Structural Mechanism of Assembly and Maturation of the HBs Antigen

Abstract

Hepatitis B surface antigen (HBsAg) is overexpressed in great excess during hepatitis B (HBV) infection in the form of noninfectious (DNA‐free) subviral particles. These particles greatly outnumber mature virus particles (virions) and presumably function as decoys for the immune system to facilitate infection by the DNA‐containing virions. These properties are exploited in the current HBV vaccine, in which recombinant HBsAg is used as a potent antigen capable to induce a strong immune response and long‐term immunological memory. Several studies have implicated that HBsAg consist of a lipid bilayer with embedded HBs subunits [1‐3]. The particles appear as pleomorphic spheres with an average diameter of ~23 nm and an empty internal cavity. Particles produced for the vaccine contain monomeric HBs subunits that subsequently cross‐link via formation of disulfide bridges within the HBsAg particles. We employed cryo‐electron tomography to image individual HBsAg particles in their native environment at different times of maturation. Reconstructed 3D tomograms showed different kinds of HBsAg particles that could be classified into three groups: (i) small spherical particles of ~16 nm in diameter, (ii) spherical particles of ~25 nm in diameter with an internal cavity and (iii) elongated oval particles with an internal cavity of ~20 nm diameter and 25‐30 nm length. The electron‐dense shell of HBsAg particles is about 5 nm thick and likely represents the lipid bilayer. The shell contains short protrusions with higher density (Figure 1) that are spaced 5‐6 nm apart and likely represent lipid‐embedded HBs subunits. Parallel measurements of proton NMR spectra indicated significant structural changes in protein backbone and aliphatic side chains of lipids during HBsAg particles maturation. Those variations support a decreased flexibility and suggested more rigid structure of the lipid bilayer during HBsAg maturation [4]. We analyzed size distribution of HBsAg particles and densities corresponding to HBs subunits within the particles to assess the molecular architecture of particles during maturation. The results provide insights into how structural changes in the lipid bilayer and cross‐linking of HBs subunits affect the molecular architecture of HBsAg particles that is directly related to immunogenicity of the HBV vaccine. This research has been funded by and performed at Sanofi Pasteur.