Characterization of influenza H1N1 Gag virus-like particles and extracellular vesicles co-produced in HEK-293SF
Autor: | Rénald Gilbert, Alina Venereo-Sanchez, Kelly M. Fulton, Amine Kamen, Olivier Henry, Susan M. Twine, Krisztina Koczka, Sven Ansorge, Parminder S. Chahal |
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Rok vydání: | 2019 |
Předmět: |
Proteomics
viruses 030231 tropical medicine Population Gene Products gag exosomes virus-like particles gag Gene Products Human Immunodeficiency Virus Cell Line Green fluorescent protein 03 medical and health sciences Influenza A Virus H1N1 Subtype 0302 clinical medicine Heat shock protein Influenza Human Humans Vaccines Virus-Like Particle 030212 general & internal medicine education Antigens Viral Host cell membrane education.field_of_study General Veterinary General Immunology and Microbiology Chemistry HEK 293 cells Public Health Environmental and Occupational Health virus diseases HEK-293SF Recombinant Proteins Microvesicles 3. Good health Cell biology HEK293 Cells Infectious Diseases Influenza Vaccines Cell culture Molecular Medicine influenza vaccine extracellular vesicles nLC-MS/MS Nucleolin |
Zdroj: | Vaccine. 37:7100-7107 |
ISSN: | 0264-410X |
Popis: | One of the concerns associated with the use of influenza virus-like particles (VLPs) as vaccine candidate or delivery system is their heterogeneous composition. Enveloped VLPs take up the host cell membrane at the budding site carrying out not only the viral antigenic proteins but also host proteins. In addition, the intrinsic nature of cells to produce membrane derived vesicles or extracellular vesicles (EVs), which have similar size to the VLPs, makes VLP purification process challenging. To further characterize these particles and identify proteins that are unique to each population, comparative proteomic analyses were completed to ultimately provide guidance for rational design of separation protocols. The VLPs were produced in suspension and serum free media by transient transfection of an inducible clone of a Human Embryonic Kidney (HEK-293SF) cells expressing HA and NA (H1N1/A/Puerto Rico/8/34), with a plasmid containing the gag gene of HIV-1 fused to GFP. EVs were produced independently from the non-transformed HEK-293SF cell line as a control for comparative studies. Both preparations were characterized for total nucleic acids and protein concentrations and extensively analyzed by nanoLC-MS/MS for their protein compositions. The proteomic analyses showed that aside from the recombinant VLP proteins, nucleolin was the most abundant host cell protein uniquely identified within VLPs (considering the MASCOT score value) while lactotransferrin and heat shock protein 90 were the most abundant proteins in EVs. Overall, this comparative study identifies potential target proteins as specific markers to guide VLP purification and discusses the biogenesis of enveloped particles released in HEK-293 cell suspension cultures emphasizing on the biological functions of host cell proteins identified. |
Databáze: | OpenAIRE |
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