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University of Manchester

1. Faqih, Layla Ahmed A. HIV neutralising antibody delivered by gene therapy with a hybrid Vaccinia/retrovirus or BacMam/retrovirus expression systems.

Degree: 2018, University of Manchester

Production of an effective vaccine and long-term treatment against human immunodeficiency virus (HIV) is elusive. In this thesis two different techniques were used in an attempt to insert HIV-neutralising monoclonal antibody (IgG1b12) sequences into a simian retroviral gene therapy agent pseudo-typed with vesicular stomatitis virus glycoprotein. Genes were encoded in either a poxvirus split-vector system or a baculovirus expression system. Both systems aim to produce replication incompetent pseudotyped virus like particles with simian origin. It is believed that the resulting non-infectious artificial lentivirus particles enter neighbouring cells, penetrate the nucleus and insert genetic material (the antibody gene) into the mammalian genome. The poxvirus split-vector system used in this project was a Vaccinia Retroviral Hybrid Vector, where recombinant modified vaccinia Ankara (MVA) is used to deliver the simian immunodeficiency virus (SIV) like particles into mammalian cells. However, the MVA system failed to express proteins of interest due to the instability of genetic insertion into the recombinant MVA genome. As an alternative strategy, two different BacMam systems were used to allow the production of VLPs, where mammalian cells are co-transduced with different recombinant baculoviruses (rBVs). VLPs were expressed either under the control of T7 RNA polymerase system or under the cytomegalovirus immediate early gene promoter. The results from the first BacMam system indicated that the T7 RNA polymerase system was not suitable to express detectable levels of proteins. The results indicated that translation of the produced mRNA by T7 promoter is inefficient, most likely because of the absence of RNA 5’ cap structure. To overcome this hybrid BV–T7 system limitation, a different system was developed. Proteins of interest from the second BacMam system were successfully expressed and detected using western blot analysis. VLPs were generated and visualised under electronic microscope. IgG1b12 was secreted in the supernatant of the transduced mammalian cells. Mammalian cells were successfully transduced with multiple different recombinant BVs simultaneously. The study establishes the feasibility of antibody gene transfer, and demonstrates the use of SIV like particles production to transduce mammalian cells using BacMam technology. The technique may have application for use as an immunotherapy of HIV infection as well providing long-acting prevention of HIV infection for those not yet infected with HIV. Advisors/Committee Members: KLAPPER, PAUL P, Vallely, Pamela, Klapper, Paul.

Subjects/Keywords: HIV; SIV; Genetherapy; Immunotherapy; Vaccination; Baculovirus; BacMam

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Faqih, L. A. A. (2018). HIV neutralising antibody delivered by gene therapy with a hybrid Vaccinia/retrovirus or BacMam/retrovirus expression systems. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:315559

Chicago Manual of Style (16th Edition):

Faqih, Layla Ahmed A. “HIV neutralising antibody delivered by gene therapy with a hybrid Vaccinia/retrovirus or BacMam/retrovirus expression systems.” 2018. Doctoral Dissertation, University of Manchester. Accessed September 23, 2020. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:315559.

MLA Handbook (7th Edition):

Faqih, Layla Ahmed A. “HIV neutralising antibody delivered by gene therapy with a hybrid Vaccinia/retrovirus or BacMam/retrovirus expression systems.” 2018. Web. 23 Sep 2020.

Vancouver:

Faqih LAA. HIV neutralising antibody delivered by gene therapy with a hybrid Vaccinia/retrovirus or BacMam/retrovirus expression systems. [Internet] [Doctoral dissertation]. University of Manchester; 2018. [cited 2020 Sep 23]. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:315559.

Council of Science Editors:

Faqih LAA. HIV neutralising antibody delivered by gene therapy with a hybrid Vaccinia/retrovirus or BacMam/retrovirus expression systems. [Doctoral Dissertation]. University of Manchester; 2018. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:315559

2. Faqih, Layla. HIV neutralising antibody delivered by gene therapy with a hybrid Vaccinia/retrovirus or BacMam/retrovirus expression systems.

