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Background and Aims: Colonic epithelial barrier dysfunction is one of the early events in ulcerative colitis (UC) and microRNAs (miRNAs) participate in its regulation. However, cell type-specific miRNome during UC is still unknown. Thus, we aimed to explore miRNA expression patterns in colon tissue and epithelial cells at active and quiescent UC., Methods: Small RNA-sequencing in colon tissue, crypt-bottom (CD44+), and crypt-top (CD66a+) colonic epithelial cells from two cohorts of UC patients (n=74) and healthy individuals (n=50) was performed. Data analysis encompassed differential expression, weighted gene co-expression network, correlation, gene-set enrichment analyses., Results: Differentially expressed colonic tissue miRNAs showed potential involvement in regulation of interleukin-4 and interleukin-13 signalling during UC. As this pathway plays role in intestinal barrier regulation, consecutive analysis of spatially distinct colonic epithelial cell populations was performed. Cell-type (crypt-top and crypt-bottom) specific miRNA expression patterns were identified in both active and quiescent UC. Target genes of differentially expressed epithelial miRNAs at different disease activity were overrepresented in epithelial cell migration and therefore intestinal barrier integrity regulation. The pro-inflammatory miRNA co-expression module M1 correlated with endoscopic disease activity and successfully distinguished active and quiescent UC not only in both epithelial cell populations, but also in the colon tissue. The anti-inflammatory module M2 was specific to crypt-bottom cells and significantly enriched in the quiescent UC patients., Conclusions: miRNA expression was specific to colonic epithelial cell populations and UC state, reflecting endoscopic disease activity. Irrespective of the UC state, deregulated epithelial miRNAs were associated with regulation of intestinal barrier integrity., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation.)

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., Competing Interests: Laimis Silimavicius is employed by UAB Imunodiagnostika, the Lithuanian company that specializes in the development of microarray-based assays and allergy in vitro diagnostics., (©2024 Rudokas et al.)

In this study, we are reporting an electrochemical biosensor for the determination of three different clones of monoclonal antibodies (mAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recombinant nucleocapsid protein (rN). The nucleocapsid protein was chosen as a system component identifying and discriminating antibodies that occur after virus infection instead of S protein used in serological tests to measure antibodies raised after vaccination and infection. The sensing platform was based on a screen-printed carbon electrode (SPCE) covered with gold nanoparticles (AuNP) and subsequently modified with a self-assembled monolayer (SAM) to ensure the covalent immobilization of the rN. The interaction between the protein and three clones of mAbs against SARS-CoV-2 rN with clone numbers 4G6, 7F10, and 1A6, were electrochemically registered in the range of concentrations. Three techniques, cyclic voltammetry (CV), differential pulse voltammetry (DPV), and pulse amperometric detection (PAD) were used for the detection. A gradual change in the responses with an increase in mAbs concentration for all techniques was observed. To assess the performance of the developed electrochemical biosensor, 'complexation constant' (K C ), limit of detection (LOD), and limit of quantification (LOQ) were calculated for all assessed clones of mAbs and all used techniques. Our results indicated that DPV possessing higher fitting accuracy illustrated more significant differences in K C constants and LOD/LOQ values. According to the DPV results, 7F10 clone was characterized with the highest K C value of 1.47 ± 0.07 μg/mL while the lowest LOD and LOQ values belonged to the 4G6 clone and equaled 0.08 ± 0.01 and 0.25 ± 0.01 μg/mL, respectively. Overall, these results demonstrate the potential of electrochemical techniques for the detection and distinguishing of different clones of mAbs against SARS-CoV-2 nucleocapsid protein., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The serologic diagnosis of coronavirus disease 2019 (COVID-19) and the evaluation of vaccination effectiveness are identified by the presence of antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this paper, we present the electrochemical-based biosensing technique for the detection of antibodies specific to the SARS-CoV-2 proteins. Recombinant SARS-CoV-2 spike proteins (rSpike) were immobilised on the surface of a gold electrode modified by a self-assembled monolayer (SAM). This modified electrode was used as a sensitive element for the detection of polyclonal mouse antibodies against the rSpike (anti-rSpike). Electrochemical impedance spectroscopy (EIS) was used to observe the formation of immunocomplexes while cyclic voltammetry (CV) was used for additional analysis of the surface modifications. It was revealed that the impedimetric method and the elaborate experimental conditions are appropriate for the further development of electrochemical biosensors for the serological diagnosis of COVID-19 and/or the confirmation of successful vaccination against SARS-CoV-2.

Zoonotic transmission of Hendra virus (HeV) from primary hosts (pteropid bats) to horses, and, occasionally, onward adventitious spread to humans, is associated with high mortality rates in both affected secondary species. The introduction of an effective recombinant G protein vaccine for use in horses has been a major advance for the suppression of disease risk. However, equine HeV vaccination induces neutralising antibody that is indistinguishable from a post infection immune response when using most first line serology assays (eg. VNT and some ELISAs). We have constructed and evaluated an IgM antibody capture (MAC) ELISA which employs yeast expressed HeV nucleoprotein (N). All other serology tests use the G protein which does not detect early infection and is present in the current Hendra virus vaccine and may cause ambiguity in interpretation of results. Thus, this is the first test developed using a N protein which can successfully detect a recent (primarily within the last four weeks) infection of horses with HeV and is not affected by vaccination induced antibody. Testing a limited panel (21 samples) of post infection sera, a normal serum panel (288 samples) and a post vaccination panel (163 samples), we have estimated DSe to be 100 % (95 % CI, 83.9-100.0 %) and DSp to be 98.4 % (95 % CI, 96.8-99.4 %) relative to assigned serology results (VNT, ELISA and Luminex) for the test panels. The HeV IgM MAC ELISA is intended to supplement other molecular and serology test results, with selective use, and is the only serology test which can provide an indication for recent infection which is otherwise not available., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

