The pandemic of COVID-19 caused by SARS-CoV-2 has made a worldwide health emergency. Despite the fact that current vaccines are readily available, several SARSCoV-2 variants affecting the existing vaccine are to be less effective due to the mutations in the structural proteins. Furthermore, the appearance of the new variants cannot be easily predicted in the future. Therefore, the attempts to construct new vaccines or to modify the current vaccines are still pivotal works for preventing the spread of the virus. In the present investigation, the computational analysis through immunoinformatics, molecular docking, and molecular dynamics (MD) simulation is employed to construct an effective vaccine against SARS-CoV2. The structural proteins of SARS-CoV2 are utilized to create a multiepitope-based vaccine (MEV). According to our findings presented by systematic procedures in the current investigation, the MEV construct may be able to trigger a strong immunological response against the virus. Therefore, the designed MEV could be a potential vaccine candidate against SARS-CoV-2, and also it is expected to be effective for other variants.

• MeSH
• COVID-19 * prevence a kontrola   MeSH
• epitopy B-lymfocytární chemie   genetika   MeSH
• epitopy T-lymfocytární chemie   genetika   MeSH
• imunogenicita vakcíny MeSH
• kvantitativní vztahy mezi strukturou a aktivitou MeSH
• lidé MeSH
• RNA virová MeSH
• SARS-CoV-2 * MeSH
• simulace molekulární dynamiky MeSH
• simulace molekulového dockingu MeSH
• subjednotkové vakcíny chemie   MeSH
• vakcíny proti COVID-19 MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Recent studies have shown a significant level of T cell immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in convalescent coronavirus disease 2019 (COVID-19) patients and unexposed healthy individuals. Also, SARS-CoV-2-reactive T memory cells occur in unexposed healthy individuals from endemic coronaviruses that cause the 'common cold.' The finding of the expression of adaptive SARS-CoV-2-reactive T memory cells in unexposed healthy individuals may be due to multiple cross-reactive viral protein targets following previous exposure to endemic human coronavirus infections. The opinion of the authors is that determination of protein sequence homologies across seemingly disparate viral protein libraries may provide epitope-matching data that link SARS-CoV-2-reactive T memory cell signatures to prior administration of cross-reacting vaccines to common viral pathogens. Exposure to SARS-CoV-2 initiates diverse cellular immune responses, including the associated 'cytokine storm'. Therefore, it is possible that the intact virus possesses a required degree of conformational matching, or stereoselectivity, to effectively target its receptor on multiple cell types. Therefore, conformational matching may be viewed as an evolving mechanism of viral infection and viral replication by an evolutionary modification of the angiotensin-converting enzyme 2 (ACE2) receptor required for SARS-CoV-2 binding and host cell entry. The authors propose that convalescent memory T cell immunity in individuals with mild or asymptomatic SARS-CoV-2 infection may result from an evolutionarily adapted immune response to coronavirus and the 'common cold'.

• MeSH
• angiotensin konvertující enzym 2 genetika   MeSH
• asymptomatické infekce * MeSH
• buněčná imunita genetika   MeSH
• COVID-19 krev   diagnóza   imunologie   virologie   MeSH
• epitopy T-lymfocytární genetika   imunologie   MeSH
• imunogenicita vakcíny MeSH
• imunologická paměť genetika   MeSH
• internalizace viru MeSH
• lidé MeSH
• molekulární evoluce MeSH
• nachlazení imunologie   prevence a kontrola   virologie   MeSH
• protilátky virové MeSH
• replikace viru genetika   imunologie   MeSH
• Rhinovirus genetika   imunologie   MeSH
• SARS-CoV-2 imunologie   patogenita   MeSH
• sekvenční homologie MeSH
• stupeň závažnosti nemoci MeSH
• T-lymfocyty - podskupiny imunologie   MeSH
• T-lymfocyty imunologie   MeSH
• virové proteiny genetika   imunologie   MeSH
• virové vakcíny aplikace a dávkování   imunologie   MeSH
• zkřížené reakce genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Adoptive T cell transfer has been shown to be an effective method used to boost tumor-specific immune responses in several types of malignancies. In this study, we set out to optimize the ACT protocol for the experimental treatment of prostate cancer. The protocol includes a pre-stimulation step whereby T cells were primed with autologous dendritic cells loaded with the high hydrostatic pressure-treated prostate cancer cell line, LNCaP. Primed T cells were further expanded in vitro with anti-CD3/CD28 Dynabeads in the WAVE bioreactor 2/10 system and tested for cytotoxicity. Our data indicates that the combination of pre-stimulation and expansion steps resulted in the induction and enrichment of tumor-responsive CD4(+) and CD8(+) T cells at clinically relevant numbers. The majority of both CD4(+) and CD8(+) IFN-γ producing cells were CD62L, CCR7 and CD57 negative but CD28 and CD27 positive, indicating an early antigen experienced phenotype in non-terminal differentiation phase. Expanded T cells showed significantly greater cytotoxicity against LNCaP cells compared to the control SKOV-3, an ovarian cancer line. In summary, our results suggest that the ACT approach together with LNCaP-loaded dendritic cells provides a viable way to generate prostate cancer reactive T cell effectors that are capable of mounting efficient and targeted antitumor responses and can be thus considered for further testing in a clinical setting.

