INTRODUCTION

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It has been concluded that human infection with a given serovar of Chlamydia trachomatis confers serovar-specific immunity, but broadly cross-reactive genus-specific epitopes are likely to elicit immunopathological injury (23, 36). This conclusion is based on several observations. Jawetz et al. found in a small trial that humans recovering from inclusion conjunctivitis were immune to rechallenge with the same serovar but were susceptible to rechallenge with a heterologous serovar (18). Additionally, early vaccine trials established that whole inactivated elementary bodies (EBs) administered parenterally yielded brief (lasting 1.5 to 3 years) protective immunity to ocular infection that was limited to the immunizing serovar (15-17, 37). In nonhuman primate models of trachoma and pelvic inflammatory disease, immunity is short lived and serovar specific (16, 27). Lastly, antibodies that neutralize chlamydial infectivity in vitro are usually directed against serovar-specific epitopes (reviewed in references 3, 6, 23, and 36). Collectively, one could assume from these findings that immunity resulting from prior chlamydial infection, if it exists at all, leads to serovar-specific immunity. However, it has not been definitively determined that prior genital tract infection in humans leads to protective immunity or, if protective immunity exists at this anatomical site, whether it is serovar specific (4, 22, 32).

That immunopathological injury results from previous or persistent exposure to broadly cross-reacting chlamydial antigens is also a well-accepted theory. Although serovar-specific immunity was elicited by a killed EB vaccine, it appeared to potentiate the disease state upon exposure to a heterologous serovar (15, 17). Further in vivo evidence is the observation that chlamydial Hsp60, which has 90% homology across the genus Chlamydia, elicited damaging inflammation in the eyes of guinea pigs (24) and the fallopian tubes of monkeys (27) that had been previously sensitized. Regarding murine chlamydial disease, mouse strains susceptible to infertility following chlamydial genital tract infection are also characterized as immunological responders to Hsp60, whereas strains resistant to the development of infertility are nonresponders (6, 12, 13, 33, 34, 38). Lastly, and perhaps more relevant, is the observation that a high incidence of antibody to Hsp60 epitopes is observed in women with ectopic pregnancy and tubal infertility (6, 14, 36). To summarize, the consensus has been that effective immunoprophylaxis would need to include and elicit immune responses against epitopes that are specific to each of the serovars that circulate in the vaccinated population, presumably those of the major outer membrane protein (MOMP). The same vaccine would need to omit epitopes that could possibly provide cross-serovar protection, because they may lead to untoward hypersensitizing responses against chlamydial antigens (e.g., hsp60).

Two murine models for the study of disease and immunity resulting from chlamydial genital tract infection have been established. One version of this model uses human serovars of C. trachomatis either inoculated directly into the uterus or ovarian bursa of mice or instilled intravaginally (36). Alternatively, a more commonly used model is intravaginal inoculation of mice with the mouse pneumonitis (MoPn) biovar of C. trachomatis (2). In the first case, successful infection of mice with human serovars is highly dependent upon prior treatment of mice with progesterone (P4); the MoPn model is less dependent on P4, especially if multiple infecting inoculations are conducted on two or three consecutive days or higher doses are used. However, mice sustain a more consistent infection with one MoPn inoculation if the estrus cycle is arrested with prior treatment with P4. Additionally, approximately 2 log scales fewer chlamydiae are recovered from mice inoculated with human serovars at the peak of infection, and less inflammation is observed than in those infected with MoPn (12). One may reasonably conclude from these findings that human C. trachomatis is less virulent in mice than is MoPn.

It has been found that following resolution of primary infection, mice rechallenged intravaginally with MoPn are immune for a limited period. Of those animals that shed viable organisms after challenge infection, each shed smaller quantities for an abbreviated period of time (20, 28). Of equal importance is the finding that viable MoPn organisms were required to establish protection against intravaginal challenge regardless of the route of administration (intranasal, oral, vaginal, or subcutaneous), whereas UV-inactivated organisms by any route were ineffective in this regard (20). Hence, at present, any study of protective immunity in mice requires viable chlamydiae.