During the pandemic, different methods for SARS-CoV-2 detection and COVID-19 diagnostics were developed, including antibody and antigen tests. For a better understanding of the interaction mechanism between SARS-CoV-2 virus proteins and specific antibodies, total internal reflection ellipsometry based evaluation of the interaction between SARS-CoV-2 nucleoprotein (SCoV2-rN) and anti-SCoV2-rN antibodies was performed. Results show that the appropriate mathematical model, which takes into account the formation of an intermediate complex, can be applied for the evaluation of SCoV2-rN/anti-SCoV2-rN complex formation kinetics. The calculated steric factor indicated that SCoV2-rN/anti-SCoV2-rN complex formation has very strict steric requirements. Estimated Gibbs free energy (ΔG Assoc ) for SCoV-rN and anti-SCoV-rN binding was determined as -34 kJ/mol. The reported findings are useful for the design of new analytical systems for the determination of anti-SCoV2-rN antibodies and for the development of new anti-SARS-CoV-2 medications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Monoclonal and recombinant antibodies are widely used for the diagnostics and therapy of cancer. They are generated to interact with cell surface proteins which are usually involved in the development and progression of cancer. Carbonic anhydrase XII (CA XII) contributes to the survival of tumors under hypoxic conditions thus is considered a candidate target for antibody-based therapy. In this study, we have generated a novel collection of monoclonal antibodies (MAbs) against the recombinant extracellular domain of CA XII produced in HEK-293 cells. Eighteen out of 24 MAbs were reactive with cellular CA XII on the surface of live kidney and lung cancer cells as determined by flow cytometry. One MAb 14D6 also inhibited the enzymatic activity of recombinant CA XII as measured by the stopped-flow assay. MAb 14D6 showed the migrastatic effect on human lung carcinoma A549 and renal carcinoma A498 cell lines in a 'wound healing' assay. It did not reduce the growth of multicellular lung and renal cancer spheroids but reduced the cell viability by the ATP Bioluminescence assay. Epitope mapping revealed the surface-exposed amino acid sequence (35-FGPDGENS-42) close to the catalytic center of CA XII recognized by the MAb 14D6. The variable regions of the heavy and light chains of MAb 14D6 were sequenced and their complementarity-determining regions were defined. The obtained variable sequences were used to generate recombinant antibodies in two formats: single-chain fragment variable (scFv) expressed in E. coli and scFv fused to human IgG1 Fc fragment (scFv-Fc) expressed in Chinese Hamster Ovary (CHO) cells. Both recombinant antibodies maintained the same specificity for CA XII as the parental MAb 14D6. The novel antibodies may represent promising tools for CA XII-related cancer research and immunotherapy.

Background: The most popular miRNA quantitation technique is RQ-PCR with relative gene expression method that requires an endogenous control (EC) gene for data normalization. However, there are insufficient data and selection criteria on the most suitable ECs for miRNA expression studies in many cancer types including papillary thyroid carcinoma (PTC). Therefore, in this study we evaluated the impact of chosen EC and archival formalin-fixed, paraffin-embedded (FFPE) PTC tissue age on estimated target miRNA expression., Methods: RQ-PCR was used to determine expression levels of five miRNAs (miR-146b, miR-222, miR-21, miR-221 and miR-181b) and three different endogenous controls (RNU48, let-7a, miR-16), which were used to normalize the data. In total, 400 FFPE PTC tissues were analyzed that have been stored from 1 to 15 years., Results: The stability of commonly used ECs RNU48 and let-7a significantly differs from the stability of target miRNA in archival FFPE PTC tissues. Moreover, these differences have a great impact on miRNA expression results when FFPE tissue samples have been stored for a different period of time., Conclusions: It is important to select an ECs not only stable in the tissue of interest but also with similar stability to target miRNA, especially when working with samples of different age.

The pathogenicity of many bacteria, including Streptococcus pneumoniae, depends on pore-forming toxins (PFTs) that cause host cell lysis by forming large pores in cholesterol-containing cell membranes. Therefore, PFTs-neutralising antibodies may provide useful tools for reducing S. pneumoniae pathogenic effects. This study aimed at the development and characterisation of monoclonal antibodies (MAbs) with neutralising activity to S. pneumoniae PFT pneumolysin (PLY). Five out of 10 produced MAbs were able to neutralise the cytolytic activity of PLY on a lung epithelial cell line. Epitope mapping with a series of recombinant overlapping PLY fragments revealed that neutralising MAbs are directed against PLY loops L1 and L3 within domain 4. The epitopes of MAbs 3A9, 6E5 and 12F11 located at L1 loop (aa 454-471) were crucial for PLY binding to the immobilised cholesterol. In contrast, the MAb 12D10 recognising L3 (aa 403-423) and the MAb 3F3 against the conformational epitope did not interfere with PLY-cholesterol interaction. Due to conformation-dependent binding, the approach to use overlapping peptides for fine epitope mapping of the neutralising MAbs was unsuccessful. Therefore, the epitopes recognised by the MAbs were analysed using computational methods. This study provides new data on PLY sites involved in functional activity.

Many miRNAs and cancer-related mutations have been proposed as promising molecular markers of papillary thyroid carcinoma (PTC). However, there are limited data on the correlation between miRNA expression, BRAF V600E mutation, and PTC recurrence. Therefore, to evaluate the potential of BRAF V600E mutation and five selected miRNAs (-146b, -222, -21, -221, -181b) in predicting PTC recurrence, these molecular markers were analyzed in 400 formalin-fixed, paraffin-embedded PTC tissue specimens. The expression levels of miRNAs were measured using qRT-PCR. It was demonstrated that expression levels of all analyzed miRNAs are significantly higher in recurrent PTC than in non-recurrent PTC ( p < 0.05). Moreover, higher expression levels of miR-146b, miR-222, miR-21, and miR-221 were associated with other clinicopathologic features of PTC, such as tumor size and lymph node metastases at initial surgery ( p < 0.05). No significant differences in the frequency of BRAF V600E mutation in recurrent PTC and non-recurrent PTC were determined. Our results suggest that miRNA expression profile differs in PTC that is prone to recurrence when compared to PTC that does not reoccur after the initial surgery while BRAF V600E mutation frequency does not reflect the PTC recurrence status. However, the prognostic value of the analyzed miRNAs is rather limited in individual cases as the pattern of miRNA expression is highly overlapping between recurrent and non-recurrent PTC.

Background: Diagnosis of human bocavirus 1 (HBoV1) has been based on qualitative PCRs detecting HBoV1 DNA or detection of HBoV1 mRNA., Objective: This study aims to assess whether a rapid and automated HBoV1 antigen test is suitable for diagnosis of acute HBoV1 infection., Study Design: HBoV1 antigen detection has been compared with quantitative HBoV1 DNA PCR and HBoV1 mRNA RT-PCR., Results and Conclusion: We conclude that HBoV1 antigen detection has higher clinical specificity and positive predictive value than HBoV1 DNA qualitative PCRs, yet a lower sensitivity than HBoV1 mRNA detection. Additionally, HBoV1 antigen detection is beneficial in its rapidity and availability as a point-of-care test., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Monoclonal antibodies (MAbs) selectively targeting tumor-associated antigens such as carbonic anhydrase IX (CA IX) can significantly contribute to research, diagnostics, and treatment of CA IX-related cancers. CA IX is overexpressed in numerous hypoxic cancers where it promotes tumor progression. Therefore, it is considered as a promising tumor biomarker. A novel collection of MAbs against recombinant CA IX was developed and evaluated in different immunoassays for studying CA IX expression. The reactivity of MAbs with native cell surface protein was confirmed by flow cytometry and the presence of hypoxia-inducible CA IX was investigated in several human cancer cell lines. In addition, the applicability of MAbs for visualization of CA IX-positive tumor cells by immunofluorescence microscopy was demonstrated. MAb H7 was identified as the most promising MAb for different immunoassays. It recognized a linear epitope covering CA IX sequence of 12 amino acid residues 55-GEDDPLGEEDLP-66 within the proteoglycan domain. The MAb H7 was the only one of the collection to immunoprecipitate CA IX protein from cell lysates and detect the denatured CA IX with near-infrared fluorescence Western blot. It was also employed in sandwich enzyme-linked immunosorbent assay to detect a soluble form of CA IX in growth medium of tumor cells and blood plasma samples. The diagnostic potential of the MAb H7 was confirmed on formalin-fixed and paraffin-embedded tissue specimen of cervical carcinoma in situ by immunohistochemistry. The generated MAbs, in particularly clone H7, have great potential in diagnostics and research of CA IX-related cancers.

