Establishment Of A Chronic HBV Replication Mouse Model Using Immunosuppression Treatment And Its Mechanism

HBV infection is still a public health problem worldwide, and China is also a highincidence of hepatitis B virus infection. New therapeutic approaches for chronic HBVinfection are urgently needed, considering the limited success rate of the current treatmentregimes with recombinant interferon (IFN)-and the inability of antiviral drugs based onnucleotide analogues to clear HBV completely.As far,It is not clear that the immunopathogenesis during HBV infection.The lack of asuitable small animal model is a major obstacle for studies on HBV infection. The naturalinfection models like Pekin duck and woodchuck models are useful for studies on themolecular biology of hepadnaviruses and for testing of antiviral drugs and vaccines.However,there was only limited resources and tools available for both animal models. TheChimpanzee model is very expensive,but also has the problem with the ethic justification.So, the mouse models with HBV replication like transgenic mouse strains and mousemodels based on hydrodynamic injection (HI) are useful alternatives for many researchapproaches. We established the mouse models with transient and persistent HBV replication by injection a replication-competent HBV DNA into the tail veins of the mouse. In2002,Yang et al. demonstrated that HBV replication could take place in liver tissue after HIand was terminated with the appearance of HBV-specific immune responses. It could beshown that the choice of the backbone vectors and mouse strains determines the clearanceor persistence of HBV replication after HI in mice. It became clear that an intact immunesystem is essential for the timely HBV clearance in the HBV mouse model based on HI.It iswell known that HBV may persist in immunosuppressed or immunocompromised patients.We asked whether HBV mouse model based HI could be used to mimic this clinicalsituation. So we treated BALB/c mice with immunosupressive drugs,then received HI.Wemonitoring of HBV markers of these mice,detcted T cell response during acute and chronicHBV infection in order to reveal the interaction of T cell response and infectous outcome.Objective1. To observe anti-viral effect of a few immunosupressive drugs on HepG2.2.15cell whichwas treated with immunosupression drugs under safety concentration.2. To establish a persist HBV infection animal model with BALB/c mouse treated with4immunosuppressive drugs respectively started one week before HI and continued untilweek10after HI,juaged by HBV marker,HBV antibody and HBVDNA in serum forfurther study of the immunosuppression role for HBV infection persistence.3. Treated the BALB/c mouse with Saline continuously and RAPA continuouslyrespectively. And we explore the interaction of RAPA and HBV persistence;detected Tcell response,especially responses of regulatory T cell,in order to find the associationbetween the frequency of Treg in PBMC and interliver infiltrating lymphocytes and Tcell response,analysis the impact of the early activation of Treg on infectous outcomeand T cell response,then provide experimental evidence for the early immune responseduring acute HBV infection and the mechanism of chronic HBV infection. Methods1. HepG2.2.15cell were treated with fersh medium containly FK506,RAPA, DEX, andCYP, respectively at different dosage: Four hours before incubation terminal at4days,added MTT20ul with concentration5g/l,4hours later added dimethyl sulfoxideand readed OD570nm;determined it’s toxic by the survial rate of cell more than95percents,found the safety concertration.2. HepG2.2.15cell was treated with immunosupression drugs under safety concentrationrespectively,then collected cell culture supernatant and cells after4days,dected HBsAgand HBeAg in supertant by ELISA and HBV replication by Southern Blot.3. Female BALB/c mice,6-8weeks old, were treated with CsA, DEX, CYP or Saline asabove. One week later, mice received a fitted skin graft from C57BL/6mice. The graftswere inspected daily until rejection, which was defined as>90%necrosis of the graftepithelium.4. Female BALB/c mice,6-8weeks old.Mice were treated with CsA, DEX, and CYP,respectively at the indicated dosage: DEX, CYP, FK506,RAPA and Anti-asialo GM1. Alldrugs were diluted with Saline and injected intraperitoneally in a volume of0.25mLexcept RAPA with0.5mL. Mice treated with0.25ml NS every two days were used ascontrol. One week later, mice were challenged by HI of pAAV/HBV1.2.10g ofplasmid in a volume of0.9%NaCl solution equivalent to0.1ml/g of the mouse bodyweight were injected into the tail veins of mice within5-8seconds.Monitor the titer ofHBsAg, HBsAb and HBcAb in the serum using ECLIA. In addition, extraction theHBV DNA from the serum and measured the viremia by Real-Time