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.
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.
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.
The focus of the Children's Vaccine Initiative is to encourage the discovery of technology that will make vaccines more readily available to developing countries. Our strategy has been to genetically engineer plants so that they can be used as inexpensive alternatives to fermentation systems for production of subunit antigens. In this paper we report on the immunological response elicited in vivo by using recombinant hepatitis B surface antigen (rHBsAg) purified from transgenic tobacco leaves. The anti-hepatitis B response to the tobacco-derived rHBsAg was qualitatively similar to that obtained by immunizing mice with yeast-derived rHBsAg (commercial vaccine). Additionally, T cells obtained from mice primed with the tobacco-derived rHBsAg could be stimulated in vitro by the tobacco-derived rHBsAg, yeast-derived rHBsAg, and by a synthetic peptide that represents part of the a determinant located in the S region (139-147) of HBsAg. Further support for the integrity of the T-cell epitope of the tobacco-derived rHBsAg was obtained by testing the ability of the primed T cells to proliferate in vitro after stimulation with a monoclonal anti-idiotype and an anti-idiotype-derived peptide, both of which mimic the group-specific a determinant of HBsAg. In total, we have conclusively demonstrated that both B-and T-cell epitopes of HBsAg are preserved when the antigen is expressed in a transgenic plant.
Rationale: Previous studies from our laboratory have shown that peripheral blood mononuclear cells (PBMCs) from patients with chronic obstructive pulmonary disease (COPD) prone to exacerbations with nontypeable Haemophilus influenzae have impaired responses to lipoprotein P6. We hypothesized that an underlying immunosuppressive network could be responsible for the defective antibacterial immunity observed in these patients. We Objectives: We performed an in-depth characterization of Tregs, T effector cells, and MDSC in COPD and correlated their levels and function with disease severity.Methods: Treg, effector T cell, and MDSC frequency from patients with COPD and healthy subjects' PBMCs were analyzed by flow cytometry. Treg immunosuppressive capacity was measured by in vitro suppression assay. The frequency of interferon-g producing T cells and T-cell proliferation were measured after blocking CTLA-4 and PD-1. Plasma proinflammatory and immunosuppressive cytokine levels were measured.Measurements and Main Results: Significantly increased levels of Tregs, MDSC, and PD-1 1 exhausted effector T cells were present in patients with COPD compared with healthy subjects. Tregs from patients with COPD suppressed P6-specific T-cell proliferation to a greater extent than Tregs from healthy subjects. Plasma levels of Treg-generated cytokines, IL-10, and transforming growth factor-b were elevated. Blockade of CTLA-4 resulted in significant augmentation of T-cell IFN-g production in patients with COPD.Conclusions: Functionally suppressive Tregs, MDSCs, and exhausted PD-1 1 T cells contribute to effector T-cell dysfunction in COPD.
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