Characterization of hepatitis B virus surface and core antigens using a baculovirus expression system: Potential as carriers for foreign epitopes.

Abstract

Hepatitis B Virus surface (HBsAg) and core antigens (HBcAg), and a variety of derivatives, were expressed from recombinant baculoviruses in insect cells cultured in monolayer. It was initially established that HBsAg was secreted from insect cells at low efficiency. HBsAg self-assembles into 22 nm spherical lipoprotein particles. Full-length and (four) truncated HBsAg genes were fused in-frame to segments encoding portions of the rabies virus glycoprotein (rabies G). Three different rabies G segments were utilized in various combinations with HBsAg. The products were analyzed for their ability to form chimeric 22 nm particles for potential vaccine use. No fusion proteins were secreted, and all were found to be insoluble in nonionic detergent. Expression of some fusion proteins in mammalian COS cells did not alleviate the inhibition of secretion. To further investigate the assembly and secretion of HBsAg in insect cells, a variety of mutants and fusions were designed to test the importance of the first hydrophobic transmembrane (TM) domain (domain I) of HBsAg. Domain I appeared to increase the rate of membrane insertion at the endoplasmic reticulum (E.R.). Also, a negatively-charged residue immediately preceding domain I was important to particle secretion. It was found that domain I could be replaced by a heterologous TM domain provided the heterologous domain was proceeded by a negatively-charged residue. HBcAg self-assembles into 27 nm protein particles. HBcAg is released from infected insect cells by an unknown mechanism. The normally secretory HBV e antigen (HBeAg), expressed from the core plus precore gene, was not released. We found that processing of the precore signal peptide (SP) was defective in insect cells. This SP could be functionally replaced by the SP from honeybee prepromellitin. Proteolytic cleavage appeared to be occurring subsequent to mellitin SP cleavage, but its nature is unknown. Phosphorylation of HBcAg was normal, while phosphorylation of mellitin SP fusion proteins suggested restriction to fully-cleaved species. A segment of the precore domain which normally remains after SP cleavage during HBeAg synthesis was important to phosphorylation and cleavage patterns. Electron microscopy revealed no apparent mechanism for the release of HBcAg which was present as spherical particles in the cytoplasm. Mellitin SP fusion proteins were observed as spherical particles in distended rough E.R. A gene encoding three peptides derived from HIV-1 env and gag proteins was fused in-frame to both the N- and C-termini of HBcAg. Chimeric 27 nm particles were formed from both fusions, and were demonstrated to be cross-reactive to both HIV-1 p17 and gp120 proteins. Morphological and structural features suggested that the HIV-1 peptide was interior to the particles for the C-terminal fusion and exterior to the particle for the N-terminal fusion.