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Cervical cancer remains an important cause of women morbidity and mortality. The progression of cervical pathology correlates with the HPV integration into the host genome. However, the data on the viral integration status in cervical dysplasias are controversial. The aim of the current study was to evaluate the status of HPV integration in two types of cervical pathology – invasive and non invasive cervical cancer (e.g. carcinoma in situ). 156 women were included in the study: 66 women were diagnosed with invasive cervical cancer (CC) and 90 with non invasive cervical cancer (carcinoma in situ, CIS). 74.2% [95% PI: 63.64÷84.76] of specimens collected from women with diagnosed CC and 85.6% [95% PI: 85.53÷92.85] of CIS specimens were positive for HPV. The most prevalent HPV genotype in both groups was HPV16. To evaluate HPV integration, three selected HPV16 E2 gene fragments were analyzed by PCR. In the majority of CC and CIS specimens the amplification of all three HPV16 E2 gene fragments was observed. The episomal HPV16 form was detected in the majority of CC and CIS specimens. The deletion of all three HPV16 E2 gene fragments was detected in 9.4% of CC specimens and 2.2% of CIS specimens. Finally, integration status could not be used as diagnostical additional test to distinguish between invasive and non invasive cervical cancer.

Abstract Background Gardnerella vaginalis is identified as the predominant colonist of the vaginal tracts of women diagnosed with bacterial vaginosis (BV). G. vaginalis can be isolated from healthy women, and an asymptomatic BV state is also recognised. The association of G. vaginalis with different clinical phenotypes could be explained by different cytotoxicity of the strains, presumably based on disparate gene content. The contribution of horizontal gene transfer to shaping the genomes of G. vaginalis is acknowledged. The CRISPR loci of the recently discovered CRISPR/Cas microbial defence system provide a historical view of the exposure of prokaryotes to a variety of foreign genetic elements. Results The CRISPR/Cas loci were analysed using available sequence data from three G. vaginalis complete genomes and 18 G. vaginalis draft genomes in the NCBI database, as well as PCR amplicons of the genomic DNA of 17 clinical isolates. The cas genes in the CRISPR/Cas loci of G. vaginalis belong to the E. coli subtype. Approximately 20% of the spacers had matches in the GenBank database. Sequence analysis of the CRISPR arrays revealed that nearly half of the spacers matched G. vaginalis chromosomal sequences. The spacers that matched G. vaginalis chromosomal sequences were determined to not be self-targeting and were presumably neither constituents of mobile-element-associated genes nor derived from plasmids/viruses. The protospacers targeted by these spacers displayed conserved protospacer-adjacent motifs. Conclusions The CRISPR/Cas system has been identified in about one half of the analysed G. vaginalis strains. Our analysis of CRISPR sequences did not reveal a potential link between their presence and the virulence of the G. vaginalis strains. Based on the origins of the spacers found in the G. vaginalis CRISPR arrays, we hypothesise that the transfer of genetic material among G. vaginalis strains could be regulated by the CRISPR/Cas mechanism. The present study is the first attempt to determine and analyse the CRISPR loci of bacteria isolated from the human vaginal tract.

Abstract 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.

Abstract Background Gardnerella vaginalis is identified as the predominant colonist of the vaginal tract in women with bacterial vaginosis. Vaginolysin (VLY) is a protein toxin released by G. vaginalis. VLY possesses cytolytic activity and is considered as a main virulence factor of G. vaginalis. Inhibition of VLY-mediated cell lysis by antibodies may have important physiological relevance. Results Single-chain variable fragments of immunoglobulins (scFvs) were cloned from two hybridoma cell lines producing neutralizing antibodies against VLY and expressed as active proteins in E. coli. For each hybridoma, two variants of anti-VLY scFv consisting of either VL-VH or VH-VL linked with a 20 aa-long linker sequence (G4S)4 were constructed. Recovery of scFvs from inclusion bodies with subsequent purification by metal-chelate chromatography resulted in VLY-binding proteins that were predominantly monomeric. The antigen-binding activity of purified scFvs was verified by an indirect ELISA. The neutralizing activity was investigated by in vitro hemolytic assay and cytolytic assay using HeLa cell line. Calculated apparent Kd values and neutralizing potency of scFvs were in agreement with those of parental full-length antibodies. VH-VL and VL-VH variants of scFvs showed similar affinity and neutralizing potency. The anti-VLY scFvs derived from hybridoma clone 9B4 exhibited high VLY-neutralizing activity both on human erythrocytes and cervical epithelial HeLa cells. Conclusions Hybridoma-derived scFvs with VLY-binding activity were expressed in E. coli. Recombinant anti-VLY scFvs inhibited VLY-mediated cell lysis. The monovalent scFvs showed reduced affinity and neutralizing potency as compared to the respective full-length antibodies. The loss of avidity could be restored by generating scFv constructs with multivalent binding properties. Generated scFvs is the first example of recombinant single-chain antibodies with VLY-neutralizing activity produced in prokaryote expression system. G. vaginalis caused infections continue to be a world-wide problem, therefore neutralizing recombinant antibodies may provide novel therapeutic agents useful in the treatment of bacterial vaginosis and other diseases caused by G. vaginalis.

