Bolivian hemorrhagic fever (BHF) caused by Machupo virus (MACV) is a New World arenavirus having a reported mortality rate of 25-35%. The BHF starts with fever, followed by headache, and nausea which rapidly progresses to severe hemorrhagic phase within 7 days of disease onset. One of the key promoters for MACV viral entry into the cell followed by viral propagation is performed by the viral glycoprotein (GPC). GPC is post-transcriptionally cleaved into GP1, GP2 and a signal peptide. These proteins all take part in the viral infection in host body. Therefore, GPC protein is an ideal target for developing therapeutics against MACV infection. In this study, GPC protein was considered to design a multi-epitope, multivalent vaccine containing antigenic and immunogenic CTL and HTL epitopes. Different structural validations and physicochemical properties were analysed to validate the vaccine. Docking and molecular dynamics simulations were conducted to understand the interactions of the vaccine with various immune receptors. Finally, the vaccine was codon optimised in silico and along with which immune simulation studies was performed in order to evaluate the vaccine's effectiveness in triggering an efficacious immune response against MACV.

• MeSH
• americká hemoragická horečka * imunologie   prevence a kontrola   MeSH
• arenaviry Nového světa imunologie   MeSH
• epitopy imunologie   chemie   MeSH
• lidé MeSH
• simulace molekulární dynamiky MeSH
• simulace molekulového dockingu MeSH
• virové vakcíny * imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The COVID-19 pandemic caused by SARS-CoV-2 virus has created a global damage and has exposed the vulnerable side of scientific research towards novel diseases. The intensity of the pandemic is huge, with mortality rates of more than 6 million people worldwide in a span of 2 years. Considering the gravity of the situation, scientists all across the world are continuously attempting to create successful therapeutic solutions to combat the virus. Various vaccination strategies are being devised to ensure effective immunization against SARS-CoV-2 infection. SARS-CoV-2 spreads very rapidly, and the infection rate is remarkably high than other respiratory tract viruses. The viral entry and recognition of the host cell is facilitated by S protein of the virus. N protein along with NSP3 is majorly responsible for viral genome assembly and NSP12 performs polymerase activity for RNA synthesis. In this study, we have designed a multi-epitope, chimeric vaccine considering the two structural (S and N protein) and two non-structural proteins (NSP3 and NSP12) of SARS-CoV-2 virus. The aim is to induce immune response by generating antibodies against these proteins to target the viral entry and viral replication in the host cell. In this study, computational tools were used, and the reliability of the vaccine was verified using molecular docking, molecular dynamics simulation and immune simulation studies in silico. These studies demonstrate that the vaccine designed shows steady interaction with Toll like receptors with good stability and will be effective in inducing a strong and specific immune response in the body.Communicated by Ramaswamy H. Sarma.

• MeSH
• COVID-19 * prevence a kontrola   MeSH
• epitopy B-lymfocytární MeSH
• lidé MeSH
• pandemie prevence a kontrola   MeSH
• reprodukovatelnost výsledků MeSH
• SARS-CoV-2 metabolismus   MeSH
• simulace molekulového dockingu MeSH
• vakcíny proti COVID-19 MeSH
• virové vakcíny * chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

First step in developing an epitope-based vaccine is to predict peptide binding to the major histocompatibility complex (MHC) molecules. We performed computational analysis of unique available EgA31 sequence to locate appropriate antigenic propensity positions. T-cell epitopes with best binding affinity values of < 50% inhibitory concentration were selected using different available servers (Propred and IEDB). Peptides with 100% population coverage were selected. A DNA fragment corresponding to the furin linker enriched in Golgi apparatus was inserted sequentially between each epitope sequences in a synthetic DNA in order to cleave the chimeric protein into four separated peptides. Subsequently, the synthetic DNA was cloned into the pGEX4T-1 and pEGFP-N1 vectors and GST-ChEgA31 was expressed in E. coli strain BL21-DE3. The recombinant protein was detected by western blotting using an HRP-conjugated polyclonal anti-GST antibody. Fusion protein purified by affinity chromatography was used to raise antisera in rabbits. Results in agar gel immunodiffusion assay indicated induction of specific antibodies against multiepitope antigen in the tested rabbits. Cytokine assay was carried out in C57Bl/6 mice and the levels of cytokines were analyzed by sandwich ELISA. Interestingly, production of specific IFN-gamma was prominently higher in mice immunized with GST-ChEgA31 and pEGFP-ChEgA31 (650-1300 pg/ml) compared to control groups. No difference was observed in the level of IL-10 and IL-4 in immunized and GST control group. Challenge study with 500 live protoscolices of Echinococcus granulosus on immunized mice demonstrated protectivity level (50-60%). Based on our results, it appeared that the chimeric protein in the study was able to stimulate T-helper cell-1 (Th1) development and high level of cell mediated immunity in mice.

