Humoral immune response to chlamydial genital infection of mice with the agent of mouse pneumonitis.

The objective of this study was to characterize the humoral immune response to chlamydial genital infection of mice with the mouse pneumonitis agent (MoPn). With an enzyme-linked immunoabsorbent assay, immunoglobulin G antibodies to MoPn were first detected in plasma by day 14. Peak plasma antibody concentrations were reached by day 49, and this response did not decline significantly throughout the 300-day monitoring period. Immunoglobulin A against MoPn could first be detected in pooled vaginal washes by day 21 after infection and had reached peak concentrations by day 28, but anti-MoPn immunoglobulin G was not consistently present in secretions. The antibody response in secretions had declined slightly by day 300. Immunoblot analysis revealed that the early phase of the plasma antibody response to MoPn as a result of genital infection was against lipopolysaccharide, the major outer membrane protein, and a 62-kilodalton (kDa) protein. In secretions, early-phase immunoglobulin A antibodies were directed to the major outer membrane protein and lipopolysaccharide. Late reactions to 15-, 22-, and 83-kDa proteins in plasma were noted. Late reactions to the 62-kDa protein in secretions were also noted. The cause of these late responses remains unexplained. When mice were challenged intravaginally with MoPn at 50-day intervals after the primary infection, it was found that mice inoculated on day 100 or after were susceptible to reinfection. Susceptibility could not be related to a decline in the antibody concentration in plasma or secretions or in the antibody response to specific components of MoPn as measured by immunoblot analysis.     

Salmonella flagellin is not a dominant protective antigen in oral immunization with attenuated live vaccine strains

We found that oral immunization with flagellum-defective mutant strains of Salmonella enterica serovar Typhimurium with the ClpXP-deficient background protected mice against oral challenge with the virulent strain. These data indicate that Salmonella flagellin is not a dominant protective antigen in oral immunization with attenuated live vaccine strains.     

Analysis of the humoral immune response to chlamydial genital infection in guinea pigs.

Studies using the guinea pig model of chlamydial genital infection with the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis (GPIC) have shown that serum and local antibodies play a role both in the resolution of infection and in protection against reinfection. Thus, this model is suited for further exploration of immune mechanisms and for vaccine studies with chlamydial macromolecules. We have further characterized the model by assessing the antigen-specific antibody response to experimental genital infection by using immunoblotting to assay both genital secretions and serum. The GPIC agent was characterized by analysis of outer membrane proteins, which indicated that the GPIC agent possessed a major outer membrane protein (MOMP), with a molecular mass of 39 kilodaltons (kDa), and a 61-kDa protein, analogous to cysteine-rich 60-kDa proteins or doublets of Chlamydia trachomatis strains. As indicated by immunoblotting, most infected animals produced serum immunoglobulin G antibodies to MOMP, the 61-kDa proteins, an 84-kDa outer membrane protein, and lipopolysaccharide. Such serum antibodies persisted for at least 813 days after primary genital infection. Immunoglobulin A antibodies against the 61-kDa proteins, lipopolysaccharide, and MOMP, but not the 84-kDa protein, were detected in secretions. Animals challenged with GPIC 825 days after primary infection became infected again despite the presence of serum antibodies, but the period of chlamydial shedding was significantly shorter and less intense than in primary infections. Although the specific mechanism is not known, these data suggest that a long-lasting immune effect is capable of altering the course of infection late after primary infection. Correlation of the antigen-specific antibody response and other immune parameters with the duration and degree of protective immunity induced by infection or vaccination may be helpful in further understanding the nature of such protective immunity.     

Immunologic uniqueness of the genital tract: challenge for vaccine development

Although the genital tract is considered to be a component of the mucosal immune system, it displays several distinct features not shared by other typical mucosal tissues and external secretions. Both male and female genital tract tissues lack inductive mucosal sites analogous to intestinal Peyer's patches. Consequently, local humoral and cellular immune responses stimulated by infections [with e.g. Neisseria gonorrhoeae, Chlamydia trachomatis, papilloma virus, and human immunodeficiency virus (HIV-1)] are weak or absent, and repeated local intravaginal immunizations result in minimal humoral responses. In contrast to typical external secretions such as intestinal fluid that contain secretory immunoglobulin A (S-IgA) as the dominant isotype, semen and cervico-vaginal fluid contain more IgG than IgA. Furthermore, irrespective of the route of infection, humoral immune responses to HIV-1 are dominated by specific IgG and low or absent IgA antibodies in all external secretions. Because a significant proportion of IgG in genital tract secretions is derived from the circulation, systemic immunization may provide protective IgG antibody-mediated immunity in the genital tract. Furthermore, combined systemic and mucosal (oral, rectal, and especially intranasal) immunization may induce protective humoral responses in both the systemic and mucosal compartments of the immune system.     

