Impact of Truncation of the L1 Protein on VLP Capsids in Human Papilloma virus (HPV) Virus-Like Particles (VLP)

Abstract

Virus-like particles (VLPs) are nanoscale structures formed by the spontaneous self-assembly of a discrete numbers of protein subunits. Molecules of this type have begun to see broad usage in vaccines as well as therapeutics. These particles adopt native confirmations despite the absence of genetic material or accessory proteins. In most systems, intact VLPs exist in solution equilibrium with partially formed capsomeres and fully disassembled subunits. Several factors can influence this equilibrium, including pH, salt concentration, impurities, and posttranslational modifications such as truncations. Viruses have evolved various mechanisms to maintain structural stability including direct interactions with nucleic acids, accessory proteins, and lipid components. However, in the production of engineered VLPs, these structural safeguards are intentionally not in place. Because of this, instability or changes to the VLP during production can lead to modifications to the capsid and/or unnecessary yield loss. Previous testing on HPV has demonstrated that minor truncations of the major capsid proteins can form intact VLP capsids, but larger truncations can preclude their ability to self-assemble. This testing, however, did not determine the ability of truncated proteins to incorporate in the presence of native proteins, but analysis performed prior to the start of this project indicated this may be possible. The work presented here describes efforts to characterize the potential impact of truncations on HPV capsid formation. In this testing, protein separation analysis techniques like western blot and reverse phase high performance liquid chromatography (RP-HPLC) were used to determine the presence of truncations in purified products. Analytics such as size exclusion chromatography, multi-angle light scattering, and cIEF were then used to determine the state of capsids formed in the presence of these truncations. The data presented here demonstrate that intact VLPs can be formed from truncated HPV L1 proteins in the presence of intact L1. Additionally, these data suggest that the capsids generated with differing ratios of truncated proteins have varied biophysical properties including assembly state and charge. The presence of these alternative VLPs is linked with increased yield loss during manufacturing and may impact the functional activity of the final product. Increasing the understanding of these varied states may lead to improvements in VLP production results including increased product yields and long-term stability.