Preventive And Therapeutic Effecacy Of Autophagosome Derived From HepG2.2.15 Cells (HBV~+Dribbles) For HBV Infection And Their Immunological Mechanisms

Cross-presentation of antigens is important for induction of adaptive immunity against tumor cells and infectious pathogens. Autophagosomes derived from tumor cells were efficient antigen carriers for cross-presentation of tumor antigens and their safety and efficacy are tested currently in phase Ⅰ and Ⅱ clinical trials.HBV primarily infects hepatocytes. However, hepatocytes lack of costimulators and the ability to present the HBV antigens for inducing CD8+T-cell response. Dendritic cells elicit HBV-specific CD8+T cell response by cross-presentation of HBV antigens. Such a T cell response is exhausted and functionally impaired in patients with chronic HBV infection. It may be caused by lack of efficient cross-presentation of HBV antigens. We generated a HBV therapeutic vaccine (HBV-DRibbles) from a HBV-expressing cell line for testing its prophylactic and therapeutic efficacy and exploring immunological mechanisims.ObjectiveUsing autophagosomes derived from HepG2.2.15 cells as a HBV vaccine and pAAV/HBV1.2 plasmid hydrodynamically injected C57BL/6 mouse as a HBV infected animal model, we investigated the prophylactic and therapeutic effecacy and immunological mechanisms of HBV+ DRibbles against HBV infection.Methods1. Preparation and identification of HBV+ DRibbles1.1 Different drugs (Rapamycin, Bortizomib and NH4Cl) were added during logarithmic phase of HepG2.2.15 cells. DRibbles were extracted by differential centrifugation, preserved in -20℃;1.2 The expression of HBsAg and/or LC3 were detected in cell lysates and HBV+ DRibbles by Western blot;1.3 Morphology analysis of HBV+DRibbles was done under transmission electron microscopy;1.4 Levels of HBsAg and HBeAg in HBV+ DRibbles were measured by ELISA.2. Cross-presentation of HBV antigens in vitro induced by HBV+ DRibblesRecombinant HBsAg was administrated into C57BL/6 mice. To determine T-cell mediated immune responses, the anti-HBs antibody positive mice were sacrificed and the lymphocytes were isolated and restimulated with HBV+ DRibbles, soluble HBsAg. The levels of IFN-y in the supernatants were measured by ELISA. To further determine-the CD4+ and CD8+ T-cell responses, CD4+ and CD8+ T-cell were sorted from HBsAg-vaccinated mice by the magnetic bead sorting and restimulated with dendritic cells preloaded above-mentioned antigens. The levels of IFN-y in the culture supernatants were determined by ELISA.3. HBV-specific T cell responses induced by HBV+ DRibblesC57BL/6 mice were immunized with different doses of HBV+ DRibbles or HBV-DRibbles or PBS via intra-nodal injection. Seven days later, lymphocytes were harvested and restimulated with HBV antigens or peptides.The number of IFN-γ producing cells was detected by ELISPOT assay.4. Establishment of the mouse model of persistent infection with HBVC57BL/6 mice were injected hydrodynamically with pAAV/HBV1.2 plasmid DNA as discribed in reference. After injection, the mice were regularly bled or sacrificed. The serum levels of HBsAg, HBeAg, HBV DNA, ALT and AST were measured by ELISA, real-time PCR and enzymatic methods, respectively. Liver tissues were stained with hematoxylin and eosin and were further assayed for the HBcAg.5. Investigation of the prophylactic effect of HBV+ DRibblesC57BL/6 mice were immunized with HBV+ DRibbles, HBV- DRibbles or PBS via intra-nodal injection. Seven days later, pAAV/HBV1.2 plasmid DNA was injected via tail vein. Serum samples were collected at day 14 for detection of HBeAg by ELISA and HBV DNA with real-time PCR. Liver tissues were collected intracellular HBcAg expression was detected by immunohistochemical stain method;6. Exploring the immunological mechanisms of preventive effect of HBV+ DRibbles6.1 Mice were vaccinated with HBV+ DRibbles or PBS. On day 8, after injection of pAAV/HBV1.2 DNA into the mice, anti-CD4 mAb or/and anti-CD8 mAb were administered intraperitoneally. At day 14, HBeAg and HBV DNA copy number in the serum samples and intrahepatic HBcAg in liver tissues were detected as described above;6.2 To determine the function of effector T cells, lymphocytes were harvested from HBV+ DRibbles vaccinated mice and cocultured with hepatocytes from mice injected with pAAV/HBV1.2 plasmid DNA. The supernatants were collected for detection of ALT, AST by enzymatic method or for detection of IFN-y by ELISA.7. Examination of the therapeutic efficacy of HBV+ DRibbles