• MeSH
• antigeny helmintové imunologie   MeSH
• cytokiny genetika   metabolismus   MeSH
• DNA helmintů MeSH
• Echinococcus granulosus imunologie   MeSH
• ELISA MeSH
• epitopy T-lymfocytární genetika   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• králíci MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• regulace genové exprese imunologie   MeSH
• rekombinantní proteiny MeSH
• Th1 buňky imunologie   MeSH
• vakcíny imunologie   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

There is a raising demand for sensitive and high throughput MS based methods for screening purposes especially tailored to the detection of allergen contaminants in different food commodities. A challenging issue is represented by complex food matrices where the antibody-based kits commercially available might encounter objective limitations consequently to epitope masking phenomena due to a multitude of interfering compounds arising from the matrix. The performance of a method duly optimized for the extraction and simultaneous detection of soy, egg and milk allergens in a cookie food matrix by microHPLC-ESI-MS/MS, is herein reported. Thanks to the innovative configuration and the versatility shown by the dual cell linear ion trap MS used, the most intense and reliable peptide markers were first identified by untargeted survey experiment, and subsequently employed to design an ad hoc multi-target SRM method, based on the most intense transitions recorded for each selected precursor peptide. A sample extraction and purification protocol was optimized also including an additional step based on sonication, which resulted in a considerable improvement in the detection of milk allergen peptides. Data Dependent™ Acquisition scheme allowed to fill out a tentative list of potential peptide markers, which were further filtered upon fulfilling specific requirements. A total of eleven peptides were monitored simultaneously for confirmation purposes of each allergenic contaminant and the two most sensitive peptide markers/protein were selected in order to retrieve quantitative information. Relevant LODs were found to range from 0.1μg/g for milk to 0.3μg/g for egg and 2μg/g for soy.

• MeSH
• alergeny izolace a purifikace   MeSH
• analýza potravin metody   MeSH
• lidé MeSH
• limita detekce MeSH
• mléčné bílkoviny izolace a purifikace   MeSH
• mléko chemie   MeSH
• mouka analýza   MeSH
• peptidy analýza   MeSH
• proteiny ze sójových bobů izolace a purifikace   MeSH
• sójové potraviny analýza   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vaječné proteiny izolace a purifikace   MeSH
• vejce analýza   MeSH
• vibrace ultrazvukové MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Adoptive transfer of multivirus-specific T cell lines (MVST) is an advanced tool for immunotherapy of virus infections after hematopoietic stem cell transplantation (HSCT). Their preparation includes activation of donor virus-specific T cells by the mixture of oligopeptides derived from immunodominant antigens of several most harmful viruses, i.e. human cytomegalovirus (HCMV), polyomavirus BK (BKV), Epstein-Barr virus (EBV) and adenovirus (ADV). The aim of our study was to find out whether antigenic competition may have an impact on the expansion of virus-specific T cells. MVST from several heathy blood donors were generated using a pulse of overlapping oligopeptides (PepMixes™, derived from the IE1 and pp65 CMV antigens, VP1 and LTAG BKV antigens, BZLF1 and EBNA1 proteins of EBV and hexon protein from ADV) and short time culture in the presence of IL-7 and IL-4. The amount of virus-specific T cells in MVST was measured by ELISPOT and flow cytometry after re-stimulation with individual antigens. To evaluate antigenic competition, MVST were expanded either with a complete set of antigens or with the mixture lacking some of them. MVST expanded with the antigen mixture including CMV antigens contained a lower proportion of the T cells of other antigen specificities. A similar inhibitory effect was not apparent for EBV-derived peptides. The competitive effect of CMV antigens was most pronounced in MVST from CMV-seropositive donors and was mediated by both IE1 and pp65-derived peptides. Antigenic competition did not influence the phenotype of either CMV- or BKV-specific T cells. Both T cell populations had an effector memory phenotype (CD45RO+, CD27-, CCR7-). However, CMV-specific T cells preferentially consist of CD8+ while in BKV-specific T cells, the CD4+ phenotype predominated. Modification of the MVST manufacture protocol to prevent antigenic competition may increase the efficacy of MVST in therapy of BKV infections in HSCT recipients.