In the present study, we combined the mouse-MoPn and mouse-human serovar intravaginal infection models to determine if protective immunity is biovar or serovar specific and to determine the extent of cross-protective immunity resulting from infection of mice with the mouse and human biovars.

	MATERIALS AND METHODS

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C. trachomatis. All chlamydial strains were grown in HeLa 229 cells and partially purified by differential centrifugation. Stocks were assessed for viable chlamydiae by culture of sequential 10-fold dilutions in HeLa 229 monolayers and subsequent enumeration of inclusions by indirect immunofluorescence (10). The remainder were frozen at 70°C in SPG buffer (0.25 M sucrose, 10 mM phosphate, and 5 mM L-glutamic acid [pH 7.3]) until needed. MoPn (Weiss strain) was originally obtained from Todd Cotter, who acquired it from stocks maintained in the laboratory of Harlan Caldwell (Rocky Mountain Laboratory, Hamilton, Mont.).

Mice. Five- to 6-week-old C3H/HeN mice were obtained from Harlan Sprague-Dawley, Indianapolis, Ind. Mice were given food and water ad libitum and were housed under a 14:10 dark-light cycle and allowed to acclimate for 10 days prior to inclusion in experiments. C3H/HeN mice were used as the model strain due to their increased susceptibility to chlamydial infection and disease compared to other strains of mice (7, 12, 13, 33, 34, 38).

Intravaginal infection and challenge of mice. Although not essential for establishing MoPn genital tract infection in mice, progesterone pretreatment was used to ensure a 100% infection rate in these studies. Mice were treated subcutaneously with 2.5 mg of medroxyprogesterone acetate (P4 [Depo-Provera; UpJohn, Kalamazoo, Mich.]) after 10 days of acclimation, i.e., at 6 to 7 weeks old, and again 1 week later, which correlated with 10 and 3 days prior to infection, respectively (10). Three days following the second P4 treatment, mice were infected intravaginally with 200 50% infective doses (ID₅₀) (correlating to 10⁴ inclusion-forming units [IFU] for MoPn and 10⁶ IFU for serovar E) of either MoPn or human serovar E C. trachomatis in 10 µl of SPG. Following culture-confirmed resolution of infection, at 46 and 53 days postinfection, mice were again pretreated with P4 as described above. At day 56 postinfection, mice were challenged intravaginally with 2,000 ID₅₀ of either MoPn or human C. trachomatis serovar E or L2.

Assessment of infection. Infection was assessed by sequential collection of cervical-vaginal swabs (Calgiswab, type 1; Spectrum Diagnostics, Houston, Tex.) at 4, 7, 10, and 14 days postinfection and every 7 days thereafter until cessation of chlamydial shedding. Swab specimens were cultured immediately for detection of viable chlamydia or were frozen at 70°C immediately. Once thawed, specimens were vortexed vigorously and swab fluid was diluted 1:4 in minimal essential medium and inoculated onto HeLa 229 cell monolayers in 24-well plates. After centrifugation at 1,500 × g for 1 h at 37°C, followed by an additional 2 h at 36.5°C, the swab fluid was replaced with medium and the monolayers were incubated at 36.5°C in a humidified atmosphere of 5% CO₂. Following an incubation time appropriate for each serovar or strain tested, monolayers were fixed in methanol and stained by indirect immunofluorescence antibody and examined with an inverted fluorescence microscope. A detailed description of the isolation procedure and the protocol for staining and enumeration of chlamydial inclusions has been provided elsewhere (11).

Preparation of chlamydial EBs for use as antigen in immunological assays. Chlamydial EBs were purified from HeLa 229 cells by ultracentrifugation over a Renograffin density gradient, as previously described (8). Purified EBs were then inactivated with UV light (28), standardized according to protein content (BCA protein assay kit; Pierce Chemical Co., Rockford, Ill.), and frozen in sterile phosphate-buffered saline (PBS) at 80°C.

