Vol. 59, No. 4

INFECTION AND IMMUNITY, Apr. 1991, p. 1465-1469 0019-9567/91/041465-05$02.00/0 Copyright ©D 1991, American Society for Microbiology

Induction of Antibody Response to Chlamydia trachomatis in the Genital Tract by Oral Immunization ZHI-DUAN CUI, DEBRA TRISTRAM,* LEONARD J. LASCOLEA, TERRY KWIATKOWSKI, JR., SAMIR KOPTI, AND PEARAY L. OGRA Departments of Pediatrics and Microbiology, Division of Infectious Diseases, State University of New York at Buffalo, The Children's Hospital of Bi{ffalo, Buffalo, Newt, York 14222 Received 2 August 1990/Accepted 17 January 1991

Groups of BALB/c mice were orally immunized with Chlamydia trachomatis serovar L2/434/Bu in order to characterize the nature and kinetics of the chlamydial antibody response in the cervix and other mucosal sites. These animals were subsequently challenged intravaginally to determine whether oral immunization offers protection against chlamydial antigen shedding in the genital tract. Following oral immunization, immunoglobulin A antibody activity was detected in the genital tract as well as other mucosal sites. Subsequent intravaginal challenges exhibited booster effects on preexisting antibody activity in the genital tract. Significant protection against challenge infection in the genital tract was observed by oral immunization. This was indicated by the absence of any chlamydial antigen shedding in cervical secretions. On the other hand, passively administered chlamydial-specific serum immunoglobulin G antibody did not significantly influence the course of cervical shedding of the organism and did not confer any protection against a subsequent intravaginal challenge. It is concluded that prior oral immunization can induce a secretory antibody response in the genital tract and provide protection against subsequent infection.

Chiamydia trachomatis is the etiological agent of a wide range of human and animal infections, with recognition as a sexually transmitted disease of major significance (1, 2, 17-19). The range of human chlamydial infections includes mostly ocular, genital tract, and lung diseases (1, 2, 5, 7, 17-20). Oral immunization has been used to protect the intestinal and respiratory mucosae against poliovirus infection and adenovirus pneumonia. Such immunization appears to mimic immunologic events occurring after natural infection and was highly successful in inducing normal immunoglobulin A (IgA) antibody response in the respiratory and intestinal tracts. Oral vaccines have not been used previously for the prevention of sexually transmitted diseases. However, previous studies by Nichols et al. (14) indicated that resistance to Chlamydia psittaci infection in the ocular and genital tract tissues could be effectively induced by enteric immunization with the guinea pig inclusion conjunctivitis (GPIC) agent. Furthermore, local antibodies in humans and several animal systems have been implicated as having a protective role in chlamydial ocular and genital tract infections (1, 2, 6, 7, 11-13, 15). Recent studies have provided extensive characterization of the gut-associated lymphoid tissue (GALT) and its role in providing antigen-reactive IgA B cells to other peripheral mucosal sites, such as the genital tract (for a review, see reference 10). The present studies were undertaken to determine whether a chlamydial antibody response could be induced in the genital tract after oral immunization. An attempt was also made to determine whether such immunization offered any protection against subsequent intravaginal challenge with live chlamydial organisms.

*

MATERIALS AND METHODS Chlamydial strains. The prototype strain LGV-434-II was used in all studies reported here. Immunization. Groups of 4- to 6-week-old BALB/c mice (158 total) seronegative for chlamydial antibody were included in these studies. Group 1 consisted of 48 control mice sham immunized with uninfected McCoy cell cultures inoculated intragastrically by using a curled flat-tip needle attached to a 1-ml syringe introduced directly into the esophagus. Group 2 consisted of 84 animals immunized with a single dose of 0.1 ml of chlamydial strain LGV-434-II (109 IFU/ml) administered intragastrically as described above. Group 3 consisted of 26 mice who received intravenously 0.2 ml of immune serum globulin (ISG) containing high titers of chlamydial antibody (IgG [1:256] and IgA [1:16]). This ISG was derived from mice immunized intravenously with 108 infection-forming units (IFU) of strain LGV-II-434 per ml. The serum was pooled and incubated at 56°C for 30 min prior to intravenous use. Challenge. Following sham (group 1) or chlamydial (group 2) immunization or passive immunoprophylaxis with ISG (group 3), the animals were challenged intravaginally while under metofan anesthesia with 2 doses of 0.1 ml of 109 IFU of live chlamydia inocula per ml. In order to prevent the loss of target cervical epithelial cells during a 5-day estrous cycle, a progesterone preparation (2.5 mg per dose) was administered 1 week prior to and at the time of intravaginal challenge (22). Intravaginal challenges were administered on days 12 and 24 postimmunization (groups 1 and 2) or 5 h after passive immunoprophylaxis (group 3). The animals were sacrificed at different intervals before and after challenge. Specimen procurement. Specimens of serum, intestinal washings, cervical secretions, and tissue homogenates were collected at regular intervals before and after immunizations and intravaginal challenges. At least six mice were used for each determination. Antigen detection and culture. (i) Detection of antigen. Chlamydial antigen was measured in tissue homogenates and