Degree: PhD, 2018, University of Manchester

Production of an effective vaccine and long-term treatment against human immunodeficiency virus (HIV) is elusive. In this thesis two different techniques were used in an attempt to insert HIV-neutralising monoclonal antibody (IgG1b12) sequences into a simian retroviral gene therapy agent pseudo-typed with vesicular stomatitis virus glycoprotein. Genes were encoded in either a poxvirus split-vector system or a baculovirus expression system. Both systems aim to produce replication incompetent pseudotyped virus like particles with simian origin. It is believed that the resulting non-infectious artificial lentivirus particles enter neighbouring cells, penetrate the nucleus and insert genetic material (the antibody gene) into the mammalian genome. The poxvirus split-vector system used in this project was a Vaccinia Retroviral Hybrid Vector, where recombinant modified vaccinia Ankara (MVA) is used to deliver the simian immunodeficiency virus (SIV) like particles into mammalian cells. However, the MVA system failed to express proteins of interest due to the instability of genetic insertion into the recombinant MVA genome. As an alternative strategy, two different BacMam systems were used to allow the production of VLPs, where mammalian cells are co-transduced with different recombinant baculoviruses (rBVs). VLPs were expressed either under the control of T7 RNA polymerase system or under the cytomegalovirus immediate early gene promoter. The results from the first BacMam system indicated that the T7 RNA polymerase system was not suitable to express detectable levels of proteins. The results indicated that translation of the produced mRNA by T7 promoter is inefficient, most likely because of the absence of RNA 5’ cap structure. To overcome this hybrid BV–T7 system limitation, a different system was developed. Proteins of interest from the second BacMam system were successfully expressed and detected using western blot analysis. VLPs were generated and visualised under electronic microscope. IgG1b12 was secreted in the supernatant of the transduced mammalian cells. Mammalian cells were successfully transduced with multiple different recombinant BVs simultaneously. The study establishes the feasibility of antibody gene transfer, and demonstrates the use of SIV like particles production to transduce mammalian cells using BacMam technology. The technique may have application for use as an immunotherapy of HIV infection as well providing long-acting prevention of HIV infection for those not yet infected with HIV.

Subjects/Keywords: Vaccination; Baculovirus; BacMam; Genetherapy; SIV; HIV; Immunotherapy

Record DetailsSimilar RecordsGoogle PlusoneFacebookTwitterCiteULikeMendeleyreddit

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Faqih, L. (2018). HIV neutralising antibody delivered by gene therapy with a hybrid Vaccinia/retrovirus or BacMam/retrovirus expression systems. (Doctoral Dissertation). University of Manchester. Retrieved from https://www.research.manchester.ac.uk/portal/en/theses/hiv-neutralising-antibody-delivered-by-gene-therapy-with-a-hybrid-vacciniaretrovirus-or-bacmamretrovirus-expression-systems(b540de64-0484-461c-9ccf-a59ae3681f7f).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.756895

Chicago Manual of Style (16th Edition):

Faqih, Layla. “HIV neutralising antibody delivered by gene therapy with a hybrid Vaccinia/retrovirus or BacMam/retrovirus expression systems.” 2018. Doctoral Dissertation, University of Manchester. Accessed September 23, 2020. https://www.research.manchester.ac.uk/portal/en/theses/hiv-neutralising-antibody-delivered-by-gene-therapy-with-a-hybrid-vacciniaretrovirus-or-bacmamretrovirus-expression-systems(b540de64-0484-461c-9ccf-a59ae3681f7f).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.756895.

MLA Handbook (7th Edition):

Faqih, Layla. “HIV neutralising antibody delivered by gene therapy with a hybrid Vaccinia/retrovirus or BacMam/retrovirus expression systems.” 2018. Web. 23 Sep 2020.

Vancouver:

Faqih L. HIV neutralising antibody delivered by gene therapy with a hybrid Vaccinia/retrovirus or BacMam/retrovirus expression systems. [Internet] [Doctoral dissertation]. University of Manchester; 2018. [cited 2020 Sep 23]. Available from: https://www.research.manchester.ac.uk/portal/en/theses/hiv-neutralising-antibody-delivered-by-gene-therapy-with-a-hybrid-vacciniaretrovirus-or-bacmamretrovirus-expression-systems(b540de64-0484-461c-9ccf-a59ae3681f7f).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.756895.

Council of Science Editors:

Faqih L. HIV neutralising antibody delivered by gene therapy with a hybrid Vaccinia/retrovirus or BacMam/retrovirus expression systems. [Doctoral Dissertation]. University of Manchester; 2018. Available from: https://www.research.manchester.ac.uk/portal/en/theses/hiv-neutralising-antibody-delivered-by-gene-therapy-with-a-hybrid-vacciniaretrovirus-or-bacmamretrovirus-expression-systems(b540de64-0484-461c-9ccf-a59ae3681f7f).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.756895

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