The Efficiency And Mechanisms Of Poly I:C As An Adjuvant In Combination With RHBVvac In CHB

Object:HBV infection is a major public health problem in the world.Chronic hepatitis B(CHB)infection is the main cause of liver injury and inflammation.Without timely and effective treatment,roughly 25%of patients with CHB could develop into fibrosis,cirrhosis,liver failure and hepatocellular carci-noma(HCC)and even death.CHB is difficult to cure,mainly because of the immuno-suppressive microenvironment formed in the liver of CHB patients with high proportion of regulatory T cells(Tregs)and myeloid-derived suppressor cells(MDSCs),which provide T cells with inhibitory signals and disturb T cell-mediated anti-HBV functions,resulting in the exhaustion of HBV-specific CD8+ T cells.Currently,clinical therapy for CHB include nucleoside/nucleo-tide(NA)-based therapy and interferon(IFN)therapy.Unfortunately,none of these clinical therapies achieve long-term virological control in majority of patients with CHB.Therefore,there is an urgent need to develop new therapies to improve HBsAg clearance and virological cure.In order to overcome the dilemma of current clinical CHB treatment,researchers are currently developing new therapeutic drugs to achieve sustained virologic remission,and the HBV therapeutic vaccine is the research hotspot of CHB treatment in 21th century.HBV therapeutic vaccine is based on recombinant HBV proteins or HBV-envelope sub-viral particles,aiming to break chronic HBV infection-induced systemic tolerance,improve the HBV-specific CD4+ T cells,CD8+ T cells and humoral immune response,and finally achieve sustained virologic remission.Immune adjuvants are substances which can augment specific immune response in conjunction with antigens(Ag).However,there are just four Food and Drug Administration(FDA)-approved adjuvants:aluminum salts,MF-59,AS03,and AS04,limiting the progress of HBV therapeutic vaccines.Therefore,it is a challenging task to find new adjuvants for HBV therapeutic vaccines for clinical treatment of CHB.Pathogen-associated molecular patterns(PAMPs)in microbes can be recognized by pattern recognition receptors expressed on cell surface or in cytoplasm,including TLR,RIG-I-like receptor,NOD-like receptor and so on.Recent studies showed PAMPs could be applied as immune adjuvants to promote the activation of APCs.Polyinosinic-polycytidylic acid sodium salt(poly I:C)is a synthetic dsRNA mimetic TLR3 ligand.Poly I:C initiates a TRIF-dependent signaling cascade that progresses through the activation of NF-kB,MAP kinases and IRF3,which results in the production of type I IFNs and anti-HBV activity.Since cross-presenting DCs express high levels of the TLR3,the cross-priming of endogenous antigens is expected to be augmented by poly I:C stimulation,inducing HBV-specific CD8+ T cell responses.These findings suggest that poly I:C may be a potential vaccine adjuvant for use in antivirus therapy such as HBV therapeutic vaccine.Although the application of poly I:C was reported to induce HBV clearance by hydrodynamic injection,the properties of HBV-specific CD8+ T cells and the role of different subsets of CD8+ T cells in poly I:C-induced anti-HBV effects still remain unclear.Moreover,there is no description about whether poly I:C could induce the immune memory or achieve long-term virological control.Furthermore,the application of poly I:C for HBV therapy need to be optimized to avoid the occurance of autoimmunity.Based on the characteristics of immune tolerance induced by chronic HBV infection,we employed poly I:C as an adjuvant for HBV therapeutic vaccine,which was referred to as pHBV-vaccine.This prime-booster vaccination was administered via subcutaneous injection,and the efficiency of pHBV-vaccine in CHB treatment were evaluated in HBV-carrier mice.Meanwhile,we revealed the role and internal mechanism of short lived effector cells(SLECs)in HBV clearance.Our study provide a new theoretical and experimental basis for clinical CHB therapy.Methods:1.HBV-carrier mice and HBV-carrier-CDllc-DTR mice were established by hydrodynamic injection of pAAV/HBV 1.2 plasmids via the tail vein.2.HBV-carrier mice were immunized subcutaneously once weekly for 3 weeks using prime-booster vaccination strategy.3.The levels of serum HBsAg and anti-HBs were tested by CLIA and ELISA,respectively.Serum HBV DNA levels were analyzed by Real-time PCR.Intrahepatic capsid-associated HBV cccDNA,HBV DNA,HBV total RNA and HBV-3.5kb-RNA were analyzed by Real-time PCR.4.The levels of intrahepatic capsid-associated HBV cccDNA were analyzed by Southern blot.5.The levels of serum IL-12 were tested by ELISA.6.The levels of serum alanine aminotransferase(ALT)were measured using commercially available kit.7.Expression of HBcAg in liver sections was detected by immunohistochemical staining.H&E staining for pathologic analysis of liver injury.8.Bone marrow-derived dendritic cells(BMDCs)were generated by IL-4 and GM-CSF in vitro.9.HBV-carrier-CD11c-DTR mice were intraperitoneally inj ected with Diphtheria toxin to deplete DCs.10.Hepatocytes were isolated by in situ hepatic irrigation,then the expression of EGFP in hepatocytes was detected by immunofluorescence and FACS.11.Single-cell suspension of splenocytes were sorted by FACS,then these cells were intravenously transferred into recipient HBV-carrier mice.12.The expression of extracellular molecules,intracellular molecules and nuclear transcription factors were analyzed by FACS.13.The proliferation of splenocytes was analyzed by MTT and CFSE.14.For long-term immune memory assay,HBV-carrier mice treated as above were re-challenged with pAAV/HBV 1.2 via hydrodynamic injection.Results:Part 1:Multiple HBV DNA intermediates presented in HBV-carrier mice1.The pAAV-HBV plasmid entered into the liver and was continuously expressedafter hydrodynamic injectionThe pAAV-HBV plasmid entered into the liver and was continuously expressed after hydrodynamic inj ection.2.Multiple HBV DNA intermediates presented in HBV-carrier miceDifferent HBV DNA intermediates were detected in HBV-carrier mice hydrodynamically injected with pAAV/HBV 1.2 vector,including the HBV cccDNA,HBV rcDNA,ss HBV DNA as well as the dsl AAV-HBV.Part 2:Poly I:C-based HBV vaccine limited HBV successfully and safely1.Poly I:C or rHBV vaccine alone could not efficiently eliminate HBVCompared with Untr group,poly I:C or rHBVvac alone could not efficiently decline HBV cccDNA,HBsAg,HBV DNA,HBV total RNA and HBV-3.5kb-RNA in HBV-carrier mice,and didn t promote the production of anti-HBs.Moreover,poly I:C or rHBVvac alone did not show protective effects against HBV re-challenge.2.pHBV-vaccine could efficiently eliminate HBVCompared with Untr group,pHBV-vaccine remarkably decreased the levels of serum HBsAg,and the therapeutic effects could maintain for at least 4 weeks.Furthermore,serum HBV DNA and intrahepatic HBV DNA,HBV RNA,especially HBV cccDNA were nearly undetectable by pHBV-vaccine treatment.And we also observed reduced HBcAg-positive hepatocytes after the pHBV-vaccine therapy.Moreover,the anti-HBs levels after the pHBV-vaccine therapy showed a significant upregulation trend on week 5 after the treatment.3.pHBV-vaccine therapy could not induce significant liver injuryAlthough mononuclear cells infiltrated in liver after pHBV-vaccine treatment,the pathological analysis of liver tissues and ALT test did not show significant liver injury in mice treated with pHBV-vaccine when compared with Untr group.Part 3:pHBV-vaccine promoted maturation and antigen presentation ability of DCs1.pHBV-vaccine promoted maturation and antigen presentation ability of DCsCompared with Untr group,the percentage of CD11c+ DCs was increased in peripheral blood and draining lymph nodes(dLNs)by both poly I:C alone and pHBV-vaccine treatment,accompanied with the elevation of MHC-Ⅱ,CD86 and CD80.However,rHBVvac alone did not show significant effect on CD11c+ DCs.Compared with the negative control,poly I:C enhanced DC-mediated phagocytosis of FITC-OVA protein in vitro and elevated the expression of MHC-Ⅱ,CD80 and CD86 on DCs in a dose-dependent manner.2.The antiviral effect induced by pHBV-vaccine dependent on the participation of DCsAfter the treatment of DTx,the level of CD11c+ subset,especially the CD11c+MHC-Ⅱ+ subset,was markedly reduced.Notably,pHBV-vaccine-mediated HBV clearance was dampened significantly in HBV-carrier-CD11c-DTR mice treated with DTx.Part 4:pHBV-vaccine could improve the immune suppression microenvironmentCompared with Untr group,the expression of PD-L1 on hepatocytes was also dramatically decreased by pHBV-vaccine.In addition,pHBV-vaccine reduced both the percentage of Tregs and MDSCs in CHB mice.Part 5:pHBV-vaccine-induced HBV-specific CD11ahi CD8αlo T cells contributed to HBV clearance1.CD11alo CD8αhi T cells were HBV-specific cellsThe frequency of total CD3+ T cells,CD4+ and CD8+ T cells in spleen and liver,especially the absolute number of CD8+ T cells,increased on week 3 after the treatment when compared with Untr mice,and this effect lasted for at least 3 weeks.Moreover,the expression of CD69 on CD8+ T cells was increased by pHBV-vaccine,along with elevation of IFN-γ-production and TNF-α-production.CFSE-labelled whole splenocytes,CD8+ T cells and CD11ahi CD8αlo cells displayed significant antigen-specific proliferation in vivo,while the CD11alo CD8αhi cells did not,and the proliferated cells among the splenocytes and CD8+ T cells were indentified as CD11ahi CD8αlo.CFSE-labeled splenocytes from HBV-carrier mice treated with PBS,rHBVvac alone,poly I:C alone and pHBV-vaccine were co-cultured with BMDCs in presence of HBsAg for 72 h in vitro,the CD11ahi CD8αol cells displayed significant antigen-specific proliferation,while the CD11alo CD8αhi cells did not2.pHBV-vaccine induced the generation of HBV-specific CDllalo CD8αhi T cellsThe frequency of HBV-specific CD11ahi CD8αlo cells was increased by pHBV-vaccine treatment.Splenocytes derived from pHBV-vaccine-treated HBV-carrier mice showed antigen-specific proliferation when stimulated by HBsAg in vitro.The expression of Ki-67 was elevated by pHBV-vaccine treatment.However,the frequency of CD11ahi CD8αlo cells was not significantly influenced by rHBVvac or poly I:C alone treatment.CD11alo CD8αhi cells did not influence the serum HBsAg in HBV-carrier mice,while CD11ahi CD8αlo cells efficiently decreased the levels of serum HBsAg and HBV DNA,with similar therapeutic efficacy as whole splenocytes and CD8+ T cells.Furthermore,HBcAg-positive hepatocytes were nearly undetectable in CD11ahi CD8αlo cell-treated mice3.pHBV-vaccine induced the generation of HBV-specific CD11ahi CD49dhi CD4+T cells which contributed to CD8+ T cell responsespHBV-vaccine increased the expression of IFN-γ and TNF-α by CD4+T cells.In addition,the HBV-specific CD11ahi CD49dhi CD4+ T cells were also significantly increased in the spleen after the pHBV-vaccine therapy.Part 6:pHBV-vaccine restored the exhaustion of HBV-specific CD8+ T cells containing KLRG1+ CD8+ subset1.pHBV-vaccine restored the exhaustion of HBV-specific CD8+ T cellsBy pHBV-vaccine therapy,we found the expression of LAG-3,CTLA-4,TIGIT,PD-1 and Tim-3 on HBV-specific CDllahi CD8alo cells from liver and spleen was decreased,which accompanied with the downregulation of Eomes.Furthermore,the total frequency of polyfunctional CD84+ T cells expressing two kinds of cytokines(IFN-γ+ IL-2+,IFN-γ+ TNF-α+ and IL-2+ TNF-α+)was higher in pHBV-vaccine-treated mice than in Untr mice.Meanwhile,the total frequency of CD8+ T cells expressing one kind of cytotoxicity associated molecules(CD107a+,granzyme B+,perforin+)was increased by pHBV-vaccine treatment.2.pHBV-vaccine augmented the percentage of KLRG1+ CD8+ T cellspHBV-vaccine augmented the percentage of KLRG1+ CD8+ T cells and expression level of KLRG1 on CD8+ T cells in peripheral blood,which was maintained at stable level.Meanwhile,KLRG1+ CD8+ T cells in liver and spleen were also increased by pHBV-vaccine therapy.Moreover,KLRG1 was only expressed on HBV-specific CD11ahi CD8αlo cells but not on CD11alo CD8ahi subset.Part 7:pHBV-vaccine promoted terminally differentiated effector memory responses1.pHBV-vaccine enhanced the differentiation of SLECspHBV-vaccine promoted CD8+ T cell memory differentiation,exhibiting high frequency and absolute number of SLECs in liver,spleen and peripheral blood.Compared with MPECs,SLECs in liver and spleen showed enhanced expression of Ki-67.However,rHBVvac or poly I:C alone did not enhance the differentiation of SLECs.2.SLECs induced by pHBV-vaccine mediated HBV clearance.SLECs contained significantly higher frequency of IFN-y-producing and CD 107a-producing cells as well as granzyme B and polyfunctional cells(granzyme B/perforin-double positive)compared with MPECs.Meanwhile,the expression of inhibitory molecule Tim-3 was decreased on SLECs.After CD11ahi CD8alo cells from HBV-carrier mice treated with pHBV-vaccine were intravenously transferred into recipient HBV-carrier mice,we found that proportion of SLECs increased in blood,spleen and liver,which was not observed in recipient HBV-carrier mice transferred with CD11alo CD8αhi cells.3.pHBV-vaccine-therapy reduced expression of Eomes in HBV-specific CDllahi CD8alo cells,promoting the terminally differentiated effector memory responsesEomes levels in hepatic and splenic SLECs were lower than in MPECs of HBV-carrier mice.Importantly,pHBV-vaccine reduced the expression of Eomes in hepatic and splenic MPECs.Part 8:HBV-specific SLECs induced by pHBV-vaccine protected against HBV re-challenge1.pHBV-vaccine-therapy could protect against HBV re-challengeCompared with Untr mice,we did not detect the serum HBsAg in pHBV-vaccine-treated mice re-challenged with HBV,and more than 50%of pHBV-vaccine-treated mice produced high levels of protective anti-HBs.Moreover,intrahepatic HBV cccDNA,HBV DNA,HBV RNA and HBcAg in hepatocytes or the serum HBV DNA from pHBV-vaccine—treated mice were nearly undetectable.Meanwhile,the proportion of CXCR5+ PD-1+ follicular helper T cells was increased in response to HBV re-challenge.2.SLECs played a critical role in HBV re-challenge.The frequency and absolute number of SLECs strikingly increased in liver and spleen of pHBV-vaccine treated HBV-carrier mice compared with Untr mice,while MPECs did not show any significant change.Moreover,the frequency of SLECs in liver showed negative correlation with the levels of serum HBsAg on day 5 after HBV re-challenge(p<0.01).Consistent with these results,the expression of KLRG1 on CD8+ T cells also correlated negatively with the levels of serum HBsAg(p<0.05).Compared with Untr mice,MPECs from mice treated with pHBV-vaccine expressed high levels of CD62L,along with increased serum IL-12 levels.Conclusions and Significance:1.In this study,we found pHBV-vaccine safely and efficiently eliminated HBV in HBV-casrrier mice.2.pHBV-vaccine overcame chronic HBV infection-induced systemic tolerance,improved the functions of DCs and reversed the exhaustion of HBV-specific CD8+ T cells.3.The long-term immune memory was induced by pHBV-vaccine-therapy,and short-lived effector cells(SLECs)induced by pHBV-vaccine might play a crucial role in protecting from HBV reinfection.4.pHBV-vaccine might be a potential candidate for clinical CHB therapy and could well protect from the recurrence of HBV infection after the clinical treatment.