A chlamydial type III-secreted effector protein (Tarp) is predominantly recognized by antibodies from humans infected with Chlamydia trachomatis and induces protective immunity against upper genital tract pathologies in mice

Chlamydia trachomatis genome is predicted to encode a type III secretion system consisting of more than 40 open reading frames (ORFs). To test whether these ORFs are expressed and immunogenic during chlamydial infection in humans, we expressed 55 chlamydial ORFs covering all putative type III secretion components plus control molecules as fusion proteins and measured the reactivity of these fusion proteins with antibodies from patients infected with C. trachomatis in the urogenital tract (24 antisera) or in the ocular tissue (8 antisera). Forty-five of the 55 proteins were recognized by at least 1 of the 32 human antisera, suggesting that these proteins are both expressed and immunogenic during chlamydial infection in humans. Tarp, a putative type III secretion effector protein, was identified as a novel immunodominant antigen due to its reactivity with the human antisera at high frequency and titer. The expression and immunogenicity of Tarp were confirmed in cell culture and mouse systems. Tarp was mainly associated with the infectious form of chlamydial organisms and became undetectable between 13 and 24 h during the infection cycle in cell culture. Mice intravaginally infected with C. muridarum developed Tarp-specific humoral and cellular immune responses. More importantly, immunization of mice with Tarp induced Th1-dominant immunity that significantly reduced the shedding of live organisms from the lower genital tract and attenuated inflammatory pathologies in the fallopian tube tissues. These observations have demonstrated that Tarp, an immunodominant antigen identified by human antisera, can induce protective immunity against chlamydial infection and pathology in mice.     

Intragastric primary infection sensitizes to lung reinfection in a Chlamydia pneumoniae mouse model

The most frequently used infection route in Chlamydia pneumoniae animal models is intranasal (i.n.), while the intragastric (i.g.) infection route has not been studied previously. The aim of the present study was to examine the course of Chlamydia pneumoniae infection in mice infected via the i.g. route and to compare the outcome of i.n. reinfection in these mice to i.n. reinfection of primarily i.n. infected mice. C57BL/6JBom mice were used, and the infection was monitored using chlamydial culture, C. pneumoniae PCR and histological examination of several tissues, and antibody measurements. C. pneumoniae was able to disseminate from the gastrointestinal tract to other organs, and i.g. inoculation led to an immunological response. In addition, the primary i.g. challenge made mice more susceptible to i.n. reinfection. In conclusion, the results suggest that the nature of the immune response to a previous C. pneumoniae infection affects the outcome of reinfection.     

The potential to use PspA and other pneumococcal proteins to elicit protection against pneumococcal infection

Pneumococcal proteins, alone, in combination with each other, or in combination with capsular polysaccharide-protein conjugates may be useful pneumococcal vaccine components. Four proteins with a potential for use in vaccines are PspA, pneumolysin, PsaA, and PspC. In a mouse model of carriage, PsaA and PspC were the most efficacious vaccine proteins. Of these, PsaA was the best at eliciting protection against carriage. However, a combination of PspA and pneumolysin may elicit stronger immunity to pulmonary infection and possibly sepsis than either protein alone. Recently, a phase one trial of a recombinant family 1 PspA was completed in man. PspA was observed to be safe and immunogenic. Injection of 0.1 ml of immune serum diluted to 1/400 was able to protect mice from fatal infection with S. pneumoniae. Under these conditions, pre-immune serum was not protective. The immune human serum protected mice from infections with pneumococci expressing either of the major PspA families (1 and 2) and both of the pneumococcal capsular types tested: 3 and 6.     

Recombinant heat shock protein 65 carrying hepatitis B core antigen induces HBcAg-specific CTL response

Many studies have provided evidence that heat shock protein 65 (Hsp65) can elicit potent specific cellular adaptive immune responses (e.g. CD8(+) cytotoxic T-cell effectors or classic CTLs) based on their ability to chaperone antigenic peptides. Hsp65 is thus an effective carrier for heterologous peptide epitopes for therapeutic vaccines against cancer or chronic infectious diseases. The core antigen of hepatitis B virus (HBcAg) is extremely immunogenic, and functions as both a T-cell-dependent and a T-cell-independent antigen. Therefore, HBcAg may be a promising candidate target for therapeutic vaccine control of chronic HBV infection. Here, a chimeric protein, Hsp65Bc, was created by fusing the HBcAg sequence to the carboxyl terminus of the Hsp65 sequence in E. coli. Analysis of its antigenicity and immunogenicity revealed that HBc epitopes are surface accessible. Hsp65Bc induced moderate anti-HBc immune responses as well as a strong specific T-cell response in BALB/c mice. These results indicate that Hsp65Bc may have potential as a vaccine for treatment of HBV chronic infection.     

