Anti-HBV PreS2 Monoclonal antibody, Clone T0

The classical hepatitis B virus (HBV) DNA vaccination plasmid only encodes for a single viral antigen, either the S or the PreS2/S antigen. Many strategies have been employed to improve the effect of these DNA vaccines. Our previous study identified that the fusion gene, HBV S-ecd cluster of differentiation 40 ligand (CD40L), may promote the activation of dendritic cells (DCs) and enhance their function in vitro. In the current study, the effect of HBV S-ecdCD40L vaccine therapy on liver DCs was investigated, and its therapeutic potential in HBV transgenic (HBV-Tg) mice was evaluated. The eukaryotic expression plasmid, pcDNA3.1-S-ecdCD40L, was constructed by inserting the HBV S gene and mouse CD40L gene into the vector, pcDNA3.1 (+). HBV-Tg mice were immunized with pcDNA3.1-S-ecdCD40L, pcDNA3.1-S, pcDNA3.1 or PBS. Following this, immunophenotyping, cytokine production and T-cell activation were analyzed in the CD11c-enriched DC population obtained from the liver. Vaccine efficacy was further assessed by the detection of serological and biochemical parameters. When comparing with other control groups, DCs from HBV-Tg mice immunized with pcDNA3.1-S-ecdCD40L exhibited increased expression of immunologically important cell molecules (CD86 and major histocompatibility complex class II), pro-inflammatory cytokines (interleukin-12), and enhanced capacity to promote allogeneic T-cell proliferation. Furthermore, the HBV S-ecdCD40L vaccine resulted in a significant inhibition of HBV DNA replication and down-regulation of the hepatitis B virus surface antigen (HBsAg) in HBV-Tg mice, without obvious liver injury. In conclusion, the HBV S-ecdCD40L vaccine may be a feasible strategy for chronic HBV immunotherapy via promoting DC activation and function.

PreS domain of Hepatitis B virus (HBV) surface antigen is a good candidate for an effective vaccine as it activates both B and T cells besides binding to hepatocytes. This report deals with overexpression and purification of adr subtype of surface antigen that is more prevalent in Pakistan. PreS region, comprising 119 aa preS1 region plus a 55 aa preS2 region plus 11 aa from the N-terminal S region, was inserted in pET21a+ vector, cloned in E. coli DH5 alpha cells and expressed in E. coli BL21 codon+ cells. The conditions for over expression were optimized using different concentrations of IPTG (0.01-5 mM), and incubating the cells at different temperatures (23-41 degrees C) for different durations (0-6 h). The cells were grown under the given optimized conditions (0.5 mM IPTG concentration at 37 degrees C for 4 h), lysed by sonication and the protein was purified by ion exchange chromatography. On the average, 24.5 mg of recombinant protein was purified per liter of culture. The purified protein was later lyophilized and stored at -800 degrees C.