Corresponding author. 1465

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body fluids employing an enzyme-linked immunosorbent assay (ELISA) (Abbott Diagnostics, Chicago, Ill.) as described elsewhere in detail (4). The reagents in the assay utilized specific rabbit antibody against C. trachomatis elementary bodies and horseradish peroxidase-conjugated antirabbit IgG. (ii) Bacterial culture and quantitation. Cervical tissue homogenates were cultured for other aerobic and anaerobic bacteria by conventional methods. Assessment of antibody production. For the determination of chlamydial IgA and IgG antibody levels in serum and mucosal secretions, an ELISA was developed. This assay involved Renografin- and serial centrifugation-purified elementary bodies of C. trachomatis LGV-434-II as the antigen in the solid phase, which is in accordance with the method of Caldwell et al. (3) with minor modifications. Such chlamydial antigen preparations containing 400 ,ug of protein per ml were diluted 1:100 in 0.05 M carbonate buffer prior to coating the microtiter plates, which were then incubated overnight at 4°C, and rinsed with phosphate-buffered saline (PBS)-Tween 20 three times. Plates were again washed with PBS-Tween 20, and serially diluted test samples were added and incubated at 37°C for 1 h. Subsequently, affinity-purified horseradish peroxidase conjugate with goat anti-mouse IgA (oxchain) or IgG was added at a dilution of 1:800 in PBS-Tween 20 with 1.5% fetal calf serum and incubated 37°C for 1 h. The appropriate substrate was then added, and the color change was measured by means of a microELISA spectrophotometer at an optical density of 490 nm. Endpoints of positivity were considered to be optical densities with >2 standard deviations of the negative and antigen control values. For the testing of specificity, antibody- and conjugate-blocking controls were performed by absorption with chlamydial antigen and purified mouse IgA and IgG, respectively. Additional negative controls consisted of uninfected McCoy cells (400 ,ug of protein per ml diluted 1:100), specimens of serum, and the cervix, and intestinal washings of uninfected mice. The antibody titers were represented as geometric means with standard deviations of the last positive dilution. RESULTS Sham immunization with uninfected McCoy cells was not associated with the development of any antibody response to chlamydia in the cervix or at other mucosal sites and resulted in no chlamydial shedding (Fig. 1). Subsequent intravaginal challenge with LGV in the sham-immunized mice resulted in continuous chlamydial shedding in the cervix, which appeared in vaginal secretions after 3 days and persisted for at least 10 days. Chlamydial shedding could not be detected in the gut and serum. Intravaginal challenge of sham-immunized animals resulted in a minimal IgA antibody response (peak antibody titers of 26.7 ± 8.3, 11.3 ± 5.3, and 29.7 + 8.9) and little or no IgG response (peak antibody titers of 2.0 ± 2.2, 5.3 + 2.1, and 21.3 ± 8.3) in the cervix, intestine, and serum, respectively. Animals immunized orally with LGV developed a high level of chlamydia-specific IgA antibody response in the cervix as well as the in the gut and serum. However, the levels of IgA antibody in the intestine were four- to eightfold higher than those observed in the cervix. The kinetics of the antibody response in different mucosal secretions is presented in Fig. 2. IgG antibody was also detected at all body sites but usually at lower levels (Fig. 2). Animals immunized orally with LGV exhibited chlamydial antigen shedding in only the intestine for up to 4 days following such immuniza-