Early local cytokine profiles in strains of mice with different outcomes from chlamydial genital tract infection

In this study, we expand on the examination of genetically determined differences in host responses that correlate with clearance of Chlamydia trachomatis from the genital tract. We infected C57BL/6, BALB/c, and C3H/HeN mice with the mouse pneumonitis agent of C. trachomatis (MoPn). C57BL/6 mice had the shortest course of infection (22 days) and the lowest incidence of severe hydrosalpinx. BALB/c mice also had a short course of infection (25 days), but all developed hydrosalpinx. C3H/HeN mice had the longest course of infection (38 days), and all developed severe hydrosalpinx. Determination of local cytokine responses by enzyme-linked immunosorbent assay (ELISA) of genital tract secretions revealed that the levels of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) were significantly increased in the C57BL/6 and BALB/c strains compared to those in the C3H/HeN strain whereas the level of IL-6 was not different. The level of the neutrophil chemokine macrophage inflammatory protein 2 (MIP-2) was increased during the first week of infection in all three strains but was significantly higher in the BALB/c strain, the strain with the most rapid influx of neutrophils into the genital tract. Prolonged detection of MIP-2 in C3H/HeN mice was associated with a protracted presence of neutrophils in the genital tract. Early increases in the levels of the proinflammatory cytokines TNF-alpha and IL-1beta are associated with earlier eradication of infection in the C57BL/6 and BALB/c strains than in the C3H/HeN strain. Increased levels of MIP-2 and neutrophils in BALB/c and C3H/HeN mice relative to C57BL/6 mice suggest that these responses may contribute to pathology.     

Characterization of the protective T-cell response generated in CD4-deficient mice by a live attenuated Mycobacterium tuberculosis vaccine

The global epidemic of tuberculosis, fuelled by acquired immune-deficiency syndrome, necessitates the development of a safe and effective vaccine. We have constructed a DeltaRD1DeltapanCD mutant of Mycobacterium tuberculosis (mc(2)6030) that undergoes limited replication and is severely attenuated in immunocompromised mice, yet induces significant protection against tuberculosis in wild-type mice and even in mice that completely lack CD4(+) T cells as a result of targeted disruption of their CD4 genes (CD4(-/-) mice). Ex vivo studies of T cells from mc(2)6030-immunized mice showed that these immune cells responded to protein antigens of M. tuberculosis in a major histocompatibility complex (MHC) class II-restricted manner. Antibody depletion experiments showed that antituberculosis protective responses in the lung were not diminished by removal of CD8(+), T-cell receptor gammadelta (TCR-gammadelta(+)) and NK1.1(+) T cells from vaccinated CD4(-/-) mice before challenge, implying that the observed recall and immune effector functions resulting from vaccination of CD4(-/-) mice with mc(2)6030 were attributable to a population of CD4(-) CD8(-) (double-negative) TCR-alphabeta(+), TCR-gammadelta(-), NK1.1(-) T cells. Transfer of highly enriched double-negative TCR-alphabeta(+) T cells from mc(2)6030-immunized CD4(-/-) mice into naive CD4(-/-) mice resulted in significant protection against an aerosol tuberculosis challenge. Enriched pulmonary double-negative T cells transcribed significantly more interferon-gamma and interleukin-2 mRNA than double-negative T cells from naive mice after a tuberculous challenge. These results confirmed previous findings on the potential for a subset of MHC class II-restricted T cells to develop and function without expression of CD4 and suggest novel vaccination strategies to assist in the control of tuberculosis in human immunodeficiency virus-infected humans who have chronic depletion of their CD4(+) T cells.     

