Nonpathogenic Colonization with Chlamydia in the Gastrointestinal Tract as Oral Vaccination for Inducing Transmucosal Protection

Intragastric immunization elicits rapid and durable protective immunity to genital tract challenge. C57BL/6J mice with (n = 5) or without (n = 5) prior intragastric immunization with 2 ×10⁵ IFU of CM-mCherry for 1 week (1W) (a) or 20 weeks (20W) (b) were challenged vaginally with clone G13.32.1. The mice were monitored for C. muridarum shedding by evaluation of both vaginal and rectal (not shown) swab specimens on days 3 and 7 postinfection (3′ and 7′, respectively) and weekly thereafter. The results are expressed as the log₁₀ number of IFU per swab specimen. Mice were significantly resistant to a genital tract challenge only 1 week after immunization in the GI tract (*, P < 0.05, Wilcoxon rank-sum test, AUC), and the resistance increased and lasted for up to 20 weeks (**, P < 0.01, Wilcoxon rank-sum test, AUC). All mice were sacrificed on day 56 after the challenge infection, and the upper genital tract was evaluated for the incidence (in percent) of hydrosalpinx and the severity score (mean ± standard deviation). Immunization via the GI tract resulted in significant protection against hydrosalpinx induced by the vaginal infection (#, P < 0.05, Fisher's exact test; *, P < 0.05, Wilcoxon rank-sum test; **, P < 0.01, Wilcoxon rank-sum test).

The durable transmucosal protection induced by intragastric immunization is not dependent on long-term gastrointestinal infection. Groups of C57BL/6J mice immunized intragastrically with 2 × 10⁵ IFU of CM-mCherry (n = 5 for panel a and n = 7 for panel b) or not immunized (n = 6) (c) were treated on day 28 with doxycycline (20 μg/kg of body weight intragastrically once daily) for 2 weeks (days 28 to 42) (b and c) or were not treated with doxycycline (a). The doxycycline-treated mice were then rested for 2 weeks (days 43 to 56). On day 56 after immunization in the GI tract, all mice were intravaginally challenged with 2 × 10⁵ IFU of clone G13.32.1. (A) Mice were monitored for the shedding of chlamydiae by evaluation of both vaginal (a to c) and rectal (a1 to c1) swab specimens over the course of infection (the days after challenge infection are designated 3′ to 56′ in parentheses). Results are expressed as the log₁₀ number of IFU per swab specimen. Mice in the immunization plus doxycycline treatment group displayed no IFU in the rectal swab specimens prior to the intravaginal challenge (days 31 to 56) (b) but maintained transmucosal protection against chlamydial infection in the genital tract (*, P < 0.05, Wilcoxon rank-sum test, for panel b1 versus panel c1), equivalent to the findings for immunized mice not treated with doxycycline (*, P < 0.05, Wilcoxon rank-sum test, for panel a1 versus panel c1). These two groups maintained similar levels of protection (*, P > 0.05, Wilcoxon rank-sum test, for panel b1 versus panel a1). (b and c) The genital tract G13.32.1 organisms spread to the GI tracts. (a and a1) Black bars, G13.32.1 alone; dark red bars; both CM-mCherry and G13.32.1. (B) On day 114 after intragastric immunization, all mice were sacrificed to evaluate the upper genital tract pathology macroscopically. Representative images of the entire genital tracts from the groups receiving immunization without doxycycline treatment (a2), immunization plus doxycycline treatment (b2), or doxycycline treatment without immunization (c2) are shown. White arrows, oviducts positive for hydrosalpinges. Magnified images of oviduct/ovary regions are shown on the right of the overall genital tract images, with the white numbers indicating the hydrosalpinx scores. Both the incidence of hydrosalpinx and the hydrosalpinx severity score (mean ± standard deviation) are listed above the corresponding images. Regardless of doxycycline treatment, immunized mice were significantly protected from the development of hydrosalpinx (*, P < 0.05, Wilcoxon rank-sum test, for the immunization alone group in panel a2 versus panel c2 and for the immunization plus doxycycline treatment group in panel b2 versus panel c2).

