High-Yield Expression and Purification of Recombinant Influenza Virus Proteins from Stably-Transfected Mammalian Cell Lines

Abstract

Influenza viruses infect millions of people each year, resulting in significant morbidity and mortality in the human population. Therefore, generation of a universal influenza virus vaccine is an urgent need and would greatly benefit public health. Recombinant protein technology is an established vaccine platform and has resulted in several commercially available vaccines. Herein, we describe the approach for developing stable transfected human cell lines for the expression of recombinant influenza virus hemagglutinin (HA) and recombinant influenza virus neuraminidase (NA) proteins for the purpose of in vitro and in vivo vaccine development. HA and NA are the main surface glycoproteins on influenza virions and the major antibody targets. The benefits for using recombinant proteins for in vitro and in vivo assays include the ease of use, high level of purity and the ability to scale-up production. This work provides guidelines on how to produce and purify recombinant proteins produced in mammalian cell lines through either transient transfection or generation of stable cell lines from plasmid creation through the isolation step via Immobilized Metal Affinity Chromatography (IMAC). Collectively, the establishment of this pipeline has facilitated large-scale production of recombinant HA and NA proteins to high purity and with consistent yields, including glycosylation patterns that are very similar to proteins produced in a human host.

Convergent antibody evolution and clonotype expansion following influenza virus vaccination

“…Enzyme-linked immunosorbent assays (ELISAs) were performed both on the human polyclonal and mAbs, the latter expressed in this study following the procedures previously utilized in our laboratory [ 20 – 22 ]. Briefly, plates were coated with 50 ng/well of recombinant hemagglutinnin (rHA) [ 23 , 24 ], blocked, antibodies were serially diluted starting from a 1:500 dilution for polyclonal sera, and from a concentration of 20 μg/mL for the mAbs. HA-specific IgG antibodies were detected, absorbance measured at 414 nm, and HA-specific IgG equivalent concentrations were calculated based on an 8-point standard curve generated using a human IgG reference protein (Athens Research and Technology, Athens, GA, USA).…”

Next Generation of Computationally Optimized Broadly Reactive HA Vaccines Elicited Cross-Reactive Immune Responses and Provided Protection against H1N1 Virus Infection

“…Soluble HA proteins were obtained by transfecting truncated HA genes that were cloned into the pcDNA3.1+ plasmid into HEK293T suspension cells as previously described [ 26 ]. The truncated HA genes were generated by replacing the transmembrane domain with a T4 fold-on domain, an Avitag, and a 6× His-tag for purification [ 26 ]. The concentration of the soluble HA proteins was determined by conventional bicinchoninic acid assay (BCA) according to the manufacture’s instruction.…”

Impaired memory B-cell recall responses in the elderly following recurrent influenza vaccination

“…Human PBMC (~5 x 10 6 live cells) were stained on ice for 30 min with fluorochrome conjugated rHA probes (180–350 pM) in 100 μL of staining buffer (PBS/2% fetal bovine serum [FBS]) as previously described [ 8 , 30 , 54 , 78 , 79 ]. Human PBMC were first treated with Fc receptor blocking solution (BioLegend, Dedham, MA, USA) then stained for 30 min on ice using titrated quantities of fluorescently conjugated monoclonal antibodies ( S1 Table ).…”