Background/Objectives: Acinetobacter baumannii is an opportunistic nosocomial pathogen characterized by its multidrug-resistant (MDR) phenotype, increasing patient mortality and healthcare costs as a result. Previously, we constructed an immunoinformatics-based Acinetobacter Multi-Epitope Vaccine (AMEV2) candidate and demonstrated robust protection against this MDR pathogen. In this study, we delineate the mechanisms of AMEV2-mediated protective immunity. Methods: In vivo passive immunization with AMEV2 antisera and in vitro opsonophagocytic killing assays (OPKAs) were used to assess the critical role of antibody-mediated protection induced by AMEV2 vaccination. Results: The passive transfer of AMEV2 immune sera to naïve mice afforded 67% protection in a pulmonary challenge mouse model. Although AMEV2 sera reacts with bacterial antigens, it is not bactericidal on its own and does not enhance the complement-mediated direct killing of A. baumannii. However, OPKAs demonstrate AMEV2 sera enhancement of the killing of A. baumannii in the presence of primary bone marrow-derived macrophages. This killing occurs via complement and Fc gamma receptor-mediated phagocytosis. A highly immunogenic AMEV2 component peptide, pTonB, elicits pTonB-specific antibodies and protection in vivo. The depletion of pTonB antibodies from AMEV2 immune sera by pTonB absorption significantly reduced the opsonophagocytic killing of A. baumannii in vitro. Conclusions: The data presented here demonstrate the importance of humoral immunity and its protective mechanisms against A. baumannii. These findings further expand the in vivo evaluation of in silico-designed vaccines as a viable alternative to combat the current global MDR pathogen health crisis.

We have demonstrated previously that TNF-α-producing CD8+ T cells mediate chlamydial pathogenesis, likely in an antigen (Ag)-specific fashion. Here we hypothesize that inhibition of Ag-specific CD8+ T cell response after immunization and/or challenge would correlate with protection against oviduct pathology induced by a protective vaccine regimen. Intranasal (i.n.) live chlamydial elementary body (EB), intramuscular (i.m.) live EB, or i.n. irrelevant antigen, bovine serum albumin (BSA), immunized animals induced near-total protection, 50% protection, or no protection, respectively against oviduct pathology following i.vag. C. muridarum challenge. In these models, we evaluated Ag-specific CD8+ T cell cytokine response at various time-periods after immunization or challenge. The results show protective efficacy of vaccine regimens correlated with reduction of Ag-specific CD8+ T cell TNF-α responses following i.vag. chlamydial challenge, not after immunization. Depletion of CD4+ T cells abrogated, whereas adoptive transfer of Ag-specific CD4+ T cells induced the significant reduction of Ag-specific CD8+ T cell TNF-α response after chlamydial challenge. In conclusion, protective anti-chlamydial vaccine regimens induce Ag-specific CD4+ T cell response that mediate early inhibition of pathogenic CD8+ T cell response following challenge and may serve as a predictive biomarker of protection against Chlamydia -induced chronic pathologies.

