INFECTION

AND

IMMUNITY, Dec. 1991, p. 4343-4348

Vol. 59, No. 12

0019-9567/91/124343-06$02.00/0 Copyright © 1991, American Society for Microbiology

Immunization of Guinea Pigs with Recombinant TmpB Antigen Induces Protection against Challenge Infection with Treponema pallidum Nichols KONRAD WICHER,l* LEO M. SCHOULS,2 VICTORIA WICHER,' JAN D. A. VAN EMBDEN 2 AND SHAHEEN S. NAKEEB3

Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201-05091; National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands2; and Department of Pathology, School of Medicine, State University of New York, Buffalo, New York 142143 Received 21 June 1991/Accepted 16 September 1991

Treponema pallidum-susceptible guinea pigs of strain C4D were immunized with recombinant T. pallidum antigens TmpA, TmpB, TmpC, and TmpA plus TmpB plus TmpC; with Escherichia coli membranes; or with adjuvant alone. Animals in groups of five received six immunizing injections, each of 100 ,ug of antigen incorporated in RIBI adjuvant. After the sixth immunization, all experimental and nonimmunized controls were intradermally challenged with 3 x 106 T. paUidum Nichols freshly extracted from infected rabbit testes. Although high titers of antitreponemal antibodies in the fluorescent-treponemal-antibody test or an enzymelinked immunosorbent assay were evoked in all animals immunized with recombinant antigens, only guinea pigs receiving TmpB antigen demonstrated protection expressed by the development of significantly (P < 0.01) smaller, atypical lesions of significantly (P < 0.01) shorter duration and devoid of or containing fewer T. pallidum organisms than lesions in the remaining immunized and control animals. Untreated natural or experimental infection with Treponema pallidum subsp. pallidum (T. pallidum) leads to development of immunity to reinfection (2, 17, 18, 26, 33). Most attempts to induce protective immunity by various native antigens and protocols of active immunization have failed (4, 6, 14, 16, 19, 20), except in studies conducted by Metzger et al. (21, 22) and Miller (24). The nature of the immunity-inducing substances(s) in T. pallidum has been a matter of speculation. McLeod (19) and Miller (23) suggested that it is a polysaccharide, whereas Metzger et al. (21) presented evidence that the immunogenic activity of treponemes is related to a heat-labile protein component. The last decade of syphilis investigation has been enriched with recombinant DNA technology. One of the benefits of recombinant technology is that it can provide large quantities of purified and well-characterized T. pallidum antigens sufficient for serological, immunological, and immunochemical work. Of the 26 recombinant T. pallidum antigens identified (28, 31), only a few were examined by various protocols for their potentials to induce protective immunity. Borenstein et al. (3) immunized rabbits with a protease-resistant 190-kDa T. pallidum antigen known as 4D incorporated in Freund's or RIBI adjuvants; the latter contained trehalose dimycolate (TDM) plus cell wall skeleton (CWS). The investigators, who used various protocols for immunization, found that intravenous immunization with the 4D antigen in RIBI adjuvant was the best protocol, leading to alteration of the course of infection in rabbits challenged intradermally with 103 virulent T. pallidum. In preliminary experiments on two rabbits immunized with a 24-kDa recombinant T. pallidum antigen, Hsu et al. (13) found no protection against a challenge infection with 103 virulent T. pallidum. In a previous study (34), we have used recombinant T. pallidum membrane proteins designated TmpA, TmpB, and

*

Corresponding author. 4343

TmpC (11) for immunization of inbred strain 2 guinea pigs. A mixture of crude TmpAB and purified TmpC fractions incorporated into RIBI adjuvant without CWS were injected into the animals' hind footpads. A total of 300 ,ug of antigen was administered in three injections given at 3-week intervals. In spite of substantial production of treponemal antibodies, the animals were not protected against a challenge infective dose of 108 T. pallidum Nichols. In the present report, the protocol of immunization differs substantially from the previous one. TmpA, TmpB, and TmpC antigens purified to near homogeneity and incorporated into RIBI adjuvant containing CWS were administered in six injections (total of 600 ,ug of antigen) to highly susceptible (50% infective dose, 102 organisms) C4D guinea pigs (35). The results showed that only guinea pigs immunized with the TmpB antigen demonstrated a considerable protection against challenge with 3 x 106 virulent T. pallidum Nichols organisms.