T-lymphocyte blast transformation. At 56 days post-primary infection, mice were sacrificed and spleens and iliac lymph nodes (ILN) were excised. Single-cell suspensions were prepared by mincing tissues over stainless steel 40 to 60 mesh screens in cold Hanks balanced salt solutions, washed by centrifugation, and passed over nylon wool-packed columns in order to enrich for T cells. Nylon wool-enriched T lymphocytes were washed three times by centrifugation and resuspended in culture medium (RPMI 1640 with 10% fetal bovine serum, penicillin, streptomycin, L-glutamine, nonessential amino acids, and sodium pyruvate) to 2 × 10⁶/ml. One hundred microliters of this suspension per well was placed in 96-well culture plates with an equal concentration and volume of mitomycin C-treated syngeneic splenic feeder cells that had been previously pulsed with 5 µg of UV-inactivated EBs per ml. T-lymphocyte proliferation was determined by incorporation of [³H]thymidine for the last 18 to 24 h of a 5-day culture period and expressed as mean counts per minute of quadruplicate cultures. A detailed description of chlamydial antigen-driven T-lymphocyte blast transformation assays is provided elsewhere (29).

Assessment of delayed-type hypersensitivity (DTH) to chlamydial antigen. At 56 days post-primary infection, 5 µg of UV-inactivated chlamydial antigen in 50 µl of sterile pyrogen-free PBS was injected into the hind footpad of mice. The contralateral hind footpad received 5 µg of HeLa 229 cell antigen in 50 µl of PBS. Footpad thickness measurements were made with a dial micrometer (MTI, Aurora, Ill.) just prior to injection and at 4 to 6, 12, 24, 48, and 72 h postinjection (1, 30). Increases in footpad thickness were assessed by subtracting footpad thickness just prior to injection of antigen from subsequent measurements and are expressed as mean increases in footpad thickness of five animals.

Immunoblotting. Purified chlamydial antigens were solubilized and resolved by electrophoresis according to the manufacturer's instructions (Nupage electrophoresis system; Novex, San Diego, Calif.). Samples were loaded onto 4 to 12% gradient bis-Tris gels at 1.5 µg/well. Gel conditions were 200 V (constant) for 55 min. Resolved proteins were electrophoretically transferred to a 0.2-µm-pore-size nitrocellulose membrane (Novex) at 25 V (constant) for 120 min. Membranes were blocked in casein blocking buffer overnight at 4°C with gentle rocking. Immune sera were diluted 1:50 and 1:400 in casein blocking buffer for serovar E- and MoPn-immune animals, respectively. Samples were incubated on membranes with gentle rocking for 60 min at room temperature, followed by three wash steps with 0.5% Tween in PBS. Secondary antibody (peroxidase-conjugated goat anti-mouse immunoglobulin G [IgG]; Kirkegaard and Perry Laboratories, Gaithersburg, Md.) was diluted to 1:10,000 in casein blocking buffer and incubated on the membranes for an additional 60 min at room temperature. The membranes were then washed five times with PBS-Tween for 10 minutes each, followed by a wash with PBS only. Membranes were developed with SuperSignal chemiluminescent substrate solution, according to the manufacturer's instructions (Pierce Chemical Co.), for 5 min, rinsed briefly in distilled water, and placed on glass plates. Bound antibody was detected by exposure of blots to Biomax MS film (Kodak, Rochester, N.Y.) for 30 s.

Statistics. The two-tailed t test was used to determine significant differences in IFU counts, [³H]thymidine incorporation, and footpad swelling. Differences were considered significant at P of <0.05.