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tion. No chlamydial antigen was detected in blood or in lung, kidney, genital tract, spleen, or liver tissue after oral immunization, indicating that inoculation of live chlamydia by the oral route did not lead to a low-grade systemic infection. Orally immunized animals were challenged intravaginally with chlamydia on days 12 and 24 postimmunization. The first challenge on day 12 exhibited a marked booster effect on preexisting IgA antibody in the cervix as well as the serum, but no increase was observed in the intestine (Fig. 2). Following the first challenge, there was a marked decrease in antigen shedding in the genital tract from a minimum of 10 days in the unimmunized genital tract to 4 days (Fig. 2). With the second (or day 24) intravaginal challenge, a further booster effect on IgA antibody levels was observed to a lessor extent in the cervix and serum, with the intestine still remaining constant (Fig. 2). The IgG antibody response was detected at all mucosal sites but at minimal levels. However, following the second intravaginal challenge, there was a total lack of chlamydial antigen shedding from the cervix. Mice passively inoculated intravenously with ISG (group 3) exhibited no chlamydial antibody prior to intravenous infusion. However, after infusion with ISG, small amounts of IgA and IgG antibody were detected in serum (13.2 ± 9.7 and 29.3 ± 18.7, respectively). However, no antibody was detected in the cervix (Fig. 3). Five hours after infusion, mice were intravaginally challenged. Three days postintravaginal challenge, IgA antibody in the cervix was still undetectable, whereas in serum, IgA and IgG remained constant. On days 7 to 11 postchallenge, IgA was detected in the cervix at substantial levels (19.4 ± 9.3) versus IgG (2.0 ± 2.2); serum antibody levels remained constant (Fig. 3).

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DISCUSSION The findings summarized in this report indicate that oral immunization induces a secretory antibody response in the genital tract and that such immunization inhibits chlamydial shedding after intravaginal challenge. Subsequent intravaginal challenge exhibited booster effects on preexisting antibody levels in the genital tract. The production of specific antibody in multiple mucosal sites by oral immunization with chlamydia was most likely a result of the migration of antigen-reactive IgA B cells and not the result of a low-grade systemic infection. This conclusion is based on the observation that no chlamydial antigen was detected in blood or in lung, kidney, genital tract, spleen, or liver tissue after such immunization. Furthermore, studies undertaken with parenteral administration of ISG indicated that the presence or the levels of passively acquired specific IgG antibody in serum alone did not significantly influence the course of cervical shedding of the organism and did not confer any protection against subsequent intravaginal challenge. The immunologic reactivity induced in the female genital tract bears several similarities to the secretory immune system of GALT, bronchus-associated lymphoid tissue (BALT), and the mammary glands (10). It now appears that all mucosal surfaces in direct contact with the external

FIG. 3. Passive immunization with ISG administered intravenously in BALB/c mice and the effects of subsequent intravaginal challenge (I.V.) with C. trachomatis LGV-434-II on the kinetics of LGV-specific IgA and IgG antibody responses. Ag, Antigen; G.M., geometric mean; SD, standard deviation.

environment including the genital tract have a common immune system with selective traffic of B and T lymphocytes from the GALT and BALT. This response may be independent of the response in the serum and is mostly associated with secretory IgA. Recent observations indicate that the immunocompetent precursor cells in the genital tract are largely derived from the BALT and GALT, which suggests that effective development of secretory IgA antibody activity in the genital tract must rely on primary stimulation of the antigen-reactive precursor cells in the gastrointestinal or respiratory tract (10). Information concerning the development of immunity to C. trachomatis infection and reinfection in humans is limited. The concept of protective immunity has been suggested because the rate of chlamydial isolation is much higher among males experiencing a first attack of nongonococcal urethritis than among those with a history of two or more