(Part of the information in this paper was presented at the 1990 Annual Meeting of the American Society for Microbiology [33a].) MATERIALS AND METHODS

Animals. Young adult (2- to 3-month-old) male C4D guinea pigs obtained from our institution's animal facilities were caged individually in air-conditioned quarters (18 to 22°C) and fed antibiotic-free food and water ad libitum. Adjuvant. RIBI (RIBI Immunochemical Research, Inc., Hamilton, Mo.) adjuvant consists of nontoxic monophosphoryl lipid A (MPL) produced from endotoxin of Salmonella minnesota R595, TDM, and CWS; the last two are products of Mycobacterium phlei (29). Antigens. The production and purification of the TmpA (43-kDa) and TmpB (34-kDa) antigens have been described elsewhere (32). The production and purification of TmpC (35

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kDa) was done by the method of Schouls and associates (unpublished data). Briefly, exponentially growing K-12 Escherichia coli cells carrying recombinant plasmid pRIT9070 were heat induced for production of the recombinant proteins. Cells were harvested by centrifugation and disrupted by successive treatment with EDTA, lysozyme, and ultrasonication. Intact cells were removed by low-speed centrifugation, and membranes were sedimented by centrifugation at 100,000 x g for 60 min. The TmpC protein was then selectively solubilized from the membranes by treatment with the Zwittergent N-tetradecyl-N,N-dimethyl ammonio-1-propane sulfonate (SB-14) in the presence of MgCl2. Membrane residue was removed by centrifugation, and the solubilized protein, containing 90% of the recombinant protein, was further purified on an anion-exchange column. The fractions containing the recombinant proteins were pooled, and TmpC was purified to near homogeneity by preparative isoelectric focusing. Immunization. In the first set of experiments, guinea pigs assigned randomly to seven groups of five animals each were immunized with TmpA, TmpB, TmpC, TmpA plus TmpB plus TmpC (TmpABC), or E. coli membranes. Five animals received adjuvant in phosphate-buffered saline, and five guinea pigs served as nonimmunized control. In the second, repeat experiments, five additional guinea pigs were immunized with TmpB antigen by using the same protocol and antigen concentration; for challenge with T. pallidum, an additional five age-matched controls were included. For each immunization, the adjuvant-antigen mixture was prepared as recommended by RIBI Immunochemical Research. Briefly, the sealed vials containing adjuvant were kept for 10 to 15 min in a water bath (42°C). Into each vial, 2.5 ml of properly diluted antigen was injected, and the antigen was vortexed for 5 min. Each animal received six injections, each of 100 ,ug of antigen in a 0.4-ml volume, distributed in two subcutaneous injections (0.15 ml each) in the inguinal lymph nodes areas and one intraperitoneal injection (0.1 ml). The mixture of TmpABC contained 33 ,ug of each antigen. The adjuvant-PBS mixture was injected in the same volume and by the same route as the antigens. The age-matched nonimmunized control animals were kept in the same animal quarters as the immunized guinea pigs. Three weeks after each injection, blood was taken from all animals. Challenge infection. A week after immunizing injection 6, all animals, including untreated controls, were injected intradermally in a hind leg with 100 Rl of suspension containing 3 X 106 T. pallidum Nichols freshly extracted from rabbit testes. After infection, the animals were examined for immediate, late, and delayed-type hypersensitivity (DTH) reactions. Blood was collected at various intervals postinfection to monitor antibody production. All manipulations with the guinea pigs were done with the animals under sedation (Ketaset, Bristol Laboratory, Syracuse, N.Y.). At the end of the experiments, the animals were sacrificed with euthanasia agent T-61 (American Hoechst, Sommerville, N.J.). The animal procedures have been approved by the Institutional Animal Care and Use Committee of the Wadsworth Center for Laboratories and Research. Immunologic examination of sera. Pre- and postimmunization and postinfection sera were examined for treponemal antibodies by the fluorescent-treponemal-antibody (FTAABS) test and by the enzyme-linked immunosorbent assay (ELISA). For the latter, solubilized (2% sodium-N-lauryl sarcosine, 37°C, 30 min) T. pallidum, sonicated T. phagedenis Reiter, TmpA, TmpB, TmpC, or E. coli as antigens were used for coating the microplates in concentrations of 0.5

INFECT. IMMUN.

