Is bacterial vaginosis associated with autoimmune antibody positivity?

OBJECTIVE: To evaluate the association between bacterial vaginosis (BV) and autoimmune antibody positivity. METHOD: We evaluated Papanicolaou-stained cervicovaginal smears of 210 patients with poor obstetric history who were admitted to a special preconception counselling programme. Cytological specimens with various types of microorganisms except for BV, epithelial cell abnormalities and other non-neoplastic findings, including inflammation were excluded from the cohort in addition to patients with autoimmune and chronic inflammatory diseases. The remaining study population (n = 121) was divided into two groups of patients with autoimmune antibody positivity (study group, n = 80) and patients without antibody positivity (control group, n = 41). RESULTS: The rate of BV was demonstrated to be 13.8% and 2.4% in the study and control groups respectively (P = .042). We also demonstrated that the anti-nuclear antibody was positive in 58.3% of the cases with BV. CONCLUSION: BV was found more frequently in patients with autoimmune antibody positivity to a statistically significant degree.

Production and characterization of a monoclonal antibody against the sialidase of Gardnerella vaginalis using a synthetic peptide in a MAP8 format.

Bacterial vaginosis is one of the most frequent vaginal infections. Its main etiological agent is Gardnerella vaginalis, which produces several virulence factors involved in vaginal infection and colonization, in particular, sialidase (SLD), a potential clinical biomarker that participates in immune response modulation and mucus degradation. The main objective of this work was the production and evaluation of a monoclonal antibody against G. vaginalis sialidase and its validation in immunoassays. For immunization of mice, a synthetic multiantigenic peptide was used, and hybridomas were generated. After fusion, hybridomas were evaluated for antibody production and cloned by limited dilution. One clone producing IgG1 was selected and characterized by indirect ELISA, dot blot, and Western blot, and we also tested clinical isolates and HeLa cells infected with G. vaginalis. The results showed that the anti-SLD antibody recognized a single protein of ~90 kDa that correlated with the estimated molecular weight of SLD. In addition, anti-SLD antibody recognized SLD from complete bacteria and from culture supernatants of infected Hela cells. In conclusion, our results showed that the anti-SLD antibody recognized SLD from different sources and could be considered a new tool for the diagnosis of bacterial vaginosis. KEY POINTS: • Anti-sialidase mAb was generated using a synthetic peptide • The mAb recognizes synthetic peptide and intact protein from multiple sources • The antibody was characterized by several immunological methods.

The vaginal microbiota associates with the regression of untreated cervical intraepithelial neoplasia 2 lesions.

Emerging evidence suggests associations between the vaginal microbiota (VMB) composition, human papillomavirus (HPV) infection, and cervical intraepithelial neoplasia (CIN); however, causal inference remains uncertain. Here, we use bacterial DNA sequencing from serially collected vaginal samples from a cohort of 87 adolescent and young women aged 16-26 years with histologically confirmed, untreated CIN2 lesions to determine whether VMB composition affects rates of regression over 24 months. We show that women with a Lactobacillus-dominant microbiome at baseline are more likely to have regressive disease at 12 months. Lactobacillus spp. depletion and presence of specific anaerobic taxa including Megasphaera, Prevotella timonensis and Gardnerella vaginalis are associated with CIN2 persistence and slower regression. These findings suggest that VMB composition may be a future useful biomarker in predicting disease outcome and tailoring surveillance, whilst it may offer rational targets for the development of new prevention and treatment strategies.

Apoptosis of vaginal epithelial cells in clinical samples from women with diagnosed bacterial vaginosis.

Bacterial vaginosis (BV) is one of the most common vaginal infections among women of childbearing age. Gardnerella vaginalis (G. vaginalis) is a keystone microorganism present in more than 95% of all BV cases. The first step of the infection process in BV is mediated by interaction of microorganisms with epithelial cells (ECs). However, the role of these cells in BV pathogenesis is largely unknown. The present study aimed to investigate the vaginal EC response during BV. Twenty healthy women and 34 women with BV were enrolled in this study. The number of ECs in the vaginal swab was counted and analyzed for intracellular signals and apoptosis by flow cytometry. Cell damage was evaluated by lactate dehydrogenase assay. Compared to that in healthy donors, the percentage of exfoliated vaginal ECs was increased in women with BV, and an absence of neutrophils was observed in both groups. Activation signals, such as p-IκBα and c-Fos were unmodulated in the vaginal ECs of women with BV. Moreover, EC damage and apoptosis were significantly increased in patients with BV. Apoptosis was related to caspase-3 activation and the presence of G. vaginalis. This study provides the first evidence of a direct involvement of G. vaginalis in the apoptotic process of vaginal ECs during BV. This effect was mediated by caspase-3 activation, and G. vaginalis appeared to be one of causes for inducing EC apoptosis in BV. Hence, our findings suggest a possible explanation for the increased exfoliation of ECs in the vagina during BV.

Cervicovaginal cytokines, sialidase activity and bacterial load in reproductive-aged women with intermediate vaginal flora.