Rat hepatitis E virus (HEV) is an orthohepevirus which is related to other HEV found in humans and other mammals. It was first identified in Norway rats (Rattus norvegicus) from Germany in 2010, and later it has been detected in Black rats (Rattus rattus) and Norway rats from USA, China, Indonesia, Vietnam and many European countries. In this study, we describe molecular and serological investigations of Black and Norway rats trapped in Lithuania, Eastern Europe, for infections with rat HEV and human HEV genotypes 1-4. Rat HEV-specific real-time reverse transcription-PCR (RT-qPCR) analysis of rat liver samples revealed the presence of rat HEV in 9 of 109 (8.3%) samples. In contrast, a RT-qPCR specific for HEV genotypes 1-4 did not reveal any positive samples. A nested broad spectrum RT-PCR was used for a confirmation of rat HEV infection with a subsequent sequencing of the amplified rat HEV genome fragment. Phylogenetic analysis revealed a clustering of all newly identified rat HEV sequences with Norway rat-derived rat HEV sequences from Germany within the species Orthohepevirus C. An indirect ELISA using a yeast-expressed truncated rat HEV capsid protein variant revealed 31.2% seropositive samples indicating a high rate of rat HEV circulation in the rat population examined. In conclusion, the current investigation confirms rat HEV infections in Norway and Black rats in Lithuania, Eastern Europe, and the non-persistent nature of HEV infection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

Gardnerella vaginalis is a facultative anaerobic bacterium that inhabits the genitourinary tract of both healthy women and those with bacterial vaginosis. We report a case of G. vaginalis bacteremia associated with severe toxic encephalopathy in a young woman. Anaerobic blood cultures yielded pure growth of small gram-variable rods later identified as G. vaginalis by both rapid biochemical tests and 16S rRNA gene sequencing. The patient recovered after treatment with amoxicillin-clavulanate according to the in vitro susceptibility testing. The complete genome of G. vaginalis isolate from blood cultures was determined. In vitro G. vaginalis isolate produced elevated amounts of a pore-forming toxin vaginolysin compared to control G. vaginalis isolates. We hypothesize that this toxin, if produced in high amounts in blood, is able to disrupt the blood-brain barrier and exert a toxic activity on brain cells., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Clinically relevant diagnosis of human bocavirus 1 (HBoV1) is challenging, as the virus is frequently detected in asymptomatic patients, and cofindings with other respiratory viruses are common. The clinical value of current diagnostic methods, such as PCR, is therefore low, and alternative diagnostic strategies are needed. We describe for the first time the use of an antigen detection assay for the rapid identification of HBoV1 in a paediatric patient with respiratory tract infection symptoms. We estimate the duration of active HBoV1 infection to be 6 days.

Hepatitis B virus (HBV) surface antigen (HBsAg) is considered to be the most important target for the diagnosis and immune prophylaxis of HBV infection. HBsAg-specific monoclonal antibodies (MAbs) are extensively used for studying the complex structure of the HBsAg, mapping the neutralizing epitopes and development of HBV diagnostic tests. However, the efficiency of anti-HBV binding strongly depends on the epitope structure and MAb capability to recognize different HBV variants. In the current study, 9 MAbs against yeast-expressed HBsAg of ayw2 serotype were generated and 7 of them were shown to recognize a linear epitope comprising amino acid (aa) residues 119-GPCRTCT-125 within the main antigenic "a" determinant of HBsAg. One MAb of the highest affinity (clone HB1) was selected for detailed cross-reactivity studies, generation of recombinant single-chain antibody (scFv) and molecular modelling of antibody-epitope interaction. The importance of each aa residue within the identified MAb epitope was determined by alanine substitution study that revealed aa residues C(121), T(123), C(124) and T(125) as essential for binding. These aa residues are highly conserved among HBV variants. In contrast, alanine substitution of G119, P120 and R122 had no or minor influence on the reactivity with the MAb. Certain aa residues at position 122 (either R or K) define different HBV serotypes (either d or y), therefore, the affinity of the MAb HB1 for the epitope with R122K substitution was determined to evaluate its diagnostic potential. The MAb recognized both epitope variants with high affinity. Sequence alignment of the MAb epitope within different HBV strains demonstrated that the shortest peptide recognized by the MAb 121-CR(K)TCT-125 is identical among different human HBV genotypes (HBV A-F, H) and monkey HBV species (HBVCP, HBVGO, HBVGB, WMHBV). In line with these data, the MAb HB1 was cross-reactive in Western blot with a large panel of antigens derived from different HBV genotypes. Recombinant scFv consisting of immunoglobulin VH and VL regions joined by a 20 aa-long linker was generated by cloning the respective cDNA sequences from hybridoma HB1. The recombinant scFv generated in Escherichia coli recognized the same epitope as the parental MAb HB1. Cloning of HB1 VH and VL regions allowed determination of their primary structure and subsequent computer modeling of antibody-epitope interaction. The generated molecular models of HB1 variable region with its target peptides were in accordance with experimental data showing the importance of certain aa residues in antibody binding. In conclusion, the current study describes new HBsAg-specific antibodies with HBV-neutralizing potency and a broad cross-reactivity against different HBV strains. The generated MAb HB1 will be of great value in diagnostic and research settings, while the recombinant HB1-derived scFv represents a promising "building block" for producing anti-HBV tools with a potential biopharmaceutical application., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Epigenetic alterations of human papillomavirus (HPV) genome play an important role in virus life cycle and carcinogenic progression. The aim of the current study was to investigate the correlation between the grade of cervical pathology and DNA methylation status within the L1 gene and the long control region (LCR) of HPV16, HPV18 and HPV51. HPV genomes were analyzed using bisulfite DNA modification procedure with the subsequent amplification of target DNA regions and sequencing. A collection of 202 cervical specimens was analyzed: 157 HPV16-positive specimens, 21 HPV18-positive specimens and 24 HPV51-positive specimens. This study revealed that methylation of CpG was significantly more prevalent in L1 gene as compared to LCR region of all three studied HPV types and the degree of DNA methylation level correlated with the severity of cervical neoplasia. An increased DNA methylation level of HPV16 promoter region in case of cervical cancer was determined., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Virus-like particles (VLPs) can be efficiently produced by heterologous expression of viral structural proteins in yeast. Due to their repetitive structure, VLPs are extensively used for protein engineering and generation of chimeric VLPs with inserted foreign epitopes. Hamster polyomavirus VP1 represents a promising epitope carrier. However, insertion of large sized protein sequences may interfere with its self-assembly competence. The co-expression of polyomavirus capsid protein VP1 with minor capsid protein VP2 or its fusion protein may result in pseudotype VLPs where an intact VP1 protein mediates VLP formation. In the current study, the capacity of VP1 protein to self-assemble to VLPs and interact with the modified VP2 protein has been exploited to generate pseudotype VLPs displaying large-sized antibody molecules., Results: Polyomavirus-derived pseudotype VLPs harbouring a surface-exposed functionally active neutralizing antibody specific to hepatitis B virus (HBV) surface antigen (HBsAg) have been generated. The pseudotype VLPs consisting of an intact hamster polyomavirus (HaPyV) major capsid protein VP1 and minor capsid protein VP2 fused with the anti-HBsAg molecule were efficiently produced in yeast Saccharomyces cerevisiae and purified by density-gradient centrifugation. Formation of VLPs was confirmed by electron microscopy. Two types of pseudotype VLPs were generated harbouring either the single-chain fragment variable (scFv) or Fc-engineered scFv on the VLP surface. The antigen-binding activity of the purified pseudotype VLPs was evaluated by ELISA and virus-neutralization assay on HBV-susceptible primary hepatocytes from Tupaia belangeri. Both types of the pseudotype VLPs were functionally active and showed a potent HBV-neutralizing activity comparable to that of the parental monoclonal antibody. The VP2-fused scFv molecules were incorporated into the VLPs with higher efficiency as compared to the VP2-fused Fc-scFv. However, the pseudotype VLPs with displayed VP2-fused Fc-scFv molecule showed higher antigen-binding activity and HBV-neutralizing capacity that might be explained by a better accessibility of the Fc-engineered scFv of the VLP surface., Conclusions: Polyomavirus-derived pseudotype VLPs harbouring multiple functionally active antibody molecules with virus-neutralizing capability may represent a novel platform for developing therapeutic tools with a potential application for post-exposure or therapeutic treatment of viral infections.

Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes.

Human papillomavirus (HPV) is the main cause of cervical cancer. Therefore, the detection of oncogenic HPV types is important in predicting the risk of cervical cancer. The aim of the current study was to estimate the prevalence of 16 carcinogenic and potentially carcinogenic HPV types in the study group of Lithuanian women with various grades of cervical pathology in comparison to healthy women. A total of 824 cervical specimens were investigated for HPV DNA: 547 specimens of women with abnormal cytology and 277 specimens of healthy women. Cytological diagnosis was confirmed by histology. For the detection of HPV infection, HPV DNA was amplified by PCR using three different primer systems. HPV DNA was detected in 67.6% of specimens collected from women with abnormal cytology and 24.2% of specimens collected from healthy women. The frequency of HPV-positive specimens correlated with the severity of cervical pathology: it ranged from 50.0% in the subgroup of atypical squamous cells to 80.6% in cervical cancer. In cases confirmed by histology the frequency of HPV-positive specimens ranged from 68.6% in the subgroup of cervical intraepithelial neoplasia grade 1 to 89.2% in cervical intraepithelial neoplasia grade 3 or carcinoma in situ. HPV DNA-positive samples were further investigated for the presence of 16 HPV types by multiplex PCR. The most common HPV type was HPV 16 (detected in 42.3% of HPV-positive specimens) followed by HPV 31 (10.1%), HPV 33 (8.2%), and HPV 56 (5.7%). In contrast, the frequency of HPV 18 was lower as compared to other countries., (© 2014 Wiley Periodicals, Inc.)

Gardnerella vaginalis produces cytolysin vaginolysin (VLY), which has been suggested to be a contributor to bacterial vaginosis pathogenesis. VLY along with intermedilysin (ILY) from Streptococcus intermedius have been attributed to a group of cholesterol-dependent cytolysins (CDCs) whose pore-forming activity depends on human CD59 (hCD59). Here, we show that different types of cells lacking hCD59 are susceptible to VLY-mediated lysis, albeit to different extents. We analyze the effects of both hCD59 and cholesterol on VLY cytolytic activity. We show that VLY binds to cholesterol-rich membranes of non-human cells, while VLY with an impaired cholesterol recognition site retains binding to the hCD59-containing cells. We further demonstrate that cholesterol binding by VLY is sufficient to trigger the formation of oligomeric complexes on cholesterol rich-liposomes lacking hCD59. Thus, VLY may induce cell lysis following two alternative pathways. One requires only cholesterol and does not depend on hCD59. The second pathway involves hCD59 contribution similarly to ILY. Apparently, under physiological conditions VLY acts in the most effective way by accepting the assistance of hCD59.

Background: Porcine circovirus type 2 (PCV2) is considered to be an important emerging pathogen associated with a number of different syndromes and diseases in pigs known as PCV2-associated diseases. It has been responsible for significant mortality among pigs and remains a serious economic problem to the swine industry worldwide leading to significant negative impacts on profitability of pork production., Results: In this study we have demonstrated that PCV2 capsid (Cap) protein based virus-like particles (VLPs) were efficiently produced in yeast S. cerevisiae and induced production of monoclonal antibodies (MAbs) reactive with virus-infected cells. Moreover, PCV2 Cap VLPs served as a highly specific recombinant antigen for the development of an indirect IgG PCV2 Cap VLP-based ELISA for the detection of virus-specific IgG antibodies in swine sera. Four hundred-nine serum samples collected from pigs in Lithuania were tested for PCV2-specific IgG to determine the sensitivity and specificity of the newly developed ELISA in parallel using a commercial SERELISA test as a gold standard. From 409 tested serum samples, 297 samples were positive by both assays. Thirty-nine sera from 112 serum samples were determined as negative by SERELISA but were found to be positive both in the newly developed indirect IgG PCV2 Cap VLP-based ELISA and the PCR test., Conclusions: We have demonstrated that S. cerevisiae expression system is an alternative to insect/baculovirus expression system for production of homogenous in size and shape PCV2 Cap protein-based VLPs similar to native virions. Yeast expression system tolerated native virus genes encoding PCV2 Cap protein variants as well as the codon-optimized gene. Moreover, yeast-derived PCV2 Cap VLPs were capable to induce the generation of PCV2-specific MAbs that did not show any cross-reactivity with PCV1-infected cells. The high sensitivity and specificity of the indirect IgG PCV2 Cap VLP-based ELISA clearly suggested that this assay is potentially useful diagnostic tool for screening PCV2-suspected samples.

Abstract Human carbonic anhydrase XII (CA XII) is a single-pass transmembrane protein with an extracellular catalytic domain. This enzyme is being recognized as a potential biomarker for different tumours. The current study was aimed to generate monoclonal antibodies (MAbs) neutralizing the enzymatic activity of CA XII. Bioinformatics analysis of CA XII structure revealed surface-exposed sequences located in a proximity of its catalytic centre. Two MAbs against the selected antigenic peptide spanning 167-180 aa sequence of CA XII were generated. The MAbs were reactive with recombinant catalytic domain of CA XII expressed either in E. coli or mammalian cells. Inhibitory activity of the MAbs was demonstrated by a stopped flow CO2 hydration assay. The study provides new data on the surface-exposed linear CA XII epitope that may serve as a target for inhibitory antibodies with a potential immunotherapeutic application.

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.