[Lg/100 ,l in each well. The sera were also examined by the Western blot (immunoblot) technique. All procedures were described earlier (34). Examination of lesions. Punch biopsies (3-mm diameter) of dermal reactions or lesions were cut in half, and duplicate imprints of each half in 5 RI of phosphate-buffered saline were made on microscope slides, covered with coverslips, and examined by dark-field microscopy. Fifty to 100 fields (x 1,000) were examined, and the number of organisms were calculated as described before (27). Formalin-fixed and paraffin-embedded tissue sections from punch biopsies were stained with hematoxylin-eosin for histopathologic examination and by the Warthin-Starry silver stain (7) for spirochetes. Recognition of protection. In this paper, protection is defined as the failure to elicit, after challenge infection, the ulcerative lesions observed in all control animals. In the protected animals, the character and duration of lesions must differ significantly from those in the controls. Statistical analysis. The numbers of typical and atypical lesions developing in the immunized and control groups were compared by x2 for goodness of fit. The maximum diameters and durations of lesions in immunized groups were compared with lesions of controls (infected only) by the Mann-Whitney U test or by Student's t test (5). RESULTS Animals and course of immunization. After immunizing injection 1, a slightly erythematous papule (5 to 7 mm) remained in the groin area for over 2 weeks. After each following injection, erythema (24 to 72 h) of various intensities and indurations with subsequent necrosis developed in all animals at the site of injection. The reaction subsided within 2 to 3 weeks. The reaction was milder and disappeared faster in animals receiving adjuvant alone. Challenge infection. Except for the nonimmunized and adjuvant-receiving animals, infection with T. pallidum evoked after 24 h a DTH reaction without ulceration, which waned after several days. The strongest DTH reaction, measured on day 3 postchallenge, was observed among TmpB- and TmpABC-immunized animals. The onset of the primary lesion in most immunized animals overlapped with the subsiding DTH reaction, although as well as we could assess, the incubation time of a typical primary lesion was similar in untreated controls (range, 7 to 14 days). The clinical course of infection, however, differed significantly among the various groups (Table 1, Fig. 1). Thus, while one of five TmpB-immunized animals (first experiment) developed a small (7-mm) ulcerative lesion lasting 34 days (Fig. 1A), four of five TmpA- or TmpC-immunized animals, all of five animals immunized with TmpABC, E. coli (Fig. 1B), or adjuvant alone (Fig. 1C), and all untreated controls (Fig. 1D) developed typical chancres (size range, 7 to 22 mm) lasting from 27 to >160 days. In the repeat experiments, two of five guinea pigs immunized with TmpB developed small ulcerative lesions not exceeding 7 mm in diameter, one developed induration only, and two did not develop lesions. The lesions disappeared after 15 and 34 days; one was negative for T. pallidum, and the other showed three organisms in 200 fields examined. The group mean duration for lesions was 20.2 days. All five controls in the second experiment developed large ulcerative chancres, with a group mean duration of 108 days and the presence of T. pallidum (49 to 75 organisms in 50 fields; Tables 1 and 2). Silver-stained sections prepared on days 4 and 18 after

RECOMBINANT TmpB-INDUCED PROTECTION AGAINST T. PALLIDUM

VOL. 59, 1991

TABLE 2. Presence of T. pallidum in early and advanced stages of lesion development examined by Warthin-Starry silver staining

TABLE 1. Courses of infection in immunized and control guinea pigs Lesions

Group

ofDuain(ys Duration (days) Size animals Character No.

TmpA

4 1 1 4 2 1 2 4 1 5 5 5 5 5

TmpBl1

TmpB2b TmpC

TmpABC E. coli Adjuvant Control 1 Control 2b

Chancre Induration Chancre Induration Chancre Induration Negative Chancre Induration Chancre Chancre Chancre Chancre Chancre

(mm)

Range

8-10 8 7 6-8 5, 6 6-8

27-34 20 34 14-20 15, 34 18-34

7-10 8 8-17 9-12 8-22 7-10 9-15

20-48 34 20-160 27-56 53-84 42-130 46-150

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Group mean

Group

Animal no.

