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" Influenza Vaccine Development: "
Makarkov, Alexander I.
Ward, Brian J.
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Latin Dissertation
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| Language of Document
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English
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| Record Number
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1109150
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| Doc. No
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TLpq2470595496
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| Main Entry
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Makarkov, Alexander I.
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Ward, Brian J.
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| Title & Author
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Influenza Vaccine Development:\ Makarkov, Alexander I.Ward, Brian J.
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| College
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McGill University (Canada)
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| Date
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2019
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| student score
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2019
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| Degree
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Ph.D.
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| Page No
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318
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| Abstract
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Influenza viruses cause significant morbidity and mortality worldwide. Vaccines are the best tools available to reduce the disease burden; however, vaccine effectiveness varies significantly between years, target populations and strains. Medicago Inc. has developed a highly efficient platform to produce plant-derived virus-like particle (VLP) vaccines bearing influenza hemagglutinin (HA) that have been shown to elicit strong humoral and CD4+ T cell responses in both pre-clinical and clinical studies. To better understand the immunogenicity of these vaccines, we studied the early interactions of VLPs with antigen-presenting cells (APC) in vitro. We demonstrated that VLPs bind to human monocytoid U-937 cells and monocyte-derived macrophages (MDMs) in a sialic acid-dependent manner. VLP attachment to the cell surface led to internalization, trafficking to acidic cell compartments and fusion of the VLP lipid envelope with endosomal membranes. Incubation of MDMs with VLPs bearing H1 (HA sequence from A/California/07/2009 (H1N1) strain) but not H5 (HA sequence from A/Indonesia/05/2005 (H5N1) induced proliferation of autologous lymphoid cells suggesting antigen processing by MDMs and stimulation of a memory T cell response. Pulse-exposure of MDMs with H1-VLPs resulted in a rapid and massive intracellular accumulation of HA that was driven by clathrin-mediated and clathrin-independent endocytosis as well as macropinocytosis/phagocytosis. The H1-VLPs endosomal distribution pattern suggested that HA delivered by VLP had entered both high-degradative late (supporting major histocompatibility complex (MHC) II-restricted antigen presentation) and low-degradative static early and/or recycling (favoring MHC I-restricted antigen cross-presentation) endosomal pathways. High-resolution tandem mass spectrometry identified a large number of HA-derived peptides associated with MHC I in the H1-VLP-treated MDMs. In addition, many host-derived MHC I peptides were identified in VLP-treated samples. These peptides were mainly processed by matrix metalloproteinases and cathepsins. The host proteins associated with these peptides were primarily involved in pathways modulating inflammation (i.e. stimulation and attenuation), innate and adaptive immunity, clathrin-mediated endocytosis, protein synthesis and endo-lysosomal degradation. Finally, tools we used while studying endosome-lysosome fusion led to the development of a novel serological assay for influenza based on 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD) fluorescence dequenching. This assay measures 'functional' influenza antibody titers, is free from observer bias and has the potential to be fully automated. In summary, we demonstrated that HA delivery to APCs in a form of plant-derived VLPs facilitates antigen uptake, endosomal processing, presentation and cross-presentation. These observations may help to explain the broad and cross-reactive immune responses generated by VLP vaccines. The new DiD fluorescence dequenching assay we developed may give new insights into the spectrum of antibodies produced in response to influenza infection or vaccination.
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Immunology
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Medicine
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