Here we present data showing oral immunogenicity of recombinant hepatitis B surface antigen (HBsAg) in preclinical animal trials. Mice fed transgenic HBsAg potato tubers showed a primary immune response (increases in HBsAg-specific serum antibody) that could be greatly boosted by intraperitoneal delivery of a single subimmunogenic dose of commercial HBsAg vaccine, indicating that plants expressing HBsAg in edible tissues may be a new means for oral hepatitis B immunization. However, attainment of such a goal will require higher HBsAg expression than was observed for the potatoes used in this study. We conducted a systematic analysis of factors influencing the accumulation of HBsAg in transgenic potato, including 5' and 3' flanking elements and protein targeting within plant cells. The most striking improvements resulted from (1) alternative polyadenylation signals, and (2) fusion proteins containing targeting signals designed to enhance integration or retention of HBsAg in the endoplasmic reticulum (ER) of plant cells.

The extent to which T cell-mediated immune surveillance is impaired in human cancer remains a question of major importance, given its potential impact on the development of generalized treatments for advanced disease where the highest degree of heterogeneity exists. Here we report the first global analysis of immune dysfunction in patients with advanced hepatocellular carcinoma (HCC). Using multi-parameter FACS analysis, we quantified the cumulative frequency of T regulatory cells (Tregs), exhausted CD4+ helper T cells, and myeloid-derived suppressor cells (MDSC) to gain concurrent views on the overall level of immune dysfunction in these inoperable patients. We documented augmented numbers of Tregs, MDSC, PD-1+ exhausted T cells and increased levels of immunosuppressive cytokines in HCC patients, compared to normal controls, revealing a network of potential mechanisms of immune dysregulation in HCC patients. In dampening T cell-mediated anti-tumor immunity, we hypothesized that these processes may facilitate HCC progression and thwart the efficacy of immunotherapeutic interventions. In testing this hypothesis, we demonstrated that combined regimens to deplete Tregs, MDSC, and PD-1+ T cells in advanced HCC patients restored production of granzyme B by CD8+ T cells, reaching levels observed in normal controls, and also modestly increased the number of IFN-γ producing CD4+ T cells. These clinical findings encourage efforts to restore T cell function in patients with advanced stage disease, by highlighting combined approaches to deplete endogenous suppressor cell populations that can also expand effector T cell populations.

Oral immunogenicity of recombinant hepatitis B surface antigen (HBsAg) derived from yeast (purified product) or in transgenic potatoes (uncooked unprocessed sample) was compared. An oral adjuvant, cholera toxin, was used to increase immune responses. Transgenic plant material containing HBsAg was the superior means of both inducing a primary immune response and priming the mice to respond to a subsequent parenteral injection of HBsAg. Electron microscopy of transgenic plant samples revealed evidence that the HBsAg accumulated intracellularly; we conclude that natural bioencapsulation of the antigen may provide protection from degradation in the digestive tract until plant cell degradation occurs near an immune effector site in the gut. The correlate of protection from hepatitis B virus infection is serum antibody titers induced by vaccination; the protective level in humans is 10 milliunits͞ml or greater. Mice fed HBsAg-transgenic potatoes produced HBsAg-specific serum antibodies that exceeded the protective level and, on parenteral boosting, generated a strong longlasting secondary antibody response. We have also shown the effectiveness of oral delivery by using a parenteral prime-oral boost immunization schedule. The demonstrated success of oral immunization for hepatitis B virus with an ''edible vaccine'' provides a strategy for contributing a means to achieve global immunization for hepatitis B prevention and eradication.