28.4

TmpBlc

104 105 108

21.6

TmpB2d

272b

20.2

TmpC

TmpA

48.0 61.2 79.2 108.0

E. coli Adjuvant Control 1C

Control 2d

a Data from first experiment b Data from second experiment

pallidum in 50 fields

4 18 4 18 4 18 4 18 4 18 4 18 18 18 18 18 18 18

64 15 oa 10 oa 3 68 20 12 15 25 28 24 10 65 100 49 75

112b

TmpABC 34.0 87.2

Duration of infection (days)

275 114 113 120 118 125 123 100 101 148 150 309 310

No. of T.

One hundred fields in two slides were counted. guinea pigs developed a chancre. c Data from first experiment. d Data from second experiment. a

b These

challenge from two animals in each group indicated that with the exception of guinea pigs 108 (Fig. 2b) and 272 (TmpB immunized), T. pallidum was present in all biopsy samples examined (Table 1; example in Fig. 2d). Hematoxylin-andeosin-stained sections of skin (site of infection) taken on day 4 of infection showed in all immunized animals a marked cellular infiltration characteristic of the DTH reaction that extended from superficial to deeper layers of the dermis and consisted mainly of mononuclear cells with sparse polymorphonuclear leukocytes. A substantially higher number of mononuclear cells was observed in TmpB-immune animals (Fig. 2a) than in other groups, though mononuclear cell infiltration was also evident in the E. coli-immunized animals (Fig. 2c). Day 18 dermal lesions showed a substantially higher number of infiltrating macrophages in the antigenimmunized guinea pigs than in the nonimmunized controls (average, 180 versus 80 per high-power field). Antibody response. Production of antibodies detected by the FTA-ABS test was relatively high in TmpA-, TmpB-, and TmpABC-immunized animals, with peak titers ranging from 5.120 to 6.400 after immunization 3. TmpC-immunized

___ _ IC

_ .

_

......... ..

0~*~~ .._

FIG. 1. Dermal lesions (maximum size) developed after challenge with 3 X 106 T. pallidum in representative animals. (A) TmpB immunized; (B) E. coli immunized; (C) adjuvant; (D) nonimmunized control.

animals, however, did not respond until after immunizing injection 4 and then with very low titers (17 + 21), which did not increase after immunization 5 (Fig. 3). Pooled sera obtained from each group after immunizations 1 and 5 and 2 and 12 weeks after infection were examined by ELISA for their reactions to solubilized T. pallidum membrane proteins; sonicated Treponema phagedenis biotype Reiter; and TmpA, TmpB, TmpC, and E. coli antigens. Although the highest levels of antibody in each group, and particularly in TmpC-immunized animals, were directed against the homologous antigen (titer, 76.800 after immunization 5), there was cross-reactivity between recombinants (apparently due to the presence of E. coli) and treponemal antigens (T. pallidum membranes and T. phagedenis). The cross-reactivity was further confirmed by Western blot (data not shown). The reactivity against T. pallidum peptides evoked in the various groups of animals was readily detectable after immunization 1 in the TmpA- and TmpB-immunized animals and after immunization 5 in the TmpC-immunized animals. It also shows that animals immunized with E. coli or adjuvant elicited antibodies cross-reacting with single treponemal peptides (Fig. 4). DISCUSSION

In view of the increasing incidence of syphilis, we are reminded that successful serodiagnosis of syphilis and subsequent penicillin treatment are not sufficient to control the disease. At such a juncture, active immunization against T. pallidum infection is looked upon as a solution. Unfortunately, too little work has been done in this area to indicate whether vaccination against syphilis is attainable. Early attempts to develop a treponemal vaccine have been well discussed by Miller (25) and Lukehart (15), who also outlined in detail the difficulties to be overcome in production and implementation of a vaccine for syphilis. The two successful attempts by Metzger et al. (21) and by

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WICHER ET AL.

,,

..

INFECT. IMMUN.

'C

JI.

. -,

I

A ..V-4-A.

I

41 FIG. 2. Cell infiltration (hematoxylin and eosin) and T. pallidum (silver staining) 4 days postinfection in TmpB-immunized (no. 108; a and b) and control E. coli-immunized (c and d) animals. Larger numbers of infiltrating cells at the site of infection were observed in the TmpB group (a) compared with the adjuvant (not shown) and E. coli control (c) groups. Similar differences were observed in the presence of T. pallidum (arrows).

