Arch Virol (1992) [Suppl] 4: 133-136

© Springer-Verlag 1992

Deletion and insertion mutants of HBsAg particles U. Machein, R. Nagel, R. Prange, A. Clemen, and R. E. Streeck Institute for Medical Microbiology, University of Mainz, Federal Republic of Germany

Summary. We have found previously that hybrid 22-nm HBsAg particles can be created by insertion of short antigenic sequences into the HBV major envelope protein [1]. We have now performed a detailed deletion mutagenesis of the S gene of HBV encoding HBsAg. Deletion of the 51 C-terminal amino acids including most of the third and all of the fourth hydrophobic domain of the S protein did not affect particle assembly and secretion. However, secretion of 22-nm particles was abolished by minor deletions in the N-terminal region. Insertion and deletion/substitution mutants carrying a poliovirus epitope at the N-terminus and the preS1 region at the C-terminus have been characterized. Introduction During an HBV infection, large amounts of 22-nm empty envelope particles carrying the surface antigen (HBsAg) are secreted from the infected hepatocytes. Transfection of cell lines with the S gene encoding the 226-amino acid sequence of the major envelope protein yields similar particles which are secreted into the cell culture medium. Such 22-nm particles from serum, cell lines, or yeast are currently used as vaccine against hepatitis B. We have previously shown by insertion of a poliovirus epitope into the major envelope protein that 22-nm particles can be used as efficient immunogenic carriers of small heterologous antigenic sequences [1]. However, assembly and secretion of such particles were strongly affected by longer inserts [2]. We have now constructed deletion mutants of the S gene to identify regions dispensable for envelope assembly and to create space for the insertion of longer sequences. Results and discussion

In the small envelope protein of HBV two hydrophilic domains (30-80, 100-155) are separated by a strongly hydrophobic region which is essential

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for the transmembrane orientation [3]. The second hydrophilic domain carries the surface antigen. Leaving the essential hydrophobic and the antigenic region unchanged, we have deleted progressively larger sequences from the carboxy terminus (176-226) and shorter sequences of five to thirty amino acids at variable places near the aminoterminus (3-80). To determine whether the mutant proteins could still be assembled into particles and secreted, HBsAg was assayed in cellular lysates and in cellular supernatants using a monoclonal ELISA (Abbott) specific for HBsAg particles. HBsAg Synthesis (ELISA) Cell. Lysate I Medium

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