Induction of humoral and cellular immune responses by intragastric immunization with C. muridarum. (A) C57BL/6J mice with intragastric immunization with CM-mCherry (solid symbols, n = 5) and naive mice (open symbols, n = 5) were monitored for fecal IgA (a) and serum IgG (b) as well as serum IgG isotypes (c) on different days after immunization (days 0 to 56 for IgA and days 0 to 140 for serum IgG). Samples collected from mice prior to immunization were defined as day 0 samples. CM-mCherry EB were used as antigens and were used to coat the ELISA plates. For the measurement of IgA, undiluted fecal suspensions (neat) from the immunized mice or fecal suspensions from the immunized mice diluted 1:2 or 1:4 along with neat samples from the control mice were tested. Serum samples from the same mice were 4-fold serially diluted, and to assess serum anti-C. muridarum IgG responses, 1:1,600, 1:6,400, and 1:25,600 dilutions were included for immunized mice and 1:1,600 dilutions were included for control mice. Immunization via the GI tract induced significantly higher levels of anti-C. muridarum IgA in the gut and IgG antibodies in the serum (*, P < 0.05, Wilcoxon rank-sum test) compared with the levels of the other antibodies. (c) For the isotyping of serum IgG, the binding of serum samples from immunized mice (at a 1:1,600 dilution) to the plate-immobilized EBs was probed by the use of HRP-conjugated goat anti-mouse IgG1, IgG2a, IgG2b, IgG2c, and IgG3. The ELISA results were expressed as raw OD values, as displayed on the y axis. Immunization via the GI tract induced significantly higher levels of anti-C. muridarum IgG2 antibodies than IgG1 and IgG3 antibodies. (B) C57BL/6J mice intragastrically immunized with 2 × 10⁵ IFU of CM-mCherry or control mice were sacrificed on days 0 (n = 2), 7 (n = 5 for immunized mice, n = 2 for control mice), 14 (n = 4 for immunized mice, n = 2 for control mice), 28 (n = 8 for immunized mice, n = 2 for control mice), or 56 (n = 10 for immunized mice, n = 2 for control mice) to detect the intracellular cytokines TNF-α, IFN-γ, IL-5, and IL-13 in CD4⁺ (left) and CD8⁺ (right) T cells from mesenteric draining lymph nodes (MLN) (top) and spleen (bottom). Results are expressed as the percentage of CD4⁺ or CD8⁺ T cells that express a given cytokine. Immunized mice developed dominant IFN-γ-producing CD4⁺ and CD8⁺ T cell responses in both the MLN and spleen. P values are for the number of IFN-γ-producing T cells versus the number of T cells that produced TNF-α, IL-5, or IL-13. *, P < 0.05, Wilcoxon rank-sum test; **, P < 0.01, Wilcoxon rank-sum test.

Effect of gene deficiency on intragastric immunization-induced transmucosal protection against genital tract infection. C57BL/6J mice without a deficiency (n = 20) (a) or with a deficiency in MHC class I (MHC-I KO; n = 10) (b), MHC class II (MHC-II KO; n = 10) (c), CD4⁺ T cells (CD4 KO; n = 10) (d), or B cells (μ chain KO; n = 10 for panel e and n = 5 for panel f) were intragastrically immunized with 2 × 10⁵ IFU of CM-mCherry (n = 5 or 10 per group) or buffer alone (control; n = 5 or 10 per group). On day 35 after the immunization, the mice were intravaginally challenged with 2 × 10⁵ IFU of clone G13.32.1 and live organism shedding was monitored by evaluation of vaginal swab specimens at various time points after the intravaginal infection (from days 3′ to 56′). Results are expressed on the y axis as the log₁₀ number of IFU per swab specimen. Intragastric immunization with C. muridarum protected against challenge infection in the genital tracts of wild-type (a), MHC-I KO (b), CD4 KO (d), or μ chain KO (e) mice (P < 0.05, Wilcoxon rank-sum test). However, MHC-II KO mice (c) remained highly susceptible to C. muridarum colonization regardless of their immunization status (P > 0.05 for immunized versus control mice). Although CD4 KO mice were also induced to develop significant protection, the protection was not as robust as that in the wild-type, MHC-I KO, or B cell KO mice (P < 0.05 for immunized CD4 KO mice versus immunized wild-type, MHC-I KO, or μ chain KO mice). (f) Anti-CD4 antibody was applied to a group of immunized μ chain KO mice 5 days prior to the intravaginal challenge infection and then twice every week for a total of 5 weeks, which completely blunted the B cell-independent transmucosal protection.