The early innate immune response to coccidioidomycosis has proven to be pivotal in directing the adaptive immune response and disease outcome in mice and humans but is unexplored in dogs. The objectives of this study were to evaluate the innate immune profile of dogs with coccidioidomycosis and determine if differences exist based on the extent of infection (i.e., pulmonary or disseminated). A total of 28 dogs with coccidioidomycosis (pulmonary, n = 16; disseminated, n = 12) and 10 seronegative healthy controls were enrolled. Immunologic testing was performed immediately, without ex vivo incubation (i.e., constitutive), and after coccidioidal antigen stimulation of whole blood cultures. Whole blood cultures were incubated with a phosphate-buffered solution (PBS) (negative control) or a coccidioidal antigen (rCTS1 (105–310); 10 µg/mL) for 24 h. A validated canine-specific multiplex bead-based assay was used to measure 12 cytokines in plasma and cell culture supernatant. Serum C-reactive protein (CRP) was measured with an ELISA assay. Leukocyte expression of toll-like receptors (TLRs)2 and TLR4 was measured using flow cytometry. Dogs with coccidioidomycosis had higher constitutive plasma keratinocyte chemotactic (KC)-like concentrations (p = 0.02) and serum CRP concentrations compared to controls (p < 0.001). Moreover, dogs with pulmonary coccidioidomycosis had higher serum CRP concentrations than those with dissemination (p = 0.001). Peripheral blood leukocytes from dogs with coccidioidomycosis produced higher concentrations of tumor necrosis factor (TNF)-α (p = 0.0003), interleukin (IL)-6 (p = 0.04), interferon (IFN)-γ (p = 0.03), monocyte chemoattractant protein (MCP)-1 (p = 0.02), IL-10 (p = 0.02), and lower IL-8 (p = 0.003) in supernatants following coccidioidal antigen stimulation when compared to those from control dogs. There was no detectable difference between dogs with pulmonary and disseminated disease. No differences in constitutive or stimulated leukocyte TLR2 and TLR4 expression were found. These results provide information about the constitutive and coccidioidal antigen-specific stimulated immune profile in dogs with naturally acquired coccidioidomycosis.

Understanding the immune response to SARS-CoV-2 is important for development of effective diagnostics and vaccines. We report here a broad antibody response to SARS-CoV-2 spike protein receptor binding domain (RBD) in 100 convalescent patient plasma samples. Antibody isotypes IgA, IgM, and IgG exhibited significantly higher anti-RBD titers when compared to SARS-CoV-2 negative controls. IgG subtyping indicated IgG1 and IgG3 to be most abundant. Greater than 90 % of SARS-CoV-2 positive plasma samples tested exhibited significant neutralization capacity using a surrogate virus neutralization assay. Of the IgG subclasses, IgG1 and IgG3 exhibited the highest viral neutralization capacity; whereas, IgG2 and IgG4 viral neutralization was not observed. Comparison of SARS-CoV-2 elicited total IgG binding to emerging variant (alpha, beta, and delta) RBDs indicated decreased binding. Furthermore, neutralization by SARS-CoV-2 convalescent plasma of delta and omicron variant RBDs was significantly decreased suggesting that neutralizing antibodies in convalescent plasma are less effective in inhibiting variants currently in circulation.

Background Stiffer aortas are associated with a faster rate of aortic root (AoR) dilation and higher risk of aortic dissection in patients with Marfan syndrome. We have previously shown that mild aerobic exercise reduces aortic stiffness and rate of AoR dilation in a Marfan mouse model. In this study, we investigated if these results could be translated to pediatric patients with Marfan syndrome. Methods and Results We enrolled 24 patients with Marfan syndrome aged 8 to 19 years to participate in a 6‐month physical activity intervention, excluding those with ventricular dysfunction or prior history of aortic surgery. We instructed patients to take 10 000 steps per day, tracked by an activity tracker. At baseline and 6 months, we measured AoR dimension, arterial stiffness, endothelial function, physical activity indices, inflammatory biomarkers, and coping scores. Controls consisted of 15 age‐matched patients with Marfan syndrome. Twenty‐four patients with Marfan syndrome (median age, 14.4 years [interquartile range {IQR}, 12.2–16.8], 14 male patients) were enrolled. Baseline assessment demonstrated that the majority of these patients were sedentary and had abnormal arterial health. Twenty‐two patients completed the intervention and took an average of 7709±2177 steps per day (median, 7627 [IQR, 6344–9671]). Patients wore their Garmin trackers at a median of 92.8% (IQR, 84%–97%) of their intervention days. AoR Z score in the intervention group had a significantly lower rate of change per year compared with the controls (rate of change, −0.24 versus +0.008; P=0.01). Conclusions In this clinical intervention in pediatric patients with Marfan syndrome, we demonstrated that a simple physical activity intervention was feasible in this population and has the potential to decrease the AoR dilation rate. REGISTRATION URL: https://www.clinicaltrials.gov; Unique identifier: NCT03567460.

