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Post-transcriptional modification of mRNA molecules in viruses of the Poxviridae family
Jakešová, Kristýna ; Vopálenský, Václav (advisor) ; Šmahel, Michal (referee)
Post-transcriptional modifications of mRNA molecules in viruses belonging to the Poxviridae family, including the vaccinia virus, play a key role in regulating viral gene expression and also influence virus-host cell interactions. These modifications include the synthesis of a 7-methylguanosine cap, 2'-O-methylation, 3' polyadenylation of the mRNA strand, hydrolysis of the 7-methylguanosine cap, and 5' polyadenylation of the mRNA strand. Studying post-transcriptional or co-transcriptional mRNA modifications in Poxviridae viruses can contribute to a better understanding of viral pathogenesis and the development of new strategies for treating infections caused by viruses in this family. The aim of this bachelor's thesis is to summarize current knowledge of co-transcriptional or post-transcriptional mRNA modifications in Poxviridae viruses, with a special focus on vaccinia virus.
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Expression of sTGFbeta RII-Fc-Jun from recombinant vaccinia virus
Samková, Zuzana ; Němečková, Šárka (advisor) ; Španielová, Hana (referee)
Expression of sTGFbetaRII-Fc-Jun from recombinant vaccinia virus TGFß has a biphasic role in tumorigenesis. In early phases it acts as tumor sup-pressor. However, in late phases when cells have escaped selectively from the antimito-genic response of TGFß, it may act as a promoter of tumor progression and invasion. One way of control tumor formation and progression is blocking of TGFß signalling pathways in late phases of tumorigenesis. We have constructed recombinant vaccinia virus P13 expressing soluble TGFbeta type II receptor fused with the Fc fragment of IgG1 and with Jun fragment (sTbetaRII-Fc-Jun). This sTbetaRII-Fc-Jun is supposed to increase the effect of antitumor vaccinia virus vaccine expressing SigE7LAMP, which is investigated for the treatment of the HPV-16 associated cervical cancer. Binding of sTbetaRII-Fc-Jun to protein G were tested by SDS-PAGE and by im-munoblotting. We found that Jun fragment and sTbetaRII fragment do not block Fc bind-ing site for protein G. sTbetaRII-Fc-Jun was characterised using SDS-PAGE and immunoblot analysis. We observed that the amount of sTbetaRII-Fc-Jun was higher in cell supernatans of in-fected cells in comparison to cell lysates. In cell lysates we observed higher amount of sTbetaRII than sTbetaRII-Fc-Jun. The expression of sTbetaRII-Fc-Jun was stronger under...
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Experimental and clinically used vaccines based on vaccinia virus
Pilná, Hana ; Mělková, Zora (advisor) ; Šroller, Vojtěch (referee)
Vaccinia virus (VACV) is an enveloped DNA virus belonging in the Orthopoxviridae genus. It is a laboratory virus in which the natural host and exact origin remain unclear. However it is of great significance for human kind. First of all, different VACV strains were used for preparation of vaccines used in the smallpox eradication campaign. Even today a significant effort is made to prepare more efficient and safer vaccines against smallpox, namely because of still remaining concerns that variola virus - causative agent of smallpox - could be misused as a biological weapon. Advances in genetic engineering allowed use of VACV for additional purposes, namely as a vaccination and expression vector. VACV enables insertion of large pieces of foreign DNA into its genome and expression of this DNA in a host. Furthermore VACV replicates exclusively in a cytoplasm, decreasing a risk of incorporation of the viral DNA into the host genome. These and other features make VACV an ideal candidate as a vector for preparation of recombinant vaccines against various infectious and oncological diseases. This thesis provides a summary of both clinically used and experimental vaccines derived from VACV. Powered by TCPDF (www.tcpdf.org)
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Effects of the Interferon regulatory factor 3 on immune responses to vaccinia virus in the atopic organism
Pilná, Hana ; Mělková, Zora (advisor) ; Drbal, Karel (referee)
Vaccinia virus (VACV) is an enveloped DNA virus, member of the Orthopoxviridae genus. VACV genome size is about 200 kbp. This huge genome capacity allows VACV to encode a set of factors that are non-essential for virus replication and spread in vitro. While these factors are needed for interfering with host immune responses, VACV remains strongly immunogenic. Cell-mediated and humoral immune responses in atopic disorders are deregulated to a certain extent, leading to complications in case of infection or vaccination with vaccines based on replicating viruses, such as eczema vaccinatum caused by VACV. VACV effects on immune responses consist among others in the inhibition of expression of type I interferon (IFN) at various levels - for example in a specific inhibition of phosphorylation of the interferon regulatory factor-3 (IRF-3) via inhibition of the activity of TANK-binding kinase 1 (TBK 1) that normally phosphorylates IRF-3. Phosphorylation allows IRF-3 to translocate into the nucleus where it initiates transcription of IFNβ followed by induction of expression of IFN and interferon stimulated genes. Expression of these genes is shut down when IRF-3 activity is inhibited. To overcome this block, a recombinant VACV expressing murine IRF-3 under VACV p7.5 promotor (WR-IRF3) was generated....