Comparison of colons from mice with C. muridarum colonization and mice without C. muridarum colonization. (A) For macroscopic comparison, colons collected from female C57BL/6J mice with (+) (b, d and f) or without (−) (a, c, and e) intragastric immunization with 2 ×10⁵ IFU of CM-mCherry on days 7, 28, and 56 following infection were examined for signs of colitis. Images of the cecum, colon, and rectum from one mouse in each group are shown with the cecum on top. The length (in centimeters) of the colon from each group (mean ± standard deviation) is listed below the corresponding images. Data are for 5 mice in each group (each treatment at each time point). The lengths of the colons remained similar regardless of whether the mice were immunized or when the colon samples were collected. (B) Some of the colon tissues described above were selected for microscopic examination. Colonic sections from mice sacrificed on day 7 (n = 5 mice colonized with CM-mCherry, n = 4 mice not colonized with CM-mCherry) or 28 (n = 5 mice colonized with CM-mCherry, n = 4 mice not colonized with CM-mCherry) were subjected to immunofluorescence (a, a1, c, and c1) or H&E (b, b1, d, and d1) staining. Representative images from one immunized mouse sacrificed on day 7 (a and b) and one immunized mouse sacrificed on day 28 (c and d) are presented. C. muridarum inclusions labeled with a rabbit anti-C. muridarum antibody (Anti-CM, green) and a mouse anti-chlamydial HSP60 monoclonal antibody (Anti-cHSP60, red) are indicated with green arrows. (a1 and c1) A selected area from the images visualized with a 10× objective lens (a and c) was magnified with a 100× objective lens. (a2 and c2) An infected cell from the images visualized with a 100× objective lens was further magnified digitally as an overlay, and images of anti-CM labeling (a3 and c3) (green) and anti-cHSP60 labeling (a4 and c4) (red) are shown. The adjacent sections were subjected to H&E staining and visualized under a 10× (b and d) and a 100× (b1 and d1) objective lens. The images visualized under the 100× objective lens were taken from the areas marked with white squares in the images visualized with a 10× objective lens. Putative inflammatory cells in the images visualized with a 100× objective lens are marked with white arrows. Representative longitudinal and cross-section crypts are indicated with asterisks in both the images with immunofluorescence staining (a and c) and the images with H&E staining (b and d). When both the number of C. muridarum inclusions and the extent of colonic inflammatory infiltration were semiquantitatively scored, no significant difference was found between mice with Cm-mCherry colonization and mice without Cm-mCherry colonization.

Effect of C. muridarum gastrointestinal colonization on gut microbiota. Fecal samples were collected from C57BL/6J female mice with (+; n = 5) or without (−; n = 5) C. muridarum colonization in the GI tract for various lengths of time, as described in the Fig. 6 legend. Fecal samples collected prior to intragastric inoculation (day 0) were used for the baseline. Total genomic DNA extracted from the fecal samples was used for quantitation of 16S rRNA genes by qPCR using primers specific for 7 bacteria phyla, including the 4 classes of the Proteobacteria phylum, listed in the key on the right. (a) The relative abundance of each phylum or class is expressed in percent, as shown along the y axis. All mice maintained a stable ratio of Firmicutes to Bacteroidetes throughout the time course, regardless of whether the mice were colonized with C. muridarum. (b) To display the phyla occupying less than 2% abundance, the portion of the overall plot containing the less abundant phyla was magnified with a maximal y axis scale setting of 2%. C. muridarum colonization did not affect the time-dependent acquisition of the phyla “Candidatus Saccharibacteria” and Tenericutes but slowed the increase in the Actinobacteria (*, P < 0.05, Wilcoxon rank-sum test) and the Gammaproteobacteria and Deltaproteobacteria (*, P < 0.05, Wilcoxon rank-sum test).

Effect of C. muridarum gastrointestinal colonization on intestinal immune responses to nonchlamydial infection. C57BL/6J female mice without (top; Control) or with (bottom, C. muridarum) CM-mCherry colonization in the GI tracts for 22 days were intravenously injected with 10,000 naive CD8⁺ T cells purified from P14 mice (Donor P14) and immediately infected intraperitoneally with 2 × 10⁵ PFU of LCMV. Seventeen days after LCMV infection, the mice were sacrificed and splenocytes and intestinal intraepithelial lymphocytes (IEL) were isolated. Splenocytes were used for monitoring the donor P14 mouse and endogenous CD8⁺ T cell populations (A), while the small intestine (SI) IELs were used for measuring LCMV epitope-specific gut-resident memory cells using flow cytometry (B). Representative fluorescence-activated cell sorting profiles are shown. Three mice from each group were analyzed, and similar results were observed for each mouse. LCMV infection induced similar numbers of P14 IELs positive for both CD103 and CD69 (gut-resident memory CD8⁺ T cells) in mice with or without C. muridarum colonization.