Intranasal administration of chlamydial outer protein N (CopN) induces protection against pulmonary Chlamydia pneumoniae infection in a mouse model

Chlamydia pneumoniae is an intracellular pathogen that grows inside a vacuole, referred to as an inclusion. C. pneumoniae possess a type III secretion system (TTSS), which allows them to secrete effector molecules into the inclusion membrane and to the host cell cytosol. Proteins such as chlamydial outer protein N (CopN) that associate with the inclusion membrane are potential targets for the host's MHC-dependent antigen presentation, thereby representing ideal antigen candidates for T cell-based vaccination. The results of this study showed that intranasal immunization of BALB/c mice with heat-aggregated CopN protein and an Escherichia coli heat-labile toxin (LT) induced a strong immune response, detected as antigen-specific antibody production, lymphocyte proliferation and IFN-gamma production. Furthermore, the immunization induced statistically significant protection against intranasal C. pneumoniae challenge, the level of which correlated with the magnitude of CopN-specific lymphocyte proliferation. Both heat-aggregation of the antigen and the presence of LT adjuvant were required for maximal protective effect.     

Recombinant Vibrio cholerae ghosts as a delivery vehicle for vaccinating against Chlamydia trachomatis

An efficacious vaccine is needed to control the morbidity and burden of rising healthcare costs associated with genital Chlamydia trachomatis infection. Despite considerable efforts, the development of reliable chlamydial vaccines using conventional strategies has proven to be elusive. The 40kDa major outer membrane protein (MOMP) of C. trachomatis is so far the most promising candidate for a subunit vaccine. The lack of satisfactory protective immunity with MOMP-based vaccine regimens to date would suggest that either MOMP alone is inadequate as a vaccine candidate or better delivery systems are needed to optimize the effect of MOMP. Recombinant Vibrio cholerae ghosts (rVCG) are attractive for use as non-living vaccines because they possess strong adjuvant properties and are excellent vehicles for delivery of antigens of vaccine relevance to mucosal sites. The suitability of the ghost technology for designing an anti-chlamydial vaccine was evaluated by constructing a rVCG vector-based candidate vaccine expressing MOMP (rVCG-MOMP) and assessing vaccine efficacy in a murine model of C. trachomatis genital infection. Intramuscular delivery of the rVCG-MOMP vaccine induced elevated local genital mucosal as well as systemic Th1 responses. In addition, immune T cells from immunized mice could transfer partial protection against a C. trachomatis genital challenge to nai;ve mice. These results suggest that rVCG expressing chlamydial proteins may constitute a suitable subunit vaccine for inducing an efficient mucosal T cell response that protects against C. trachomatis infection. Altogether, the potency and relatively low production cost of rVCG offer a significant technical advantage as a chlamydial vaccine.     

Identification of immunodominant antigens of Chlamydia trachomatis using proteome microarrays

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen in the world. In order to control this infection there is an urgent need to formulate a vaccine. Identification of protective antigens is required to implement a subunit vaccine. To identify potential antigen vaccine candidates, three strains of mice, BALB/c, C3H/HeN and C57BL/6, were inoculated with live and inactivated C. trachomatis mouse pneumonitis (MoPn) by different routes of immunization. Using a protein microarray, serum samples collected after immunization were tested for the presence of antibodies against specific chlamydial antigens. A total of 225 open reading frames (ORF) of the C. trachomatis genome were cloned, expressed, and printed in the microarray. Using this protein microarray, a total of seven C. trachomatis dominant antigens were identified (TC0052, TC0189, TC0582, TC0660, TC0726, TC0816 and, TC0828) as recognized by IgG antibodies from all three strains of animals after immunization. In addition, the microarray was probed to determine if the antibody response exhibited a Th1 or Th2 bias. Animals immunized with live organisms mounted a predominant Th1 response against most of the chlamydial antigens while mice immunized with inactivated Chlamydia mounted a Th2-biased response. In conclusion, using a high throughput protein microarray we have identified a set of novel proteins that can be tested for their ability to protect against a chlamydial infection.     