against acute HBV infection.7.1 C57BL/6 mice were injected hydrodynamically with pAAV/HBV1.2 plasmid DNA and were vaccinated with HBV+ DRibbles, HBsAg or PBS via intra-nodal injection 2 days later. The lymphocytes were restimulated with recombinant HBV antigens and peptides. The number of IFN-y producing cells was detected by ELISPOT;7.2 C57BL/6 mice were immunized as above mentioned. The serum samples were collected for measurement of ALT, AST, HBeAg, HBsAg, anti-HBs, HBV DNA and liver tissues were collected for detection of expression of HBcAg in hepatocytes and the liver sections were stained with hematoxylin-eosin.8. Examination of the therapeutic efficacy of HBV+ DRibbles in chronic HBV infection.8.1 Determination of the tolerance toward HBsAg in HBV carrier mice. Sixty days after hydrodynamic injection of pAAV/HBV1.2 or PBS, the mice were vaccinated with HBsAg as above. The levels of serum anti-HBs antibody after immunization were determined by ELISA;8.2 The HBsAg tolerant mice were selected and then immunized with HBV+ DRibbles or HBsAg or PBS, respectively. Lymphocytes were restimulated with HBV antigens and peptides. The number of IFN-y producing cells was measured by ELISPOT;8.3 The HBsAg tolerant mice were immunized as above mentioned. Lymphocytes from vaccinated HBsAg tolerant mice were adoptive transferred intravenously into each of the new set of 60-day HBV carrier mice or control mice. Serum samples were collected after adoptive transfer for detection of HBsAg, anti-HBs, HBV DNA, ALT and AST. Liver tissues were collected for detection of expression of HBcAg in hepatocytes and the liver sections were stained with hematoxylin-eosin.Results1. Preparation and identification of HBV+ DRibbles1.1 HB V DRibbles were isolated and kept in-20℃;1.2 In cell lysates, the levels of major HBsAg protein did not differ after different treatments, and Bortizomib treatment led to appearance of HBsAg of lower molecular weight products and addition of NH4Cl further increased the appearance of these bands. LC3 protein was detected in cells lysates and HBV+ DRibbles after different, treatments. However, accumulation of LC3-Ⅰ and LC3-Ⅱ in cell lysates and predominant LC3-Ⅱ in the DRibbles was found when NH4Cl was included in addition to Bortizomib and Rapamycin treatment;1.3 Transmission electron microscopy analysis showed that DRibbles had a double membrane structure and their size was ranged from 100 to 1000 nm when all three drugs were combined;1.4 ELISA analysis showed that the amount of HBsAg and HBeAg in the HBV+ DRibbles were significantly higher when all three drugs were combined.2. Cross-presentation of HBV antigens in vitro induced by HBV+ DRibbles When HBsAg-specific lymphocytes were restimulated, the HBV+ DRibbles produced much higher amount of IFN-γ than soluble HBsAg. Then, HBsAg-specific lymphocytes were sorted into CD4+ and CD8+T cells before they were restimulated with DC preloaded HBV+ DRibbles or soluble HBsAg. ELISA assay showed that only the HBV+ DRibbles-loaded DC stimulated a significant higher levels of IFN-γ.3. HB V-specific T cell responses induced by HBV+ DRibblesELISPOT assay revealed that lymphocytes from mice immunized with 30μg of HBV+ DRibbles restimulated with HBV antigens and peptides contained the highest number of HBsAg or HBcAg specific IFN-y producing cells. Injection of 10μg of DRibbles was insufficient, while 100μg showed a high-dose suppression. Lymphocytes from 30μg HBV+ DRibbles vaccinated mice generated much higher number of IFN-y producing cells than that from 30μg HBV- DRibbles-vaccinated mice when restimulated with either HBV antigens or HBV peptides;4. Establishment of the mouse model of persistent infection with HBVC57BL/6 mice were injected hydrodynamically with pAAV/HBV1.2 plasmid DNA as discribed in reference. At different time points after injection, the level of ALT is similar to that of WT mice or PBS injected control mice, however, the AST was significantly higher than that of WT mice only on day 3. Levels of serum HBsAg, HBeAg were high at day 3, and then dropped at later time and were undetectable in some samples at day 63. The viral titers were high on average 180.9±98.61×104 copies per milliliter of sera at day 3 and were mesurable at 102×104 or 34.1×104 copies per milliliter of sera from HBsAg-positive mice on day 63. Compared with the normal liver architecture of WT mice on day 3, the livers from PBS injected mice showed a small amount of foci of inflammatory cell infiltration and a mild necrosis, however, the