Chlamydia trachomatis is the leading pathogen in sexually transmitted bacterial infections across the globe. The development of a selective treatment against this pathogen could be an attractive therapeutic option that will reduce the overuse of broad-spectrum antibiotics. Previously, we reported some sulfonylpyridine-based compounds that showed selectivity against C. trachomatis. Here, we describe a set of related compounds that display enhanced anti-chlamydial potency when compared to our early leads. We found that the active molecules are bactericidal and have no impact on Staphylococcus aureus or Escherichia coli strains. Importantly, the molecules were not toxic to mammalian cells. Furthermore, a combination of molecule 20 (the most active molecule) and azithromycin at subinhibitory concentrations acted synergistically to inhibit chlamydial growth. Molecule 20 also eradicated Chlamydia in a 3D infection model and accelerated the recovery of Chlamydia-infected mice. This work presents compounds that could be further developed to be used alone or in combination with existing treatment regimens against chlamydial infections.

Multiple Sclerosis (MS) is the most common progressive neurologic disease among young adults worldwide; yet the pathogenesis remains poorly understood. The myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalitis (EAE) in mice is a robust model and has been extensively used to understand the pathophysiology of MS. The expression of connexin 43 (CX43), a gap junction protein, has been shown to be enhanced in the choroid plexus during the development of acute EAE, with significantly increased CX43 expression at the peak of the disease. However, the contribution of such increased CX43 expression to the development of EAE has yet to be fully characterized. Using the Cre-Lox platform, we generated mice with conditional deletion of CX43 in the ciliated columnar epithelium of the choroid plexus (Foxj1Cre-CX43 KO mice; referred to as CX43 KO mice hereafter). We employed the MOG-induced EAE model in both male and female CX43 KO mice to understand the role of CX43 in the pathogenesis of EAE. Male CX43 and WT mice displayed comparable peak EAE clinical disease scores of 1.83±0.33 and 1.75±0.66, respectively. Female CX43 KO, however, displayed a significantly reduced peak EAE clinical disease score of 0.33±0.24 compared to 1.15±0.39 in female WT animals. These results suggest a role for CX43 in the ciliated columnar epithelium of choroid plexus to the development of EAE in female, not male, mice and underscore the need for further exploration of mechanism(s) underpinning the role of CX43 in the pathogenesis of EAE.

The term lung disease describes a broad category of disorders that impair lung function. More than 35 million Americans have a preventable chronic lung disease with high mortality rates due to limited treatment efficacy. The recent increase in patients with lung disease highlights the need to increase our understanding of mechanisms driving lung inflammation. Connexins, gap junction proteins, and more specifically connexin 43 (Cx43), are abundantly expressed in the lung and are known to play a role in lung diseases. This review focuses on the role of Cx43 in pathology associated with acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD) and asthma. Additionally, we discuss the role of Cx43 in preventing disease through the transfer of mitochondria between cells. We aim to highlight the need to better understand what cell types are expressing Cx43 and how this expression influences lung disease.

We have demonstrated previously that CD4 T cells are protective whereas CD8 T cells cause reproductive pathology following genital chlamydial infection. In this study, we explored the interactions of these cell types in context of vaccine-mediated protection. We evaluated three immunization regimens: live-chlamydial elementary body intranasal (EB-i.n.), live-EB i.m. (EB-i.m.), and irrelevant antigen bovine serum albumin i.n. (BSA-i.n.) that induced near-total (90%), partial (50%), and no (0%) protection, respectively against oviduct pathology following i.vag. C. muridarum challenge in C57BL6/J animals. We found no differences in Chlamydia-specific IFN-g, TNF-α, or IL-17 response from purified splenic CD8 T cells on days 7, 14, and 21 after immunization. We further evaluated Chlamydia-specific splenic CD8 T cell responses on days 3, 6, 9, and 12 following i.vag. chlamydial challenge. All groups of challenged animals displayed comparable induction of Ag-specific CD8 T cell cytokine responses on day 6 after challenge. As early as day 9 after challenge, Ag-specific IFN-g and TNF-α production from CD8 T cells was significantly reduced in EB-i.n.-immunized mice, and on day 12, both EB-i.n.- and EB-i.m.-immunized animals displayed minimal Ag-specific TNF-a production, compared to enhanced TNF-a production from CD8 T cells in BSA-i.n. immunized animals. Furthermore, adoptive transfer of Chlamydia-specific CD4 T cells at the time of genital infection induced significant reduction in Chlamydia-specific splenic CD8 T cell response on day 12 after challenge. In summary, these results suggest that Ag-specific CD4 T cells subvert a pathogenic Chlamydia-specific CD8 T cell response to protect against reproductive pathology.

TNF-α producing antigen (Ag)-specific CD8 T cells induce upper genital tract (UGT) pathology following primary C. muridarum infection; therefore, we hypothesized that a vaccine regimen must subvert Ag-specific CD8 T cell response following immunization, and/or challenge to attenuate Chlamydia-induced UGT pathology. We evaluated three immunization regimens: live-chlamydial elementary body intranasal (EB-i.n.), live-EB i.m. (EB-i. m.), and irrelevant antigen bovine serum albumin i.n. (BSA-i.n.) that induced near-total (90%), partial (50%), and no (0% ) protection, respectively against oviduct pathology following i.vag. C. muridarum challenge in C57BL6/J animals. Live-EB-specific IFN-γ, TNF-α, and IL-17 response from purified splenic CD8 T cells was evaluated on days 7, 14, and 21 after immunization. There was no detectable reduction of Ag-specific CD8 T cell response following immunization with protective vaccine regimens. We evaluated splenic live-EB-specific CD8 T cell responses on days 3, 6, 9, and 12 following i.vag. chlamydial challenge. All groups of challenged animals displayed comparable induction of Ag-specific CD8 T cell cytokine responses on day 6 after challenge. As early as day 9 after challenge, Ag-specific IFN-γ and TNF-α production from CD8 T cells was significantly reduced in EB-i.n.-, compared to the EB-i.m.- or BSA-i.n.-immunized animals. On day 12, both EB-i.n.- and EB-i.m.-immunized animals displayed minimal Ag-specific TNF-α production, compared to enhanced TNF-α production from CD8 T cells in BSA-i.n. immunized animals. These results suggest that TNF-α producing CD8 T cell response could serve as a predictive biomarker of anti-chlamydia vaccine efficacy against reproductive pathology.

We have shown previously that intranasal vaccination with recombinant chlamydial protease-like activity factor (rCPAF: antigen) and interleukin-12 (IL-12) as an adjuvant induces robust protection against pathological consequences of female genital tract infection with Chlamydia muridarum , a closely related species and a rodent model for the human pathogen Chlamydia trachomatis . Another related species Chlamydia pneumoniae , a human respiratory pathogen, has been associated with exacerbation of atherosclerotic pathology. CPAF is highly conserved among Chlamydia spp. leading us to hypothesize that immunization with rCPAF with IL-12 will protect against high-fat diet (HFD) and C. pneumoniae -induced acceleration of atherosclerosis. rCPAF ± IL-12 immunization induced robust splenic antigen (Ag)-specific IFN-γ and TNF-α production and significantly elevated serum total anti-CPAF Ab, IgG2c, and IgG1 antibody levels compared to mock or IL-12 alone groups. The addition of IL-12 to rCPAF significantly elevated splenic Ag-specific IFN-γ production and IgG2c/IgG1 anti-CPAF antibody ratio. Following intranasal C. pneumoniae challenge and HFD feeding, rCPAF ± IL-12-immunized mice displayed significantly enhanced splenic IFN-γ, not TNF-α, response on days 6 and 9 after challenge, and significantly reduced lung chlamydial burden on day 9 post-challenge compared to mock- or IL-12-immunized mice. Importantly, rCPAF ± IL-12-immunized mice displayed significantly reduced atherosclerotic pathology in the aortas after C. pneumoniae challenge. Serum cholesterol levels were comparable between the groups suggesting that the observed differences in pathology were due to protective immunity against the infection. Together, these results confirm and extend our previous observations that CPAF is a promising candidate antigen for a multisubunit vaccine regimen to protect against Chlamydia -induced pathologies, including atherosclerosis.

Chlamydia trachomatis genital infections lead to severe immunopathological consequences in the upper genital tract (UGT), including pelvic inflammatory disease and infertility, in a small subset of infected women. Heat shock protein 70 (Hsp70) is an evolutionarily conserved stress-induced protein and has been shown to exhibit significant cytoprotective and immunoregulatory activities. However, the role of Hsp70 in genital chlamydial pathologies had not been examined. Hsp70 transgenic (Hsp70 Tg) mice were created by injecting a villin promoter-driven human Hsp70 targeting vector into C57BL/6 oocytes. The villin promoter is active only in the epithelium of the mouse oviduct but not in other parts of the female mouse genital tract. Female, 6-8 week old, Hsp70 Tg and non-transgenic (NTG) littermates were infected intravaginally with 5×104 IFU of C. muridarum. Vaginal chlamydial shedding, bacterial burden in the upper genital tract including oviducts and uterine horns, Chlamydia-specific IFN-γ, TNF-α, and IL-17 production, and serum anti-Chlamydia antibody levels were comparable between NTG and Hsp70 Tg mice. However, at day 80 after inoculation, the incidence and severity of pathology in oviducts, not neighboring uterine horns, was significantly reduced in Hsp70 Tg mice compared to NTG mice. Given the highly polymorphic nature of hsp-70 gene and associated susceptibility to various inflammatory conditions, and the occurrence of immunopathology in only a subset of Chlamydia-infected women, these results also underscore the need to further evaluate the role of hsp-70 in chlamydial immunopathogenesis in the female upper reproductive tract.

The host immune responses that mediate Chlamydia-induced chronic disease sequelae are incompletely understood. The role of TNF-α, TNF receptor 1 (TNFR1), and TNF receptor 2 (TNFR2), in Chlamydia pneumoniae (CPN)-induced atherosclerosis was studied using the high-fat diet-fed male C57BL/6J mouse model. Following intranasal CPN infection, TNF-α knockout (KO), TNFR1 KO, TNFR2 KO, and TNFR 1/2 double-knockout, displayed comparable serum anti-chlamydial antibody response, splenic antigen-specific cytokine response, and serum cholesterol profiles compared to wild type (WT) animals. However, atherosclerotic pathology in each CPN-infected KO mouse group was reduced significantly compared to WT mice, suggesting that both TNFR1 and TNFR2 promote CPN-induced atherosclerosis.

OBJECTIVE:This study aims to eliminate Mycoplasma spp. contamination from laboratory stocks of Chlamydia spp. by in vivo passage or by plaque assay. RESULTS:We have described two methods of eliminating Mycoplasma contamination from Chlamydia laboratory stocks. We conclude that Mycoplasma species commonly contaminating chlamydial stocks do not survive passage in mice. Chlamydia may also be derived Mycoplasma-free by plaque assay.

We recently demonstrated that Chlamydia-specific TNF-α producing and TNF receptor 2 bearing CD8+ T cells mediate chlamydial pathogenesis in the mouse female upper genital tract (UGT). Additionally, TNF-α production from CD8+ T lymphocytes is necessary for pathogenesis; however, CD8+ T lymphocytes are not a sufficient source of TNF-α production for pathogenesis. Myeloid cells including neutrophils are important sources of TNF-α production and have been implicated in chlamydial pathogenesis. Herein, we evaluated frequency of neutrophils in Chlamydia muridarum-infected female genital tracts of C57BL/6J (WT) mice, mice deficient in Chlamydia-specific CD8+ T cells (OT-1 mice) and TNFR2 knockout (KO) mice. Frequencies of Gr-1+ cells (neutrophils) in UGT were evaluated on days 4, 9, 15, and 24 following genital Chlamydia muridarum infection. OT-1 and TNFR2 KO displayed significant reduction in neutrophil frequency compared to WT animals on day 9 following chlamydial inoculation. Furthermore, we found that OT-1 mice replete with WT CD8+ T cells at the time of chlamydial inoculation display restoration of neutrophil (Ly-6G) frequency to WT levels. These results suggest that Chlamydia-specific CD8+ T cells engage other inflammatory cells such as neutrophils in infected genital tracts in order to cause chronic pathologies in the upper reproductive tract.