• MeSH
• aktivace lymfocytů MeSH
• antigeny nádorové imunologie   MeSH
• bioreaktory MeSH
• dendritické buňky imunologie   MeSH
• epitopy T-lymfocytární imunologie   MeSH
• imunoterapie adoptivní metody   MeSH
• interferon gama biosyntéza   imunologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prostaty imunologie   MeSH
• nádory vaječníků imunologie   MeSH
• regulační T-lymfocyty imunologie   MeSH
• studie případů a kontrol MeSH
• T-lymfocyty - podskupiny imunologie   MeSH
• T-lymfocyty imunologie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Aurora kinase A (AURKA) is a centrosomal protein that is overexpressed in a number of human malignancies and can contribute to tumor progression. As we used this protein as a target of DNA immunization, we increased its immunogenicity by the addition of the PADRE helper epitope and decreased its potential oncogenicity by mutagenesis of the kinase domain. For in vitro analysis of induced immune responses in mice, we identified the Aurka(220-228) nonapeptide representing an H-2Kb epitope. As DNA vaccination against the Aurka self-antigen by a gene gun did not show any antitumor effect, we combined DNA immunization with anti-CD25 treatment that depletes mainly regulatory T cells. Whereas 1 anti-CD25 dose injected before DNA vaccination did not enhance the activation of Aurka-specific splenocytes, 3 doses administered on days of immunizations augmented about 10-fold immunity against Aurka. However, an opposite effect was found for antitumor immunity-only 1 anti-CD25 dose combined with DNA vaccination reduced tumor growth. Moreover, the administration of 3 doses of anti-CD25 antibody alone accelerated tumor growth. Analysis of tumor-infiltrating cells showed that 3 anti-CD25 doses not only efficiently depleted regulatory T cells but also activated helper T cells and CD3(-)CD25(+) cells. Next, we found that blockade of the PD-1 receptor initiated 1 week after the first immunization was necessary for significant inhibition of tumor growth with therapeutic DNA vaccination against Aurka combined with depletion of CD25 cells. Our results suggest that combined cancer immunotherapy should be carefully evaluated to achieve the optimal antitumor effect.

• MeSH
• aktivace lymfocytů MeSH
• antigeny CD279 antagonisté a inhibitory   MeSH
• aurora kinasa A genetika   imunologie   metabolismus   MeSH
• buňky - růstové procesy účinky léků   MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• DNA vakcíny MeSH
• epitopy T-lymfocytární genetika   imunologie   metabolismus   MeSH
• H-2 antigeny metabolismus   MeSH
• HEK293 buňky MeSH
• imunizace MeSH
• imunoterapie * MeSH
• kombinovaná terapie MeSH
• lidé MeSH
• monoklonální protilátky terapeutické užití   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• protinádorové vakcíny imunologie   MeSH
• receptor interleukinu-2 - alfa-podjednotka imunologie   MeSH
• regulační T-lymfocyty imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Our previous studies on intranasal tolerance induction demonstrated reduction of allergic responses with different allergen constructs. The underlying mechanisms varied depending on their conformation or size. OBJECTIVE: The aim of the present study was to compare the uptake of two structurally different allergen molecules within the respiratory tract following intranasal application. METHODS: The three-dimensional Bet v 1 (Bv1-Protein) and the T cell epitope peptide of Bet v 1 (Bv1-Peptide) were labelled with 5,6-Carboxyfluorescein (FAM) and their uptake was investigated in lung cells and cells of the nasal associated lymphoid tissue from naive and sensitised BALB/c mice. Phenotypic characterisation of FAM+ lung cells after antigen incubation in vitro and after intranasal application was performed by flow cytometry. Impact of Bv1-Protein and Bv1-Peptide on cytokine profiles and gene expression in vivo or in an alveolar epithelial type II (ATII) cell line were assessed in mono- and co-cultures with monocytes using ELISA and quantitative real-time PCR. RESULTS: Both antigens were taken up preferably by ATII-like cells (ATII-LCs) in naive mice, and by macrophages in sensitised mice. After intranasal application, Bv1-Peptide was taken up faster and more efficiently than Bv1-Protein. In vivo and in vitro experiments revealed that Bv1-Protein induced the transcription of thymic stromal lymphopoietin mRNA while Bv1-Peptide induced the transcription of IL-10 and MCP1 mRNA in ATII-LCs. CONCLUSION AND CLINICAL RELEVANCE: Both tested antigens were taken up by ATII-LCs under steady state conditions and induced different polarisation of the immune responses. These data may have an important impact for the generation of novel and more effective prophylactic or therapeutic tools targeting the respiratory mucosa.