Failure of serology in diagnosing chlamydial infections of the female genital tract.

Chlamydia trachomatis was recoved from 20% (36/180) of women attending a venereal disease clinic. All infected women had chlamydial antibodies in their serum and cervical secretions. However, the background rates of chlamydial antibody in chlamydia-negative women were very high. Measurement of antibodies in serum (complement fixation or immunoglobulin G [IgG] and IgM by microimmunofluorescence) or cervical secretion (IgG, IgM, IgA or secretory IgA classes) did not result in predictive values of greater than 32%. It is concluded that the detection of chlamydial antibodies in serum or cervical secretions cannot be substituted for agent isolation in diagnosing these infections.     

The development of live attenuated cold-adapted influenza virus vaccine for humans

A procedure to attenuate live influenza virus of type A and type B was developed using adaptation of the virus to grow at 25 degrees C (cold adaptation; ca). Through a series of stepwise passages, two stable mutants were obtained and designated as 'Master' strains, one for type A influenza virus (A/Ann Arbor/6/60-H2N2) and one for type B influenza virus (B/Ann Arbor/1/66). These mutants were used in genetic reassortment using either the classical method or more recently described reverse genetics to update the relevant surface antigens of the circulating strains of influenza virus. The derivation is based on the concept of 6/2 where 6 signifies the six internal genes of the master strain and 2 refers to the two genes coding for the two surface glycoproteins HA and NA of the circulating influenza virus. The advantages of this vaccine were demonstrated to be (1) proper level of attenuation, (2) non-transmissibility, (3) genetic stability, (4) presence of the ca and ts markers and (5) immunogenicity involving both local and the cell-mediated immune responses. The clinical trials in infants, children, adults and elderly have provided the necessary data for eventual licensing of this vaccine. The ease of administration (intranasal) safety and high efficacy make this vaccine suitable to prevent influenza virus infection in all age groups.     

Criteria of primary screening of attenuated strains for live vaccine against Influenza A

Reassortant strains for live influenza vaccine (LIV) were selected using two additional markers: intensity of cytopathic effect (CPE) at 40 degrees C in MDCK cells and toxicity for mice (induction of acute hemorrhagic pulmonary edema after intranasal challenge with undiluted virus). All wild-type viruses induced a high CPE in MDCK cells, while the reassortants differed by this sign. Only vaccine strains and attenuation donors were characterized by a low CPE. Modern epidemic viruses are highly toxic for mice, causing the death of 60-100% animals from hemorrhagic pulmonary edema on days 3-4 after intranasal infection. Attenuation donors and vaccine strains were not toxic for mice, the level of toxic effect correlating with CPE in MDCK culture. Evaluation of CPE in MDCK culture and toxicity for mice can be used for primary screening of candidates for LIV.     

Epidemiology of chlamydial infection of the human genital tract: Evidence for the existence of latent infections

A group of 536 women attending a clinic for sexually transmitted diseases was investigated for cervical infection byNeisseria gonorrhoeae andChlamydia trachomatis. Conclusions have been reached concerning the existence and significance of latent and subclinical chlamydial infection of the female genital tract, and on the sexual infectivity ofChlamydia trachomatis to women. The results of the study indicate (1) demonstrable chlamydial infection in 25 % of all women attending the clinic for the first time, and in 11 % of those with unknown contact history; (2) sexual transmission ofChlamydia trachomatis; 45 % of women exposed to chlamydiae contract the infection compared with 75 % of those exposed toNeisseria gonorrhoeae; (3) the possibility of reactivation of latent chlamydial infection byNeisseria gonorrhoeae in some women.     

Further studies on the development of a live attenuated vaccine against turkey rhinotracheitis

Turkey rhinotracheitis (TRT) virus attenuated by passaging in Vero cells was tested at two different passage levels (15 or 25 passages) and two dose levels [10(3) or 10(4) TCID50 (50% tissue culture infectious doses) per bird] to determine the efficacy in protecting turkey poults against experimental challenge with virulent TRT virus. Following administration by the eyedrop route at 10 days of age, all four preparations proved successful in providing protection against clinical disease and establishment of challenge virus in the trachea when challenged with virulent virus 3 weeks later. Twelve-day-old poults given the 25th Vero passage TRT virus at a dose of 10(3.5) TCID50 per bird were protected against experimental challenge with virulent virus for at least 22 weeks post-primary inoculation. The 25th passage virus was tested for safety by administering ten times the dose (10(4.5) TCID50 per bird) used in the previous trial to a group of 10-day-old turkey poults. None of the birds showed any clinical signs during 21 days post-inoculation. Attempts to back-passage the virus from bird to bird were unsuccessful.     

