HBV X protein-based therapeutic vaccine accelerates viral antigen clearance by mobilizing monocyte infiltration into the liver in HBV carrier mice.

BACKGROUND: Hepatitis B virus (HBV) persistently infected about 250 million people worldwide, and a curative treatment remains an unmet medical need. Among many approaches to treat chronic hepatitis B (CHB), therapeutic vaccines have been developed for two decades, but none have yielded promising results in clinical trials. Therefore, dissection of HBV clearance mechanisms during therapeutic vaccination in appropriate models, which could give rise to new curative therapies, is urgently needed. Growing evidence indicates that prolonged and intensive exposure of antigen-specific T cells to viral antigens is a major cause of T cell exhaustion, and decreases anti-HBV immunity efficacy of therapeutic vaccination. HBV X protein (HBx) is expressed at low levels, and the understanding of its immunogenicity and potential in therapeutic CHB vaccines is limited. METHODS: HBV genome sequences from CHB patients were cloned into a pAAV plasmid backbone and transfected into immunocompetent mouse hepatocytes through hydrodynamic injection. Mice carrying > 500 IU/mL serum HBV surface antigen (HBs) for more than 4 weeks were considered HBV carriers mimicking human CHB and received 3 doses of weekly HBx vaccine by subcutaneous immunization. Serum HBV clearance was evaluated by monitoring serum HBs and HBV-DNA titers. Residual HBV in the liver was evaluated by western blotting for HBV core antigen. The splenic antigen-specific T cell response was quantified by a 15-mer overlapping peptide-stimulated interferon-γ enzyme-linked immunospot assay. Blood and hepatic immune cells were quantified by flow cytometric analysis. RESULTS: Our HBx-based vaccine induced systemic HBx-specific CD4+ and CD8+ T cell responses in HBV carrier mice and demonstrated significant HBs and HBV-DNA elimination. The protective effect persisted for at least 30 days without additional booster immunization. Different infiltrating myeloid cell subsets, each with distinctive roles during immune-mediated HBV clearance, were found in the liver of vaccinated mice. During vaccine therapy, inflammatory monocyte depletion resulted in sustained HBV clearance inhibition, whereas phagocytic monocyte-derived macrophage and Kupffer cell elimination resulted in only transient inhibition of vaccine-induced HBV clearance. CONCLUSIONS: We report the potential role of HBx as a major immunogen in an HBV therapeutic vaccine and the significance of a liver-infiltrating monocyte subset during hepatic viral clearance.

A genetic approach to inactivating chemokine receptors using a modified viral protein.

We have developed a genetic system, called degrakine, that specifically and stably inactivates chemokine receptors (CKR) by redirecting the ability of the HIV-1 protein, Vpu, to degrade CD4 in the endoplasmic reticulum (ER) via the host proteasome machinery. To harness Vpu's proteolytic targeting capability to degrade new receptors, we fused a chemokine with the C terminal region of Vpu. The fusion protein, or degrakine, accumulates in the ER, trapping and functionally inactivating its target CKR. We have demonstrated that degrakines based on SDF-1 (CXCL12), MDC (CCL22) and RANTES (CCL5) specifically inactivate their respective receptor functions. Using a retroviral vector expressing the SDF-1 degrakine, we have established that CXCR4 is required for the homing of hematopoietic stem/progenitor cells (HSPC) to the bone marrow immediately after transplantation. Thus the degrakine provides an effective genetic tool to dissect receptor functions in a number of biological systems in vitro and in vivo.

Cationic nanoglycolipidic particles as vector and adjuvant for the study of the immunogenicity of SIV Nef protein.

The objective of this study was to test the immunogenicity of SIV Nef protein formulated in cationic nanoglycolipidic particles of 100nm of diameter. In parallel, the adjuvant effect of these nanoglycolipidic particles was compared in similar experiments using GST-Nef in association with the commonly strongest used complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant in association with MDP or MDP alone. Our results showed that these particles do not alter the integrity of our immunogen GST-Nef, which remains stable for more than three months at 4°C. We demonstrated that in the presence of nanoglycolipidic particles antibodies against Nef were produced since the first injection and remained stable after the third injection with high titers for long lasting periods as observed with CFA and IFA/MDP adjuvant. The analysis of immunoglobulin isotype profiles of antibodies generated by the different protocols of immunization showed the preponderance of IgG1 isotypes suggesting the predominance of Th2-type immune response.