Induction of immune memory by a multisubunit chlamydial vaccine

We tested the hypothesis that intramuscular immunization with a multisubunit chlamydial vaccine candidate will induce long lasting immune responses in mice. Accordingly, groups of female C57BL/6 mice were immunized intramuscularly with Vibrio cholerae ghosts (VCG) expressing the Poring B and polymorphic membrane protein-D proteins of Chlamydia trachomatis or a control antigen. Humoral and cell-mediated immune responses were evaluated following immunization and after live chlamydial infection. Immunization induced an anamnestic response characterized by chlamydial-specific IgG2a and IgA antibodies in sera and vaginal lavage as well as specific genital and splenic T cell responses. The results also revealed that the local mucosal and systemic cellular and humoral immune effectors induced in mice following immunization with the vaccine candidate are long lasting. Vaccinated mice cleared intravaginal challenge with 10⁵ chlamydial inclusion forming units within 12 days compared to control mice, which shed up to 2 × 10³ IFUs at this time point. Moreover, rechallenge of mice 98 days after resolution of the primary infection resulted in the recall and retention of a relatively high frequency of chlamydial-specific Th1 cells and IgG2a in the genital mucosa. These results provide the first evidence that a VCG-based multisubunit chlamydial vaccine is capable of effectively stimulating anamnestic systemic and mucosal immune responses in mice. The data support further vaccine evaluation and testing for induction of long-term protective immunity.     

Immunization with the Chlamydia trachomatis major outer membrane protein,using the outer surface protein A of Borrelia burgdorferi as an adjuvant,can induce protection against a chlamydial genital challenge

Two strains of mice C3H/HeN (H-2(k)) and BALB/c (H-2(d)) were immunized with the Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) using the Borrelia burgdorferi outer surface protein A (OspA) as an adjuvant. As a control, groups of mice were inoculated with ovalbumin instead of MOMP. Female mice were immunized using three different routes: intramuscular (i.m.) plus subcutaneous (s.c.), intranasal (i.n.) and perivaginal and perisacral (p.vag.+p.sac.). Significant humoral and cell mediated immune responses developed particularly in mice inoculated by the i.m.+s.c. routes as determined by the levels of chlamydial specific antibody in the serum and genital secretions and a T-cell proliferative assay. Following immunization the animals were challenged in the genital tract with C. trachomatis MoPn and the course of the infection followed by vaginal cultures. Significant protection against infection was achieved in the C3H/HeN mice inoculated i.m.+s.c. with MOMP+OspA, as shown by the intensity and duration of vaginal cultures, and by the number of mice with positive cultures. On the other hand in BALB/c mice there was only a decrease in the number of animals with positive vaginal cultures. Six weeks after the challenge the mice were mated and the outcome of the pregnancy evaluated. In both the C3H/HeN and the BALB/c mice immunized i.m.+s.c. with MOMP+OspA there was significant protection against infertility as shown by the number of animals with bilateral fertility and number of embryos per uterine horn. In conclusion, immunization using C. trachomatis MOMP, and B. burgdorferi OspA as an adjuvant, can induce significant protection against a chlamydial genital challenge.     

Induction of protective immunity against Chlamydia muridarum intracervical infection in DBA/1j mice

We previously reported that intracervical inoculation with Chlamydia muridarum induced hydrosalpinx in DBA/1j mice, but intravaginal inoculation failed to do so. In the current study, we found unexpectedly that intrabursal inoculation of live chlamydial organisms via the oviduct failed to induce significant hydrosalpinx. We further tested whether primary infection via intravaginal or intrabursal inoculation could induce protective immunity against hydrosalpinx following intracervical challenge infection. Mice infected intravaginally with C. muridarum were fully protected from developing hydrosalpinx, while intrabursal inoculation offered partial protection. We then compared immune responses induced by the two genital tract inoculations. Both inoculations induced high IFNγ and IL-17 T cell responses although the ratio of IgG2a versus IgG1 in intravaginally infected mice was significantly higher than in mice infected intrabursally. When the antigen-specificities of antibody responses were compared, both groups of mice dominantly recognized 24 C. muridarum antigens, while each group preferentially recognized unique sets of antigens. Thus, we have demonstrated that intrabursal inoculation is neither effective for causing hydrosalpinx nor efficient in inducing protective immunity in DBA/1j mice. Intravaginal immunization, in combination with intracervical challenge infection in DBA/1j mice, can be a useful model for understanding mechanisms of chlamydial pathogenicity and protective immunity.     

