Subclinical chlamydial infection of the female mouse genital tract generates a potent protective immune response: implications for development of live attenuated chlamydial vaccine strains

Abstract

Chlamydia trachomatis is a major cause of sexually transmitted disease (STD) for which a vaccine is needed. CD4(+) T-helper type 1 (Th1) cell-mediated immunity is an important component of protective immunity against murine chlamydial genital infection. Conventional vaccine approaches have not proven effective in eliciting chlamydial-specific CD4 Th1 immunity at the genital mucosa. Thus, it is possible that the development of a highly efficacious vaccine against genital infection will depend on the generation of a live attenuated C. trachomatis vaccine. Attenuated strains of C. trachomatis do not exist, so their potential utility as vaccines cannot be tested in animal models of infection. We have developed a surrogate model to study the effect of chlamydial attenuation on infection and immunity of the female genital tract by treating mice with a subchlamydiacidal concentration of oxytetracycline following vaginal infection. Compared to untreated control mice, antibiotic-treated mice shed significantly fewer infectious organisms (3 log(10)) from the cervico-vagina, produced a minimal inflammatory response in urogenital tissue, and did not experience infection-related sequelae. Antibiotic-treated mice generated levels of chlamydia-specific antibody and cell-mediated immunity equivalent to those of control mice. Importantly, antibiotic-treated mice were found to be as immune as control untreated mice when rechallenged vaginally. These findings demonstrate that subclinical chlamydial infection of the murine female genital tract is sufficient to stimulate a potent protective immune response. They also present indirect evidence supporting the possible use of live attenuated chlamydial organisms in the development of vaccines against chlamydial STDs.

FIG. 1

Chlamydial clearance from the genital tracts of untreated or oxytetracycline-treated female mice. Groups of 10 mice were infected intravaginally with chlamydiae. One group was injected subcutaneously with oxytetracycline (1.2 mg/mouse) on the day of challenge and at 2-day intervals for the next 14 days, and the other group was not treated. Mice were cultured at different time points postinfection, and the numbers of infectious organisms shed from the cervicovagina were determined by calculating recoverable IFUs on monolayers of HeLa 229 cells. The values are means ± the standard errors of the means.

FIG. 2

Histopathological analyses of genital tissues of chlamydia-infected mice untreated or treated with oxytetracycline following intravaginal infection. Inflammatory scores were recorded for the cervix, uterus, and oviduct following infection. Panels: A, 6 days postchallenge; B, 12 days postchallenge. The scores shown are the mean inflammatory scores of tissues from three separate mice at each time point. Inflammation scores (0 to 4+) were assigned by a veterinary pathologist as described in Materials and Methods.

FIG. 3

Incidence of hydrosalpinx in untreated or oxytetracycline-treated mice. Mice (five per group) were sacrificed 40 days following clearance of the primary infection and scored for the presence of bilateral hydrosalpinx. The results shown are the percentage of mice in each group exhibiting bilateral hydrosalpinx.

FIG. 4

Serum antichlamydial IgG titers of untreated and oxytetracycline-treated mice. The sera of 10 mice were assayed individually by ELISA for IgG antibodies specific to formalin-killed MoPn EBs. End point titers were calculated as the highest dilution of serum producing an optical density of 0.2 or greater. The results are expressed as the mean antibody titer ± the standard deviation.

FIG. 5

IgA antichlamydial antibodies in the vaginal washes of untreated and oxytetracycline-treated mice. Washes were collected by injecting two 60-μl volumes of sterile phosphate-buffered saline containing 0.5% bovine serum albumin into the vaginal vault of each mouse. The washes of individual mice (5 to 10 per treatment group) were tested for IgA antibodies specific to formalin-killed MoPn EBs. The results shown are means ± the standard deviations.

FIG. 6

Cytokine profiles of splenocytes from untreated and oxytetracycline-treated mice. Splenocytes (pooled from the spleens of three mice in each experimental group) were cultured in the presence of heat-inactivated MoPn EBs for 72 h. Culture supernatants were collected and assayed for IFN-γ and IL-10 by ELISA. Individual samples were tested in triplicate, and the results are expressed as means ± the standard deviations. Ag, antigen.

FIG. 7

Chlamydial rechallenge of untreated and oxytetracycline-treated mice. Mice were challenged with 100 ID₅₀ of MoPn 40 days after clearance of a primary infection. Naive mice were infected intravaginally, and chlamydial shedding in these mice was compared to that of secondarily rechallenged oxytetracycline-treated and untreated animals. Mice (10 per group) were cultured for chlamydiae at different time points after rechallenge, and the numbers of infectious organisms shed from the vaginas of the mice were determined by calculating the numbers of recoverable IFUs. The values are means ± the standard errors of the means.