Immunity to Syphilis: Passive Transfer in Rabbits Using

INFECTION AND IMMUNITY, May 1976, p. 1402-1407 Copyright 1976 American Society for Microbiology Vol. 13, No. 5 Printed in USA. Immunity to Syphilis: Passive Transfer in Rabbits Using Serial Doses of Immune Serum RUSSELL S. WEISER,* DAVID ERICKSON, PETER L. PERINE, AND NANCY N. PEARSALL Department of Microbiology and Immunology, University of Washington School of Medicine, Seattle, Washington 98195 Received for publication 23 February 1976 The influence of prolonged treatment with serial doses of immune serum initiated either 2 or 48 h before intracutaneous challenge with Treponema pallidum on the subsequent appearance and development of lesions was studied. Whereas treatment with large doses of immune serum initiated 48 h before treponemal challenge caused a delay of 9 to 20 days in the onset of lesions and marked suppression of lesion development, treatment initiated 2 h before treponemal challenge failed to delay the onset of lesions and only moderately suppressed their development. Lesion development was suppressed as long as treatment was continued. The significance of the results is discussed. Although man possesses little or no immunity to primary infection with Treponema pallidum, a substantial degree of immunity to superinfection develops late in the course of the disease. The forces responsible for this immunity have not been elucidated. For many years it has been known that humoral antibodies (Ab) specific for T. pallidum are produced by infected animals and humans; however, their possible contribution to acquired immunity remains in question. In 1973, three groups of investigators, Sepetjian et al. (6), Perine et al. (5), and Turner et al. (8), demonstrated that systemic-acquired immunity to experimental syphilis in the rabbit can be passively transferred with large volumes of immune serum. Sepetjian et al. (6) showed that numerous large serial intravenous doses of immune serum initiated at the time of infection with 4.8 x 105 treponemes and extending over a period of 14 days delayed the appearance of lesions by 2 days, markedly suppressed their severity, and accelerated their healing. In contrast to lesions on control animals, which ulcerated and were dark-field positive, the lesions on the treated animals did not ulcerate and were dark-field negative. Perine et al. (5) showed that the intraperitoneal administration of a single large dose of immune serum (80 ml/kg) to rabbits bearing early cutaneous chancres (day 5) caused temporary arrest of the lesions; however, the lesions subsequently enlarged and ran a near-normal, albeit late, course, ending in healing. When immune serum was administered 24 h before intracutaneous challenge with 103 T. pallidum, 1402 the onset of lesions was delayed for 3 days; moreover, they were attenuated and healed earlier than lesions in control animals. Turner et al. (8) reported similar observations. In certain experiments of theirs in which a large dose of immune serum (50 ml/kg) was administered intraperitoneally 24 h before challenge with 500 T. pallidum, the appearance of lesions was delayed for 6 days. The chancres were attenuated and healed earlier than chancres in control animals; moreover, metastatic lesions in the testes occurred less frequently. One of the most notable aspects of the above reports is that even the largest doses of immune serum did not rid the animals of all of the injected treponemes, despite their marked capacity to suppress the development of lesions. The present experiments were conducted to determine whether treatment with large serial doses of immune serum initiated as long as 48 h before treponema challenge would result in greatly prolonged or permanent suppression of lesion development and possibly rid the animals of infectious treponemes. MATERIALS AND METHODS The methods employed were similar to those used earlier (5). Male New Zealand rabbits weighing 2.0 to 5.0 kg were screened for T. cuniculi infection, using the fluorescent treponemal antibody absorption test. Since previous experience in our laboratories had indicated that rabbits showing rapid hair growth exhibit increased resistance to cutaneous infection with the Nichols strain of T. pallidum, the backs of all rabbits were clipped and observed for a few days before use to screen out the "rapid hair growers." All animals were caged individually in an air-conditioned room in which the ambient tempera-