Immunity to Chlamydia pneumoniae induced by vaccination with DNA vectors expressing a cytoplasmic protein (Hsp60) or outer membrane proteins (MOMP and Omp2)

Immune responses induced by intramuscular DNA immunization with Chlamydia pneumoniae genes coding for the major outer membrane protein (MOMP), cysteine-rich outer membrane protein 2 (Omp2) or the heat shock protein 60 (Hsp60) were studied. BALB/c mice were vaccinated intramuscularly three times at 3-week intervals and challenged intranasally 2 weeks after the last injection. Immunization with pmomp or phsp60 showed 1.2-1.5 log reduction in the mean lung bacterial counts after the challenge. Specific antibodies were detected only in sera of the mice immunized with pomp2 and phsp60. Although immunization with pomp2 resulted in a strong serum antibody response against Omp2 protein, it failed to protect the mice. Immunization with any of the three vaccines did not reduce the severity of histologically assessed pneumonia, but resulted in significantly higher lymphoid reaction in the lung indicating immunological memory.     

Potent antigen-specific immunity to Toxoplasma gondii in adjuvant-free vaccination system using Rop2-Leishmania infantum Hsp83 fusion protein

The results of this study describe the immunostimulatory properties of Leishmania infantum Hsp83 (83) to elicit humoral and cellular response against the Toxoplasma gondii Rop2 protein in an adjuvant-free vaccination system. The analysis was performed by immunizing three different mice strains (BALB/c, C57BL/6 and C3H). Mice immunized with fusion Rop2-83 elicited a stronger humoral and cellular response in comparison to mice immunized with Rop2 alone, or a mix of LiHsp83 and Rop2. The fusion protein induced a Th1 type response, with predominance of specific IgG2a/IgG2c isotype and IFN-gamma secretions, whereas Rop2 alone or mixed with LiHsp83 produced a Th1/Th2 mixed response. Vaccination with fusion protein conferred a remarkable resistance against oral infection with ME49 cysts in C57BL/6 and C3H mice, in comparison to mice immunized with Rop2 alone or the protein mixture. Following lethal challenge, a significant survival rate was observed in Rop2-83 immunized Balb/c and C57BL/6 mice in comparison to control groups.     

Resistance to mycobacterial infection: A pattern of early immune responses leads to a better control of pulmonary infection in C57BL/6 compared with BALB/c mice

In this study, we have compared the immunological responses associated with early pulmonary mycobacterial infection in two mouse strains, BALB/c and C57BL/6 known to exhibit distinct differences in susceptibility to infection with several pathogens. We infected mice via the intranasal route. We have demonstrated that BALB/c was less able to control mycobacterial growth in the lungs during the early phase of pulmonary infection. Our results showed that during the early phase (day 3 to week 1), BALB/c mice exhibited a delay in the production of TNF and IFN-γ in the lungs compared to C57BL/6 mice. Levels of IL-12 and soluble TNF receptors (sTNFR) were comparable between the mouse strains. The cellular subset distribution in these mice before and after infection showed a higher increase in CD11b+ cells in the lungs of C57BL/6, compared to BALB/c as early as day 3 postinfection. At early time points, higher levels of monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein 1 (MIP)-α were detected in C57BL/6 than BALB/c mice. In vitro, BCG-infected bone marrow derived macrophages (BMM) from both mouse strains displayed similar capacities to either phagocytose bacteria or produce soluble mediators such as TNF, IL-12 and nitric oxide (NO). Although IFN-γ stimulation of infected BMM in both mouse strains resulted in the induction of antimycobacterial activity, BALB/c mice had a reduced capacity to kill ingested bacteria. The above observations indicate that the chain of early, possibly innate immunological events occurring during pulmonary mycobacterial infection may directly impact on increased susceptibility or resistance to infection.     

PsaA (pneumococcal surface adhesin A) and PspA (pneumococcal surface protein A) DNA vaccines induce humoral and cellular immune responses against Streptococcus pneumoniae.

