A modular protocol for virosome display of subunit vaccine antigens

Abstract

Antigen array increases B cell receptor (BCR) triggering and the titer of antibodies elicited by subunit vaccines. Here, we present a protocol for multivalent antigen display by synthetic virosomes: preformed liposomes bearing glycoprotein spike proteins from enveloped viruses. We describe how to customize lipid stoichiometry within preformed liposomes and attach user-defined antigens via covalent and/or non-covalent interactions. In addition to generating vaccine research tools, this protocol demonstrates how two-dimensional membrane array resolves and activates exceptionally weak but critical virus-receptor interactions.

Keywords: antibody; immunology; material sciences; molecular biology; protein expression and purification.

Graphical abstract

Figure 1

Ectodomain sequence of A/ New Caledonia/20/1999 hemagglutinin (HA) The HA ectodomain (cyan) fused to the trimeric Foldon of T4 fibritin (pink) followed by a 6× Histidine tag (yellow) with or without a C-terminal Cysteine.

Figure 2

Overview of the Tangential Flow Filtration (TFF) system

Figure 3

Three-Dimensional profile of WT HA and HA dRBS (A) WT HA is trimeric in solution as seen by size exclusion chromatography on a Superdex 200 increase column. (B) Insertion of an N-linked glycan at position 190 does not alter the 3-dimensional profile of dRBS HA as compared to WT.

Figure 4

Virosomes are isolated at the 2.5%–0% iodixanol interface after flotation in an iodixanol density gradient (A) The virosome fraction can be visualized by white light and then fractionated. (B) Fractionated gradient fractions are resolved by SDS PAGE and silver staining. Free unbound protein will not have the buoyancy to float in this gradient. (C) Visual representation of a virosome bearing HA dRBS.

Figure 5

Two dimensional array of HA in virosomes ‘activates’ the millimolar affinity sialic acid binding by this viral protein (A) Membrane attachment of WT HA (with lectin activity for sialyl-oligosaccharide intact) results in virosome agglutination; contrasts the trimeric in solution profile of WT HA as a soluble three-dimensional protein (see Figure 3). (B) Virosomes bearing dRBS HA (where an N-glycan is inserted at position 190 to block lectin activity) do not agglutinate, indicating that membrane array of HA ‘activates’ the otherwise weak affinity for sialyl-oligosaccharide, the primary receptor for influenza virus.