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previous attacks (56 versus 12%) (1). Chlamydial antibody has been detected in cervical secretions of women with pelvic inflammatory disease (17), the tears of infants with inclusion conjunctivitis, and the nasopharyngeal secretions of infants with pneumonia (5). These findings suggest that in humans, secretory IgA in C. trachomatis infections may be of particular importance, as there is a significant inverse correlation between the titer of this antibody in genital secretions and the presence of chlamydia isolated from the cervix (2). Furthermore, sufficient information is available to suggest a possible protective role of local antibody in human trachoma. While serum antibody did not appear to confer immunity to C. trachomatis in trachoma, immunoglobulin from the ocular secretions of children with trachoma has been demonstrated to neutralize this organism when inoculated into the eyes of owl monkeys and in cell culture (9, 15, 16). Additionally, monoclonal antibodies to major outer membrane protein determinants passively neutralize chlamydial toxicity in mice (23). In animals, resistance to reinfection is observed with guinea pigs and the GPIC agent (13) and with mice and the agent of murine pneumonitis (22). Antibody, serum or local, appears important in mediating this response. In the guinea pig model with GPIC agent, the duration and intensity of infection was increased with estradiol, which suppresses the local antibody response in the genital tract (21). Other reports (7, 8) have implicated the protective response of secretory immunoglobulin to GPIC infections of the conjunctiva and the genital tract. Murray et al. (11-13) observed that the GPIC immunity to conjunctival challenge developed only in those guinea pigs that developed specific secretory IgA in eye secretions in addition to serum antibodies. This finding contrasts those with animals that developed serum antibodies alone. Support for the findings of this report, that exposure of the intestinal mucosal surface to chlamydial organisms induces a specific antibody response at the primary site of immunization as well as other distal mucosal sites with the modulation of chlamydial shedding and disease, comes from recent studies in our laboratory (4) and the guinea pig model (8). The appearance of IgA in cervical secretions in increased quantity following intestinal priming and subsequent intravaginal challenge is consistent with trafficking of antigensensitized B cells from the gut to the genital tract prior to genital challenge. The time lag between challenge and subsequent genital IgA response may relate to further proliferation of antigen-sensitized homed cells. However, further studies would be necessary to confirm this possibility. Cui et al. (4) observed that oral immunization by using live C. trachomatis in the mouse model led to the production of secretory antibody in the lung and protection from chlamydial pulmonary infection. Murray et al. (13) observed that primary conjunctival infection produced immunity to vaginal or male urethral challenge. Conversely, a primary vaginal or male urethral infection produced detectable immunity to a subsequent conjunctival challenge (11). Further support for the findings of this report is provided by an elegant study conducted by Nichols et al. (14), in which it was demonstrated in the GPIC agent model that resistance to chlamydial infections in the eye and the genital tract was induced by enteric vaccines. ACKNOWLEDGMENTS We thank Robert C. Welliver for comments and Mary Anne Borruso for excellent secretarial assistance.

INFECT. IMMUN. This work was supported in part by grants from the National Institute of Allergy and Infectious Diseases (AI-15939-10) and the National Institute of Child Health and Human Development (HD-

19679-04).