Specific immune response in the human respiratory tract following oral immunization with live typhoid vaccine

Specific antibody responses in the lower respiratory tract of human subjects to orally administered Salmonella typhi Ty21a are reported. These responses, predominantly of the immunoglobulin G class, were determined to be a transudate from serum. These results were supported by the similarity in responses to parenteral administration of heat-killed typhoid vaccine. Specific immunoglobulin A antibody was a poor contributor to the respiratory antibody response to either vaccine.     

Characterization of lymphocyte response in the female genital tract during ascending Chlamydial genital infection in the guinea pig model

It is well known that pathology caused by chlamydial infection is associated closely with the host response to the organism and that both innate and adaptive host responses contribute to tissue damage. While it is likely that the organism itself initiates the acute inflammatory response by eliciting cytokine and chemokine production from the host cell, the adaptive response is the result of activation of the cell-mediated immune response. While there are several studies describing the nature of the pathologic response in primate, guinea pig, and murine models, there is less information on the kinetics of the CD4 and CD8 response following primary and challenge infections. In this study, we have quantified by flow cytometry the mononuclear cell response to genital infection with the agent of guinea pig inclusion conjunctivitis in the cervix, endometrium, and oviducts at various times following a primary intravaginal infection and after a challenge infection. Tissues from individual animals were assessed for cells expressing CD4, CD8, or Mac-1 and for B cells. Peak responses of each subset occurred 10 to 14 days after a primary infection. The number of Mac-1-expressing cells in each tissue site was found to be dependent on the size of the inoculating dose of chlamydiae. The responses of each cell type were generally stronger in the cervix than in the upper genital tract. In contrast to the murine model but consistent with the primate models, there were equal numbers of CD4 and CD8 cells present in the infiltrates. Twenty-one days after challenge infection, which was performed 50 days after the primary infection, there was a significant increase in the number of CD4, CD8, and B cells in the oviduct compared to the number of these cells at the same time after a primary infection, providing clear cellular evidence for a cell-mediated immune pathologic response.     

A gonococcal efflux pump system enhances bacterial survival in a female mouse model of genital tract infection

Active efflux of antimicrobial substances is likely to be an important bacterial defense against inhibitory host factors inherent to different body sites. Two well-characterized multidrug resistance efflux systems (MtrCDE and FarAB-MtrE) exist in Neisseria gonorrhoeae, a bacterial pathogen of the human genital mucosae. In vitro studies suggest that the MtrCDE and FarAB-MtrE efflux systems protect the gonococcus from hydrophobic antimicrobial substances that are likely to be present on mucosal surfaces. Here we report that a functional MtrCDE efflux system, but not a functional FarAB-MtrE system, enhances experimental gonococcal genital tract infection in female mice. Specifically, the recovery of mtrD and mtrE mutants, but not a farB mutant, from mice inoculated with mutant or wild-type gonococci was reduced compared with that of the wild-type strain. Competitive-infection experiments confirmed the survival disadvantage of MtrCDE-deficient gonococci. This report is the first direct evidence that a multidrug resistance efflux system enhances survival of a bacterial pathogen in the genital tract. Additionally, experiments using ovariectomized mice showed that MtrCDE-deficient gonococci were more rapidly cleared from mice that were capable of secreting gonadal hormones. MtrCDE-deficient gonococci were more sensitive to nonphysiological concentrations of progesterone in vitro than were wild-type or FarAB-MtrE-deficient gonococci. These results suggest that progesterone may play an inhibitory role in vivo. However, hormonally regulated factors rather than progesterone itself may be responsible for the more rapid clearance of mtr-deficient gonococci from intact mice.     