• MeSH
• alergeny metabolismus   MeSH
• alergie MeSH
• antigeny rostlinné chemie   MeSH
• antigeny metabolismus   MeSH
• cytokiny metabolismus   MeSH
• dýchací soustava imunologie   MeSH
• epitelové buňky cytologie   MeSH
• epitopy T-lymfocytární chemie   MeSH
• epitopy chemie   MeSH
• fenotyp MeSH
• fluoresceiny chemie   MeSH
• imunitní systém MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• plicní alveoly cytologie   MeSH
• průtoková cytometrie MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Development of DNA vaccines against transcription factors SOX2 and NANOG that are necessary for self-renewal of cancer stem cells in various human tumors. Enhancement of immunogenicity of these vaccines by the addition of cell localization signals and sequences from the tetanus toxin that encode strong helper epitopes activating Th cells. Analysis of the effect of xenogeneic genes on vaccine efficacy. Reduction of potential oncogenicity of SOX2 and NANOG by the mutagenesis of nuclear localization signals and by the removal of their initiation codons in fusion genes. Detection of immune responses induced with the constructed DNA vaccines by in vitro tests and by observation of an anti-tumor effect of the vaccines in mouse tumor models using embryonal carcinoma cell lines.

Vývoj DNA vakcín proti transkripčním faktorům SOX2 a NANOG, které jsou nezbytné pro sebeobnovu nádorových kmenových buněk u různých typů lidských nádorů. Zvýšení imunogennosti těchto vakcín přidáním buněčných lokalizačních signálů a sekvencí z genu tetanového toxinu kódujících silné pomocné epitopy aktivující buňky Th. Analýza vlivu xenogenních genů na imunogennost vakcín. Snížení potenciální onkogennosti SOX2 a NANOG mutagenezí jaderných lokalizačních signálů a odstraněním jejich iniciačních kodonů u fúzních genů. Stanovení imunitních reakcí vyvolaných připravenými DNA vakcínami testy in vitro a sledováním protinádorového účinku na myších nádorových modelech s použitím buněčných linií získaných z embryonálních karcinomů.

• MeSH
• antigeny heterofilní MeSH
• DNA vakcíny MeSH
• embryonální karcinom MeSH
• epitopy T-lymfocytární MeSH
• experimentální nádory imunologie   MeSH
• imunogenetika MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• oktamerní transkripční faktory MeSH
• proteiny - lokalizační signály MeSH
• terapeutická ekvivalence MeSH
• transkripční faktory SOXB2 MeSH
• Check Tag
• myši MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• hematologie a transfuzní lékařství
• onkologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

The asparaginyl endopeptidase legumain that is overexpressed in M2-polarized tumor-associated macrophages has been identified as a suitable target for elimination of these cells supporting tumor progression. To enhance the efficacy of DNA immunization against legumain, we performed several modifications in this protein that could improve induction of immune responses. First, we mutated the RGD motif into GGD or RGG sequences. This alteration resulted in diminished maturation of legumain and impaired cellular localization. Then, as tolerance to self-antigens can be broken by the activation of CD4 T-cell help, we tried to enhance the immunogenicity of legumain by the insertion of a foreign helper epitope, namely the p30 epitope from the tetanus toxin. Finally, the 2 modifications were combined. After gene gun DNA immunization of C57BL/6 mice with these constructs, we identified the Lgmn111-119 CD8 T-cell epitope that binds to H-2D molecules. Furthermore, we showed that mutagenesis in the RGD motif significantly enhanced the immune response against legumain. The addition of the p30 helper epitope induced the specific production of IFN-γ by T cells, but did not significantly increase legumain-specific immunity activated after mutagenesis in the RGD motif which might be caused by simultaneous activation of a Th2 response demonstrated by the production of IL-4. However, the beneficial effect of the helper epitope on legumain-specific response was proved after the depletion of regulatory T cells by antibody against CD25 that preferentially stimulated Th1 immunity. The antitumor effect of the modified legumain gene was shown in the immunization against tumors induced by MK16 cells.