Chlamydial infections lead to a number of clinically relevant diseases and induce significant morbidity in human populations. It is generally understood that certain components of the host immune response to infection also mediate such disease pathologies. A clear understanding of pathogenic mechanisms will enable us to devise better preventive and/or intervention strategies to mitigate the morbidity caused by these infections. Over the years, numerous studies have been conducted to explore the immunopathogenic mechanisms of Chlamydia-induced diseases of the eye, reproductive tract, respiratory tract, and cardiovascular systems. In this article, we provide an overview of the diseases caused by Chlamydia, animal models used to study disease pathology, and a historical context to the efforts to understand chlamydial pathogenesis. Furthermore, we discuss recent findings regarding pathogenesis, with an emphasis on the role of the adaptive immune response in the development of chlamydial disease sequelae. Finally, we summarize the key insights obtained from studies of chlamydial pathogenesis and avenues that remain to be explored in order to inform the next steps of vaccine development against chlamydial infections.

We have shown previously that intranasal vaccination with recombinant chlamydial protease‐like activity factor (rCPAF: antigen) and interleukin‐12 (IL‐12) as an adjuvant induces robust protection against pathological consequences of female genital tract infection with Chlamydia muridarum, a closely related species and a rodent model for the human pathogen Chlamydia trachomatis. Another related species Chlamydia pneumoniae, a human respiratory pathogen, has been associated with exacerbation of atherosclerotic pathology. CPAF is highly conserved among Chlamydia spp. leading us to hypothesize that immunization with rCPAF with IL‐12 will protect against high‐fat diet (HFD) and C. pneumoniae‐induced acceleration of atherosclerosis. rCPAF ± IL‐12 immunization induced robust splenic antigen (Ag)‐specific IFN‐γ and TNF‐α production and significantly elevated serum total anti‐CPAF Ab, IgG2c, and IgG1 antibody levels compared to mock or IL‐12 alone groups. The addition of IL‐12 to rCPAF significantly elevated splenic Ag‐specific IFN‐γ production and IgG2c/IgG1 anti‐CPAF antibody ratio. Following intranasal C. pneumoniae challenge and HFD feeding, rCPAF ± IL‐12‐immunized mice displayed significantly enhanced splenic IFN‐γ, not TNF‐α, response on days 6 and 9 after challenge, and significantly reduced lung chlamydial burden on day 9 post‐challenge compared to mock‐ or IL‐12‐immunized mice. Importantly, rCPAF ± IL‐12‐immunized mice displayed significantly reduced atherosclerotic pathology in the aortas after C. pneumoniae challenge. Serum cholesterol levels were comparable between the groups suggesting that the observed differences in pathology were due to protective immunity against the infection. Together, these results confirm and extend our previous observations that CPAF is a promising candidate antigen for a multisubunit vaccine regimen to protect against Chlamydia‐induced pathologies, including atherosclerosis.

