The spleens were crushed individually, strained with a 40M cell strainer and treated with 1mL ACK lysis buffer for 5 min to lysis erythrocytes. that are recruited to the liver- an approach that can be beneficial in the search for vaccines or immune-therapies to liver disease. expression10. HBV core protein was chosen as it is the major viral determinant of HBV persistence, with one of the major differences between acute and chronic human HBV infection being the detection of CTL against HBcAg in circulation11C13. Previous data from several groups suggest that these CTLs play a crucial role in the clearance of HBV during acute infection. In addition, recent findings suggest that the induction of both cellular and humoral immunogenicity against HBV antigens are important for controlling chronic infection14. Studies from our laboratory and other independent groups have shown that plasmid DNA delivery by electroporation (EP) represents an important strategy for enhancing cell or antibody mediated immune responses15C18, which we examined in this study. We report that a synthetic construct is able to induce a strong antigen-specific T cell response that mediates cytotoxicity systematically and induces robust T cell migration into the liver. In addition, a high titer antibody response capable of recognizing a native HBcAg was observed. Importantly, immunized mice were able to clear transfected HBV antigenCexpressing hepatocytes was digested with and electroporation with the CELLECTRA? adaptive constant current electroporation device (Inovio Pharmaceuticals, Blue Bell, PA). Two 0.2 Amp constant current square-wave pulses CD48 were delivered through a triangular 3-electrode array consisting of 26-gauge solid stainless steel electrodes completely inserted into muscle. Each pulse was 52 milliseconds in length with a 1 second delay between pulses. Splenocyte isolation and purification Splenocytes were isolated as Azamethiphos described elsewhere19. In brief, mice were sacrificed one week after the last immunization and spleens were harvested, placed in R10 media (RPMI media supplemented with 10% FBS and 1x Anti-Anti). The spleens were individually crushed, strained with a 40M cell strainer and treated with 1mL ACK lysis buffer for 5 min to lysis erythrocytes. The splenocytes were resuspended in a complete R10 media and used for further immunological assays. Liver perfusion and lymphocyte Isolation Lymphocytes from liver were obtained as described elsewhere 20. Briefly, each liver was perfused by directly injecting 1mL of PBS into the hepatic vein of each mouse. Livers were harvested, crushed and resuspended in 5mL of 44% isotonic percoll. The mixtures were underlied with 3mL 66% isotonic percoll and centrifuged for 20 minutes at 2000rpm for gradient separation. Lymphocytes Azamethiphos were collected and washed in 10 mL R10 and treated with ACK lysis buffer as necessary. 2.4 Cellular Response Assays Interferon-gamma ELISpot IFN- ELISpot was performed as previously described21. Splenocytes were stimulated with two pools of 15-mer peptides spanning the entire length of pMCore and over lapping by 8 amino acids. 200,000 splenocytes in R10 media were plated in a 96 well IFN- capture antibody (R&D system) coated plate and stimulated overnight in the presence of a specific peptide pool at 37C in 5% CO2. Cells were washed out and plates were incubated overnight with biotinylated anti-mouse IFN-detection antibody (R&D system). Streptavidin-alkaline phosphatase and 5-bromo-4-chloro-3-indolylphosphate cytotoxicity assay was performed as previously described23,24. Briefly, splenocytes from na?ve mice were stained with either 1M or 1nM CFDA SE (invitrogen). The labeled splenocytes were then coated with indicated peptides (1M) and 107 cells of each population intravenously injected into na?ve or immunized mice. After 24 Azamethiphos or 90 hours cells from the spleen and liver were isolated and analyzed by flow cytometry. The percent killing was calculated as follows: 100 ? ([(% relevant peptide pulsed in infected/% irrelevant peptide pulsed in infected)/(% peptide pulsed in uninfected/% irrelevant peptide pulsed in uninfected)] 100). 2.5 Humoral Response Assays Measurements of the Sera Ag-specific IgA and IgG levels High-binding ELISA plates (Costar, Corning, NY) were coated with 1}g/ml HBcAg protein in PBS, {at 4C for 24 hours and then were washed with 0.|at 4C for 24 hours Azamethiphos and were washed with 0 then.}1% PBS-Tween then and blocked with PBS containing 1% BSA for 2 hours at room temperature. {Serially diluted serum samples.|Diluted serum samples Serially.}

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