Miller (24) used intact organisms and a long immunization period. Metzger and associates found that the best immunizing effect was achieved by intravenous injection of T. pallidum that lost virulence as a result of aging at 4°C for 7 to 10 days. The effect was dose dependent; 12 x 109 organisms injected over a period of 7 weeks provided immunity against infection with 3 x 105 T. pallidum in ca. 50% of animals (5 of 10), but 3 x 109 organisms protected only 1 of 8 rabbits (22). Miller (24) achieved 1-year-long immunity in rabbits injected weekly for 37 weeks with a total of 3.7 x 109 gammairradiated noninfectious T. pallidum Nichols. Immunity against the homologous T. pallidum strain was complete, since the testes and lymph nodes of the rabbits challenged with 1.8 x 103 T. pallidum were free from T. pallidum as measured by an infectivity test. Schouls and Lukehart, using a protocol of immunization similar to the one described in this report but with partially purified TmpB antigen in RIBI adjuvant for immunization of rabbits, were unsuccessful in producing protection against a challenge infection with T. pallidum (unpublished results). In our earlier immunization experiments with strain 2 guinea pigs (34), we have also used a mixture of crude TmpAB

100 *o Tmnp A Tmp B o Tmp C 1 01 * Tmp E. ColiA+B+C

l* Adjuvant Untreated control

T

1

IMMUNIZATION

2

3

4

5

t

6

7

8

9

INFECTION

MONTHS FIG. 3. FTA-ABS test titers determined postimmunization and postinfection. High levels of antibodies were elicited by immunization with TmpA, TmpB, and TmpABC. Immunization with TmpC evoked low levels of antibodies after immunizing injection 4, and, contrary to results with the three former groups, the titers with TmpC increased significantly after infection.

RECOMBINANT TmpB-INDUCED PROTECTION AGAINST T. PALLIDUM

VOL. 59, 1991

97.4

--~~ ~

~

~

~

~

e ~~~~~~~~~~~~~~~~~~~

66.2

42.7 31.0

--------

21.5 14.4 1

2 3 4

5 6

FIG. 4. Western blots of T. pallidum reacted with pooled sera (1:50 dilution) obtained from TmpA (43-kDa; lane 1); TmpB (34kDa; lane 2); TmpC (35-kDa; lane 3); TmpABC (lane 4; slight difference in the migration of 43-kDa protein); and E. coli-(lane 5)-immunized and adjuvant-treated (lane 6) guinea pigs after immunization 5. In lanes 5 and 6, cross-reactivity (47 and 65 kDa) was

observed.

(consisting of ca. 60% of T. pallidum antigen) and purified TmpC antigen and only 300 ,ug of the antigens mixed with RIBI (MPL and TDM) adjuvant. Since the 50% infective dose for the inbred strain 2 used is approximately 105 T. pallidum, to secure 100% symptomatic infection in nonimmunized apimals, a high dose of 108 organisms was chosen for the challenge infection. Under such experimental conditions, the immunized guinea pigs showed no protection. In the present studies, we used guinea pigs with high susceptibilities to T. pallidum infection (35), the recombinant antigens were purified to near homogeneity, twice as much antigen (600 versus 300 ,Lg) was used, and RIBI adjuvant enforced with CWS (MPL, TDM, and CWS) was introduced. Those factors seem to contribute to the results obtained. The immunization of guinea pigs with TmpB antigen led to a substantial alteration in the course of T. pallidum infection. This was reflected in the development of significantly (P < 0.01) smaller atypical lesions without or with substantially lower numbers of microorganisms and significantly (P < 0.01) shorter lesion durations compared with those of control animals. The results of the first experiment with TmpB antigen have been confirmed in a repeat experiment using very similar conditions of immunization and challenge. Because the adjuvant contained mycobacterial products, our present experiments parallel those studying the effect of presensitization of animals with Mycobacterium bovis BCG or other microorganisms on resistance to T. pallidum infection (1, 8-10, 12, 30). The question therefore arose whether the aftered course of infection was due to nonspecific presensitization with the cross-reacting M. bovis and E. coli products or was specifically induced by the treponemal antigen or both. Development of large lesions in the adjuvant and the E. coli control groups and the development of much smaller lesions in the immunized groups (TmpA, TmpB, and TmpC) provided evidence that sensitization to the treponemal antigen must be considered in part responsible for the altered course of infection. Borenstein et al. (3) and other investigators (1, 8-10, 30) reported that nonspecific stimulation of the immune system does not promote resistance to T. pallidum infection. However, Hardy et al. (12) presented data showing that rabbits presensitized with strain BCG can exert an influence on the challenging infection with T.

