Physicochemical and immunological characterization of hepatitis B virus envelope particles exclusively consisting of the entire L (pre-S1 + pre-S2 + S) protein.
The hepatitis B virus (HBV) envelope (env) protein is composed of three regions; the 108- or 119-residue pre-S1 region involved in the direct interaction with hepatocytes, the 55-residue pre-S2 region associated with the polymerized albumin-mediated interaction, and the major 226-residue S protein region. Thus, to improve the immunogenic potency of conventional HB vaccines, development of a new vaccine containing the entire pre-S1 region in addition to pre-S2 and S is desired. We previously reported the efficient production of the HBV env L (pre-S1 + pre-S2 + S) protein in the recombinant yeast cells [J Biol Chem 267 (1992) 1953]. In this study, the HBV env L protein produced as nano-particles in yeast has been purified and characterized. By equilibrium sedimentation, an average molecular weight of L particle was estimated to be approximately 6.4 x 10(6), indicating that about 110 molecules of L proteins are assembled into an L particle. By atomic force microscopy in a moist atmosphere, the L particles were observed as large spherical particles with a diameter of 50-500 nm. The L particles were stable on short-time heating at a high temperature and long-time storage at a low temperature but rather unstable on repeated freezing and thawing and treatment with dithiothreitol. When immunized in mice, L particles elicited efficiently and simultaneously the anti-S, anti-pre-S2, and anti-pre-S1 antibodies. The ED(50) values in mice for the anti-S and anti-pre-S2 antibodies were similar to those elicited by the M (pre-S2 + S) particles. Furthermore, the anti-pre-S1 rabbit antibodies were found to recognize various segments of the pre-S1 region, including the pre-S1 (21-47) segment. These results show the high ability of L particles to induce all antibodies against HBV env proteins, hence promising the future application of L particles for the next generation HB vaccine.
Yamada
T
T
Department of Structural Molecular Biology, Institute of Scientific and Industrial Research (SANKEN), Osaka University, 8-1 Mihogaoka, 567-0047, Ibaraki, Japan.
Iwabuki
H
H
Kanno
T
T
Tanaka
H
H
Kawai
T
T
Fukuda
H
H
Kondo
A
A
Seno
M
M
Tanizawa
K
K
Kuroda
S
S
D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D004355:Drug Stability; D015686:Gene Products, env / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D006510:Hepatitis B Antibodies / Q000096:biosynthesis; D006514:Hepatitis B Surface Antigens / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D017325:Hepatitis B Vaccines / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D006515:Hepatitis B virus / Q000737:chemistry* / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D018625:Microscopy, Atomic Force; D017457:Receptors, Albumin / Q000737:chemistry / Q000276:immunology; D014461:Ultracentrifugation
D015686:Gene Products, env; D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D017325:Hepatitis B Vaccines; D017457:Receptors, Albumin