Abstract: 【Abstract】 Objective To compare the protective effects of the attenuated Chlamydia muridarum vaccine pGP3S-CM with premature termination of plasmid-encoded protein 3 expression administered via intragastric, intrarectal, and intravaginal mucosal routes against genital tract infection and oviductal damage following intravaginal challenge with wild-type Chlamydia muridarum （WT-CM） in female C57BL/6J mice. Methods Twenty-four C57BL/6J mice were randomly divided into 4 groups, including an intragastric immunization group, an intrarectal immunization group, an intravaginal immunization group, and a negative control group, with 6 mice in each group. Mice in the intragastric and intrarectal immunization groups were immunized with pGP3S-CM by a gavage needle via the intragastric and intrarectal routes, respectively; mice in the intravaginal immunization group were immunized by intravaginal instillation with a pipette; mice in the negative control group received no intervention. Sixty-three days later, all mice were intravaginally challenged with WT-CM; tail vein blood samples were then collected for antibody detection, and vaginal swabs were collected periodically to determine the chlamydial load. On day 63 after challenge, the mice were sacrificed, and hydrosalpinx scores and pathological inflammation scores were evaluated. Generalized estimating equations were used for overall between-group comparisons of the vaginal chlamydial loads at different time points and the serum antibody levels at different dilutions; the Kruskal-Wallis H test was used to compare the duration of infection, hydrosalpinx scores, and inflammation scores. Multiple comparisons were adjusted using the Bonferroni correction. Results After vaginal challenge with WT-CM, compared with the negative control group （M ［Q1, Q3］: 21.0 ［21.0, 21.0］ days）, the duration of WT-CM infection was not significantly shortened in the intragastric immunization group （P = 1.000）, but was significantly shortened to 3.0 （3.0, 3.0） days and 5.0 （3.0, 7.0） days in the intrarectal and intravaginal immunization groups, respectively （both P < 0.01）. Furthermore, on days 3, 7, 14, and 21 after challenge, the vaginal chlamydial loads （lg ［1 + IFU］） were significantly lower in the intrarectal and intravaginal immunization groups than in the intragastric immunization group and negative control group （P < 0.05 or < 0.001）, and the intrarectal immunization group showed the best clearance effect in the early stage of infection （day 3）. The hydrosalpinx score was 8.0 （4.0, 8.0） in the negative control group, 1.0 （0, 5.0） in the intragastric immunization group, and 0 （0, 0） in both the intrarectal and intravaginal immunization groups, with statistically significant differences between the intrarectal/intravaginal immunization group and the negative control group （both P < 0.01）. The oviduct pathological inflammation score was 8.0 （4.0, 8.0） in the negative control group, 2.0 （0, 5.0） in the intragastric immunization group, and 0 （0, 0） in both the intrarectal and intravaginal immunization groups, with statistically significant differences between the intrarectal/intravaginal immunization group and the negative control group （both P < 0.01）. Analysis of serum specific IgG antibodies showed that the serum IgG antibody levels at dilutions of 1∶100 and 1∶200 were significantly higher in the intravaginal immunization group than in the negative control group （P = 0.006, 0.007, respectively）, whereas those were not significantly higher in the intrarectal immunization group than in the negative control group at any dilution. Conclusion Among the three mucosal immunization routes for the attenuated vaccine pGP3S-CM, intragastric immunization provided the poorest protection against genital chlamydial infection and oviductal damage in mice; intrarectal and intravaginal immunization showed favorable protective efficacy against hydrosalpinx, while the protective efficacy of the vaccine showed no parallel relationship with?the serum IgG levels.

Key words: Chlamydia muridarum, Vaccines, attenuated, Chlamydia muridarum with premature termination of plasmid-encoded protein 3 expression, Intragastric immunization, Intrarectal immunization, Intravaginal immunization, Oviductal damage