4347

pallidum but only when the treponemes are injected together with a second provocative injection of BCG. The question to be answered is why only the TmpB protein caused the alteration in the course of T. pallidum infection. In contrast to the lipoproteins TmpA and TmpC, the TmpB protein is not lipid modified. The protection obtained with TmpB and not with TmpA or TmpC is remarkable, for lipid-modified proteins are considered much more immunogenic than nonlipidated proteins. TmpA and TmpC are produced as proteins with a signal sequence that is cleaved off, and both are found in the outer-membrane fraction of recombinant E. coli. TmpB, too, has a signal sequence that is cleaved off but is, in contrast, isolated from the soluble fraction of E. coli producing this antigen. It therefore seems likely that TmpB is a periplasmic protein. Furthermore, TmpB differs from TmpA and TmpC in that these antigens are acidic proteins (pl 4.5), while TmpB is a very basic protein (pl 9.5). The relatively poor antibody response to TmpB in syphilis patients compared with the responses to TmpA and TmpC seems to conflict with the protection obtained in guinea pigs. However, the high antibody levels to TmpA and TmpC obtained during infection with T. pallidum do not protect individuals against the treponeme. The RIBI adjuvant proved to be a good promoter of both cellular and humoral responses. Although both Borenstein et al. (3) and we (34) have used RIBI adjuvant (TDM-CWS or MPL-TDM), neither of the two experiments provided such high antibody titers as those found in the present studies. The FTA-ABS test antibody titers (Fig. 3) elicited already after the first injection of the TmpA and TmpB antigens exceeded by far the levels of antibodies obtained in previous experiments (34) that used RIBI adjuvant without CWS. After the third immunization, the FTA-ABS test antibody titers reached group mean values from 3,200 + 1,280 to 6,400 2,560, the highest so far reported in guinea pigs and exceeded only by a titer of >10,000 in C4D guinea pigs infected with T. pallidum extracted from C4D guinea pigs lesions (36). In order to clarify why the guinea pigs immunized with TmpC responded poorly in the FTA-ABS test but very well in the ELISA, experiments were done with the acetone treatment of T. pallidum used for the FTA-ABS test. No difference was found when acetone-treated or untreated T. pallidum were used for the FTA-ABS test. Because of the high titer to T. pallidum seen in ELISA using solubilized antigens, it may be concluded that the TmpC epitopes are buried in the T. pallidum membrane, thus remaining inaccessible to antibodies. In conclusion, comparing the lack of protection in our earlier study (34) with crude recombinant antigens with the protection observed after immunization with TmpB, it is suggested that the purity of a recombinant antigen, its treponemal concentration, and the total dose used for immunization may be very crucial in achieving protection. Repeated experiments confirmed the unique character of TmpB as a vaccinogen. Although the mechanism of protection is not known, the results presented encourage further exploration of the TmpB antigen. ±

ACKNOWLEDGMENTS This work was supported by Public Health Service grant AI 21833 from the National Institute of Allergy and Infectious Diseases to V. Wicher and K. Wicher. Leo M. Schouls was partially supported by the Netherlands Organization for Scientific Research. The skillful technical assistance of Frank Abbruscato and Marcel Barton and the competent secretarial help of Kathy Ruth are greatly

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WICHER ET AL.

appreciated. J. Terry Ulrich is acknowledged for helpful discussion on the RIBI adjuvant, and Murray King is acknowledged for his editorial work.

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Immunization of guinea pigs with recombinant TmpB antigen induces protection against challenge infection with Treponema pallidum Nichols.

Treponema pallidum-susceptible guinea pigs of strain C4D were immunized with recombinant T. pallidum antigens TmpA, TmpB, TmpC, and TmpA plus TmpB plu...
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