PCR.5. Hydrodynamic injection pAAV/HBV1.2into the tail veins of the BALB/c mice treatedwith RAPA.and NS continuously and killed the mice at the indicated time points.Monitor the Monitor the titer of HBsAg, HBsAb and HBcAb in the serum.Thefrequencies of IFN-γ secreting splenocytes were assayed by ELISPOT after HBc andHBs peptide stimulation. Extraction the RNA from liver tissue and splenocytes, and the mRNA levels of CD molecules and cytokines were measured by Real-TimePCR.Isonated lymphocytes in periphal blood,spleen and liver,then dected the frequencyof Treg in PBMC,spleocytes and LIL by FACS.Analyzed analysis the impact of theearly activation of Treg on infectous outcome and T cell response,then provideexperimental evidence for the early immune response during acute HBV infection andthe mechanism of chronic HBV infection.Results1. The safety concentration of the immunosupressive drugs were CYP0～1000ug/ml,DEX0～500ug/ml,FK5060～10ug/ml,RAPA0～0.4ng/ml,Adv0～1ug/mlrespectivly.They didn’t inhibit HBV replication and the secretion of HBsAg andHBeAg.2. After the transplantation, the graft can survival for8.67±1.86days in NS treated group,but it can prolong obviously in groups treated with the immunosuppressive drugs. Thesurvival time of the grafts in the FK506, DEX, RAPA and CYP treated groups is21.50±3.09days,21.50±3.09days,20.00±0.71days,21.40±1.52days respectively.3. The control mice treated with saline became positive for HBsAg within3days andHBV DNA after HI and completely cleared HBV DNA after4weeks and HBsAg atweek8. HBsAg and HBV DNA were persistently detectable at high levels in micetreated with immunosuppressive drugs RAPA, DEX, and CYP. The loss of HBsAg andHBV DNA occurred in some mice after the discontinuation of immunosuppressivetreatment. The majority of mice of RAPA and CYP groups were positive for HBsAgand HBV DNA up to week26, more than6months. The mice of DEX group finallycleared HBV DNA from peripheral blood at week14, however,60%of mice remainedHBsAg positive at week26. HBsAg in mice treated with Anti-asialo GM1antibody waslower than that in mice treated with Saline,and disappeared within6weeks.4. Consistent with the HBV markers, antibodies to HBsAg and HBcAg were detected incontrol mice and mice treated with FK506after witharawn FK506. The mice of DEX and CYP groups were negative for anti-HBs and anti-HBc antibodies for the completeexperimental period. Patial mice of RAPA group became positive for anti-HBc antibodybut negative for anti-HBs antibodies.5. The HBsAg level and HBVDNA in serum of mice treated with Saline was disappearedat21days and30days respectively while that of mice treated with RAPA was positive.6. The frequency of Treg in PBMC isonated from control group was increased at1dayafter HI dued to liver ingury induced by HI,and peaked at14days after HI because ofHBV sepecific T cell response,then backed to normal level. The frequency of Treg inPBMC isonated from group treated with RAPA also increased at3days after HI whichwas remained at similar level until21days after HI.7. The frequency of Treg in spleocytes isonated from control group was increased at10days and20days after HI,backed to nomal level at30days after HI. The frequency ofTreg in spleocytes isonated from group treated with RAPA has no increase at the threetime points.8. The frequency of Treg in LIL isonated from control group was at a lower level at threetime points and that of group treated with RAPA was increased at the10days and30days after HI.9. Cellular immune responses to HBcAg and HBsAg that were detectable by Elispotassays for IFN-secreting cells was higher in mice treated with RAPA than that in micetreated with Saline.10. Treatment with RAPA can inhibit the expression of some CD molecules and cytokinesin the liver tissue, including CD3, CD4,CD8, IFN-β, FasL, Perforin, TNFα, IFN-γ. Theexpression of cytokines IL-10,TGFβ and FoxP3are obviously higher in the RAPAtreated group than in the NS treated group.11. HBV persistently replicated in the liver in the RAPA treated mice. At day30afterhydrodynamic injection, HBV replicative intermediates can be detected in all the liver tissue in the RAPA treated group. The liver tissue of the control mice was negative forHBcAg at30days after HI.Conclusions1. They has no toxic to HepG2.2.15, no impact on the secretion of HBsAg, HBeAg andHBV replication which indicated that they didn’t inhibit or promote HBV replicationdirectly.2. In the hydrodynamic injection mouse model, treated the mice with RAPA, DEX orCYP can prolonged the grafts survival time and HBV replication obviously;so we hadestablished HBV persistence mouse model induced by immunosuppressive drugstreatment successfully.3. RAPA treatment can induce the HBV persistence by selectively expansion of Treg andincreased cytokines secrected by Treg.