	RESULTS

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Prior infection with the MoPn biovar of C. trachomatis protects against subsequent intravaginal challenge with human serovars. So that primary and challenge inocula could be standardized in our model, we first determined the ID₅₀ for each of the strains tested in C3H/HeN mice (data not shown). In our first experiments, all mice were first inoculated intravaginally with MoPn according to the protocol detailed in Materials and Methods. The primary infection followed a normal course of vaginal shedding of the organism, as assessed by collection of cervical-vaginal swabs and subsequent isolation in HeLa cell culture as has been described elsewhere (10). All animals were culture negative on days 42, 49, and 56 post-primary infection (data not shown). The animals were again treated with P4 at 10 and 3 days prior to challenge. Nonimmune challenge controls were similarly treated with P4 but received no primary infection. On day 56, all mice were challenged intravaginally with 10 times the ID₅₀ used in the primary inoculum.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Primary murine genital tract infection with MoPn C. trachomatis protects against homotypic and heterotypic challenge. The course of infection is shown by solid lines, with each point representing mean IFU from cervical-vaginal swabs of culture-positive mice collected at the time points indicated. Above each point is the ratio of the number of culture-positive animals to the total number of animals in each group. Symbols: , previously uninfected controls; , mice previously infected with MoPn. (A) Mice challenged with human serovar E (total of two experiments). (B) Mice challenged with human serovar L2 (total of one experiment). (C) Mice challenged with MoPn (total of three experiments).

View larger version (28K): [in this window] [in a new window]   FIG. 2.   Primary murine genital infection with C. trachomatis serovar E protects against homotypic and heterotypic challenge. The course of infection is shown by solid lines, with each point representing mean IFU from cervical-vaginal swabs of culture-positive mice collected at the time points indicated. Above each point is the ratio of the number of culture-positive animals to the total number of animals in each group. Symbols: , previously uninfected controls; , mice previously infected with serovar E. (A) Mice challenged with MoPn (total of two experiments). (B) Mice challenged with human serovar L2 (total of one experiment). (C) Mice challenged with serovar E (total of one experiment).

Species-conserved epitopes are recognized by murine T lymphocytes following infection with either the mouse biovar or a human serovar of C. trachomatis. At day 56 postinfection with MoPn or human serovar E, T-lymphocyte reactivity in the spleen or ILN in response to purified UV-inactivated MoPn or serovar A, E, or L2 EBs was assessed in order to determine the existence of T-cell epitope conservation across human serogroups and the mouse biovar. We chose day 56 in order to assess the response at the time of challenge. Spleen and ILN T cells were selected as a measure of systemic and local draining lymph node responses, respectively (20, 21). Figure 3A shows the results of blast transformation of T lymphocytes from mice previously infected with human serovar E. T lymphocytes proliferated in response to EBs of serovar E, MoPn, serovar A, and L2. Proliferative responses to serovar E, as well as heterotypic EBs, were significant compared to the proliferative response to HeLa 229 antigen or background proliferation in medium only (P of <0.0001 in each case). Likewise, Fig. 3B indicates that spleen- and ILN-derived T lymphocytes from mice previously infected with MoPn yielded significant proliferative responses to EBs from each of the human serovars tested (P of <0.0001 in each case).

View larger version (39K): [in this window] [in a new window]   FIG. 3.   Proliferative responses of T lymphocytes to homotypic and heterotypic chlamydial antigens (Ag). Proliferation was assessed by [3H]thymidine incorporation in quadruplicate cultures of nylon wool-enriched T lymphocytes derived from either spleen or ILN in response to UV-inactivated, gradient-purified EBs of C. trachomatis serovar E (closed bars), MoPn (shaded bars), serovar A (open bars), or serovar L2 (hatched bars). (A) Response at day 56 postinfection with serovar E. (B) Response at day 56 postinfection with MoPn. Data are expressed as mean counts per minute above HeLa 229 antigen stimulation for each experimental group (serovar E infected and MoPn infected) and T-cell origin (spleen and ILN). The responses to Hela 229 antigen (and background unstimulated controls) were as follows: ILN (A), 297 ± 91 (176 ± 63); spleen (A), 1,151 ± 217 (927 ± 242); ILN (B), 303 ± 143 (148 ± 54); spleen (B), 2,524 ± 760 (532 ± 158).