• MeSH
• aminokyselinové motivy genetika   MeSH
• biolistika MeSH
• buňky NIH 3T3 MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• cysteinové endopeptidasy genetika   metabolismus   MeSH
• DNA vakcíny * MeSH
• epitopy T-lymfocytární genetika   metabolismus   MeSH
• experimentální nádory imunologie   terapie   MeSH
• HEK293 buňky MeSH
• imunoterapie metody   MeSH
• interferon gama metabolismus   MeSH
• lidé MeSH
• makrofágy imunologie   MeSH
• mutace genetika   MeSH
• mutageneze cílená MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové biomarkery genetika   metabolismus   MeSH
• peptidové fragmenty genetika   metabolismus   MeSH
• protinádorové vakcíny * MeSH
• T-lymfocyty pomocné-indukující imunologie   MeSH
• tetanový toxin genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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

Human CMV infects between 50-85% of healthy individuals and can cause live-threatening infections in immunocompromised patients. Therefore, peptide vaccination is being developed as a promising immunotherapeutic approach for treatment of patients at risk of CMV disease. The enzymatically inactive toxoid of Bordetella adenylate cyclase (CyaA-AC(-)) was shown to be an efficient tool for delivery of peptide epitopes and stimulation of Ag-specific T-cell immune responses. We investigated here the capacity of two CyaA-AC(-) constructs to deliver epitopes derived from the CMV phosphoprotein pp65 for activation of human T cells in vitro. Expansion of γ-IFN-secreting CMV-specific CD8(+) T cells, as well as increase of total IFN-γ and TNF-α production by PBMCs from CMV-seropositive donors were observed after in vitro stimulation with CyaA-AC(-) constructs carrying CMV epitopes, whereas limited activation of immune response occurred with free peptides. The activation of immune response was confirmed by expansion of CMV-specific T-cell clones and anti-CMV cytotoxic effect of stimulated PBMCs. These data open the way to clinical evaluation of CyaA-AC(-) constructs as tools for detection and expansion of CMV-specific T-cell immune responses for diagnostic and immunotherapeutic applications against CMV-associated diseases.

• MeSH
• adenylátcyklasy genetika   imunologie   MeSH
• aktivace lymfocytů MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• Cytomegalovirus imunologie   MeSH
• epitopy T-lymfocytární imunologie   MeSH
• fosfoproteiny imunologie   MeSH
• lidé MeSH
• peptidové fragmenty genetika   imunologie   MeSH
• proteiny virové matrix imunologie   MeSH
• sekvence aminokyselin MeSH
• vakcíny proti cytomegalovirové infekci genetika   imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The adenylate cyclase toxin-haemolysin of Bordetella (CyaA) targets CD11b(+) myeloid phagocytes and translocates across their cytoplasmic membrane an adenylate cyclase (AC) enzyme that catalyses conversion of cytosolic ATP into cAMP. In parallel, CyaA acts as a cytolysin forming cation-selective pores, which permeabilize cell membrane and eventually provoke cell lysis. Using cytolytic activity, potassium efflux and patch-clamp assays, we show that a combination of substitutions within the pore-forming (E570Q) and acylation-bearing domain (K860R) ablates selectively the cell-permeabilizing activity of CyaA. At the same time, however, the capacity of such mutant CyaA to translocate the AC domain across cytoplasmic membrane into cytosol of macrophage cells and to elevate cellular cAMP concentrations remained intact. Moreover, the combination of E570Q+K860R substitutions suppressed the residual cytolytic activity of the enzymatically inactive CyaA/OVA/AC(-) toxoid on CD11b-expressing monocytes, while leaving unaffected the capacity of the mutant toxoid to deliver in vitro a reporter CD8(+) T cell epitope from ovalbumin (OVA) to the cytosolic pathway of dendritic cells for MHC class I-restricted presentation and induce in vivo an OVA-specific cytotoxic T cell response. CyaA, hence, employs a mechanism of AC enzyme domain translocation across cellular membrane that avoids passage across the cytolytic pore formed by toxin oligomers.

• MeSH
• adenylátcyklasový toxin genetika   metabolismus   MeSH
• biologické modely MeSH
• buněčná membrána metabolismus   MeSH
• cytotoxické T-lymfocyty imunologie   MeSH
• dendritické buňky imunologie   MeSH
• epitopy T-lymfocytární imunologie   MeSH
• kultivované buňky MeSH
• makrofágy imunologie   MeSH
• monocyty imunologie   MeSH
• mutageneze cílená MeSH
• mutantní proteiny genetika   metabolismus   MeSH
• myši MeSH
• ovalbumin imunologie   MeSH
• substituce aminokyselin MeSH
• transport proteinů MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH