients with early latent syphilis were indistinguishable from the pattern seen in secondary syphilis; however, there may be a quantitative difference in the amount of antigen detected. Late latent and late syphilis. Representative reactions of sera from five patients with late latent and five patients with late (cardiovascular) syphilis are shown in Figure 5. Again, the 45,000, 42,000, 33,000, 30,000, 16,500, and 15,500 m.w. proteins are antigenic in each case of late latent and late syphilis. Treponemal antigens of 54,000,35,500, 29,000, and 28,500 daltons were also detected. One additional polypeptide of 62,000 m.w. was demonstrable by late latent sera. Although the antibody response to antigens of greater than 45,000 m.w. is variable, these sera are strikingly free of antibody to antigens

Figure 5. Detection of T. pallidurn polypeptide antigens by sera from five patientswithlate latent and five patientswithlate (cardiovascular) syphilis. Electrophoretic transfer of separated T. pallidurn proteins was carried out as 16 hr with sera diluted1 :loo.A-E. late described, and strips were incubated for latent sera; F-J. sera from patients with cardiovascular syphilis. The numbers refer to the m.w. ( x of theproteins.

TABLE I Antigens of Treponema pallidurn Molecular weight ( x 103)

115 94 79 76 62 54 47 45 42 40 35.5 33 30 29 28.5 26.8 24.5 22.0 19.5 17.4 16.5 15.5

Sera' NHS

+

+ +

Prirnsrv

+ + + + +++ ++

+

Secondarv

Earlv latent

+ + + ++ +++ +++ ++ +++ +++

+

+++ +++

+++ +++ +++ ++ + + +

+ +

+++

+++ +++

+ + ++ +++ +++ ++ +++ +++ +++ +++ +++ +++ +++

++ +

+ + + + +++

+++

Late latent

+

+ +++ +++

+

++ ++ + +

+++ +++

Late

+ +++ +++ + ++ ++ + +

+++

+++

The reactivity of sera with individual T. pallidurn polypeptides is indicated as (++ +) strong reaction, (++) intermediate reaction, (+)weak reaction. a

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POLYPEPTIDES OF TREPONEMA PALLIDUM

sera reactivity with T. pallidurn polypeptides wereindistinguishable, treponemal proteins do not appear to stimulate VDRL antibody and thus are not involved in the FP reaction. On the basis of this study, it is evident that different stages of syphilis have unique patterns of antibody to polypeptides of T. pallidurn in addition to MAP (Table I). Serum from patients with primary syphilis characteristically detects only four of the six MAP proteins. Thus, the 42,000- and 16,500-dalton antigens appear to be markers for nonprimary syphilis. As seen in Table I, identical antigens were detected by sera from patients with secondary and early latent syphilis. These sera react with 94,000, 79,000, 76,000, 11 treponemal antigens of 115,000, 47,000, 40,000, 26,800, 24,500, 22,000, 19,500, and 17,400, in contrast to sera from late latent and late syphilitics. Further, Table I shows that the antigens detected by late latent and late syphilitic sera are also indistinguishable. The evidence is thus compelling that there is an acquisition of antibody to specific treponemal antigens as the disease progresses from primary to secondary and early latent syphilis, and a predictable loss of antibody to specific treponemal antigens with progression to late latent or late syphilis. Therefore, it is conceivable that the development, maintenance, and loss of antibody to particular antigens may explain the acquisition and retention of immunity and progression of the disease. Early experimental work with human volunteers showed that patients with primary syphilis were still susceptible to experimental infection with T. pallidurn, in contrast to later stages in which patients were immune to chancre development (2). This report details, then, the differences in antibody response to T. pallidurn polypeptides between patients with primary syphilis and patients who are “chancre immune.” Just as the development of chancre immunity is paralleled by the acquisition of antibody to specific T. pallidurn antigens, the progression of the disease to late latent and late disease appears to be marked by a loss of antibody to specific treponemal antigens. This observation is consistent with the idea that a specific loss of antibody may create a permissive environment for the progression of the disease to late syphilis. Incontrast to the human disease, oncerabbits become immune to challenge 3 mo post-infection, they remain in a latent state and fail to develop manifestations associated with latesyphilis in humans (18). In an attempt tocorrelate the maintenance of the latent state in experimental syphilis with humoral factors we have analyzed sera from infected immune rabbits for antibody to T. pallidurn antigens. These sera were previously shown to inactivate T. pallidurn in an in vitro-in vivo neutralization test (1 9). The pattern detectedwas indistinguishable from the patterns we report here for human secondary and early latent syphilis (Hanff, Bishop, Miller, and Lovett, manuscript submitted for publication). Thus, in experimental latent syphilis, reactivity with specific T. pallidurn antigens is maintained for at least 17 mo post-infection, whereas in humans, antibodies to certain antigens are lost as the disease progresses into late latent and late syphilis. Although the correlation between loss of humoral antibody andprogressionof disease isstriking, a cause-and-effect relationship remains to be proven. Indeed, cell-mediated responses may also play a role in the development and persistence of immunity (20) as well as in the progression of human syphilis to late disease. Recently, Lukehart et al. (21) have shown that cortisone administered to rabbits during healing of lesions causesthe reappearance of treponemes at the primary site. Thus, perhaps an alteration in the host immune response during latent syphilis in humans might be responsible for the