Studies have shown that not only bacterial vaginosis, but also intermediate vaginal flora has deleterious effects for women's reproductive health. However, literature still lacks information about microbiological and immunological aspects of intermediate flora. OBJECTIVE: To characterize intermediate flora regarding levels of Interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor-alpha, interleukin 1 receptor antagonist (IL-1ra), IL-10, sialidase; loads of Gardnerella vaginalis, total bacteria and to verify whether it is closer related to normal flora or bacterial vaginosis. This cross-sectional study enrolled 526 non-pregnant reproductive-aged women distributed in 3 groups according to pattern of vaginal flora using Nugent's system in normal, intermediate and bacterial vaginosis. Cervicovaginal levels of cytokines, sialidases, loads of G. vaginalis and total bacteria were assessed by ELISA, conversion of MUAN and quantitative real-time PCR, respectively. A principal component analysis(PCA) using all measured parameters was performed to compare the three different types of flora. Results showed that intermediate flora is associated with increased cervicovaginal IL-1beta in relation to normal flora(P<0.0001). When compared to bacterial vaginosis, intermediate flora has higher IL-8 and IL-10 levels(P<0.01). Sialidases were in significantly lower levels in normal and intermediate flora than bacterial vaginosis(P<0.0001). Loads of G. vaginalis and total bacterial differed among all groups(P<0.0001), being highest in bacterial vaginosis. PCA showed that normal and intermediate flora were closely scattered, while bacterial vaginosis were grouped separately. CONCLUSION: Although intermediate flora shows some differences in cytokines, sialidases and bacterial loads in relation to normal flora and bacterial vaginosis, when taken together, general microbiological and immunological pattern pattern of intermediate flora resembles the normal flora.

Gardnerella vaginalis triggers NLRP3 inflammasome recruitment in THP-1 monocytes.

Gardnerella vaginalis is a Gram-positive bacterium associated with bacterial vaginosis (BV), pelvic inflammatory disease, and preterm birth. BV is the most prevalent vaginal dysbiosis in women of childbearing age characterized by the absence of normal lactobacilli and an overgrowth of G. vaginalis and other bacteria. Although mucosal fluids from BV patients exhibit increases in proinflammatory cytokines and Toll-like receptor 2 and 4 mRNA, G. vaginalis has not been demonstrated to directly induce an inflammatory response. This study tested the hypothesis that G. vaginalis induces an inflammatory response in the human monocyte cell line, THP-1. The objectives of the study were to measure proinflammatory cytokine production, molecular mechanisms by which cytokines are produced, and whether G. vaginalis results in death of the monocytic cells. We found that G. vaginalis induced significant increases in the inflammasome-dependent cytokines IL-1ß, IL-18, as well as TNF-α in treated cells. G. vaginalis caused significant cell death by 24h post-treatment compared with untreated controls, but cells remained 66% viable. Caspase-1 cleavage in treated cells confirmed the inflammatory cell death, and NLRP3 knockdown confirmed its involvement through reduction of IL-1ß secretion. Using a stably expressing YFP-ASC THP-1 cell model with immunofluorescent staining, YFP-ASC colocalized with NLRP3 in G. vaginalis-treated cells and the addition of a caspase-1 inhibitor wholly ameliorated IL-1ß secretion. Our study provides new insight into the role of G. vaginalis in inflammatory conditions in the genital tract.

Vaginolysin drives epithelial ultrastructural responses to Gardnerella vaginalis.

Gardnerella vaginalis, the bacterial species most frequently isolated from women with bacterial vaginosis (BV), produces a cholesterol-dependent cytolysin (CDC), vaginolysin (VLY). At sublytic concentrations, CDCs may initiate complex signaling cascades crucial to target cell survival. Using live-cell imaging, we observed the rapid formation of large membrane blebs in human vaginal and cervical epithelial cells (VK2 and HeLa cells) exposed to recombinant VLY toxin and to cell-free supernatants from growing liquid cultures of G. vaginalis. Binding of VLY to its human-specific receptor (hCD59) is required for bleb formation, as antibody inhibition of either toxin or hCD59 abrogates this response, and transfection of nonhuman cells (CHO-K1) with hCD59 renders them susceptible to toxin-induced membrane blebbing. Disruption of the pore formation process (by exposure to pore-deficient toxoids or pretreatment of cells with methyl-ß-cyclodextrin) or osmotic protection of target cells inhibits VLY-induced membrane blebbing. These results indicate that the formation of functional pores drives the observed ultrastructural rearrangements. Rapid bleb formation may represent a conserved response of epithelial cells to sublytic quantities of pore-forming toxins, and VLY-induced epithelial cell membrane blebbing in the vaginal mucosa may play a role in the pathogenesis of BV.

In vitro secretion profile of pro-inflammatory cytokines IL-1ß, TNF-α, IL-6, and of human beta-defensins (HBD)-1, HBD-2, and HBD-3 from human chorioamniotic membranes after selective stimulation with Gardnerella vaginalis.