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Experimental and clinically used vaccines based on vaccinia virus
Pilná, Hana ; Mělková, Zora (advisor) ; Šroller, Vojtěch (referee)
Vaccinia virus (VACV) is an enveloped DNA virus belonging in the Orthopoxviridae genus. It is a laboratory virus in which the natural host and exact origin remain unclear. However it is of great significance for human kind. First of all, different VACV strains were used for preparation of vaccines used in the smallpox eradication campaign. Even today a significant effort is made to prepare more efficient and safer vaccines against smallpox, namely because of still remaining concerns that variola virus - causative agent of smallpox - could be misused as a biological weapon. Advances in genetic engineering allowed use of VACV for additional purposes, namely as a vaccination and expression vector. VACV enables insertion of large pieces of foreign DNA into its genome and expression of this DNA in a host. Furthermore VACV replicates exclusively in a cytoplasm, decreasing a risk of incorporation of the viral DNA into the host genome. These and other features make VACV an ideal candidate as a vector for preparation of recombinant vaccines against various infectious and oncological diseases. This thesis provides a summary of both clinically used and experimental vaccines derived from VACV. Powered by TCPDF (www.tcpdf.org)
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Pathogenesis of eczema vaccinatum
Elsterová, Jana ; Mělková, Zora (advisor) ; Forstová, Jitka (referee)
Vaccinia virus (VACV) is primarily known as a vaccine against its relative variola virus, the causative agent of smallpox. In the seventies of the 20th century, the vaccination campaign with VACV led to eradication of smallpox. Consequently, vaccination of the general population was stopped. Currently, the vaccination was reintroduced, namely among army and healthcare professionals. However, vaccination with VACV is accompanied with a high incidence of vaccination-related complications, namely among immunocompromised individuals. One of the complications is eczema vaccinatum, occuring in patients with atopic dermatitis. The laboratory of Dr. Melkova has focused on development of a model of eczema vaccinatum in mice Nc/Nga and on studies of pathogenesis of this complication. The goal of my diploma thesis is to contribute to characterization of imunopathogenesis of eczema vaccinatum in mice Nc/Nga infected either with VACV strain Western Reserve (WR) or with a recombinant VACV with the integrated cDNA for IRF-3 (Interferon Regulatory Factor 3; WR-IRF3). IRF-3 regulates the expression of interferon type I in response to viral infection. This recombinant virus has been constructed in the laboratory of Dr. Melková. The objective of my work was to verify the expression of the integrated cDNA for IRF-3 and to...
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Recombinant vaccinia virus for cancer therapy, the analysis of biological and biochemical features.
Žůrková, Kamila ; Němečková, Šárka (advisor) ; Forstová, Jitka (referee) ; Trejbalová, Kateřina (referee)
151 8 SUMMARY Recombinant vaccinia virus has been used for elicitation of the immune response against expressed heterologous proteins which has led to protection of the host organisms against the agents producing that antigen (viruses, cancer cells). In our laboratory, we designed and evaluated several vaccines against cancer caused by human papillomavirus type 16 (HPV16). Vaccinia viruses derived from replication competent strain P13 or attenuated MVA were used for construction of recombinant viruses expressing HPV16-E7 in highly immunogenic fusion construct SigE7LAMP. Recombinant viruses were used both in prophylactic and therapeutic settings in mouse tumor models using TC-1 or TC-1/A9 cells. The genes encoding stimulatory cytokines GM-CSF or Flt3 ligand were inserted into the above viruses to support the immune system and to potentiate the anticancer response. Tumor microenvironment was modified using the recombinant viruses expressing both the E7 gene and soluble receptor for TGF-β which should decrease the inhibition of immune system caused by tumor TGF-β cytokine and elicit the response against tumor cells. Intratumoral or intraperitoneal administration of viruses enhanced anticancer response in mice, the viruses expressing Flt3 ligand induced the proliferation of E7- specific cytotoxic T lymphocytes....
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Expression of sTGFbeta RII-Fc-Jun from recombinant vaccinia virus
Samková, Zuzana ; Španielová, Hana (referee) ; Němečková, Šárka (advisor)
Expression of sTGFbetaRII-Fc-Jun from recombinant vaccinia virus TGFß has a biphasic role in tumorigenesis. In early phases it acts as tumor sup-pressor. However, in late phases when cells have escaped selectively from the antimito-genic response of TGFß, it may act as a promoter of tumor progression and invasion. One way of control tumor formation and progression is blocking of TGFß signalling pathways in late phases of tumorigenesis. We have constructed recombinant vaccinia virus P13 expressing soluble TGFbeta type II receptor fused with the Fc fragment of IgG1 and with Jun fragment (sTbetaRII-Fc-Jun). This sTbetaRII-Fc-Jun is supposed to increase the effect of antitumor vaccinia virus vaccine expressing SigE7LAMP, which is investigated for the treatment of the HPV-16 associated cervical cancer. Binding of sTbetaRII-Fc-Jun to protein G were tested by SDS-PAGE and by im-munoblotting. We found that Jun fragment and sTbetaRII fragment do not block Fc bind-ing site for protein G. sTbetaRII-Fc-Jun was characterised using SDS-PAGE and immunoblot analysis. We observed that the amount of sTbetaRII-Fc-Jun was higher in cell supernatans of in-fected cells in comparison to cell lysates. In cell lysates we observed higher amount of sTbetaRII than sTbetaRII-Fc-Jun. The expression of sTbetaRII-Fc-Jun was stronger under...
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