growth and multiplicationof T. pallidurn in late syphiliticlesions. Antigenic analysis of T. pallidurn in the past has been limited by the fact that T. pallidurn could not be purified from rabbit testicular material without loss of motility and virulence (4-7), and therefore potentialloss of motility and virulence associated antigens. In earlier studies of treponemal antigens (22, 23), nonmotile and avirulent treponemes were used in conjunction with in vitro labeling and immunoprecipitation techniques. The “Western blot” methodology, utilized in this report, is a useful tool for antigenic analysis that overcomes some major problems associated with immunoprecipitation techniques (8). There is no need to radiolabel the polypeptide antigens to be studied, and the possibility of precipitating and detecting nonantigenic polypeptides that might be associated with the antigenic proteins is circumvented. Inasmuch as preparations of treponemal proteins arealways subjected to boiling in the presence of SDS and B-mercaptoethanol, it is possible that important treponemal antigenic determinants were destroyed in our study, and that nonnative determinants were exposed. We are now studying theimmune response to human syphilis using nondenaturing methods for preparation of treponemal proteins. It is recognized that treponemal antigenic determinants may reside in intact biologic structures formed by individual protein and nonprotein moieties (24, 25). Thus, we are also investigating the role of nonprotein antigens in the human immune response to T. pallidurn infection. Although the Nichols strain of T. pallidurn remains infectious for humans (26), it is also conceivable that this strainmay differ antigenically from “wild-type” strains (24). We have described the treponemal proteins involved in the immune response to human syphilitic infection. Recently, we have reported the expressionof treponemal antigens in clones of treponemal DNA in Escherichia coli (27). Recombinant sources of T. pallidurn antigens should providesufficient amounts of the key antigens defined in this report, to permit a logical approach to the induction of chancre immunity in experimental syphilis. Acknowledgments. Michael A. Lovett wishes to thank Drs. S . Falkow and K. K. Holmes for their encouragement during his senior fellowship in Medicine at the University of Washington, Seattle. REFERENCES 1. Chesney. A. M. 1926.Immunity in syphilis. Medicine 5:463. 2. Magnuson, H. J., E. W. Thomas, S. Olansky, €3.I. Kaplan, L. DeMello. and J. C. Cutler. 1956.Inoculation syphilis in human volunteers. Medicine 35:33. 3. Fieldsteel. A. H., D. L. Cox, and R . A. Moeckli. 1981.Cultivation of virulent Treponema palljdum in tissue culture. Infect. Immun. 32:908. 4. Rathlev. T., and C. J. Pfau. 1965.Purification of the pathogenic Treponema

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Clin. Lab. Invest.

171130. 5 . Schmale. J. D., D. S. Kellogg. Jr., C. E. Miller, P. Schammel. and J. D. Thayer. 1970.Separation of Treponema pallidurn from tissue debris through continuous-particle electrophoresis. Appl. Microbiol. 19:287. 6. Thomas, M. I., J. W. Clark, G. 6.Cline, N. G. Anderson, and H. Russell. 1972.Separation of Treponema pallidurn from tissue substances by contlnuous-flow zonal centrifugation. Appl. Microbiol. 23:714. 7. Baseman, J. B.. J. C. Nichols, J. W. Rumpp, and N. S. Hayes. 1974. Purification of Treponema palhdum from infected rabbit tissue: resolution Into two treponemal populations. Infect. Immun. 10:1062. 8. Renart, J., J. Reiser. G . R . Stark. 1979.Transfer of protems from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and anttgen structure. Proc. Natl Acad. Sci. USA 76:3116. 9. Towbin, H.. T. Staehelin. and J. Gordon. 1979.Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350. 10. Tramont. E. C. 1979. Syphilis. In Principles and Practice of Infectious Diseases, Vol 2. Edited by G . L. Mandell. R . G . Douglas, Jr..and J. E. Bennett. John Wiley and Sons, New York. P. 1820. 1978. Demonstration of the fn vftro 1 1 . Lukehart. S. A , . and J.N.Miller.

POLYPEPTIDES OF TREPONEMA PALLlDUM phagocytosis of Treponema palhdum by rabbit peritoneal macrophages. J. Immunol. 121:2014. 12. Marchalonis, J. J. 1969.An enzymic method for trace iodination of immunoglobulins and other proteins. Biochem. J. 1 1 3:299. 13. Laemmli, U. K. 1970.Cleavage of structural proteins duringthe assembly of the head of bacteriophage T4. Nature 227:680. 14. Fribourg-Blanc. A. 1956.Le pouvoir treponemicide nature1 du serum humain. Presse Med. 64:1396. 15. Hederstedt. B. 1974. Studies on the Treponema pallldum immobilizing actwty in normalhuman serum. 1. A method. Acta Pathol. Microbiol. Scand. (6) 82: 185. 16. Hederstedt, E. 1976. Studies onthe Treponema pallidum immobilizing activity in normal human serum. 2.Serum factors participatingin the normal serum immobilization reaction. Acta Pathol. Microbiol. Scand. (C)84:135. 17. Hederstedt, E. 1976. Studies on the Treponema pallidum immobilizing activity in normalhuman serum. 3.The kineticsof theimmobilization reaction of normal and immune sera. Acta Pathol. Microbiol. Scand. (C) 84:142. 18. Turner, T. E.. and D. H. Hollander. 1957. Immunity phenomena in the treponematoses. In Biology of the Treponematoses. World Health Organization, Geneva. P. 123. 19. Blshop. N. H.. and J.N. Miller. 1976.Humoral immunity in experimental syphilts. 11. The relationship of neutralizing factorsin immune serum to acquired reststance. J. Immunol. 1 1 7:197.

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