Monoclonal antibodies (MAbs) against viral glycoproteins have important diagnostic and therapeutic applications. In most cases, the MAbs specific to viral glycoproteins are raised against intact virus particles. The biosynthesis of viral glycoproteins in heterologous expression systems such as bacteria, yeast, insect or mammalian cells is often problematic due to their low expression level, improper folding and limited stability. To generate MAbs against hantavirus glycoprotein Gc, we have used initially a recombinant yeast-expressed full-length Puumala virus (PUUV) Gc protein. However, this approach was unsuccessful. As an alternative recombinant antigen, chimeric virus-like particles (VLPs) harboring a segment of PUUV Gc glycoprotein were generated in yeast Saccharomyces cerevisiae. A 99 amino acid (aa)-long segment of Gc protein was inserted into the major capsid protein VP1 of hamster polyomavirus at previously defined positions: either site #1 (aa 80-89) or site #4 (aa 280-289). The chimeric proteins were found to self-assemble to VLPs as evidenced by electron microscopy. Chimeric VLPs induced an efficient insert-specific antibody response in immunized mice. Monoclonal antibody (clone #10B8) of IgG isotype specific to hantavirus Gc glycoprotein was generated. It recognized recombinant full-length PUUV Gc glycoprotein both in ELISA and Western blot assay and reacted specifically with hantavirus-infected cells in immunofluorescence assay. Epitope mapping studies revealed the N-terminally located epitope highly conserved among different hantavirus strains. In conclusion, our approach to use chimeric VLPs was proven useful for the generation of virus-reactive MAb against hantavirus Gc glycoprotein. The generated broadly-reactive MAb #10B8 might be useful for various diagnostic applications.

Carbonic anhydrases (CAs) are enzymes that catalyse the reversible hydration of CO2 to bicarbonate. CA XII is considered a potential biomarker of tumor cells and a promising target for specific therapies. The aim of the current study was to develop new monoclonal antibodies (MAbs) against human recombinant CA XII and evaluate their diagnostic potential. An extracellular catalytic domain of human CA XII was expressed in E. coli and used as an immunogen. Seven stable hybridoma cell lines producing high-affinity IgG antibodies against human CA XII were generated. The majority of MAbs were highly specific to CA XII and did not cross-react with human recombinant CA I, CA II, CA VII, and CA XIII. In order to demonstrate the diagnostic value of the MAbs, they were employed for the immunohistochemistry analysis of CA XII expression in tissues. Two MAbs (15A4 and 4A6) demonstrated a strong and specific immunostaining of CA XII in human tissue specimens. Flow cytometry analysis of 5 human tumor cell lines with the MAb 15A4 revealed its immunoreactivity with cellular CA XII. In conclusion, the MAbs raised against recombinant catalytic domain of CA XII recognize cellular CA XII and represent a promising diagnostic tool for the immunodetection of CA XII-expressing cells.

Schmallenberg virus (SBV), discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. To develop improved reagents for SBV serology, a high-level yeast expression system was employed to produce recombinant SBV nucleocapsid (N) protein. Recombinant SBV N protein was investigated as an antigen in SBV-specific IgG enzyme immunoassay and used for generation of monoclonal antibodies (MAbs). Yeast-expressed SBV N protein was reactive with anti-SBV IgG-positive cow serum specimens collected from different farms of Lithuania. After immunization of mice with recombinant SBV N protein, four MAbs were generated. The MAbs raised against recombinant SBV N protein reacted with native viral nucleocapsids in SBV-infected BHK cells by immunofluorescence assay. The reactivity of recombinant N protein with SBV-positive cow serum specimens and the ability of the MAbs to recognize virus-infected cells confirm the antigenic similarity between yeast-expressed SBV N protein and native viral nucleocapsids. Our study demonstrates that yeast expression system is suitable for high-level production of recombinant SBV N protein and provides the first evidence on the presence of SBV-specific antibodies in cow serum specimens collected in Lithuania.

Functional reconstitution of the cholesterol-dependent cytolysin vaginolysin (VLY) from Gardnerella vaginalis into artificial tethered bilayer membranes (tBLMs) has been accomplished. The reconstitution of VLY was followed in real-time by electrochemical impedance spectroscopy (EIS). Changes of the EIS parameters of the tBLMs upon exposure to VLY solutions were consistent with the formation of water-filled pores in the membranes. It was found that reconstitution of VLY is a strictly cholesterol-dependent, irreversible process. At a constant cholesterol concentration reconstitution of VLY occurred in a concentration-dependent manner, thus allowing the monitoring of VLY concentration and activity in vitro and opening possibilities for tBLM utilization in bioanalysis. EIS methodology allowed us to detect VLY down to 0.5 nM (28 ng/mL) concentration. Inactivation of VLY by certain amino acid substitutions led to noticeably lesser tBLM damage. Pre-incubation of VLY with the neutralizing monoclonal antibody 9B4 inactivated the VLY membrane damage in a concentration-dependent manner, while the non-neutralizing antibody 21A5 exhibited no effect. These findings demonstrate the biological relevance of the interaction between VLY and the tBLM. The membrane-damaging interaction between VLY and tBLM was observed in the absence of the human CD59 receptor, known to strongly facilitate the hemolytic activity of VLY. Taken together, our study demonstrates the applicability of tBLMs as a bioanalytical platform for the detection of the activity of VLY and possibly other cholesterol-dependent cytolysins.

We have detected a high incidence of lymphomas in a colony of GASH:Sal Syrian golden hamsters (Mesocricetus auratus). This strain is characterised by its ability to present convulsive crises of audiogenic origin. Almost 16 % (90 males and 60 females) of the 975 animals were affected during a 5-year period by the development of a progressing lymphoid tumour and exhibited similar clinical profiles characterised by lethargy, anorexia, evident abdominal distension, and a rapid disease progression resulting in mortality within 1 to 2 weeks. A TaqMan® probe-based real-time PCR analysis of genomic DNA from different tissue samples of the affected animals revealed the presence of a DNA sequence encoding the hamster polyomavirus (HaPyV) VP1 capsid protein. Additionally, immunohistochemical analysis using HaPyV-VP1-specific monoclonal antibodies confirmed the presence of viral proteins in all hamster tumour tissues analysed within the colony. An indirect ELISA and western blot analysis confirmed the presence of antibodies against the VP1 capsid protein in sera, not only from affected and non-affected GASH:Sal hamsters but also from control hamsters from the same breeding area. The HaPyV genome that accumulated in tumour tissues typically contained deletions affecting the noncoding regulatory region and adjacent sequences coding for the N-terminal part of the capsid protein VP2.