Effect of antithymocyte serum on the course of chlamydial genital infection in female guinea pigs

The treatment of female guinea pigs, infected in the genital tract with the chlamydial agent of guinea pig inclusion conjunctivitis, with rabbit anti-guinea pig thymocyte serum extended the course of the infection by 20 to 30 days. The rabbit anti-guinea pig thymocyte serum was shown to suppress delayed hypersensitivity responses to the guinea pig inclusion conjunctivitis agent and the contact allergen oxazolone. The appearance of antibody in genital secretions was delayed, but the infection persisted at low levels even when normal serum and secretory antibody titers were attained, indicating that cell-mediated immunity may play a role in the resolution of chlamydial genital infections.     

Hydatidosis of female genital tract: a case report

Hydatidosis of female genital tract mostly occurs as secondary involvement and is very rare. A case of primary hydatid cyst of genital tract i.e., uterine cervix and parametrium in a fifty five year old lady is reported, who was admitted in hospital in emergency with acute retention of urine.     

Susceptibility to reinfection after a primary chlamydial genital infection is associated with a decrease of antigen-specific T cells in the genital tract.

We tested the hypothesis that the intensity of specific antichlamydial T cell-mediated immunity in the genital tract of female guinea pigs infected intravaginally with the chlamydial agent of guinea pig inclusion conjunctivitis would determine the resistance or susceptibility to reinfection after a primary chlamydial infection. T cell-enriched lymphocytes were isolated by collagenase treatment of genital tract tissues from either infected or control uninfected female guinea pigs at various times after infection. The nylon wool-enriched T lymphocytes were evaluated for expression of antigen-specific T cell-mediated immunity in vitro by using a blast transformation assay. Both uninfected and infected genital tracts contained T cells, as evidenced by reactivity to concanavalin A, although a greater number of T lymphocytes was detected in the genital tracts of infected animals compared with that in controls. Significant antigen-specific T-cell activity could be detected in the genital tract tissue by 7 days after a primary genital tract infection with the chlamydial agent of guinea pig inclusion conjunctivitis. When antigen-specific activity was assessed at different times after infection, the intensity of the response of genital tract-associated T lymphocytes was directly proportional to the degree of resistance of the animals to genital challenge. Thus, susceptibility of animals to reinfection by chlamydiae appears to be associated with the intensity of the local T cell-mediated immune responses in the genital tract of infected animals.     

Mouse strain-dependent variation in the course and outcome of chlamydial genital tract infection is associated with differences in host response.

Whether there is a pathogenic or protective outcome to chlamydial infection may be defined by the host response. We infected C57BL/6 (C57) and C3H/HeN (C3H) mice with the human biovar of Chlamydia trachomatis, serovar E, and, in select experiments, with the mouse pneumonitis agent of C. trachomatis (MoPn). We compared the courses of infection, histopathology, and host responses that resulted from these infections. The duration of infection with either chlamydial biovar was significantly increased in the C3H strain of mice. The intensity of infection was examined in mice infected with serovar E, and it was significantly increased in the C3H strain. Histopathology revealed the incidence of severe hydrosalpinx to be significantly greater in C3H mice than in C57 mice. In contrast, severe distention of the uterine horns was observed in all infected C57 mice compared to none of the C3H mice infected with serovar E and only 25% of those infected with MoPn. Acute inflammation was significantly increased in the uterine horns of C57 mice compared to that of C3H mice. Examination of antigen-specific responses revealed qualitatively similar responses in the two strains. Determination of gamma interferon- versus interleukin 4- producing cells revealed the predominance of a Th1 response in both strains. Serum enzyme-linked immunosorbent assays for immunoglobulin G1 (IgG1) and IgG2a revealed a predominance of IgG2a antibody in both strains, although the levels of antibody were significantly greater in C3H mice. Lymphocyte proliferation studies revealed increased proliferation in the iliac nodes of both strains at 1 to 3 weeks after infection. Because of the early eradication of infection observed in the C57 strain, we explored the relative production of tumor necrosis factor alpha (TNF-alpha) in the two strains. TNF-alpha levels were significantly increased in the genital tract secretions of C57 mice compared to that of C3H mice during the first week of infection. Increased TNF-alpha may be beneficial to the host by leading to earlier eradication of infection, thereby preventing infection of the oviduct and thus the major disease sequelae associated with chlamydial infection of the genital tract.