Genital Infiltrations of CD4+ and CD8+ T Lymphocytes, IgA+ and IgG+ Plasma Cells and Intra-Mucosal Lymphoid Follicles Associate With Protection Against Genital Chlamydia trachomatis Infection in Minipigs Intramuscularly Immunized With UV-Inactivated Bacteria Adjuvanted With CAF01

Abstract

The development of a vaccine against genital chlamydia in women is advancing, and the evaluation of in situ immune responses following vaccination and challenge infections is crucial for development of a safe and protective vaccine. This study employs the sexually mature minipig model to characterize the genital in situ immune response to Chlamydia trachomatis infection in pigs previously immunized intramuscularly with UV-inactivated C. trachomatis serovar D (UV-SvD) adjuvanted/formulated with CAF01 adjuvant compared to a CAF01-alone control group. Pigs immunized with UV-SvD were significantly protected against vaginal challenge with C. trachomatis on day 3 post inoculation and showed significantly higher cervical infiltrations of approximately equal numbers of CD4⁺ and CD8⁺ T-cells, and IgG⁺ and IgA⁺ plasma cells compared to adjuvant-alone immunized controls. These immunological signatures correspond to findings in mice and are similar to those described in female chlamydia patients. This proves important potential for the pig model in elucidating immunological in situ signatures in future translational research in chlamydia vaccinology.

Keywords: chlamydia; histology; pathology; porcine; vaccine.

FIGURE 1

Chlamydia trachomatis q-PCR detection in vaginal swabs. Detection of C. trachomatis (Ct) in vaginal swabs by q-PCR on day 3 pi. Each dot represents one animal. The gray bar shows median. Statistics: Mann-Whitney test. Two asterisks (^∗∗) indicate a P value between 0.01–0.001.

FIGURE 2

Histopathological changes including lymphocyte counts in the minipig vagina, cervix and uterus. The first and second column show representative pictures from the CAF01 and the UV-SvD/CAF01 groups, respectively. Each row represents the different anatomical locations; the vagina, cervix and uterus. The scale bar equals 200 μm. (A) In the vagina of the CAF01-group, there is a low number of lymphocytes scattered in the subepithelial stroma. (B) UV-SvD/CAF01 group, vagina. A profound perivascular infiltration of lymphocytes is noted (arrow), and also a subepithelial diffuse infiltration of lymphocytes (arrowheads). Blood vessel marked with asterisk (^∗) (C) Comparison of the semiquantitative lymphocyte infiltration scores, showing a significant difference in the lymphocyte score in the vagina of the two groups. Each dot represents one animal. Bars show median. (D) CAF01-group, cervical tissue, where a small subepithelial lymphocyte infiltration is extending into the epithelium (arrow) (E) In the cervix of the UV-SvD/CAF01 group, there is a dense subepithelial infiltration of lymphocytes (arrow) and plasma cells (insert, arrow). Intraepithelial vacuoles (arrowheads) and lymphocytes are also noted (F) Comparison of the semiquantitative lymphocyte infiltration scores, showing a highly significant difference in the lymphocyte score in the cervix of the two groups. Each dot represents one animal. Bars show median. (G) In the uterus, stromal edema is found in the CAF01-group (H) In addition to stromal edema, multiple small perivascular lymphocytic accumulations (arrowheads) are seen in the UV-SvD/CAF01 group. Blood vessel marked with asterisk (^∗) (I) Comparison of the semiquantitative lymphocyte infiltration scores, showing a significant difference in the lymphocyte score in the uterus of the two groups. Each dot represents one uterine horn. Bars show median. Statistics: Mann-Whitney test. Asterisks (^∗) indicate the level of significance: ^∗P 0.01 to 0.05, ^∗∗P 0.01–0.001, and ^∗∗∗P < 0.001.

FIGURE 3

CD3⁺, CD4⁺, and CD8⁺ T cells in the cervix along with C. trachomatis specific PBMC IFN-γ responses. Representative sections from the cervix were immunohistochemically (IHC) stained against CD3, CD4, and CD8α. All pictures and graphs are from day 14 pi. The left column shows the adjuvant (CAF01) control group IHC stains, the middle column shows the UV-SvD/CAF01 IHC stains and the right column presents C. trachomatis specific immune responses in PBMCs (G) and lymph nodes (H). CD3 (A,B): CD3⁺ cells (brown) were detected in all sections. (A) In the CAF01-group the CD3⁺ cells were mainly located intraepithelially (arrows). (B) In the UV-SvD/CAF01 group, the CD3⁺ cells were typically accumulated in the subepithelial stroma (encircled), and occasionally also intraepithelially (arrow). CD4 (C,D): CD4⁺ cells (staining red) were found in both groups, localized in subepithelial lymphoid accumulations (D, arrow), and occasionally in the epithelium; however, to a larger degree in the UV-SvD/CAF01 group (D). CD8 (E,F): CD8⁺ cells were found both intraepithelially and subepithelially in both groups (arrows), with a larger amount in the UV-SvD/CAF01 group, in a similar fashion to the CD4⁺ cells. In lymphoid accumulations, primarily present in the UV-SvD/CAF01 group, the density of CD4⁺ and CD8⁺ cells were approximate equal (also illustrated in supplementary 3). The right column shows C. trachomatis specific immune responses. Each dot represents one animal. The gray bar shows the median (G) Systemic T-cell response: IFN-γ response from PBMCs re-stimulated with C. trachomatis (H) T-cell response in the uterine draining lymph node: IFN-γ response from lymph node cells re-stimulated with C. trachomatis Statistics: Mann-Whitney test. Asterisks (^∗) indicate the level of significance: ^∗P 0.01 to 0.05, ^∗∗P 0.01–0.001, and ^∗∗∗P < 0.001.

FIGURE 4

IgG⁺ and IgA⁺ B cells in the cervix along with vaginal C. trachomatis specific IgA and IgG antibody responses. Representative sections from the cervix were immunohistochemically (IHC) stained against IgA and IgG. All pictures and graphs are from day 14 pi. The left column shows the adjuvant (CAF01) control group IHC stains, the middle column shows the UV-SvD/CAF01 IHC stains and the right column presents C. trachomatis specific immune responses in vaginal swabs (C,F). The UV-SvD/CAF01 group (B,E) contained more plasma cells than the CAF01 group (A,D) (arrows). In general, the IgA⁺ cells were more abundant than IgG⁺ cells. IgA⁺ cells were typically scattered in the subepithelial stroma (B, insert and arrows), whereas the lymphoid follicles had no, or very few IgA⁺ cells (B, lymphoid follicle encircled). IgG⁺ cells were typically found exclusively inside, or adjacent to, cellular accumulations (E, lymphoid follicle marked and inserted). The right column shows C. trachomatis specific immune responses. Each dot represents one animal. The gray bar shows the median (C) C. trachomatis specific IgA in vaginal swabs. Representative data from one experiment. (F) C. trachomatis specific IgG in vaginal swabs. Representative data from one experiment. Asterisks (^∗) indicate the level of significance: ^∗P 0.01 to 0.05, ^∗∗P 0.01–0.001, and ^∗∗∗P < 0.001.