Hybrid large hepatitis B surface protein composed of two viral genotypes C and D induces strongly cross-neutralizing antibodies.

Hepatitis B virus (HBV) vaccination is known to effectively decrease the risk of HBV infection. However, several issues need to be addressed in order to develop an improved HBV vaccine. Although the HBV vaccine has been shown to be effective, this vaccine needs to be more efficacious in defined groups, including non-responders (i.e., individuals who do not develop a protective response even after vaccination) and in health care workers and travelers who require rapid protection. Furthermore, it has been reported that universal HBV vaccination has accelerated the appearance of vaccine-escape mutants resulting from the accumulation of mutations altering the "a" determinant of the hepatitis B surface (HBs) protein. To address these problems, we have been focusing on the large HBs (LHBs) protein, which consists of three domains: pre-S1, pre-S2, and S (in N- to C-terminal order). To enhance the immunogenicity of LHBs, we developed a yeast-derived hybrid LHBs (hy-LHBs) antigen composed of the LHBs proteins from two distinct genotypes (Genotypes C and D). The levels of antibodies induced by hy-LHBs immunization were high not only against S, but also against the pre-S1 and pre-S2 domains. Additionally, hy-LHBs immunization induced significantly more strongly cross-reactive neutralizing antibodies than did small HBs (SHBs) or LHBs of any genotype alone. These findings suggested that hy-LHBs might serve as a candidate antigen for use in an improved prophylactic HBV vaccine.

Neutralization of hepatitis B virus with vaccine-escape mutations by hepatitis B vaccine with large-HBs antigen.

Although the current hepatitis B (HB) vaccine comprising small-HBs antigen (Ag) is potent and safe, attenuated prophylaxis against hepatitis B virus (HBV) with vaccine-escape mutations (VEMs) has been reported. We investigate an HB vaccine consisting of large-HBsAg that overcomes the shortcomings of the current HB vaccine. Yeast-derived large-HBsAg is immunized into rhesus macaques, and the neutralizing activities of the induced antibodies are compared with those of the current HB vaccine. Although the antibodies induced by the current HB vaccine cannot prevent HBV infection with VEMs, the large-HBsAg vaccine-induced antibodies neutralize those infections. The HBV genotypes that exhibited attenuated neutralization via these vaccines are different. Here, we show that the HB vaccine consisting of large-HBsAg is useful to compensate for the shortcomings of the current HB vaccine. The combined use of these HB vaccines may induce antibodies that can neutralize HBV strains with VEMs or multiple HBV genotypes.

Intranasal vaccination with HBs and HBc protein combined with carboxyl vinyl polymer induces strong neutralizing antibody, anti-HBs IgA, and IFNG response.

Hepatitis B virus (HBV) infection causes acute and chronic hepatitis, which is a major public health concern worldwide. Immunization methods incorporating hepatitis B surface-small (HBs-S) antigen and hepatitis B core antigen (HBc) have been proposed as candidate therapeutic vaccines, but the elimination of existing HBV infection remains a challenge. To enhance the efficacy of HBs and HBc vaccination, we investigated HBs-large (HBs-L) as an immunogen, and carboxyl vinyl polymer (CVP) as an excipient. HBs-S or HBs-L, in combination with HBc antigen, was administered subcutaneously (without CVP) or intranasally (with or without CVP) for the evaluation of immune response in the tree shrew, which is considered to be a suitable small animal model of HBV infection. Immunization with HBs-L antigen by either route induced a rapid IgG response. Intranasal immunization with HBs-S or HBs-L and HBc formulated with CVP strongly induced neutralizing antibody activity, IgA response, and HBc-specific expression of the interferon gamma-encoding gene. These data indicated the potential of HBs-L and HBc intranasal immunization with CVP, not only as a therapeutic vaccine, but also as a prophylactic vaccine candidate.

Chapter 8 - Bio-nanocapsule-liposome conjugates for in vivo pinpoint drug and gene delivery.

A bio-nanocapsule (BNC) is an ~50-nm hepatitis B virus (HBV) subviral particle comprising HBV envelope L proteins and a lipid bilayer, and is synthesized in recombinant Saccharomyces cerevisiae. When BNCs are administered intravenously in a mouse xenograft model, they can accumulate specifically in human liver-derived tissues and enter cells efficiently by the HBV-derived human liver-specific infection machinery, localized at the outer-membrane pre-S region of the L protein. BNC specificity for the human liver can be altered to other tissues by substituting the pre-S region using targeting molecules (e.g., antibodies, lectins, cytokines). BNCs can spontaneously form complexes with liposomes (LPs) by the membrane fusogenic activity of the pre-S region. LPs containing various therapeutic materials (e.g., chemicals, proteins, DNA, RNA) can therefore be covered with BNCs to form an ~150-nm BNC-LP conjugate. BNC-LP conjugates injected intravenously can deliver incorporated materials to target tissues specifically and efficiently by utilizing the HBV-derived infection machinery. The stability of BNC-LP conjugates in the blood circulation is similar to that of PEGylated LPs. In this chapter, we describe the preparation and in vivo application of BNC-LP conjugates, and the potential of BNC-LP conjugates as in vivo pinpoint drug delivery systems.