• MeSH
• Adenoviridae imunologie   patogenita   MeSH
• adenovirové infekce lidí imunologie   terapie   virologie   MeSH
• aktivace lymfocytů MeSH
• antigeny virové imunologie   MeSH
• cytomegalovirové infekce imunologie   terapie   virologie   MeSH
• Cytomegalovirus imunologie   patogenita   MeSH
• fenotyp MeSH
• imunodominantní epitopy MeSH
• imunoterapie adoptivní * MeSH
• infekce onkogenními viry imunologie   terapie   virologie   MeSH
• infekce virem Epsteina-Barrové imunologie   terapie   virologie   MeSH
• interakce hostitele a patogenu MeSH
• kultivované buňky MeSH
• lidé MeSH
• polyomavirové infekce imunologie   terapie   virologie   MeSH
• T-lymfocyty imunologie   transplantace   virologie   MeSH
• transplantace hematopoetických kmenových buněk škodlivé účinky   MeSH
• virové nemoci imunologie   terapie   virologie   MeSH
• virus BK imunologie   patogenita   MeSH
• virus Epsteinův-Barrové imunologie   patogenita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Tolerance toward tumor antigens, which are shared by normal tissues, have often limited the efficacy of cancer vaccines. However, wild type epitopes can be tweaked to activate cross-reactive T-cell clones, resulting in antitumor activity. The design of these analogs (i.e., heteroclitic peptides) can be difficult and time-consuming since no automated in silico tools are available. Hereby we describe the development of an in silico framework to improve the selection of heteroclitic peptides. The Epitope Discovery and Improvement System (EDIS) was first validated by studying the model antigen SIINFEKL. Based on artificial neural network (ANN) predictions, we selected two mutant analogs that are characterized by an increased MHC-I binding affinity (SIINFAKL) or increased TCR stimulation (SIIWFEKL). Therapeutic vaccination using optimized peptides resulted in enhanced antitumor activity and against B16.OVA melanomas in vivo. The translational potential of the EDIS platform was further demonstrated by studying the melanoma-associated antigen tyrosinase related protein 2 (TRP2). Following therapeutic immunization with the EDIS-derived epitope SVYDFFAWL, a significant reduction in the growth of established B16.F10 tumors was observed, suggesting a break in the tolerance toward the wild type epitope. Finally, we tested a multi vaccine approach, demonstrating that combination of wild type and mutant epitopes targeting both TRP2 and OVA antigens increases the antitumor response. In conclusion, by taking advantage of available prediction servers and molecular dynamics simulations, we generated an innovative platform for studying the initial sequences and selecting lead candidates with improved immunological features. Taken together, EDIS is the first automated algorithm-driven platform to speed up the design of heteroclitic peptides that can be publicly queried online.

• Publikační typ
• časopisecké články MeSH

The present study aimed to work out a peptide-based multi-epitope vaccine against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We predicted different B-cell and T-cell epitopes by using the Immune Epitopes Database (IEDB). Homology modeling of the construct was done using SWISS-MODEL and then docked with different toll-like-receptors (TLR4, TLR7, and TLR8) using PatchDock, HADDOCK, and FireDock, respectively. From the overlapped epitopes, we designed five vaccine constructs C1-C5. Based on antigenicity, allergenicity, solubility, different physiochemical properties, and molecular docking scores, we selected the vaccine construct 1 (C1) for further processing. Docking of C1 with TLR4, TLR7, and TLR8 showed striking interactions with global binding energy of -43.48, -65.88, and -60.24 Kcal/mol, respectively. The docked complex was further simulated, which revealed that both molecules remain stable with minimum RMSF. Activation of TLRs induces downstream pathways to produce pro-inflammatory cytokines against viruses and immune system simulation shows enhanced antibody production after the booster dose. In conclusion, C1 was the best vaccine candidate among all designed constructs to elicit an immune response SARS-CoV-2 and combat the coronavirus disease (COVID-19).

• Publikační typ
• časopisecké články MeSH

All renal transplant recipients should undergo a regular screening for BK viral (BKV) viremia. Gradual reduction of immunosuppression is recommended in patients with persistent plasma BKV viremia for 3 weeks after the first detection, reflecting the presence of probable or suspected BKV-associated nephropathy. Reduction of immunosuppression is also a primary intervention in biopsy proven nephropathy associated with BKV (BKVN). Thus, allograft biopsy is not required to treat patients with BKV viremia with stabilized graft function. There is a lack of proper randomised clinical trials recommending treatment in the form of switching from tacrolimus to cyclosporin-A, from mycophenolate to mTOR inhibitors or leflunomide, or the additive use of intravenous immunoglobulins, leflunomide or cidofovir. Fluoroquinolones are not recommended for prophylaxis or therapy. There are on-going studies to evaluate the possibility of using a multi-epitope anti-BKV vaccine, administration of BKV-specific T cell immunotherapy, BKV-specific human monoclonal antibody and RNA antisense oligonucleotides. Retransplantation after allograft loss due to BKVN can be successful if BKV viremia is definitively removed, regardless of allograft nephrectomy.