livers from pAAV/HBV1.2 plasmid DNA injected mice showed a moderate necrosis inflammatory cells infiltration and returned to normal architecture at day 7 and 63. In addition, HBcAg were detected both in cytoplasm and nucleus in HBsAg-positive carrier mice on day 63 after pAAV/HBV1.2 plasmid DNA injection.5. Prophylactic effect of HBV+ DRibbles against HBV infectionThe levels of sera HBeAg, HBV DNA and the percentage of HBcAg+hepatocytes in the mice immunized with HBV+ DRibbles, but not naive mice or mice immunized with HBV" DRibbles and other doses of HBV+ DRibbles, was significantly lower than control mice injected with HBV- DRibbles or PBS.6. CD8+ T cells induced by HBV+ DRibbles mediated suppression of HBV replication6.1 Non-depleted mice immunized with HBV+ DRibbles had a reduced level of serum HBeAg, DNA copy number and HBcAg+ expressing hepatocytes. The DNA copy number in mice depleted of CD8+effector T cells were not significantly different from that of control non-immunized naive mice. Depletion of both CD4+and CD8+effector T cells did not further increase the serum HBeAg or DNA copy number beyond that of mice received anti-CD8 antibody;6.2 Significantly higher levels of IFN-y were detected in the culture supernatants when effector T cells were incubated with HBV-expressing hepatocytes than control hepatocytes. Although the levels of transaminase were increased, only significantly higher amount of AST enzyme release were found in HBV-expressing hepatocytes than control hepatocytes.7. Vaccination with HBV+ DRibbles was effective against acute HBV infection7.1 Lymphocytes harvested from HBV+ DRibbles immunized mice generated a significantly higher number of IFN-y producing cells than that from PBS or HBsAg immunized mice when restimulation ex vivo with HBV antigens or peptides;7.2 The serum levels of HBeAg, HBV DNA and percentage of HBcAg+hepatocytes in the liver tissues in HBV+ DRibbles-immunized mice were greatly reduced as compared to control mice received either no vaccine or HBsAg vaccine. In addition, sera ALT and AST levels were not influenced by the vaccinations and the livers from HBV+ DRibbles vaccinated mice showed normal architecture and a mild inflammatory responses. However, the serum HBsAg levels in HBV+ DRibbles immunized mice were significantly lower than that of control mice without vaccination, but much higher than that of HBsAg immunized mice. Detection of anti-HBsAg antibody in serum from immunized mice showed that HBsAg vaccination induced significantly higher levels of anti-HBs compared with HBV+ DRibbles vaccination. The average level of serum anti-HBsAg antibody from HBV+ DRibbles-immunized mice were lower than that of HBsAg immunized mice.8. DRibbles induced therapeutic T cells and break tolerance in HBsAg tolerant mice8.1 After immunized with HBsAg, control naive C57BL/6 mice had already developed higher levels of anti-HBs, while the carrier C57BL/6 mice without anti-HBs formation;8.2 HBV+ DRibbles, but not HBsAg, induced HBV-specific IFN-γ spot-forming cells when restimulated with HBV proteins or peptides;8.3 The levels of serum HBsAg, HBV DNA and HBcAg+ hepatic cells were greatly reduced in HBsAg tolerant mice after adoptive transfer of lymphocytes from HBV+DRibbles vaccinated mice. Anti-HBsAg antibodies were detected in some mice received lymphocytes from DRibbles, but not PBS and HBsAg immunized mice. In contrast, the mice received lymphocytes from HBsAg vaccinated mice did not exhibit any measurable effect. Furthermore, the serum ALT and AST levels in HBV+ DRibbles immunized mice were similar to that of non-immunized control mice and the livers of the mice receiving HBV+ DRibbles treatment showed normal architecture with a mild inflammatory infiltrates.Conclusions:HBV+ DRibbles were efficient HBV antigen carriers for effective cross-presentation. HBV+ DRibbles elicit HBV-specific T cell responses and prophylactic effect on WT C57BL/6 mice against HBV infection. CD8+ T cells mediated the suppression of HBV replication and the clearance of HBcAg+hepatocytes. In both acute and chronic HBV-infected mouse models, HBV+ DRibbles vaccine could induce robust, multiple HBV- specific T cell responses, effectively suppress the HBV replication and clear the HBV-infected hepatocytes. HBV+ DRibbles vaccine could break the HBV tolerance and did not cause severe liver damage. Our findings have important implications for the development of therapeutic vaccines for the treatment of chronic infections of HBV or other viruses.