Characterization of influenza H1N1 Gag virus-like particles and extracellular vesicles co-produced in HEK-293SF.
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.
['Venereo-Sánchez|Alina|A|', 'Fulton|Kelly|K|', 'Koczka|Krisztina|K|', 'Twine|Susan|S|', 'Chahal|Parminder|P|', 'Ansorge|Sven|S|', 'Gilbert|Rénald|R|', 'Henry|Olivier|O|', 'Kamen|Amine|A|']
D000956:Antigens, Viral D002460:Cell Line D000067128:Extracellular Vesicles D015683:Gene Products, gag D057809:HEK293 Cells D006801:Humans D053118:Influenza A Virus, H1N1 Subtype D007252:Influenza Vaccines D007251:Influenza, Human D040901:Proteomics D011994:Recombinant Proteins D058425:Vaccines, Virus-Like Particle D054301:gag Gene Products, Human Immunodeficiency Virus
Virus-like particles Extracellular vesicles HEK-293SF Exosomes Influenza vaccine nLC-MS/MS