Background. Allergen extracts and recombinant allergens are used in allergy diagnostics and immunotherapy. Since allergen extracts from different manufacturers lack proper standardization regarding their composition, monoclonal antibodies (MAbs) against specific allergen components can be used for their identification and quantification in allergen extracts. This study aimed to generate MAbs against allergen Der p 21 of Dermatophagoides pteronyssinus for the analysis of allergen extracts. Methods. Recombinant Der p 21 was expressed in E. coli and purified using affinity chromatography. MAbs against Der p 21 were generated using hybridoma technology. House dust mite (HDM) allergen extracts were analyzed using the newly developed sandwich enzyme-linked immunosorbent assay, Western blotting and microarray immunoassay. Results. MAbs raised against recombinant Der p 21 were characterized in detail and proven to be reactive with natural Der p 21. Highly specific sandwich enzyme-linked immunosorbent assay for the quantification of Der p 21 was developed and optimized. The allergen was detected and its concentration was determined in only three of six analyzed HDM allergen extracts from different manufacturers. Conclusion. HDM analysis by MAb-based immunoassays shows their differences in allergen composition. The results demonstrate the importance of allergen-specific MAbs as a tool for the characterization of allergen extracts and the need for their appropriate standardization before their use for allergy diagnostics or immunotherapy. [ABSTRACT FROM AUTHOR]

Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3, 5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37[degrees]C. After subtraction of unspecific uptake determined in WT at 37[degrees]C or in hOCT2 at 8[degrees]C saturable specific uptake of both substrates was measured. [K.sub.m] values of hOCT2-mediated uptake of 95 [micro]M amiloride and 24 [micro]M ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [[IC.sub.50](in [micro]M) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of [Ca.sup.2+]/CaM complex (-55 [+ or -] 5%, n = 10 and -63 [+ or -] 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (-54 [+ or -] 5%, n = 14, and -31 [+ or -] 6%, n = 26) and PKA (-16 [+ or -] 5%, n = 16, and -18 [+ or -] 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 [+ or -] 6%, n = 8, and +55 [+ or -] 17%, n = 16). Inhibition of [Ca.sup.2+]/CaM complex resulted in a significant decrease of [V.sub.max] (160-99 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (-22 [+ or -] 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the [Ca.sup.2+]/CaM complex causes changes in transport capacity via hOCT2 trafficking. organic cation transport; fluorescence measurement; 4-[4-(dimethylamino)-styryl]-n-methylpyridinium; amiloride

Hepatitis E is a globally distributed human disease caused by hepatitis E virus (HEV). In Europe, it spreads through undercooked pork meat or other products and with blood components through transfusions. There are no approved or golden standard serologic systems for HEV diagnostics. Commercially available HEV tests often provide inconsistent results which may differ among the assays. In this study, we describe generation in yeast and characterization of HEV genotype 3 (HEV-3) and rat HEV capsid proteins self-assembled into virus-like particles (VLPs) and the development of HEV-specific monoclonal antibodies (MAbs). Full-length HEV-3 and rat HEV capsid proteins and their truncated variants comprising amino acids (aa) 112-608 were produced in yeast S. cerevisiae. The yeast-expressed rat HEV capsid protein was found to be glycosylated. The full-length HEV-3 capsid protein and both full-length and truncated rat HEV capsid proteins were capable to self-assemble into VLPs. All recombinant proteins contained HEV genotype-specific linear epitopes and cross-reactive conformational epitopes recognized by serum antibodies from HEV-infected reservoir animals. Two panels of MAbs against HEV-3 and rat HEV capsid proteins were generated. Their cross-reactivity pattern was investigated by Western blot, ELISA, and immunofluorescence assay on HEV-3-infected cell cultures. The analysis revealed cross-reactive, genotype-specific, and virus-reactive MAbs. MAb epitopes were localized within S, M, and P domains of HEV-3 and rat HEV capsid proteins. Yeast-generated recombinant VLPs of HEV-3 and rat HEV capsid proteins and HEV-specific MAbs might be employed to develop novel HEV detection systems. [ABSTRACT FROM AUTHOR]