Design and Assembly of VLP-Based Broadly Protective Flu Vaccines

Influenza A is a genus of contagious RNA viruses whose potential to cause severe illness makes it a major public health concern; globally, it is responsible for hundreds of thousands of deaths every year. Influenza A is capable of infecting humans using the hemagglutinin (HA) glycoprotein spikes on its viral envelope, which have two domains: a head domain that helps the virus attach to the cell membrane and a highly conserved stalk domain that enables membrane fusion. Existing influenza vaccinations that aim to reduce its spread elicit an antibody response primarily focused on the head domain, but the frequent emergence of novel viral strains through mutations in the HA head domain demands the use of numerous influenza strains in vaccines, as well as annual vaccination. The goal of this project is to demonstrate the design and assembly of a broadly protective flu vaccine using virus-like particle (VLP) technology. The VLP platform studied consisted of an engineered capsid protein core (MS2) that displayed HA on its surface by biotin-streptavidin affinity interactions. The use of multiple HA proteins on each VLP also made them polyvalent, potentially amplifying immune response. In addition to regularly oriented HA VLPs, inverted HA VLPs were assembled in an effort to elicit a broad immune response to the highly conserved stalk domain. Steric hindrance by the immunodominant HA head domains in regularly oriented HA makes inversion—the attachment of HA to the MS2 core by the head domain, with the stalk pointing outwards—a promising alternative to improve the HA stalk’s accessibility to immune cells. The VLPs were made by transforming E. Coli to express MS2, functionalizing it with biotin, producing HA in a mammalian expression system, and performing site-specific biotinylation of the AviTag at the C-terminus of the purified HA protein; in the case of VLPs displaying inverted HA, additional steps were necessary to express an HA-head-binding antibody fragment (Fab) to attach HA to the VLP in an inverted orientation. The VLP’s components were characterized using size exclusion chromatography (SEC) and SDS-PAGE. After assembly, SEC and dynamic light scattering (DLS) supported the successful assembly of VLPs with both regular and inverted HA. These results demonstrate the successful design of VLP-based vaccines with the potential to provide broad protection against seasonal influenza A strains; however, more research still needs to be done regarding these vaccines’ effectiveness in vivo.