Enhancing the Yield and Quality of Influenza Virus-like Particles (VLPs) Produced in Insect Cells by Inhibiting Cytopathic Effects of Matrix Protein M2

Abstract

To provide broader protection and eliminate the need for annual update of influenza vaccines, biomolecular engineering of influenza virus-like particles (VLPs) to display more conserved influenza proteins such as the matrix protein M2 has been explored. However, achieving high surface density of full-length M2 in influenza VLPs has been left unrealized. In this study, we show that the ion channel activity of M2 induces significant cytopathic effects in Spodoptera f rugiperda (Sf9) insect cells when expressed using M2encoding baculovirus. These effects include altered Sf9 cell morphology and reduced baculovirus replication, resulting in impaired influenza protein expression and thus VLP production. On the basis of the function of M2, we hypothesized that blocking its ion channel activity could potentially relieve these cytopathic effects, and thus restore influenza protein expression to improve VLP production. The use of the M2 inhibitor amantadine indeed improves Sf9 cellular expression not only of M2 (∼3-fold), but also of hemagglutinin (HA) (∼7-fold) and of matrix protein M1 (∼3-fold) when coexpressed to produce influenza VLPs. This increased cellular expression of all three influenza proteins further leads to ∼2-fold greater VLP yield. More importantly, the quality of the resulting influenza VLPs is significantly improved, as demonstrated by the ∼2-fold, ∼50-fold, and ∼2-fold increase in the antigen density to approximately 53 HA, 48 M1, and 156 M2 per influenza VLP, respectively. Taken together, this study represents a novel approach to enable the efficient incorporation of full-length M2 while enhancing both the yield and quality of influenza VLPs produced by Sf9 cells.

Respiratory Viruses and Virus-like Particle Vaccine Development: How Far Have We Advanced?

“…However, when the M2 inhibitor amantadine was present, there was a substantial increase in the expression of all antigen expressions. HA, M1, and M2 expressions on these VLPs were enhanced sevenfold, threefold, and threefold, each, respectively [ 52 ].…”

Pseudotyping Improves the Yield of Functional SARS-CoV-2 Virus-like Particles (VLPs) as Tools for Vaccine and Therapeutic Development

“…Previously, influenza HA and M1 genes were cloned into the XbaI/HindIII and KpnI/XmaI site in plasmid pFastBac Dual, respectively, to create plasmid pFastBac Dual-H1M1 [73]. This plasmid served as the backbone for all pseudotyped SARS-CoV-2 S/Influenza HA fusion plasmids.…”

Influenza Virus Like Particles (VLPs): Opportunities for H7N9 Vaccine Development

“…Sf9 cells are usually maintained in a serum free, animal product-free medium. Some of the chemical compounds known as M2 inhibitors, such as amantadine-enhanced expression of HA protein in VLPs by seven-fold in Sf9 expression system [62]. However, the challenge for VLPs expressed using baculovirus expression system is that it is difficult to separate VLPs from the baculovirus expression vector during vaccine manufacturing.…”

“…To overcome manufacturing challenges, technological improvements to production of H7N9 VLPs in baculovirus-Sf9 insect cell cultures have been reported [110][111][112]. The yield and quality of influenza VLPs produced in insect cells could be improved by inhibiting cytopathic effects of the protein M2 [62]. The ion channel activity of M2 induces significant cytopathic effects in Sf9 insect cells.…”

“…This increased cellular expression of all three influenza proteins further led to ∼two-fold greater VLP yield. The quality of the resulting influenza VLPs was significantly improved, as demonstrated by the ∼two-fold, ∼50-fold, and ∼two-fold increase in the antigen density to approximately 53 HA, 48 M1, and 156 M2 per influenza VLP, respectively [62].…”