We hypothesized that reactive arthritis can be induced in the knees and ankles of wild-type C57BL/6 mice following infection with Chlamydia muridarum through the involvement of inflammatory mediators. Reactive arthritis is a chronic form of arthritis, which can also result in conjunctivitis and inflammation of the genital, urinary, or gastrointestinal systems. C. muridarum is a gram-negative obligate intracellular pathogen which can infect the epithelium of the cervix, urethra, and rectum. The exact pathological mechanism of reactive arthritis is unknown. It is understood to result from CD4+ and CD8+ T cells, and factors including TNF-alpha and perforin. Five groups of C57BL/6J mice were used including a positive control group, CD4+ T cells knockout mice, TNF-alpha knockout mice, perforin knockout mice and TNFRSF knockout mice. Mice from each group were intra-vaginally infected with 5×104 inclusion forming units of C. muridarum, marked as day 0. The mice were swabbed a few days after infection to confirm C. muridarum. The mice from each group were euthanized on day 7 and day 14, and sera was collected and analyzed for cytokine expression using an antibody array. Ankles collected from C57BL/6J positive control mice were histologically processed and graded to determine pathology. C57BL/6J positive control mice showed increased synovial lining thickening 14 days following infection. Cytokine expression from TNF-alpha knockout mice, CD4+ T cells knockout mice, perforin knockout mice, and TNFRSF knockout mice was generally reduced. The results show that chlamydial induced reactive arthritis can be detected in the ankles of C57BL/6J mice and suggest that TNF-alpha and CD4+ T cells may be involved in this inflammatory process.

Chlamydia-specific CD8 T cells play a significant role in oviduct pathology, not bacterial clearance. We hypothesized that gap junction mediated antigen transport (GMAT) from infected to uninfected cells could explain the apparent targeting of uninfected epithelial cells by Chlamydia-specific CD8 T cells. Since gap junction protein connexin 43 is highly expressed in oviduct epithelium, we generated Foxj1Cre-Cx43flox mice with a conditional deficiency of CX43 only in ciliated columnar epithelia in oviducts. Foxj1Cre-Cx43flox mice displayed similar chlamydial infection, but reduced splenic Ag-specific CD8 T cell response and oviduct pathology when compared to WT mice, suggesting that CX43-mediates chlamydial pathogenesis. To evaluate the role of GMAT via CX43 in pathogenesis, HeLa S3 cells were engineered to express functional CX43 or a closed channel point-mutant CX43 (T154A). Chlamydial peptides were observed in neighboring uninfected cells at mid-developmental cycle upon infection of HeLa-CX43, not HeLa S3 or HeLa-CX43-T154A cells. Naïve mouse antigen-presenting cells (APC) optimally activated Chlamydia-specific mouse CD8 T cells upon co-culture with Chlamydia-infected HeLa-CX43, when compared to HeLa S3 or HeLa-CX43-T154A cells, demonstrating that GMAT is involved in activation of CD8 T cells. Furthermore, naïve mouse APC optimally activated Chlamydia-specific mouse CD8 T cells upon co-culture with a 1:4 mixture of Chlamydia-infected HeLa-CX43 and uninfected HeLa-CX43 cells, when compared to mixtures of infected HeLa-CX43 with uninfected HeLa-S3 or HeLa-CX43-T154A cells. These results demonstrate that GMAT via uninfected epithelial cells amplifies pathogenic Chlamydia-specific CD8 T cell response.

We have comprehensively demonstrated using the mouse model that intranasal immunization with recombinant chlamydial protease-like activity factor (rCPAF) leads to a significant reduction in bacterial burden, genital tract pathology and preserves fertility following intravaginal genital chlamydial challenge. In the present report, we evaluated the protective efficacy of rCPAF immunization in guinea pigs, a second animal model for genital chlamydial infection. Using a vaccination strategy similar to the mouse model, we intranasally immunized female guinea pigs with rCPAF plus CpG deoxynucleotides (CpG; as an adjuvant), and challenged intravaginally with C. trachomatis serovar D (CT-D). Immunization with rCPAF/CpG significantly reduced vaginal CT-D shedding and induced resolution of infection by day 24, compared with day 33 in CpG alone treated and challenged animals. Immunization induced robust anti-rCPAF serum IgG 2 weeks following the last immunization, and was sustained at a high-level 4 weeks post challenge. Upregulation of antigen-specific IFN-γ gene expression was observed in rCPAF/CpG-vaccinated splenocytes. Importantly, a significant reduction in inflammation in the genital tissue in rCPAF/CpG-immunized guinea pigs compared with CpG-immunized animals was observed. Taken together, this study provides evidence of the protective efficacy of rCPAF as a vaccine candidate in a second animal model of genital chlamydial infection.