VOL. 13, 1976 ture was held between 62 and 65 F (ca. 16.7 and 18.3 C); they were maintained on an antibiotic-free diet of commercial rabbit food (Albers Milling Co., Los Angeles, Calif.). The large quantities of serum required for the work restricted the number of recipient animals that could be used. Immune serum was prepared using blood collected by cardiac or venous aspiration from 200 rabbits immunized with several large intracutaneous doses (10") of live treponemes (Nichols strain) given at monthly intervals over a period of 3 to 6 months or longer. About 10 days after the last dose of treponemes and at three weekly intervals thereafter, the animals were bled and their sera were assembled in small pools, filtered through a membrane filter (Millipore Corp., 0.45 gm), heat inactivated at 56 C for 30 min, and stored at -70 C. Finally, the sera were thawed and mixed to form one large pool, which in turn was divided into aliquants and stored at -70 C until used. The pooled serum was thawed and administered into the marginal ear vein or intraperitoneally. Serum from normal rabbits was collected, processed, and administered in a similar manner. The fluorescent treponemal antibody absorption test and treponema-immobilizing antibody titers of the immune serum were 1:2,048 and 1:256, respectively; the normal serum pool was negative to the fluorescent treponemal antibody absorption test. The treponemes used were grown in the testicles of cortisone-treated rabbits (8), suspended in treponema immobilization test medium (3) containing 10% heat-inactivated normal rabbit serum and 10% dimethyl sulfoxide; they were stored in a liquid nitrogen bank. Immediately before use, the suspensions were thawed rapidly and held in an ice bath; viable counts were conducted under dark field, and serial dilutions were made using treponema-immobilizing medium containing 10% heat-inactivated normal rabbit serum. Each rabbit was inoculated intradermally with 103 T. pallidum at each of six or eight widely spaced sites on the closely clipped back. All inoculations were randomized and were completed within a period of 45 min after thawing the treponeme suspension. Thereafter the backs of the rabbits were examined daily for lesions and clipped frequently to keep them bare. The severity of lesions was based on several criteria because diameter alone does not fully reflect lesion development, especially in animals treated with immune serum; the height of the lesion and the presence or absence of ulceration are also important criteria. Whereas the height of lesions in control animals often reached 10 mm or more, the lesions in animals given the largest doses of immune serum remained essentially flat and were only grossly discernible because of erythema. In contrast to the bright red color of the lesions of control rabbits, the color of the lesions of treated rabbits was a dull red. The method of evaluating lesions was modeled after the method of Turner et al. (8). Lesions were assigned a score of 1+ to 5 + based on lesion diameter and ulceration. Lesions rated 1 + were small, relatively flat papules of less than 8 mm in diameter, whereas those designated 5 + were large, raised, IMMUNITY TO SYPHILIS 1403 ulcerated lesions exceeding 25 mm in diameter and 8 mm in height. The scores 2 +, 3 +, and 4 + were used to designate an intermediate continuum of raised lesions of increasing severity; those rated 2 + were 8 to 13 mm in diameter, those rated 3+ were 14 to 18 mm in diameter, and those rated 4+ were 19 to 25 mm in diameter. In the few instances in which lesions with diameters deserving 1 + or 2 + ratings showed necrosis, the readings were increased to 3+. After the initiation of healing, scoring of lesions often becomes difficult or impossible due to scab formation. With rare exception, there was little variation in the severity of lesions on any given animal at any given interval. However, the variations among lesions on different rabbits of a group at a given interval were often substantial. The extent of these variations between rabbits is depicted in Tables 1 and 3. The curves of lesion development presented in Fig. 1 and 3 encompass all variations in lesion severity, the mean and standard deviation values being determined for all lesions of the animals of each group at a given time. Rectal temperatures taken with an electronic thermometer several times during the experiment were normal. RESULTS Experiment 1. Each of the three experimental animals of group A received a daily intravenous dose of 7 ml of immune serum per kg over a period of 8 days, beginning 2 days before challenge at eight sites with 103 live T. pallidum. Each ofthe three animals ofgroup B were given the same treatment, except that the first dose of immune serum was injected 1 to 2 h before challenge. Controls consisted of five normal animals that were not given serum and one animal that was given normal serum over a period of 8 days, beginning 2 days before challenge, at the rate of 7 ml/kg per day. Lesion development in the control animal given normal serum closely paralleled lesion development in the normal control animals. It is evident from Table 1 and Fig. 1 that the onset of lesions in treated animals of group A was delayed about 9 days beyond the time of onset of lesions in control animals and, moreover, that the curves of lesion development are significantly different. Lesion development was almost completely suppressed in the treated animals of this group, and no lesion greater than 2+ arose. In contrast, there was no significant delay in the onset of lesions in the treated animals of group B; however, about 3 days after onset the lesions in the animals of this group ceased to grow for a time; after about day 23 (15 days after discontinuing treatment), they began to enlarge once more at a rate essentially equivalent to the rate of development of lesions in control animals.

