A predominant role for antibody in acquired immunity to chlamydial genital tract reinfection

Abstract

Acquired immunity to murine Chlamydia trachomatis genital tract reinfection has long been assumed to be solely dependent on cell-mediated immunity. However, in this study, we identify a previously unrecognized protective role for Ab. Immunity develops in Ab-deficient mice following the resolution of primary chlamydial genital infection. Subsequent depletion of CD4+ T cells, but not CD8+ T cells, in those immune Ab-deficient mice before secondary infectious challenge, resulted in an infection that did not resolve. Passive immunization with immune (convalescent) serum conferred a marked level of protective immunity to reinfection, which was characterized by a striking decrease in bacterial shedding, from >100,000 inclusion forming units to fewer than 10 inclusion forming units, and a shortened duration of infection. Furthermore, mAbs to the chlamydial major outer membrane protein and LPS conferred significant levels of immunity to reinfection and reduced chlamydial shedding by >100-fold. Anti-heat shock protein 60 mAb had no protective effect. In contrast to the marked protective efficacy of immune serum on reinfection, the course of primary infection was essentially unaltered by the passive transfer of immune serum. Our results convincingly demonstrate that Abs contribute importantly to immunity to chlamydial genital tract reinfection, and that Ab-mediated protection is highly dependent on CD4+ T cell-mediated adaptive changes that occur in the local genital tract tissues during primary infection. These results impact our understanding of immunity to chlamydial genital infection and may provide important insight into vaccine development.

FIGURE 1

Immune serum fails to protect CD4-depleted Ab-deficient mice during a primary chlamydial genital infection. Naive Ab-deficient mice were either untreated or depleted of CD4⁺ or CD8⁺ T cells before and during the course of primary infection (as indicated by arrowheads and described in Materials and Methods). The vertical dashed line indicates minimum duration of T cell subpopulation depletion. Animals were injected i.p. with 0.5 ml of either nonimmune or immune serum on the indicated days (*). All mice were challenged with 100 ID₅₀ of C. muridarum on day 0, and the course of infection was followed by enumerating IFUs from cervicovaginal swabs. Data are presented as the mean + SEM of IFUs recovered from five to seven mice per group. For clarity, only a single side of the SE bar is presented. Values of p < 0.05 for nondepleted mice given immune serum, compared with nondepleted mice given normal serum at all time points analyzed.

FIGURE 2

Immune serum protects against chlamydial genital tract reinfection in CD4-depleted Ab-deficient mice. Immune Ab-deficient mice (i.e., resolved primary infection) were treated with anti-CD4 as described in Materials and Methods and as indicated (arrowheads). The vertical dashed line indicates minimum duration of T cell subpopulation depletion. Groups of mice were injected i.p. with 0.5 ml of either normal serum (▲), immune serum (■), or PBS (●) at the indicated times (*). All mice were challenged with 100 ID₅₀ of C. muridarum (5 × 10⁴ IFUs) on day 56 after primary infection (arrow). A, The course of secondary infection. Data are presented as the mean number of IFUs recovered from cervicovaginal swabs collected from five or more mice per group ± SEM (for clarity, only a single side of the SE bar is presented). IFU recovery for the group of mice receiving immune sera was significantly lower (p < 0.001) than groups receiving normal serum or PBS at all enumeration time points. B, The number of culture-positive mice per total mice at each culture time point. Mice receiving immune serum completely resolved infection more quickly than mice receiving normal serum or PBS (p < 0.05 at days 14, 21, and 28 after secondary infection).

FIGURE 3

Immune serum protects against chlamydial genital tract reinfection in CD8-depleted Ab-deficient mice. Immune Ab-deficient mice were treated with anti-CD8 as described in Materials and Methods. Refer to Fig. 2 legend for explanation of injections, challenge, and data presentation. △, Normal serum; □, immune serum; ○, PBS. A, Course of secondary infection. IFU recovery for the group of mice receiving immune sera was significantly lower at days 3 (p < 0.001) and 7 (p < 0.05) after secondary infection than groups receiving normal serum or PBS. B, Number of culture-positive mice over total mice at each culture time point. No statistically significant differences in the rate of resolution of reinfection were observed between the groups of treated mice.

FIGURE 4

Effect of anti-chlamydial mAbs on the resolution of genital reinfection in immune, Ab-deficient CD4-depleted mice. Mice were injected i.p. with either anti-MOMP (▲), anti-LPS (■), or anti-hsp60 (●). Refer to Fig. 2 legend for explanation of injections, challenge, and data presentation. A, Course of secondary infection. Shedding of infectious chlamydiae was statistically significant (p < 0.01) for anti-MOMP (days 59, 63, 66, 70, 77, 84, and 91 postreinfection) and anti-LPS (days, 63, 66, 70, 77, 84, and 91 postreinfection) when compared with nontreated CD4-depleted mice or CD4-depleted mice given anti-hsp60 mAb. B, Number of culture-positive mice over total mice at each culture time point. No statistically significant differences in the rate of resolution of reinfection were observed between the groups of treated mice.

FIGURE 5

Effect of mAbs to chlamydial surface Ags on the resolution of chlamydial genital tract rein-fection in immune, Ab-deficient, CD8-depleted mice. Mice were injected i.p. with anti-MOMP (△), anti-LPS (□), or anti-hsp60 (○). Refer to Fig. 2 legend for explanation of injections, challenge, and data presentation. A, Course of reinfection. B, Number of culture-positive mice over total mice at each culture time point. No statistically significant differences were observed.

FIGURE 6

Anti-chlamydial Ab titers of immune serum and mAb preparation before and after passive transfer. Immune serum and mAb preparations were evaluated by ELISA before passive transfer (■) and at 10 days after secondary infection (). The anti-chlamydial titer for immune serum and preinjection (mAbs) represents the mean titers of triplicate determinations of the pool of convalescent serum or mAb preparation before passive transfer. At 10 days postreinfection, a time in which mice had received five injections of either immune serum or mAb, blood was collected and analyzed by ELISA of anti-chlamydia Ab. Those data represent the mean titers of at least seven mice per group. Error bars are omitted from the figure, but were always <0.5 log₂.

FIGURE 7

Anti-chlamydial Abs in vaginal wash fluid after passive transfer. Vaginal washes were collected at 9 days after secondary infection (i.e., a time when mice had received five injections of immune serum or mAb) and analyzed by ELISA for anti-chlamydial Ab. Data are presented as OD of vaginal washes (diluted 1/16) from individual mice.