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
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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