• MeSH
• imunosupresiva terapeutické užití   MeSH
• leflunomid terapeutické užití   MeSH
• lidé MeSH
• nemoci ledvin * farmakoterapie   MeSH
• polyomavirové infekce * diagnóza   farmakoterapie   MeSH
• transplantace ledvin * MeSH
• viremie diagnóza   farmakoterapie   MeSH
• virus BK * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Primary ciliary dyskinesia (PCD) is a genetic disorder associated with recurrent and chronic respiratory infections due to functional defects of motile cilia. In this study, we aimed to elucidate inflammatory and proliferative responses in PCD respiratory epithelium and evaluate the effect of Azithromycin (AZT) on these responses. Airway basal cells (BCs) were isolated from nasal samples of Wild-type (WT) epitope of healthy donors and PCD donors with bi-allelic mutations in DNAH5, DNAH11 and CCDC39. Cells were expanded in vitro and stimulated with either Lipopolysaccharide (LPS) or vehicle control. Post stimulation, cells were treated with either Azithromycin (AZT) or vehicle control. Cell proliferation was imaged in real-time. Separately, BCs from the same donors were expanded and grown at an air-liquid interface (ALI) to generate a multi-ciliated epithelium (MCE). Once fully mature, cells were stimulated with LPS, AZT, LPS + AZT or vehicle control. Inflammatory profiling was performed on collected media by cytokine Luminex assay. At baseline, there was a significantly higher mean production of pro-inflammatory cytokines by CCDC39 BCs and MCEs when compared to WT, DNAH11 and DNAH5 cells. AZT inhibited production of cytokines induced by LPS in PCD cells. Differences in cell proliferation were noted in PCD and this was also corrected with AZT treatment.

• MeSH
• azithromycin * farmakologie   MeSH
• cytokiny MeSH
• epitelové buňky MeSH
• lidé MeSH
• lipopolysacharidy toxicita   MeSH
• poruchy ciliární motility * MeSH
• proliferace buněk MeSH
• zánět farmakoterapie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

PURPOSE: About 15% of patients with common variable immunodeficiency (CVID) develop a small intestinal enteropathy, which resembles celiac disease with regard to histopathology but evolves from a distinct, poorly defined pathogenesis that has been linked in some cases to chronic norovirus (NV) infection. Interferon-driven inflammation is a prominent feature of CVID enteropathy, but it remains unknown how NV infection may contribute. METHODS: Duodenal biopsies of CVID patients, stratified according to the presence of villous atrophy (VA), IgA plasma cells (PCs), and chronic NV infection, were investigated by flow cytometry, multi-epitope-ligand cartography, bulk RNA-sequencing, and RT-qPCR of genes of interest. RESULTS: VA development was connected to the lack of intestinal (IgA+) PC, a T helper 1/T helper 17 cell imbalance, and increased recruitment of granzyme+CD8+ T cells and pro-inflammatory macrophages to the affected site. A mixed interferon type I/III and II signature occurred already in the absence of histopathological changes and increased with the severity of the disease and in the absence of (IgA+) PCs. Chronic NV infection exacerbated this signature when compared to stage-matched NV-negative samples. CONCLUSIONS: Our study suggests that increased IFN signaling and T-cell cytotoxicity are present already in mild and are aggravated in severe stages (VA) of CVID enteropathy. NV infection preempts local high IFN-driven inflammation, usually only seen in VA, at milder disease stages. Thus, revealing the impact of different drivers of the pathological mixed IFN type I/III and II signature may allow for more targeted treatment strategies in CVID enteropathy and supports the goal of viral elimination.

• MeSH
• atrofie komplikace   patologie   MeSH
• běžná variabilní imunodeficience * komplikace   imunologie   MeSH
• CD8-pozitivní T-lymfocyty MeSH
• imunoglobulin A MeSH
• infekce viry z čeledi Caliciviridae * imunologie   MeSH
• interferony MeSH
• lidé MeSH
• Norovirus * fyziologie   MeSH
• zánět komplikace   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH