Eliminating Hepatitis B By SMAC Mimetics In Mouse Model Of Chronic HBV Infection

BackgroundChronic hepatitis B virus(Hepatitis B virus,HBV)belongs to Hepadnaviridae,which can lead to serious liver diseases such as liver failure,cirrhosis,and hepatocellular carcinoma(HCC)worldwide.Inhibitor of apoptosis proteins(IAPs)are highly conserved endogenous antiapoptotic proteins.The binding of SMAC and IAPs can induce the poly-ubiquitin and degradation of IAPs,which promotes cell apoptosis.It is reported that cellular inhibitor of apoptosis protein 2(cIAP2)inhibit HBV clearance,however,the IAPs antagonist(also known as Smac mimetics)can promote HBV infection clearance,which provides us with a new strategy for the treatment of HBV.In addition,IAPs antagonists also play a key role in anti-tumor effects through immune regulation;however,whether it has an immunomodulatory effect on HBV infection remains to be studied.APG-1387,which is with independent intellectual property rights of the Ascentage Pharma Group Corp,is a novel small molecule IAPs inhibitor.Objectives1.Whether APG-1387 eliminates HBV in the mouse model of chronic HBV infection;2.Preliminary study on the mechanism of the elimination of HBV by APG-1387 in the mouse model of chronic HBV infectionMethods1.Evaluation of therapeutic effect of APG-1387 on HBV:we observed the changes of HBsAg and HBV DNA levels in serum and the expression of HBV core antigen(HBcAg)and replication of HBV DNA in liver tissue after APG-1387 injection.2.APG-1387 induced apoptosis and immunoregulatory effect:The method included Tunel staining,immunofluorescence,flow cytometry and so on.3.Statistics:Prism 6.0d software(Graph Pad Software)was used to perform statistical tests,p<0.05 was found to be statistically significant.Results1.In a mouse model by hydrodynamic injection of the plasmid(pAAV-HBV1.2)into the tail veins of C57BL/6 mice,which was used to imitate chronic HBV infection in immune-control(IC),APG-1387 promoted the clearance of HBV.1.1 The clearance of serum HBsAg and HBV DNA could be observed after four doses of APG-1387 continuously(weekly,?),what's more;some mouse could product serum HBV surface antibody(HBsAb).1.2 Expression of HBcAg and HBV replication intermediates in liver tissue couldn't detected by HBcAg immunohistochemical stainings and Southern Blots respectively.2.In a mouse model by hydrodynamic injection of the plasmid(pAAV-HBV1.2)into the tail veins of C3H/HeN mice,which was used to imitate chronic HBV infection in immune-active(IA),APG-1387 promoted the clearance of HBV.1.1 Serum HBsAg,HBeAg and HBV DNA couldn't be detected after ten doses of APG-1387 continuously(weekly,?),what's more;some mouse could product serum HBsAb.2.1 Expression of HBcAg and HBV replication intermediates in liver tissue couldn't detected by HBcAg immunohistochemical stainings and Southern Blots respectively.3.APG-1387 induced apoptosis3.1 Serum alanine transaminase(ALT)and aspartate aminotransferase(AST)elevated after one dose of APG-13 87(iv,12h),Importantly,the effect of APG-1387 on ALT and AST was transient,and within 72h,transaminase levels returned to baseline.Tunnel and Cleaved-caspase3 positive staining of frozen sections of liver tissue were observed after one dose of APG-1387(iv 12h).3.2 The colocalization of Tunel and HBcAg vertified the existence of apoptosis of HBcAg-expressing hepatocytes by using three dimensional(3 D)reconstruction technique of laser-scanning confocal microscope.4.Immunomodulatory effects of APG-1387In a mouse model with sustained HBV viremia after infection with a recombinant adeno-associated virus(AAV)carrying a replicable HBV genome(AAV/HBV),comparing the frequency and percentage of lymphocytic infiltration(CD4+and CD8+T cells)of the liver,we found that APG-1387 treatment was associated with a accumulation of CD4+and CD8+T cells in the liver,and this accumulation of CD4+ T cells reached statistical significance(p<0.05).After 5 hours of stimulation with PMA in vitro,the secretion of IFN-y and TNF-? showed an increasing trend in the experimental group compared with the control group.Conclusions1.APG-1387 can rapidly promote the clearance of HBsAg and HBV DNA in mouse model of chronic HBV infection by antagonizing IAPs,which is a new treatment strategy,and has an important value of application.2.Discussing preliminarily the mechanism of the clearance of HBV,we suggest a hypothesis that APG-1387 may enhance the sensitivity of cytotoxic effect of T cells on infected hepatocytes by HBV and promote apoptosis of infected hepatocytes and eliminates HBV ultimately.3.In conclusion,APG-1387 is promising in the treatment of HBV infection.