PROBLEM: Preterm labor associated with infection is a major clinical condition; in this work, we analyze the response of human chorioamniotic membranes stimulated with Gardnerella vaginalis. METHOD OF STUDY: Using a two-compartment experimental model, 1 × 10(6) CFU/mL of G. vaginalis were added to either the amnion or choriodecidua face or to both. Concentrations of IL-1ß, TNF-α, and IL-6, as well as human beta defensins (HBD) 1-3 were quantified by ELISA. RESULTS: In comparison with control conditions and regardless of the stimulation modality, IL-1ß and IL-6 increased 4-fold and 28-fold, respectively, in the choriodecidual compartment. HBD-1 increased 2-fold mainly in the amniotic compartment when the stimulus was applied directly to this region. HBD-2 and HBD-3 increased an average of 2- and 8-fold, respectively, in the choriodecidual region. CONCLUSIONS: Stimulation with G. vaginalis induced a tissue-specific secretion profile of 1L-1ß, IL-6, and HBD 1-3 in the chorioamniotic membranes.

Production in yeast of pseudotype virus-like particles harboring functionally active antibody fragments neutralizing the cytolytic activity of vaginolysin.

BACKGROUND: Recombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs) represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY), the main virulence factor of Gardnerella vaginalis. RESULTS: The scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs) that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody. CONCLUSIONS: Recombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the scFv-Fc molecules on the surface of pseudotype VLPs was successful and allowed generation of multivalent scFv-Fc proteins with high VLY-neutralizing potency. Our study demonstrated for the first time that large recombinant antibody molecule fused with hamster polyomavirus VP2 protein and co-expressed with VP1 protein in the form of pseudotype VLPs was properly folded and exhibited strong antigen-binding activity. The current study broadens the potential of recombinant VLPs as a highly efficient carrier for functionally active complex proteins.

Atopobium vaginae triggers an innate immune response in an in vitro model of bacterial vaginosis.

Bacterial vaginosis is the most common vaginal disorder among women of reproductive age. The pathogenesis of bacterial vaginosis is poorly understood, but is defined by a transition in the vaginal flora from the predominant Lactobacillus species to other bacterial species such as Atopobium vaginae and Gardnerella vaginalis. This change is associated with an increase in vaginal cytokine secretion. We hypothesize that vaginal epithelial cells respond to bacterial vaginosis-associated bacteria by triggering an innate immune response. We observed that vaginal epithelial cells secreted interleukin-6 and interleukin-8 in response to Atopobium vaginae and Gardnerella vaginalis, but not to Lactobacillus crispatus. Atopobium vaginae induced increased levels of interleukin-6 and interleukin-8 transcripts, as well as increased transcripts for the antimicrobial peptide beta-defensin 4. This innate immune response required live bacteria capable of protein synthesis in direct contact with vaginal epithelial cells. The response of vaginal epithelial cells was mediated by Toll-like receptor 2, required the adaptor protein MyD88, and involved activation of the NFkappaB signaling pathway. These results suggest that Atopobium vaginae stimulates an innate immune response from vaginal epithelial cells, leading to localized cytokine and defensin production, and possibly contributes to the pathogenesis of bacterial vaginosis.

Reversible deficiency of antimicrobial polypeptides in bacterial vaginosis.

Bacterial vaginosis is a common condition associated with increased risk of sexually transmitted diseases, including human immunodeficiency virus infections. In contrast, vulvovaginal candidiasis has a much weaker association with sexually transmitted diseases. We found that vaginal lavage fluid from women with bacterial vaginosis is deficient in antimicrobial polypeptides and antimicrobial activity compared to fluid from healthy women or women with vulvovaginal candidiasis. Effective treatment normalized the concentrations of antimicrobial polypeptides in both bacterial vaginosis and in vulvovaginal candidiasis, suggesting that the abnormalities were a result of the diseases. Unlike in vulvovaginal candidiasis, the neutrophil attractant chemokine interleukin-8 (IL-8) was not increased in bacterial vaginosis, accounting for low concentrations of neutrophil-derived defensins in vaginal fluid. In organotypic cultures of human vaginal epithelium containing dendritic cells, treatment with Lactobacillus jensenii, a typical vaginal resident, induced the synthesis of IL-8 mRNA and the epithelial human beta-defensin-2 mRNA, but a typical bacterial vaginosis pathogen, Gardnerella vaginalis, had no effect. When the two bacteria were combined, Gardnerella vaginalis did not interfere with the immunostimulatory effect of Lactobacillus jensenii. The loss of normal immunostimulatory flora in bacterial vaginosis is thus associated with a local deficiency of multiple innate immune factors, and this deficiency could predispose individuals to sexually transmitted diseases.

Detection of a species-specific antigen of Gardnerella vaginalis by Western blot analysis.

Western blot analysis was used to identify antigenic components of Gardnerella vaginalis. Polypeptides bound to nitrocellulose membranes were probed with murine antisera raised to two strains of G. vaginalis, and antibody-antigen complexes were detected with 125I-labelled antimouse immunoglobulin followed by autoradiography. Although there was inter-strain variation in immunogenic polypeptide profiles, all 23 strains of G. vaginalis examined contained a common antigen of molecular mass 41 kDa. This antigen was not found in any of six other bacterial genera.