Species-conserved epitopes are recognized by antibodies formed in response to murine genital tract infection with either the mouse biovar or a human serovar of C. trachomatis. On the day of infection and at the time of challenge, plasma samples were obtained to assess antibody reactivity by immunoblotting. Figure 4 shows the IgG antibody reactivity pattern at day 56 postinfection with either MoPn or human serovar E. Lanes C and D are EB antigens of MoPn and serovar E, respectively, probed with plasma from an MoPn-immune mouse. Three distinct antigens common to both organisms were detected. One antigen migrated to between 40 and 45 kDa and is most likely the MOMP, which is known to contain species-conserved epitopes (6, 19, 36). Another antigen migrated to ca. 60 kDa, and we speculate that this antigen may be chlamydial hsp60 because it displays >90% homology across the genus Chlamydia (6, 36). However, chlamydial Omp2 also comigrates with Hsp60; therefore, this band could represent antibody binding either or both of these proteins. The third antigen is of higher molecular mass (we estimate approximately 98 kDa), and its identity is unknown at this time. Lanes A and B are also resolved EB antigens of serovars E and MoPn, respectively, but were probed with immune plasma from a serovar E-immune mouse. IgG antibodies recognized serovar E MOMP, but not that of MoPn, and the ca. 60-kDa protein(s) of both strains. The high-molecular-weight antigen identified in this MoPn-infected mouse was not recognized. Similar results were obtained for four mice each of the serovar E- and MoPn-immune groups, for a total of 8 animals individually assessed. These observations clearly indicate that at least two, and possibly three, distinct antigenic components of MoPn and human serovar E share epitopes recognized by IgG antibodies produced during the course of murine genital tract infection with either strain.

View larger version (130K): [in this window] [in a new window]   FIG. 4.   IgG antibody reactivity at day 56 postinfection with a mouse or human biovar of C. trachomatis. Solubilized gradient-purified EBs of serovar E (lanes A and D) or MoPn (lanes B and C) resolved on 4 to 12% gradient gels and transferred to nitrocellulose were probed with immune plasma collected at day 56 postinfection from a mouse infected with MoPn (lanes C and D) or a mouse infected with serovar E (lanes A and B). Similar results were obtained from a total of eight mice tested (four each MoPn- and serovar E-infected animals). The exposed film was developed and scanned on a Microtek Scanmaker E3 with Photoshop 3.0.5 software. The scanned image was prepared and labeled with QuarkXpress 3.32.

	DISCUSSION

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The data in this study conclusively demonstrate that immunity resulting from chlamydial genital tract infection in the mouse is not solely restricted to the infecting serovar or biovar. The fact that we used organisms from two different human C. trachomatis biovars (lymphogranuloma venereum and trachoma) and the mouse biovar indicates that the immune mechanisms elicited during primary infection that protect against challenge infection are broadly cross-protective. It follows, therefore, that some protective antigenic epitopes recognized following primary infection are common to each of the three biovars (mouse, human oculogenital, and lymphogranuloma venereum) tested in our experiments. In support of this finding is the observation that specific T lymphocytes produced in response to primary infection with either human serovar E or the murine biovar responded to EB antigens of human serovars A, E, and L2 and the mouse biovar. This was evident by both in vivo (DTH) and in vitro (proliferative response) assessments. Additionally, the IgG antibodies produced in response to infection recognized the MOMP, a ca. 60-kDa antigen(s), and a higher-molecular-mass antigen (ca. 98 kDa) common to human and mouse C. trachomatis biovars.