REFERENCES 1. Alani, M. D., S. Darougar, D. C. M. Burns, R. N. Thin, and H. Dunn. 1977. Isolation of Chlamydia trachomatis from the male urethra. Br. J. Vener. Dis. 53:88-92. 2. Brunham, R. C., C. C. Kuo, L. Cles, and K. K. Holmes. 1983. Correlation of host immune response with quantitative recovery of Chlamydia trachomatis from the human endocervix. Infect. Immun. 39:1491-1494. 3. Caldwell, H., T. Kromhout, and J. Schachter. 1981. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect. Immun. 30:1161-1176. 4. Cui, Z.-D., L. J. LaScolea, J. Fisher, and P. L. Ogra. 1989. Immunoprophylaxis of Chlamydia trachomatis lymphogranuloma venereum pneumonitis in mice by oral immunization. Infect. Immun. 57:739-744. 5. Harrison, H. R., M. G. English, C. K. Lee, and E. R. Alexander. 1978. Chlamydia trachomatis infant pneumonitis. Comparison with matched controls and other infant pneumonitis. N. Engl. J. Med. 298:702-208. 6. Howard, L., M. O'Leary, and R. Nichols. 1976. Animal model studies of genital chlamydial infections: immunity to reinfection with guinea pig inclusion conjunctivitis agent in the urethra and eye of male guinea pigs. Br. J. Vener. Dis. 52:261-265. 7. Johnson, A. 1985. Pathogenesis and immunology of chlamydial infections of the genital tract. Rev. Infect. Dis. 7:741-745. 8. Lamont, H. C., D. Z. Semine, C. Leveille, and R. L. Nichols. 1978. Immunity to vaginal reinfection in female guinea pigs infected sexually with Chlamydia of guinea pig inclusion conjunctivitis. Infect. Immun. 19:807-813. 9. Lucero, M. E., and C.-C. Kuo. 1985. Neutralization of Chlamydia trachomatis cell culture infection by serovar-specific monoclonal antibodies. Infect. Immun. 50:595-597. 10. Mestecky, J. 1987. The common mucosal immune system and current strategies for induction of immune responses in external secretions. J. Clin. Immunol. 7:265-276. 11. Murray, E. 1977. Review of clinical epidemiological and immunological studies of guinea pig inclusion conjunctivitis infection in guinea pigs, p. 199-204. In K. K. Holmes and D. Hodson (ed.), Nongonococcal urethritis and related infections. American Society for Microbiology, Washington, D.C. 12. Murray, E. S., and L. T. Charbonnet. 1971. Experimental conjunctival infection of guinea pigs with the guinea pig inclusion conjunctivitis organism, p. 369-376. In R. L. Nichols (ed.), Trachoma and related disorders caused by chlamydial agents. Excerpta Medica, Amsterdam. 13. Murray, E. S., L. T. Charbonnet, and A. B. MacDonald. 1973. Immunity to chlamydial infections of the eye. I. The role of circulatory and secretory antibodies in resistance to reinfection with guinea pig inclusion conjunctivitis. J. Immunol. 110:15181525. 14. Nichols, R. L., E. S. Murray, and P. E. Nisson. 1978. Use of enteric vaccines in protection against chlamydial infections of the genital tract and in the eye of guinea pigs. J. Infect. Dis. 138:742-745. 15. Nichols, R. L., R. E. Oertley, C. E. 0. Fraser, A. B. MacDonald, and E. D. McComb. 1973. Immunity to chlamydial infections of the eye. VI. Homologous neutralization of trachoma infectivity for the owl-monkey conjunctiva by eye secretions from humans with trachoma. J. Infect. Dis. 127:429-432. 16. Peeling, R., I. W. Maclean, and R. C. Brunham. 1984. In vitro neutralization of Chlamydia trachomatis with monoclonal antibody to an epitope on the major outer membrane protein. Infect. Immun. 46:484-488. 17. Puolakkainen, M., E. Vesterinen, E. Purola, P. Saikku, and J. Paavonen. 1986. Persistence of chlamydial antibodies after pelvic inflammatory disease. J. Clin. Microbiol. 23:924-928.

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18. Schachter, J. 1978. Chlamydial infections. N. Engl. J. Med. 298:428-540. 19. Schachter, J., and C. Dawson. 1978. Human chlamydial infections. PSG Publishing Company, Inc., Littleton, Mass. 20. Schachter, J., J. Holt, E. Goodner, M. Grossman, R. Sweet, and J. Mills. 1979. Prospective study of chlamydial infection in neonates. Lancet ii:377-380. 21. Tuffrey, M., and D. Taylor-Robinson. 1981. Progesterone as a key factor in the development of a mouse model for genital tract

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infection with Chlamydia trachomatis. FEMS Microbiol. Lett. 12:111-115. 22. Williams, D. M., J. Schacter, B. Grubbs, and C. V. Sumaya. 1982. The role of antibody in host defense against the agent of mouse pneumonitis. J. Infect. Dis. 145:200-205. 23. Zhang, Y.-X., S. S. Steward, and H. D. Caldwell. 1989. Protective monoclonal antibodies to Chlamydia trachomatis serovarand serogroup-specific major outer membrane protein determinants. Infect. Immun. 57:636-638.

Induction of antibody response to Chlamydia trachomatis in the genital tract by oral immunization.

Groups of BALB/c mice were orally immunized with Chlamydia trachomatis serovar L2/434/Bu in order to characterize the nature and kinetics of the chlam...
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