1404 WEISER ET AL. INFECT. IMMUN. TABLE 1. Influence of the timing of immune serum treatment on the onset and development of cutaneous lesions in individual rabbits of experiment 1 Interval after treponemal challenge (days) Group and treatment Rabbit - _- - T - _ 0-11 12 14 18 21 23 128 32 35 49 60 A Immune serum (days -2 to +5; W 899 0 0 0 0 0. 2a h0 3b 7 ml/kg per day) W 888 0 0 0 0 0 0 0.1 0.5 1.0 0.8 0.7 W 900 0 0 0 0 0 0 0.2 1.0 2.2 2.7 2.0 B Immune serum (days 0 to +7; 7 W 907 0 0.5 0.9 0.2 0.7 0.7 1.6 2.5 3.0 2.2 2.1 ml/kg per day) W 892 0 0 0 0 0 0 1.4 2.6 3.2 2.7 2.8 W 893 0 0.3 1.2 0.5 0.4 0.3 1.3 2.2 2.4 3.6 2.8 a b Normal serum (days -2 to +5; 7 W 894 0 0 0.8 1.6 2.2 2.8 3.2 3.5 2.7 2.0 1.3 ml/kg per day) No serum W 903 0 0 0.7b W 906 0 0 0.8 2.4 2.6 3.2 4.0 4.0 2.6 2.3 2.6 W 905 0 0.7 1.0 2.2 2.6 3.0 3.3 3.8 3.8 2.6 2.1 W 895 0 0 1.2 2.4 2.8 2.7 3.6 4.2 4.3 3.0 2.9 W 904 0 0.3 1.1 2.3 2.5 2.7 3.6 3.9 3.8 2.7 2.0 Each number represents the mean of the scores for the eight lesions on a given rabbit. Removed from the experiment because of intercurrent infection. Glroup A Percod cf serum Ireoment Group B- Period of serum treatment 4 O-I Soandord lev 0lon * Readings were discontinued 3 5- after the onsel of hecling Co5tntrol '2 5 3 o15 ControlsGr 05~~~~~~~~~~~~r 5 C 15 20 25 30 35 40 Days post cholienge FIG. 1. Influence of the timing of immune serum treatment on the onset and development of cutaneous lesions in rabbits of experiment 1 (see Table 1 for treatment schedule). Photographs of the backs of representative animals of the various groups taken on day 22 of the experiment are presented in Fig. 2. They illustrate the striking differences in lesion development depicted in Fig. 1. In summary, the results of experiment 1 using daily doses of serum over a period of 8 days, beginning either 48 h before or 2 h before treponeme challenge, showed that both treatments were protective. However, a 9-day delay in the onset of lesions occurred when serum treatment was initiated 48 h but not 2 h before treponeme challenge. This finding is compatible with the view expressed by Turner et al. (8), namely, that time is required for the injected Ab to equilibrate between blood and tissue fluids and FIG. 2. Photograph of the backs of representative rabbits ofserum-treated and control groups ofexperiment 1 taken on day 22. Whereas the control untreated animal shows large raised lesions, the animal in seri.m-treated group B shows small flat lesions. The animal ofgroup A possessed barely visible lesions. See Fig. 1 for curves depicting lesion development. The inoculation sites are circled with ink. that high extravascular levels of Ab are needed for maximum effects because the injected organisms are deposited at extravascular sites. If this concept is true, it follows that even relatively high levels of extravascular Ab may not be rapidly lethal to all or perhaps even the majority of the injected organisms. To test this hypothesis, experiment 2 was conducted in which serial doses of immune serum at different levels were continued over longer periods of time to determine whether the development of lesions could be suppressed indefinitely by such treatment and whether lesions would arise at

VOL. 13, 1976 inoculation sites subsequent to the termination of prolonged treatment. Experiment 2. In our previous experience with some 10 control rabbits treated with large volumes of normal serum, we had never noted any indication that normal serum affords protection against skin challenge with T. pallidum. This has also been the experience of others (8). Consequently, animals treated with normal rabbit serum were not included in experiment 2. The schedule of intraperitoneal treatment with pooled immune serum is shown in Table 2. The results are presented in Table 3 and Fig. 3. They show that, whereas the ani- TABLE 2. a Schedule ofimmune serum treatment used in experiment 2 Immune serum Nof Size of pri- Size of each Group No. mary dose 48 of 13 animals subse- hbefore quent doses challenge injected at 3- (ml/kg) day intervals (mi/kg) A 1 30 30 B 3 25 15 C 2 12 6 D 3 6 3 Control 5 NSAa NSA NSA, No serum administered. IMMUNITY TO SYPHILIS 1405 mals of groups C and D that received the lowest doses of immune serum were not protected, the animals of groups A and B that received the highest doses of immune serum were markedly protected during the period of treatment, lesion development being almost completely suppressed. It is also notable that, although there was essentially no delay in the onset of lesions in animals of groups C and D, a delay of 20 and 10 days occurred in animals of groups A and B, respectively. The lesions on animal no. X 081 plateaued until about day 80 and then slowly receded. Dark-field tests on material taken from cutaneous lesions and from the testes on day 75 were positive. This animal was totally resistant to intradermal rechallenge with 106 T. pallidum on day 90, presumably because active immunity had developed in response to the smoldering lesions that existed between day 20 and 80. In contrast, the animal receiving the highest dose of serum, animal no. X 117, showed the greatest delay in lesion onset (20 days). After the treatment was discontinued, the lesions began to enlarge and from that point on showed a curve of lesion development similar to the curves shown by normal animals. Moreover, the curve of lesion development to rechallenge with 10" organisms on day 60 was similar, albeit somewhat lower than the curve shown by TABLE 3. Onset and development ofcutaneous lesions in individual rabbits ofexperiment 2 during and after prolonged treatment with varying dosages of immune seruma Group and treat- Interval after treponemal challenge (days) Gropendtret Rabbit -- -- -- mentp Rabbit 13 16 18 21 23 27 29 32 34 38 43 47 53 62 69 A X 117 0 0 0 0 0 0 0 0.1C 0.4 0.3 0.4 0.8 1.0 2.9 3.5 B X 084 0 0 0 1.0 1.0 1.0 1.1 1.0d X 081 0 0 0 0 0.5 0.6 1.0 1.0 1.0 1.0 0.5 0.4 0.4 0.8 X 083 0 0 0 1.0 1.0 1.0 1.3 1.3 1.3 1.7 1.5 1.8 1.6 1.2 C X 060 1.0 1.4 1.3 1.0 1.3 1.3 2.7 3.3 3.7 4.2 4.2 4.3 4.1 X 072 0.8 1.0 1.2 1.5 1.5 1.6 3.5 4.0 4.3 4.4 5.0 5.0 5.0 D X 088 0.6 1.0 1.5 2.0 2.3 2.9 3.7 3.7 3.5 3.3 3.7 3.6 3.5 X 085 0.2 0.6 1.3 2.5 2.7 2.7 3.8 4.0 4.4 4.6 5.0 5.0 5.0 X 075 0.4 0.5 1.7 2.1 2.0 2.5 3.3 2.7 2.1 2.0 2.1 2.0 2.0 Controls (no X 076 0.8 1.1 1.9 2.5 3.6 3.9 4.5 4.5 4.7 4.7 4.2 3.7 3.9 treatment) X 082# 1.2 1.4 1.7 2.3 2.5 3.5 3.8 4.0 3.7 3.5 3.6 3.5 3.6 X 074 0 0.3 2.2 2.8 3.4 3.5 4.2 4.3 4.4 4.7 4.6 4.6 4.5 X 073 0.3 2.3 2.5 3.0 3.8 3.8 4.4 4.3 4.2 4.0 3.7 2.9 2.5 X 126 0 0 0 0.8 1.3 2.2 2.0] 2.0 2.8 3.0 3.0 2.7 2.2 a See Table 2 for treatment schedule. The last dose of serum was given on day 36. b Groups A to D received increasing dosages of immune serum, with group A receiving the highest dosage. r Each number represents the mean of the scores for the six lesions on d a given rabbit. Removed from the experiment because of intercurrent infection. e Rabbit no. X 082 developed lesions scoring 1.0 on day 11; no other rabbit developed lesions before day 13.