Streptococcus pneumoniae is one of the most important human pathogens and improvement of the currently used polysaccharide vaccines is being pursued. We constructed DNA vaccine vectors containing either the full-length psaA (pneumococcal surface adhesin A) or a truncated pspA (pneumococcal surface protein A--pspA') gene. Both constructs showed transient expression of the antigens in vertebrate cells and induced significant antibody response to the pneumococcal antigens in BALB/c mice injected intramuscularly (i.m.). Fusion with an N-terminal cytoplasmatic SV40 T-antigen (CT-Ag), which was previously shown to stabilize poorly expressed antigens through association with Hsp73, also induced anti-PspA antibody response. The induction of antibodies with a low IgG1:IgG2a ratio and elevated gamma interferon (IFN-gamma) production by spleen cells elicited by DNA vaccination indicate preferential priming of Th1 immunity. Since induction of antibodies against both PsaA and PspA was previously shown to correlate with protection against fatal infection with S. pneumoniae and cell-mediated immune responses could contribute to protection, further evaluation of PsaA and PspA as antigens for a DNA vaccine against S. pneumoniae could be promising.     

Mucosal immunization with recombinant MOMP genetically linked with modified cholera toxin confers protection against Chlamydia trachomatis infection

Chlamydia trachomatis is a major human health pathogen due to its role in sexually transmitted diseases. Thus, there is a need to develop an effective vaccine at the mucosal surface against this pathogen. In an effort to develop a mucosal vaccine, a modified cholera toxin gene was genetically linked to the C. trachomatis MoPn NiggII MOMP gene to generate a recombinant protein with the mucosal adjuvant properties of the cholera toxin and immunological antigenicity of the chlamydial protein. The recombinant fusion protein (rMOMP) was expressed in E. coli, purified and analyzed by SDS-PAGE, immunoblot, and GM1-ELISA, and subsequently used to immunize BALB/c mice via intranasal (i.n.) and intravaginal (vag.) routes. The rMOMP protein administered via the i.n. route induced a higher concentration of anti-MOMP specific antibodies in both serum and vaginal washes as compared to mice immunized with Chlamydia or PBS. Antibody isotype analysis revealed that i.n. administration of rMOMP to mice induced higher concentrations of serum and vaginal wash IgA, IgG1, IgG2a, and IgG2b antibodies. Vaginal washes from all immunized mice following a chlamydial challenge infection were analyzed by indirect immunoflourescence to study the level of protection provided by various immunogens. Maximum protection against C. trachomatis as assessed by reduction in C. trachomatis inclusion forming units (IFU) was provided by i.n. immunization of mice with rMOMP. This is a first report using genetic fusion of cholera toxin and MOMP genes and provides a novel approach for the design and development of a mucosal vaccine against Chlamydia.     

Influence of the level of dietary ethanol in mice with murine AIDS on resistance to Streptococcus pneumoniae.

Chronic ethanol consumption impairs cellular immune functions. This may explain the increased occurrence of various opportunistic infections in heavy ethanol users. Immunological alterations associated with Acquired Immune Deficiency Syndrome (AIDS) also permit more opportunistic infections. In this study, we used a murine model of retrovirus infection induced by LP-BM5 murine leukemia virus. The combined effects of ethanol use and early retroviral infection (prior to the development of AIDS) on resistance to Streptococcus pneumoniae were investigated. Consumption of ethanol by non-retrovirus-infected mice resulted in decreased resistance to S. pneumoniae. However, retrovirus-infected mice fed a diet containing high concentrations of ethanol (6 and 7% v/v) exhibited a greater resistance to S. pneumoniae infection than retrovirus-infected mice fed diets with lower concentrations (5%) or no ethanol. The total number of white blood cells also decreased as serum ethanol levels increased. There were also fewer lymphocytes and more neutrophils and monocytes in retrovirus-infected mice fed ethanol. Diet consumption decreased as the concentration of ethanol increased in the diet. Consumption was dependent upon the dark-light cycle. The highest diet consumption was observed during the first 4 hr of the dark period. The level of ethanol in serum was influenced by the amount of the diet consumed and its ethanol concentration. Both retrovirus infection and ethanol consumption effected survival after S. pneumoniae infection.     

