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New chimeric antigen receptor (CAR) for therapy of human cytomegalovirus (HCMV) infection
Kroutilová, Marie ; Němečková, Šárka (advisor) ; Forstová, Jitka (referee)
Human cytomegalovirus (HCMV, Herpesviridae) can cause severe complications in the infected individuals undergoing hematopoietic stem cell transplantation. Nowadays, these patients are treated using antivirotics or HCMV-specific T cells derived from the seropositive graft donor. This study explored the possibility of redirecting HCMV-non-specific T cells from a seronegative donor towards HCMV-infected cells via chimeric antigen receptor (CAR), i.e. artificially designed T cell receptor. Viral glycoprotein B (gB) has been selected as a target for this receptor. Published sequence of a single chain variable fragment of a human antibody was used for the design of the CAR against gB (gBCAR). After the verification of production and surface localization in cell lines, gBCAR was being introduced into human T cells via lentiviral vectors. Human fetal lung fibroblasts (LEP) infected with HCMV were used as target cells after the expression of gB at their surface was demonstrated. gBCAR functionality was evaluated by the incubation of modified T cells with infected cells and subsequent analysis of media for IFNγ concentration, which was significantly higher in the setting of gBCAR T cells incubated with HCMV-LEP than in the control incubations. The results obtained show the specificity of gBCAR against...
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New chimeric antigen receptor (CAR) for therapy of human cytomegalovirus (HCMV) infection
Kroutilová, Marie ; Němečková, Šárka (advisor) ; Forstová, Jitka (referee)
Human cytomegalovirus (HCMV, Herpesviridae) can cause severe complications in the infected individuals undergoing hematopoietic stem cell transplantation. Nowadays, these patients are treated using antivirotics or HCMV-specific T cells derived from the seropositive graft donor. This study explored the possibility of redirecting HCMV-non-specific T cells from a seronegative donor towards HCMV-infected cells via chimeric antigen receptor (CAR), i.e. artificially designed T cell receptor. Viral glycoprotein B (gB) has been selected as a target for this receptor. Published sequence of a single chain variable fragment of a human antibody was used for the design of the CAR against gB (gBCAR). After the verification of production and surface localization in cell lines, gBCAR was being introduced into human T cells via lentiviral vectors. Human fetal lung fibroblasts (LEP) infected with HCMV were used as target cells after the expression of gB at their surface was demonstrated. gBCAR functionality was evaluated by the incubation of modified T cells with infected cells and subsequent analysis of media for IFNγ concentration, which was significantly higher in the setting of gBCAR T cells incubated with HCMV-LEP than in the control incubations. The results obtained show the specificity of gBCAR against...
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Molecules involved in immune evasion encoded within the US2-US11 genomic region of human cytomegalovirus
Kroutilová, Marie ; Němečková, Šárka (advisor) ; Lichá, Irena (referee)
Human cytomegalovirus (HCMV) from the family Herpesviridae causes lifelong latent infections in immunocompetent patients. HCMV encodes multiple molecules which interfere with host immune responses, thus helping the virus to escape from them and persist in the host. Inhibition of antigen presentation by MHC class i glycoproteins is an important immune evasion mechanism, which enables the virus to protect infected cells from recognition by CD8+ T lymphocytes. Most of the molecules inhibiting MHC class i presentation is encoded withing the US2 to US11 region of HCMV genome, including five glycoproteins (US2, US3, US6, US10 a US11) and one miRNA (US4-1). gpUS2 and gpUS3 are also able to block MHC class II antigen presentation and supress the response of CD4+ T lymphocytes. This thesis summarises current knowledge about the immune evasion molecules encoded within the US2-US11 genomic region of HCMV. Keywords: human cytomegalovirus (HCMV), immune evasion, MHC I, MHC II, US2, US3, US4-1, US6, US10, US11
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