1406 WEISER ET AL. 5 01 * Serum In jection Stondard devitiono Readings were discontinued Group CR after the onset of healing 3 57 3O-1 2,2 25-{/( Group Group B - 0) 5 ( 05-' G_ < G roup A - 5 I0 I"5 20 25 30 35 40 45 50 55 60 65 70 Days post chollenqe FIG. 3. Influence -of prolonged treatment with varying dosages of immune serum on the onset and development of cutaneous lesions in rabbits of experiment 2 (see Table 2 for treatment schedule). The arrows designate the days of serum treatment. the primary lesions. Evidently, active protective immunity did not develop in this animal during serum treatment, presumably because the late and limited development of lesions did not provide a sufficient antigenic stimulus. DISCUSSION The present results in the rabbit model confirm and extend our previous results (5) and those of others (6, 8); namely, they show that large doses of immune serum confer marked protection against intradermal challenge. Since acquired immunity to syphilis in humans is not known to result from any cause other than prior infection with T. pallidum or closely related organisms and in view of the observation that normal rabbit serum (5, 8) and serum from BCG-sensitized rabbits (unpublished data) does not convey protection against T. pallidum in rabbits, it seems highly probable that the major protective factor in immune rabbit serum is specific Ab. However, nonspecific factors, such as lysozyme, might act in concert with Ab, the latter lending specificity to protection. Assuming that protection by immune serum is due to Ab, it is important to learn what their immunoglobulin nature and specificity may be and how they may act. (Work along these lines is currently in progress in our laboratories.) Protective Ab could act through such agencies as agglutination, opsonization, and complement killing. Circumstantial evidence against a protective role for treponema-immobilizing Ab has been summarized by Sepetjian et al. (6) and by Turner et al. (8). The evidence consists princi- INFECT. IMMUN. pally of numerous reports of a lack of correlation between serum levels of treponema-immobilizing Ab and immunity to superinfection (8). In our initial publication (5) we suggested that immune serum may serve to immobilize and destroy or inhibit the growth of treponemes in challenge inocula. Turner et al. (8) expressed the view that passively transferred immune serum may kill the majority of organisms in challenge inocula and that the remainder survive because Ab does not reach them. At least one of several mechanisms can be invoked to explain the present observation that continuing high doses of immune serum can accomplish essentially complete suppression of lesion development during treatment without destroying the injected organisms, namely, that the protective agent(s) transferred is bacteriostatic, is very slowly lethal, fails to sterilize tissue because a resistant population of treponemes exists, or, lastly, fails to act on a fraction of the injected organisms because they somehow come to occupy privileged positions within tissues or cells beyond the reach of effective concentrations of the agent (8). There is limited information on the question of whether T. pallidum can perpetuate itself as an intracellular parasite or enter and persist in cells for extended periods of time (7). Although treponemes have been found within host cells, they are present in only a few cells and the majority of organisms are extracellular (7). The thesis that immunity to syphilis is principally humoral demands that, to perpetuate itself indefinitely in the face of strong immunity, T. pallidum would, perforce, have to be able to find a haven of escape from the destructive serum factor(s) in order to carry on at least limited division. Perhaps this occurs during late and latent syphilis when immunity is high and the organisms are sparse. In any consideration of acquired