Exploration Of The Role Of PD-1/PD-L1 Pathway In HBV Infection

Part 1: Programmed death-1 expression is associated with the diseasestatus in hepatitis B virus infectionAim: To define the potential role of programmed death-1 (PD-1) pathway in differenthepatitis B virus (HBV) infection disease status; we examined the expression of PD-1 onCD8~+T lymphocytes and the number of antigen specific CD8~+ cytotoxic Tlymphocytes(CTLs) in peripheral blood of patients with chronic hepatitis B (CHB) andacute exacerbation of hepatitis B (AEHB) infection.Methods: The PD-1 level on CD8~+T lymphocytes and the number of HBV specificCD8~+ CTLs in patients and healthy controls (HCs) were analyzed by staining withpentameric peptide-human leukocyte antigen2 (HLA2) complexes combined with flowcytometry. Real-time quantitative polymerase chain reaction (PCR) was used to measurethe serum HBV DNA level.Results: The level of PD-1 expression on total CD8~+T cells in CHB patients (13.86％±3.38％) was significantly higher than that in AEHB patients (6.80％±2.19％, P＜0.01) andhealthy individuals (4.63％±1.23％, P＜0.01). Compared to AEHB patients (0.81％±0.73％), lower frequency of HBV-specific CD8~+ CTLs were detected in CHB patients(0.37％±0.43％, P＜0.05). There was an inverse correlation between the number ofHBV-specific CD8~+ CTLs and the level of PD-1 expression. Besides, there was asignificant positive correlation between serum viral load and the percentage of PD-1expression on CD8~+T cells in CHB and AEHB subjects (R = 0.541, P＜0.01). However,PD-1 expression was not associated with disease flare-ups as indicated by alanine aminotransferase (ALT) levels (R = 0.066, P＞0.05).Conclusion: PD-1 expression in the patients infected with HBV is negativelyassociated with the number of HBV specific CD8~+T lymphocytes and positively associatedwith HBV DNA loads. But there is no evident correlation between the PD-1 expressionlevel and the patients' serum ALT levels. Moreover, our results confirmed that HBVspecific CD8~+ CTLs response in the peripheral blood is more intense in patients withAEHB than in CHB with persistent viral infection.Part 2: Establishment of an immunocompetent mouse model forhuman chronic HBV infectionAim: To successfully establish an immunocompetent mouse model for human chronicHBV infection.Methods: Ten micrograms of replication-competent HBV plasmid DNA,pAAV/HBV1.2 having 1.2-fold overlength of HBV DNA was injected hydrodynamicallyinto the tail vein of C57BL/6 mice in a volume of PBS equivalent to about 8％of the mousebody weight. The total Volume was delivered within 5s. The serum specimens were assayedfor HBV DNA level using Real-time PCR technology on day 1,30,90 respectively aftertail vein injection. Serum HBsAg and HBeAg were tested on day 1,14 and anti-HBc,anti-HBs 28 days after injection by using ELISA. On day 105 after tail injection,intrahepatic HBsAg expression was visualized by immunohistochemical assay of tissues.For histopathological studies, the liver sections were also stained with hematoxylin andeosin. Liver and kidney samples were collected on day 105 after tailvain injection, and thendetected for HBV DNA by real-time PCR. Results: HBsAg and HBeAg in serum could be detected on day 1 and day 14 afterinjection by ELISA in 22 of 60 mice receiveing pAAV/HBV1.2, and could not be detectedin other mice. Viral titer in the serum was on average 5.02×10~3 copies/ml at 1 daypostinjection (dpi) . Then the viral titers as well as HBeAg were undetectable in serumsamples from HBsAg negative carrier mice but remained high from HBsAg positive carriermice at 30 dpi (avg viral level is 4.48×10~4 copies/ml). The average viral titer of serumsamples from all HBsAg positive mice at 90 dpi was 4.82×10~4 copies/ml. These micefailed to produce protective neutralizing anti-HBs antibody against HBV, although theyproduced anti-HBc. A moderately high serum ALT level (avg level is 130 U/L) wasobserved on day 90 after injection(the normal range is 16～42U/L). The livers frominfected C57BL/6 mice showed moderate inflammatory response. Moreover, cytoplasmicHBsAg was positive in the hepatocytes in serum HBsAg positive mice. HBV DNA waspositive in liver and kidney tissue of model mice detected by Real-time PCR.Conclusion: The positive results of HBsAg, HBeAg and HBV DNA in serum, liverand kidney tissue of the mouse model as well as moderate inflammatory responseevidenced by hematoxylin/eosin-stained liver section and serum ALT levels indicated thatmouse model of chronic infected with HBV was successful established.Part 3: The effect of blocking PD-1/PD-L1 pathway on the viral loadsand biochemical responses in a chronic HBV infected mice modelAim: To investigate whether applying anti-PD-L1 could reinvigorate antiviralimmunity and reduce viral load in a chronic HBV infected mice model.Methods: The serum samples from hydrodynamically injected C57BL/6 mice modelwere assayed for HBV DNA levels at 90 days post tailvain injection by Real-time PCR. 18mice successfully established were partnered according to the levels of serum HBV DNA and divided into three groups: 6 mice were administered intraperitoneally with 200μg ofanti PD-L1 blocking mAb in 200μl of PBS every third day, beginning on the day 91st and94th after establishment of mice model. 12 Control mice received injection of PBS alone or200μg of rat IgG2a isotype control in 200μl of PBS in the same way. Serum HBV DNAwere analyzed by Real-time PCR, serum ALT were determined on Fully-auto ChemistryAnalyzer at 1 day before and 7,14 day post-block. Levels of HBV-DNA in liver and kidneytissue of recipient mice at 14th day after treatment were measured by real-time PCR.Intrahepatic HBsAg and PD-L1 expression were visualized by immunohistochemical assayof tissues. For histopathological studies, the liver sections were also stained withhematoxylin and eosin at 14 days post-block.Results: We observed elevated expression of PD-L1 in liver cells in infected mice atdays 105 following infection than health control mice (P＜0.05) .At the 14th day posttreatment, results showed an increase in the inflammatory activity of chronic hepatitis inmice treated with anti-PD-L1 Ab, but there were no remarkable changes in the histologicalappearance of biopsy specimens from PBS-treated and isotype control Ab-treated mice.Moreover, we found that ALT levels rose from 130U/L on the day before treatment to apeak of 580U/L on day 7 after anti-PD-L1 blocking and remained significantly abovenormal throughout our study with a value of 558U/L on day 14 after anti-PD-L1 blocking.Mice receiving anti-PD-L1 Ab showed a substantial reduction in virus level from the bloodcompared with control isotype Ab or PBS treated mice (P＜0.01) . Mice given theanti-PD-L1 blocking antibody showed a persistent decrease of virus load even aftertreatment (P＜0.01). In addition, the treated mice demonstrated accelerated clearance ofvirus in tissues including liver and kidney 14 days after treatment; while control Ab andPBS treated mice still harboured high levels of virus in these tissues (P＜0.05). Anti-PD-L1blocking antibody also inhibited the expression of HBsAg in the liver. The anti-PD-L1treated mice that alleviated the infection remained healthy, and did not display any evidentsigns of disease. Conclusion: Our data indicate that PD-L1 expression is up-regulated in the livers ofchronic Infected HBV Mice. Treatment with therapeutic anti-PD-L1 blocking Ab in vivocan restore effective antiviral immune response in mice persistently infected with HBV andlead to advanced viral control of HBV infection compared with control mice.