Background: Differences between populations might be reflected in their different genetic risk maps to complex diseases, for example, inflammatory bowel disease. We here investigated the role of known inflammatory bowel disease-associated single nucleotide polymorphisms (SNPs) in a subset of patients with ulcerative colitis (UC) from the Northeastern European countries Lithuania and Latvia and evaluated possible epistatic interactions between these genetic variants., Methods: We investigated 77 SNPs derived from 5 previously published genome-wide association studies for Crohn's disease and UC. Our study panel comprised 444 Lithuanian and Latvian patients with UC and 1154 healthy controls. Single marker case-control association and SNP-SNP epistasis analyses were performed., Results: We found 14 SNPs tagging 9 loci, including 21q21.1, NKX2-3, MST1, the HLA region, 1p36.13, IL10, JAK2, ORMDL3, and IL23R, to be associated with UC. Interestingly, the association of UC with previously identified variants in the HLA region was not the strongest association in our study (P = 4.34 × 10, odds ratio [OR] = 1.25), which is in contrast to all previously published studies. No association with any disease subphenotype was found. SNP-SNP interaction analysis showed significant epistasis between SNPs in the PTPN22 (rs2476601) and C13orf31 (rs3764147) genes and increased risk for UC (P = 1.64 × 10, OR = 2.44). The association has been confirmed in the Danish study group (P = 0.04, OR = 3.25)., Conclusions: We confirmed the association of the 9 loci (21q21.1, 1p36.13, NKX2-3, MST1, the HLA region, IL10, JAK2, ORMDL3, and IL23R) with UC in the Lithuanian-Latvian population. SNP-SNP interaction analyses showed that the combination of SNPs in the PTPN22 (rs2476601) and C13orf31 (rs3764147) genes increase the risk for UC.

Beta amyloid (Aβ) oligomers are thought to contribute to the pathogenesis of Alzheimer’s disease. However, clinical trials using Aβ immunization were unsuccessful due to strong brain inflammation, the mechanisms of which are poorly understood. In this study we tested whether monoclonal antibodies to oligomeric Aβ would prevent the neurotoxicity of Aβ oligomers in primary neuronal-glial cultures. However, surprisingly,the antibodies dramatically increased the neurotoxicity of Aβ. Antibodies bound to monomeric Aβ fragments were non-toxic to cultured neurons, while antibodies to other oligomeric proteins: hamster polyomavirus major capsid protein, human metapneumovirus nucleocapsid protein, and measles virus nucleocapsid protein, strongly potentiated the neurotoxicity of their antigens. The neurotoxicity of antibody-antibody oligomeric antigen complexes was abolished by removal of the Fc region from the antibodies or by removal of microglia from cultures, and was accompanied by inflammatory activation and proliferation of the microglia in culture. In conclusion, we find that immune complexes formed by Aβ oligomers or other oligomeric/multimeric antigens and their specific antibodies can cause death and loss of neurons in primary neuronal-glial cultures via Fc-dependent microglial activation. The results suggest that therapies resulting in antibodies to oligomeric Aβ or oligomeric brain virus proteins should be used with caution or with suppression of microglial activation., (2013 International Society for Neurochemistry)

Background: The central molecule in the pathogenesis of Alzheimer's disease (AD) is believed to be a small-sized polypeptide - beta amyloid (Aβ) which has an ability to assemble spontaneously into oligomers. Various studies concerning therapeutic and prophylactic approaches for AD are based on the immunotherapy using antibodies against Aβ. It has been suggested that either active immunization with Aβ or passive immunization with anti-Aβ antibodies might help to prevent or reduce the symptoms of the disease. However, knowledge on the mechanisms of Aβ-induced immune response is rather limited. Previous research on Aβ1-42 oligomers in rat brain cultures showed that the neurotoxicity of these oligomers considerably depends on their size. In the current study, we evaluated the dependence of immunogenicity of Aβ1-42 oligomers on the size of oligomeric particles and identified the immunodominant epitopes of the oligomers., Results: Mice were immunized with various Aβ1-42 oligomers. The analysis of serum antibodies revealed that small Aβ1-42 oligomers (1-2 nm in size) are highly immunogenic. They induced predominantly IgG2b and IgG2a responses. In contrast, larger Aβ1-42 oligomers and monomers induced weaker IgG response in immunized mice. The monoclonal antibody against 1-2 nm Aβ1-42 oligomers was generated and used for antigenic characterization of Aβ1-42 oligomers. Epitope mapping of both monoclonal and polyclonal antibodies demonstrated that the main immunodominant region of the 1-2 nm Aβ1-42 oligomers is located at the amino-terminus (N-terminus) of the peptide, between amino acids 1 and 19., Conclusions: Small Aβ1-42 oligomers of size 1-2 nm induce the strongest immune response in mice. The N-terminus of Aβ1-42 oligomers represents an immunodominant region which indicates its surface localization and accessibility to the B cells. The results of the current study may be important for further development of Aβ-based vaccination and immunotherapy strategies.

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.

Gardnerella vaginalis is considered a substantial player in the progression of bacterial vaginosis (BV). We analysed 17 G. vaginalis strains isolated from the genital tract of women diagnosed with BV to establish a potential link between genotypes/biotypes and the expression of virulence factors, vaginolysin (VLY) and sialidase, which are assumed to play a substantial role in the pathogenesis of BV. Amplified ribosomal DNA restriction analysis revealed two G. vaginalis genotypes. Gardnerella vaginalis isolates of genotype 2 appeared more complex than genotype 1 and were subdivided into three subtypes. Biochemical typing allowed us to distinguish four different biotypes. A great diversity of the level of VLY production among the isolates of G. vaginalis may be related to a different cytotoxicity level of the strains. We did not find any correlation between VLY production level and G. vaginalis genotype/biotype. In contrast, a link between G. vaginalis genotype and sialidase production was established. Our findings on the diversity of VLY expression level in different clinical isolates and linking sialidase activity with the genotype of G. vaginalis could help to evaluate the pathogenic potential of different G. vaginalis strains., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Protein engineering provides an opportunity to generate new immunogens with desired features. Previously, we have demonstrated that hamster polyomavirus major capsid protein VP1-derived virus-like particles (VLPs) are highly immunogenic and can be employed for the insertion of foreign epitopes at certain surface-exposed positions. In the current study, we have designed pseudotype VLPs consisting of an intact VP1 protein and VP2 protein fused with the target antigen--cellular marker p16(INK4A)--at its N terminus. Both proteins coexpressed in yeast were self-assembled to pseudotype VLPs harbouring the inserted antigen on the surface. The pseudotype VLPs were used for generation of antibodies against p16(INK4A) that represents a potential biomarker for cells transformed by high-risk human papillomavirus (HPV). The pseudotype VLPs induced in immunized mice a strong immune response against the target antigen. The antisera raised against pseudotype VLPs showed specific immunostaining of p16(INK4A) protein in malignant cervical tissue. Spleen cells of the immunized mice were used to generate monoclonal antibodies against p16(INK4A) protein. The specificity of antibodies was proven by the immunostaining of HPV-transformed cells. In conclusion, the current study demonstrates the potential of pseudotype VLPs with inserted target antigen as a new type of immunogens to generate antibodies of high diagnostic value.