A T cell epitope-based vaccine protects against chlamydial infection in HLA-DR4 transgenic mice

Vaccination with recombinant chlamydial protease-like activity factor (rCPAF) has been shown to provide robust protection against genital Chlamydia infection. Adoptive transfer of IFN-γ competent CPAF-specific CD4⁺ T cells was sufficient to induce early resolution of chlamydial infection and reduction of subsequent pathology in recipient IFN-γ-deficient mice indicating the importance of IFN-γ secreting CD4⁺ T cells in host defense against Chlamydia. In this study, we identify CD4⁺ T cell reactive CPAF epitopes and characterize the activation of epitope-specific CD4⁺ T cells following antigen immunization or Chlamydia challenge. Using the HLA-DR4 (HLA-DRB1*0401) transgenic mouse for screening overlapping peptides that induced T cell IFN-γ production, we identified at least 5 CPAF T cell epitopes presented by the HLA-DR4 complex. Immunization of HLA-DR4 transgenic mice with a rCPAFep fusion protein containing these 5 epitopes induced a robust cell-mediated immune response and significantly accelerated the resolution of genital and pulmonary Chlamydia infection. rCPAFep vaccination induced CPAF-specific CD4⁺ T cells in the spleen were detected using HLA-DR4/CPAF-epitope tetramers. Additionally, CPAF-specific CD4⁺ clones could be detected in the mouse spleen following Chlamydia muridarum and a human Chlamydia trachomatis strain challenge using these novel tetramers. These results provide the first direct evidence that a novel CPAF epitope vaccine can provide protection and that HLA-DR4/CPAF-epitope tetramers can detect CPAF epitope-specific CD4⁺ T cells in HLA-DR4 mice following C. muridarum or C. trachomatis infection. Such tetramers could be a useful tool for monitoring CD4⁺ T cells in immunity to Chlamydia infection and in developing epitope-based human vaccines using the murine model.     

Participation of Chlamydia pneumoniae in an epidemic of respiratory infections in Slovakia

In human sera collected in three regions of Slovakia during an epidemic of respiratory infections, both genus-specific chlamydial and species-specific anti-Chlamydia pneumoniae antibodies, as detected by enzyme-linked immunosorbent assay and microimmunofluorescence test, respectively, were found. Based on seroconversion or significant rise of antibody titers and detection of antibodies of IgM class, an acute C. pneumoniae infection was indicated in 21 of 298 (7.0%) patients tested. The results obtained bring the first evidence on the role of C. pneumoniae as respiratory pathogen in Slovakia.     

Heat denatured enzymatically inactive recombinant chlamydial protease-like activity factor induces robust protective immunity against genital chlamydial challenge

We have shown previously that vaccination with recombinant chlamydial protease-like activity factor (rCPAF) plus interleukin-12 as an adjuvant induces robust protective immunity against primary genital Chlamydia muridarum challenge in mice. Since CPAF is a protease, we compared the effects of enzymatically active and inactive (heat denatured) rCPAF to determine whether proteolytic activity is expendable for the induction of protective immunity against chlamydial challenge. Active, but not inactive, rCPAF immunization induced high levels of anti-active CPAF antibody, whereas both induced robust splenic CPAF-specific IFN-γ production. Vaccination with active or inactive rCPAF induced enhanced vaginal chlamydial clearance as early as day 6 with complete resolution of infection by day 18, compared to day 30 in mock-vaccinated and challenged animals. Importantly, significant and comparable reductions in oviduct pathology were observed in active and inactive rCPAF-vaccinated mice compared to mock-vaccinated animals. Thus, rCPAF induced anti-chlamydial immunity is largely independent of enzymatic activity and secondary or higher order protein conformation.     

In silico identification and in vivo analysis of a novel T-cell antigen from Chlamydia, NrdB

Chlamydial infections are a serious economic burden and health threat to developed and developing countries. Development of an efficacious vaccine is thought to be the most convenient, potentially reliable and cost effective option to control chlamydial infection and disease complications. Currently there are very few efficacious vaccine candidates that have been identified and characterized. In this study we have identified a number of unique vaccine candidates using a novel in silico approach. The chlamydial genome was screened for proteins containing epitopes predicted to bind multiple HLA class II molecules (i.e. 'promiscuous' epitopes). A selection of target proteins were cloned, expressed, and purified. Recombinant proteins were screened against sera samples from patients with Chlamydia trachomatis genital tract infections. Two proteins, hypothetical protein CT425 and ribonucleotide reductase small chain protein (NrdB) were identified as being immunoreactive. Using a mouse model, we found that intranasal immunization with NrdB conferred a CD4+ T-cell driven degree of protection similar to that seen with CD4+ T-cells primed from a whole organism, live challenge. In addition, serum from immunized mice was found to neutralize chlamydial infection of a cell monolayer in vitro. NrdB is a highly conserved chlamydial protein with an essential role in the replication of chlamydiae and could be a useful component of a multi-subunit vaccine against chlamydial genital tract infections.