resistance to syphilis, the relative efficiency of local as well as systemic forces of immunity should be kept in mind. It is possible that effective specific immunity, either cellular and/or humoral, can be mounted locally in the primary lesion and initiate healing before strong systemic immunity develops. With respect to the protective Ab of humoral immunity, it is notable that plasma cells are abundant in local lesions; thus, effective local immunity could develop, due to intense local production of Ab, before high levels of circulating Ab are attained. It is possible that the strong systemic immunity of late disease depends more on memory cells than on circulating Ab levels at the time of challenge, i.e., on

VOL. 13, 1976 IMMUNITY TO SYPHILIS 1407 memory B cells and possibly helper T cells that can quickly mobilize to sites of infection and set up intense local production of Ab. If the anamnestic response is the principal determinant in resistance to superinfection, it could account for the need for high doses of immune serum to convey passive immunity in the virgin animal lacking memory. Also, in the actively immunized animal it could account for the reported lack of correlation between the serum levels of Ab and resistance to treponemal challenge. Support for the widely held concept that specific immunity to syphilis is primarily cell mediated is circumstantial, and at present there is no convincing evidence that the classical effector cell of antimicrobial cell-mediated immunity, the activated macrophage, operates against T. pallidum. An alternative possibility is that cell-mediated immunity against T. pallidum is effected extracellularly by lymphokines secreted by immune lymphocytes stimulated by specific antigen (4). The high incidence of relapses during early and latent syphilis is difficult to fit with any theory of immunity without invoking the concept that successive periods of natural immunosuppression (2) occur or that the organism undergoes phase variation in its antigenic composition. Present evidence indicates that acquired resistance to syphilis is complex and that it probably involves forces of both humoral and cellular immunity. ACKNOWLEDGMENTS This work was supported by Public Health Service research grant 1-ROl-AI 11384 from the Institute of Allergy and Infectious Diseases. The technical assistance of Marilyn Backman is gratefully acknowledged. LITERATURE CITED 1. Hardy, P. H., Jr., and E. E. Nell. 1957. Study of the antigenic structure of Treponema pallidum by specific agglutination. Am. J. Hyg. 66:160-172. 2. Levene, G. M., J. L. Turk, D. J. M. Wright, and A. G. S. Grimble. 1969. Reduced lymphocyte transformation due to a plasma factor in patients with active syphilis. Lancet 2:246-247. 3. Ovcinnikov, N. M. 1965. A simplified method of performing the Treponema pallidum immobilization test. Bull. W.H.O. 33:197-208. 4. Pearsall, N. N., J. Sundsmo, and R. S. Weiser. 1973. Lymphokine toxicity for yeast cells. J. Immunol. 110:1444-1446. 5. Perine, P. L., R. S. Weiser, and S. J. Klebanoff. 1973. Immunity to syphilis. I. Passive transfer in rabbits with hyperimmune serum. Infect. Immun. 8:787-790. 6. Sepetjian, M., D. Salussalo, and J. Thivolet. 1973. Attempt to protect rabbits against experimental syphilis by passive immunization. Br. J. Vener. Dis. 49:335-337. 7. Sykes, J. A., J. N. Miller, and A. J. Kalan. 1974. Treponema pallidum within cells of a primary chancre from a human female. Br. J. Vener. Dis. 50:40-44. 8. Turner, T. B., P. H. Hardy, Jr., B. Newman, and E. E. Nell. 1973. Effects of passive immunization on experimental syphilis in the rabbit. Johns Hopkins Med. J. 133:241-251.