The objective of this proposal is to assess the feasibility of using recombinant virus-like-particles (VLPs) to elicit neutralizing antibodies and prime CD4+/CD8+ T cells reactive with hepatitis B viral (HBV) antigens as candidate immunotherapeutics for chronic HBV infection. For this purpose, selected well- defined neutralizing B cell epitopes representing HBV envelope Pre-S (1) and (2) antigens are being inserted onto a species variant of the HBV core protein, namely the woodchuck hepatitis core antigen (WHcAg). Pre-S B cell epitopes were chosen because of their preferential expression on HBV virions. We have previously developed the WHcAg as a VLP vaccine carrier for a number of heterologous B and T cell epitopes and use in this particular application will permit circumvention of the T cell immune tolerance that exists in chronically infected patients. The WHO estimates that more than 400 million individuals are chronically infected with HBV and approximately 20-40% will develop serious complication such as cirrhosis, liver failure and hepatocellular carcinoma. Although a safe and efficacious preventative vaccine for HBV has been available for over 20 years, HBV infections continue (with more than 50 million HBV infections per year) to be a major health problem and no effective treatments for chronic infection exist. Antiviral drugs such as lamivudine, adefovir, entecavir and pegylated interferon-alpha have improved the therapeutic options for chronic HBV, but, their efficacy remains limited due to reactivation of HBV replication upon drug withdrawal. Because chronic HBV infection is an immune-mediated disease and adoptive transfer of immunity to HBV through bone marrow transplantation has achieved resolution of chronic HBV infection, vaccine-based immunotherapy has been suggested as a possible monotherapy or as a combination therapy with antiviral drugs. To this end a number of clinical trials have been conducted using the HBV envelope antigens (i.e., HBsAg, HBsAg- PreS(2) and HBsAg-PreS(2)-PreS(1) containing subviral particles) either singly or in combination delivered as proteins in adjuvant or as DNA constructs, all with rather disappointing results. The most important factor to explain the defective T cell responses to the HBsAgs in chronic HBV carriers is immune tolerance. To circumvent the obstacle of immune tolerance in HBV chronic carriers, we have chosen to construct hybrid VLPs consisting of HBsAg Pre-S neutralizing B cell epitopes inserted onto the WHcAg, which is approximately 66-68% homologous with the HBcAg. The WHcAg and the HBcAg are not crossreactive at the B cell level and, just as importantly for our purposes, are only partially crossreactive at the CD4+ T cell level based on unique WHcAg-specific T cell sites and shared WHcAg/HBcAg-specific T cell sites in regions of the WHcAg that are conserved between WHcAg and HBcAg. Therefore, CD4+ T cells specific for WHcAg-unique T cell sites will provide cognate T-B cell help for anti-PreS antibody production and will not be curtailed by immune tolerance to HBcAg-specific T cell sites. In fact, in preliminary studies in HBcAg-Tg mice, which are tolerant to HBcAg, immunization with hybrid WHcAg-PreS VLPs elicits equivalent high titer anti-PreS antibodies in wildtype and HBcAg-Tg mice. Another advantage of immunizing with a closely related species variant of the HBcAg is that CD4+ and possibly CD8+ T cell sites within the WHcAg that differ only marginally from the sites within the HBcAg (i.e., 1 or 2 amino acids) may elicit so-called """"""""bystander"""""""" T cell help and may also even break T cell tolerance of the HBV patient's HBcAg-specific T cells by virtue of higher avidity T cell crossreactivity. Historically, breaking T cell tolerance to self antigens has been accomplished by immunization with closely related species variant proteins (induction of experimental autoimmune arthritis, diabetes, encephalitis etc.). Specifically, in Aim 1 we propose to insert 3 HBsAg-PreS(1) and 1 HBsAg-PreS(2) neutralizing B cell epitopes onto the WHcAg VLP carrier and optimize the constructs based on assembly, yield, stability and immunogenicity.
In Aim 2 the therapeutic efficacy of the VLP-based vaccine candidates will be evaluated in a transgenic (Tg) mouse model of HBV replication, in which HBV replicates in hepatocytes at levels comparable to that in the infected livers of chronic HBV patients without evidence of cytopathology. The hybrid WHcAg-PreS VLP candidates have the potential to reduce HBV load in at least 3 ways: (1) anti-PreS antibodies should clear serum HBV through immune complexes;(2) WHc/HBcAg-crossreactive CD4+ T cells may reduce HBV replication in the liver through inflammatory cytokine production;and (3) WHc/HBcAg-crossreactive CD8+ T cells may reduce viral replication in the liver through cytokine production as well as through direct hepatocyte cytolysis. The ability of immunization with the WHcAg-PreS VLP candidates to reduce HBV load through all of these mechanisms will be monitored in the HBV-Tg mouse model (Aim 2). It is anticipated that the combination of these two technologies, the ability to produce hybrid-VLPs (VLP Biotech) and the murine model of chronic HBV infection (TSRI), will enable us to produce and test the therapeutic efficacy of at least 4 hybrid WHcAg-PreS VLPs in a two-year timeframe.
It is estimated by the WHO that worldwide more than 400 million people are chronically infected with the hepatitis B virus (HBV) and approximately 20-40% will develop serious complications such as cirrhosis, liver failure and hepatocellular carcinoma. Although a safe and effective preventative vaccine for HBV is available, the existing treatments for chronic infection are unsatisfactory for a number of reasons. This is a proposal to develop virus-like-particles (VLPs) capable of eliciting neutralizing anti-HBV antibodies and priming CD4+/CD8+ T cells reactive with HBV antigens as a candidate therapeutic vaccine for chronic HBV infection.