Cohort studies are of great importance in defining the mechanism responsible for the development of allergy-associated diseases, such as atopic dermatitis, allergic asthma, and allergic rhinoconjunctivitis. Although these disorders share genetic and environmental risk factors, it is still under debate whether they are linked or develop sequentially along an atopic pathway. The current study was aimed to determine the pattern of allergy sensitization in the Lithuanian birth cohort "Alergemol" (n = 1558) established as a part of the multicenter European birth cohort "EuroPrevall". Early sensitization to food allergens in the "Alergemol" birth cohort was analysed. The analysis revealed 1.3% and 2.8% of symptomatic-sensitized subjects at 6 and 12 months of age, respectively. The sensitization pattern in response to different allergens in the group of infants with food allergy symptoms was studied using allergological methods in vivo and in vitro. The impact of maternal and environmental risk factors on the early development of food allergy in at 6 and 12 months of age was evaluated. Our data showed that maternal diet, diseases, the use of antibiotics, and tobacco smoke during pregnancy had no significant impact on the early sensitization to food allergens. However, infants of atopic mothers were significantly more often sensitized to egg as compared to the infants of nonatopic mothers.

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.

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.

Puumala virus (PUUV) is the predominant hantavirus species in Germany causing large numbers of mild to moderate cases of haemorrhagic fever with renal syndrome (HFRS). During an outbreak in South-East Germany in 2004 a novel PUUV subtype designated Bavaria was identified as the causative agent of HFRS in humans [1]. Here we present a molecular characterization of this PUUV strain by investigating novel partial and almost entire nucleocapsid (N) protein-encoding small (S-) segment sequences and partial medium (M-) segment sequences from bank voles (Myodes glareolus) trapped in Lower Bavaria during 2004 and 2005. Phylogenetic analyses confirmed their classification as subtype Bavaria, which is further subdivided into four geographical clusters. The entire N protein, harbouring an amino-terminal hexahistidine tag, of the Bavarian strain was produced in yeast Saccharomyces cerevisiae and showed a slightly different reactivity with N-specific monoclonal antibodies, compared to the yeast-expressed N protein of the PUUV strain Vranica/Hällnäs. Endpoint titration of human sera from different parts of Germany and from Finland revealed only very slight differences in the diagnostic value of the different recombinant proteins. Based on the novel N antigen indirect and monoclonal antibody capture IgG-ELISAs were established. By using serum panels from Germany and Finland their validation demonstrated a high sensitivity and specificity. In summary, our investigations demonstrated the Bavarian PUUV strain to be genetically divergent from other PUUV strains and the potential of its N protein for diagnostic applications.

In contrast to most RNA viruses, influenza viruses replicate their genome in the nucleus of infected cells. As a result, newly-synthesized vRNA genomes, in the form of viral ribonucleoprotein complexes (vRNPs), must be exported to the cytoplasm for productive infection. To characterize the composition of vRNP export complexes and their interplay with the nucleus of infected cells, we affinity-purified tagged vRNPs from biochemically fractionated infected nuclei. After treatment of infected cells with leptomycin B, a potent inhibitor of Crm1-mediated export, we isolated vRNP export complexes which, unexpectedly, were tethered to the host-cell chromatin with very high affinity. At late time points of infection, the cellular export receptor Crm1 also accumulated at the same regions of the chromatin as vRNPs, which led to a decrease in the export of other nuclear Crm1 substrates from the nucleus. Interestingly, chromatin targeting of vRNP export complexes brought them into association with Rcc1, the Ran guanine exchange factor responsible for generating RanGTP and driving Crm1-dependent nuclear export. Thus, influenza viruses gain preferential access to newly-generated host cell export machinery by targeting vRNP export complexes at the sites of Ran regeneration.

Background & Aims: Interactions between genetic and environmental factors are believed to be involved in onset and initiation of inflammatory bowel disease. We analyzed the interaction between gastrointestinal mucosal microbiota and host genes in twin pairs discordant for ulcerative colitis (UC) to study the functional interaction between microbiota and mucosal epithelium., Methods: Biopsy were collected from sigmoid colon of UC patients and their healthy twins (discordant twin pairs) and from twins without UC. Microbiota profiles were determined from analysis of 16S ribosomal DNA libraries; messenger RNA profiles were determined by microarray analysis., Results: Patients with UC had dysbiotic microbiota, characterized by less bacterial diversity and more Actinobacteria and Proteobacteria than that of their healthy siblings; healthy siblings from discordant twins had more bacteria from the Lachnospiraceae and Ruminococcaceae families than twins who were both healthy. In twins who were both healthy, 34 mucosal transcripts correlated with bacterial genera, whereas only 25 and 11 correlated with bacteria genera in healthy individuals and their twins with UC, respectively. Transcripts related to oxidative and immune responses were differentially expressed between patients with UC and their healthy twins., Conclusions: The transcriptional profile of the mucosa appears to interact with the colonic microbiota; this interaction appears to be lost in colon of patients with UC. Bacterial functions, such as butyrate production, might affect mucosal gene expression. Patients with UC had different gene expression profiles and lower levels of biodiversity than their healthy twins, as well as unusual aerobic bacteria. Patients with UC had lower percentages of potentially protective bacterial species than their healthy twins., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Monoclonal antibodies are important tools for various applications in hantavirus diagnostics. Recently, we generated Puumala virus (PUUV)-reactive monoclonal antibodies (mAbs) by immunisation of mice with chimeric polyomavirus-derived virus-like particles (VLPs) harbouring the 120-amino-acid-long amino-terminal region of the PUUV nucleocapsid (N) protein. Here, we describe the generation of two mAbs by co-immunisation of mice with hexahistidine-tagged full-length N proteins of Sin Nombre virus (SNV) and Andes virus (ANDV), their characterization by different immunoassays and comparison with the previously generated mAbs raised against a segment of PUUV N protein inserted into VLPs. All of the mAbs reacted strongly in ELISA and western blot tests with the antigens used for immunization and cross-reacted to varying extents with N proteins of other hantaviruses. All mAbs raised against a segment of the PUUV N protein presented on chimeric VLPs and both mAbs raised against the full-length AND/SNV N protein reacted with Vero cells infected with different hantaviruses. The reactivity of mAbs with native viral nucleocapsids was also confirmed by their reactivity in immunohistochemistry assays with kidney tissue specimens from experimentally SNV-infected rodents and human heart tissue specimens from hantavirus cardiopulmonary syndrome patients. Therefore, the described mAbs represent useful tools for the immunodetection of hantavirus infection.