Our results support previous studies that determined that primary infection with human serovars in the murine genital tract tends to be more brief in duration than primary infection with MoPn by about 7 days (12). Additionally, primary infection with human serovars yields approximately 2 logs fewer IFU at its peak than primary infection with MoPn. Lastly, we found that the ID₅₀ of human serovar E in mice is on the order of 2 logs higher than that of MoPn. One could reasonably conclude from these comparisons that the human chlamydial biovars are relatively weak pathogens in mice compared to the mouse biovar. Hence, it would be easier to induce heterotypic protection against challenge infection with human serovars when mice are infected first with a strain more virulent in the mouse. A higher antigenic burden and possibly a further degree of dissemination in this scenario would yield a broader and more potent immune response that would provide more protection against challenge with the weaker human pathogen. Further supporting this explanation is the fact that primary infection with a human serovar apparently provides a lesser degree of protection against the more virulent mouse biovar (Fig. 2A). Indeed, we have found that antibody levels in response to human serovar infection in mice are much lower than those following MoPn infection (unpublished data). However, it should be emphasized that in each case, the challenge inoculum was increased by 1 log scale over the primary infecting inoculum on an ID₅₀ basis, thus skewing the challenge in favor of reinfection. Therefore, the exceptionally high challenge inocula may have overcome an otherwise protective immune response, and a greater degree of protection might be observed at lower challenge doses.

In humans, repeated genital infections with C. trachomatis are common. However, persons repeatedly exposed to infection display reduced shedding of viable organisms (4). This reduction in the isolation rate is not prominent if the interval between positive cultures is greater than 6 months. In human ocular infections, protection from infection is observed only if the infecting or immunizing serovar is the same as the challenge serovar (18). Thus, it has been assumed that immunity to natural infection in humans is brief, and many believe it is mediated by serovar-specific antibody (3). However, it is commonly accepted that immunopathological injury as a result of chlamydial infection arises from antigens conserved among serovars and even among the chlamydial genus (6). An unexpected observation in our study was that homotypic challenge with MoPn provided a lesser degree of protection than what was observed following heterotypic challenge. While we are not able to provide an explanation of these results, they were consistent for a total of three experiments.

Whether the nature of the cross-biovar protection observed in the present study was antibody mediated, T cell mediated, or both cannot reasonably be correlated with the type of data provided. Due to the observed cross-reactive antibody, one could speculate that antibody played a contributing role in immune protection in this study. However, other studies have demonstrated that, although capable of neutralizing chlamydia in vitro (4, 5, 9, 25), the role played by antibody in mice is not critical to protection because mice deficient in B cells resolve infection in a manner similar to B-cell-intact mice (30). Nonetheless, Cotter et al. found a contribution of passively acquired antibody in protection from ascending infection, prevention of infection following very low doses (5 or fewer ID₅₀), and reduction in inflammation (10).

In the present study, where high challenge doses were used, it is more likely that the observed cross-protective immunity was a T-cell-mediated effect, because previous studies of immune mechanisms of chlamydial infection of mice have proven an essential role of T cells in resolution of infection (29-31). Further support of this hypothesis is that most antibody epitopes defined to date have been localized to variable regions of the MOMP that are unique among serovars or serogroups, as reviewed in reference 36. However, T-cell epitopes are often localized to regions of high amino acid sequence conservation within species (19, 26). It is likely that several broadly cross-reacting T-cell epitopes exist within the MOMP. In support of this hypothesis, Kaltenboek et al. identified candidate T-cell epitopes within the MOMP that are common between the human and mouse biovars (19). Similarly, Ortiz and coworkers found a single immunodominant MOMP sequence containing up to six possible epitopes that elicit HLA-restricted responses in the majority of infected female patients (26). Comparison of these two studies indicates that the sequence identified by Ortiz et al. is conserved in the MoPn MOMP. Interestingly, parsimony analysis of the MOMP indicates that MoPn may be an ancestral strain of human serovars of C. trachomatis (19).

In summary, we conclude that broadly cross-protective immunity against C. trachomatis in mice arises following genital tract infection. This immunity is not limited to the biovar of the primary infection but protects against challenge with other biovars as well. Future explorations with this model of heterotypic immunity in mice should include determination of whether the immunity elicited protects against the disease process (e.g., inflammation and infertility) and definition of the T-cell epitopes elicited as a result of primary infection that are conserved among the human and mouse biovars.