PMC

Vaginal Chlamydia load following genital challenge. Mice were immunized twice with a mixture of Hirep1, CTH93, and CAF01 with 2-week intervals using either the subcutaneous (SC) or simultaneous (SIM) immunization strategy. A CAF01 adjuvant control was included. Five weeks after the immunization protocol (Figure ), mice were challenged intravaginally with Chlamydia trachomatis, and on days 3, 10, and 16, postinfection cervicovaginal swabs were collected and inclusion-forming units were counted. Graphs show mean ± SEM, and statistical analyses are done by Kruskal–Wallis (non-parametric)/Dunn’s test. **p < 0.01, ****p < 0.0001.

T and B cell frequencies in lungs following immunization. Mice were immunized twice with a mix of Hirep1, CTH93, and CAF01 with 2-week intervals using either the intranasal (IN), subcutaneous (SC), or simultaneous (SIM) immunization strategy. Two weeks after the last immunization, B cell- and T cell-mediated immune responses were depicted. (A,B) Frequencies of anti-CD45.2^{−(in vivo)} anti-B220⁺, and anti-IgA⁺ B cells were assessed by flow cytometry (FACS) on pools of lung cells (n = 8). Pools of lung cells (n = 8) were restimulated in vitro with a mixture of Hirep1 and CTH93 and frequencies of interferon gamma (IFNγ) and interleukin-17-producing antigen (Ag)-specific T cells (CD4⁺CD44^high and anti-CD45.2^{−(in vivo)}) were assessed by intracellular flowcytometry (C,D) or frequencies of IFNγ-producing Ag-specific T cells (CD4⁺CD44^high) are depicted as FACS plot (E–G).

T cell-mediated responses in genital tract (GT) following vaginal Chlamydia trachomatis (C.t.) challenge. Mice were immunized twice with a mixture of Hirep1, CTH93, and CAF01 with 2-week intervals using either the subcutaneous (SC) or simultaneous (SIM) immunization strategy. A CAF01 adjuvant control was included. Five weeks after the immunization protocol described in Figure , mice were challenged intravaginally with C.t., and on days 0, 1, 3, 6, 21, and 35 postinfection pools of cells from the GT, or iliac LNs of four mice were restimulated in vitro with a mixture of Hirep1 and CTH93. (A,C) Supernatants from restimulated iliac LNs were harvested and interferon gamma (IFNγ) and interleukin (IL)-17 levels determined by enzyme-linked immunosorbent assay. (B,D) Frequencies of IFNγ and IL-17-producing antigen-specific T cells (CD4⁺CD44^high) were assessed by intracellular flow cytometry.

Antibody (Ab) and cell-mediated immune responses following simultaneous (SIM) and prime-boost (PB) immunization. Mice received three immunizations of CTH522 and CAF01 with 2-week intervals using the subcutaneous (SC) or SIM immunization strategy, or they received three SC immunizations followed by two intranasal immunizations using the PB strategy. Five weeks after the last immunization, Ab- and cell-mediated immune responses were depicted. (A,B) Splenocytes were restimulated in vitro with CTH522 and frequencies of interferon gamma (IFNγ) or interleukin (IL)-17-producing antigen-specific T cells (CD4⁺CD44^high cells) were assessed by intracellular flow cytometry (n = 4). (C,E) Serum titers of CTH522-specific IgG1 and IgA were determined by enzyme-linked immunosorbent assay (ELISA) (n = 4). A 2,000-fold serum dilution is depicted for IgG1 and 625-fold dilution for IgA. (D,F) CTH522-specific IgG1 and IgA titers were determined for pools of lung supernatants by ELISA (n = 4). A 20-fold serum dilution is depicted for IgG1 and 5-fold dilution for IgA. Dilutions represent the linear phase of the titration curves for all immunization groups. (A–C,E) Graphs show mean ± SEM, and statistical analyses are done by Kruskal–Wallis (non-parametric)/Dunn’s test. *p < 0.05.

Antibody responses and B and T cell frequencies in genital tract (GT) following immunization. Mice were immunized twice with a mix of Hirep1, CTH93, and CAF01 with 2-week intervals using either the intranasal (IN), subcutaneous (SC), or simultaneous (SIM) immunization strategy. Two weeks after last immunization, (A,B) Hirep1- and CTH93-specific IgG1 and IgA titers were determined in vaginal swabs by enzyme-linked immunosorbent assay (n = 8). A 10-fold vaginal swab dilution is depicted for IgG1 and 25-fold dilution for IgA. Dilutions represent the linear phase of the titration curves for all immunization groups. Graphs show mean ± SEM, and statistical analyses are done by Kruskal–Wallis (non-parametric)/Dunn’s test. *p < 0.05, **p < 0.01. (C,D) Frequencies of anti-CD45.2^{− (in vivo)} B220⁺ and IgA⁺ B cells were assessed by flow cytometry (FACS) on pools of GT cells (n = 8). (E–I) Pools of GT cells (n = 8) were restimulated in vitro with a mixture of Hirep1 and CTH93 and frequencies of interferon gamma (IFNγ) and IL-17 producing antigen (Ag)-specific T cells (CD4⁺CD44^high and anti-CD45.2^{− (in vivo)}) were assessed by intracellular flow cytometry (E,F) or frequencies of IFNγ producing Ag-specific T cells (CD4⁺CD44^high) are depicted as FACS plot (G–I).

Hirep1- and CTH93-specific antibody responses and IgA plasma cells in genital tract (GT) following vaginal Chlamydia trachomatis (C.t.) challenge. Mice were immunized twice with a mixture of Hirep1, CTH93, and CAF01 with 2-week intervals using either the subcutaneous (SC) or simultaneous (SIM) immunization strategy. A CAF01 adjuvant control was included. (A,B) Five weeks after the immunization protocol, mice were challenged vaginally with C.t., and vaginal swabs were collected on days 3, 7, and 10 postinfection (p.i.). Hirep1- and CTH93-specific IgG1 and IgA titers were determined in vaginal swabs by enzyme-linked immunosorbent assay (n = 12). A 10-fold vaginal swab dilution is depicted for IgG1 and 5-fold dilution for IgA. Dilutions represent the linear phase of the titration curves for all immunization groups. Graphs show mean ± SEM, and statistical analysis are done by Kruskal–Wallis (non-parametric)/Dunn’s test. (C) Seven days p.i., Hirep1- and CTH93-specific IgA ELISPOT was performed on GT cells from three to four pools of two to four mice. Graphs show mean ± SEM, and statistical analyses are done by Kruskal–Wallis (non-parametric)/Dunn’s test. **p < 0.01, ***p < 0.001, ****p < 0.0001.