Vaginolysin (VLY) is a protein toxin released by Gardnerella vaginalis. VLY belongs to the group of cholesterol-dependent cytolysins (CDCs). We have generated a panel of novel monoclonal antibodies (MAbs) against VLY. For the generation of MAbs, we have used recombinant VLY expressed in Escherichia coli. The functional activity of recombinant VLY was confirmed by an in vitro hemolytic assay using human erythrocytes. The MAbs raised against recombinant VLY were reactive with VLY from G. vaginalis both by Western blot and ELISA. The cross-reactivity of MAbs with other CDCs was investigated. For this purpose, recombinant cytolysins perfringolysin, listeriolysin, intermedilysin, pneumolysin and streptolysin were expressed in E. coli. The MAbs were specific exclusively to VLY and did not react with other CDCs. All MAbs were studied for the ability to neutralize hemolytic activity of VLY in vitro and several neutralizing MAbs were identified. The MAb produced by clone 9B4 showed the most potent neutralizing activity. The epitope for this MAb was localized near the N-terminus of VLY, between amino acid (aa) residues 112 and 268. The region recognized by the neutralizing MAb 9B4 includes the conserved motif (VAARMQYD, aa 189-196) supposed to be involved in VLY oligomerization. Selected MAbs were employed to develop a sandwich ELISA for VLY quantification. The MAb-based immunoassay was suitable for the detection of VLY in the cultures of G. vaginalis. In conclusion, the MAbs described in the current study may be useful for structural and functional studies of VLY as well as immunodetection of VLY in biological specimens., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Aim: To investigate the frequency of NOD2, IL23R and ATG16L1 genetic variants in a case-control panel for inflammatory bowel disease (IBD) from Lithuania., Methods: One hundred and eighty unrelated IBD patients [57 Crohn's disease (CD) and 123 ulcerative colitis (UC)] and 186 healthy controls were genotyped for the following known genetic susceptibility variants: NOD2 - Arg702Trp (rs2066844), Gly908Arg (rs2066845) and Leu1007insC (rs2066847), as well as IL23R - Arg381Gln (rs11209026) and ATG16L1 - Thr300Ala (rs2241880)., Results: The effect that carriership of at least one NOD2 risk allele predisposes to CD was replicated in the Lithuanian population (41.1% CD vs 16.9% controls, P = 2 x 10(-4), OR = 3.48, 95% CI: 1.81-6.72). In the allelic single marker analysis, Leu1007insC was strongly associated with CD (21.4% CD vs 4.7% controls, P = 3.687 x 10(-8), OR = 5.54, 95% CI: 2.85-10.75). Neither the other two NOD2 variants, nor the known variants in IL23R and ATG16L1 were found to be risk factors for CD, UC or IBD. However, our relatively small study population was underpowered to demonstrate such weak to moderate disease associations., Conclusion: The results support a strong association between CD susceptibility and the Leu1007insC variant in NOD2 in the Lithuanian study population.

Menangle virus (MenV) is a member of the family Paramyxoviridae isolated in Australia that causes a reproductive disease of pigs. There is a need for specific immunoassays for virus detection to facilitate the diagnosis of MenV infection. Three novel monoclonal antibodies (MAbs) of the IgG1 subtype were generated by immunizing mice with recombinant yeast-expressed MenV nucleocapsid (N) protein self-assembled to nucleocapsid-like structures. One MAb was cross-reactive with recombinant N protein of Tioman virus. The epitopes of MAbs were mapped using a series of truncated MenV N proteins lacking the 29-119 carboxy-terminal amino acid (aa) residues. The epitopes of two MAbs were mapped to aa 430-460 of the MenV N protein, whilst the epitope of one MAb was mapped to residues 460-490. All three MAbs specifically recognized MenV, as indicated by immunohistochemical staining of brain tissue isolated from a field case (a stillborn piglet) of MenV infection. The MAbs against MenV N protein may be a useful tool for immunohistological diagnosis of MenV infection.

Tioman virus (TioV) was isolated from a number of pooled urine samples of Tioman Island flying foxes (Pteropus hypomelanus) during the search for the reservoir host of Nipah virus. Studies have established TioV as a new virus in the family Paramyxoviridae. This novel paramyxovirus is antigenically related to Menangle virus that was isolated in Australia in 1997 during disease outbreak in pigs. TioV causes mild disease in pigs and has a predilection for lymphoid tissues. Recent serosurvey showed that 1.8% of Tioman Islanders had neutralizing antibodies against TioV, indicating probable past infection. For the development of convenient serological tests for this virus, recombinant TioV nucleocapsid (N) protein was expressed in the yeast Saccharomyces cerevisiae. High yields of recombinant TioV N protein were obtained. Electron microscopy demonstrated that purified recombinant N protein self-assembled into nucleocapsid-like particles which were identical in density and morphology to authentic nucleocapsids from paramyxovirus-infected cells. Different size nucleocapsid-like particles were stable and readily purified by CsCl gradient ultracentrifugation. Polyclonal sera raised in rabbits after immunization with recombinant TioV N protein reacted reliably with TioV infected tissues in immunohistochemistry tests. It confirmed that the antigenic properties of yeast derived TioV N protein are identical to authentic viral protein.

Phenotypic variation between individuals, such as different mRNA expression levels, is influenced by genetic and nongenetic factors. Although several studies have addressed the interplay between genotypes and expression profiles in various model organisms in the recent years, the detailed and relative contributions of genetic and nongenetic factors in regulating plasticity of gene expression in barrier organs (e.g., skin, gut), which are exposed to continuous environmental challenge, are still poorly understood. Here we systematically monitored the level of genetic control over genomewide mRNA expression profiles in the healthy intestinal mucosa of 10 monozygotic and 10 dizygotic human twin pairs with microarray analyses. Our results, which are supported by real-time PCR and analysis of molecular phylogenetic conservation, indicate that genes associated with energy metabolism and cell and tissue regeneration pathways are under strong genetic control. Conversely, genes associated with immune response seem to be mainly controlled by exogenous factors. Further insights into the relative extent of genetic and nongenetic determinants of transcriptomal profiles and their influence on physiological and pathophysiological mechanisms are crucial to understanding the key role played by gene-environment interactions in health and disease.

Human parainfluenza virus type 3 (hPIV3) is a respiratory tract pathogen. The current study aimed to investigate immunodominant regions of hPIV3 nucleocapsid (N) protein by using monoclonal antibodies (mAbs) raised against recombinant N protein and human serum specimens from hPIV3-infected individuals. A panel of murine mAbs was generated following immunization with yeast-expressed hPIV3 N protein self-assembled to nucleocapsid-like particles. All mAbs recognized native viral nucleocapsids in hPIV3-infected cells as confirmed by an indirect immunofluorescence analysis. Antigenic sites recognized by the mAbs were mapped using recombinant overlapping N protein fragments. One major immunodominant site was identified in the carboxy-terminal region (amino acids [aa] 397-486) of hPIV3 N protein. Further analysis with smaller N protein fragments and a synthetic peptide revealed one linear epitope representing aa 437-446 of the N protein located within this antigenic site. This epitope was reactive with 46% of hPIV3 IgG-positive sera. These results suggest that the above antigenic site on the N protein is important in eliciting a humoral immune response against hPIV3.

Human parainfluenza virus types 1 and 3 (HPIV1 and HPIV3, respectively), members of the virus family Paramyxoviridae, are common causes of lower respiratory tract infections in infants, young children, the immunocompromised, the chronically ill, and the elderly. In order to synthesize recombinant HPIV1 and HPIV3 nucleocapsid proteins, the coding sequences were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of recombinant virus nucleocapsid proteins expression (20-24 mg l(-1) of yeast culture) was obtained. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. These structures contained host RNA, which was resistant to RNase treatment. The nucleocapsid proteins were stable in yeast and were easily purified by caesium chloride gradient ultracentrifugation. Therefore, this system proved to be simple, efficient and cost-effective, suitable for high-level production of parainfluenza virus nucleocapsids as nucleocapsid-like particles. When used as coating antigens in an indirect ELISA, the recombinant N proteins reacted with sera of patients infected with HPIV1 or 3. Serological assays to detect HPIV-specific antibodies could be designed on this basis.