Purification of monoclonal antibody from hybridoma

Chapter II: Materials and Methods

2.12 Purification of monoclonal antibody from hybridoma

Cell line G4 which secreted monoclonal antibody against mouse PD-1 were cultured in RPMI1640 medium containing 10% FBS and antibiotics. The culture supernatants were collected for further purification. Protein G sepharose column was washed with 100mM Tris-HCL pH 8.0 buffer. The culture supernatant were passed through protein G column.

The column was washed and eluted with 50 mM glycine(pH3.0). The antibody concentration was determined for further applications.

Chapter III

Results

3.1 Inhibitory receptor PD-1 in chronic viral infection

Hepatitis B virus (HBV) causes acute and chronic inflammatory liver diseases and subsequent hepatic cirrhosis and hepatocellular carcinoma (HCC). During chronic HBV infection, a dynamic balance between viral replication and the host immune response is pivotal to the pathogenesis of liver disease. It is widely accepted that adaptive immune responses, particularly cellular immune responses, mediate the clearance of HBV (Chisari and Ferrari, 1995; Jung and Pape, 2002) Unfortunately, HBV-specific T-cell function is impaired in patients with chronic HBV infection characterized by low levels of antiviral cytokines, impaired cytotoxic T lymphocyte activity, and persistent viremia (Iwai et al., 2003). However, the mechanism underlying this T-cell malfunction in chronic HBV infection is not completely understood.

The immunologic receptor, programmed death (PD)-1, a 55-kDa transmembrane protein containing an immunologic receptor tyrosine-based inhibitory motif, was originally isolated from a T-cell line exhibiting a high sensitivity to apoptosis (Ishida et al., 1992).

The PD-1/PD-L1 pathway is well documented to play a negative role in regulating activation and proliferation of T-cells and production of cytokines (Chen, 2004; Nurieva et al., 2006). There is evidence that the PD-1 pathway plays an important role in inhibiting the function of virus-specific CD8⁺ T-cells in chronic viral infection involving human immunodeficiency virus (HIV) (Day et al., 2006), hepatitis C virus

(HCV) (Golden-Mason et al., 2007), and HBV (Boni et al., 2007). Although reports are available on changes in expression levels of PD-1 and T-cell responses in patients with HBV infection, the pattern of change of PD-1 expression in the natural course of chronic HBV infection has not yet been presented.

3.2 Increased PD-1-expressing CD8⁺ and CD4⁺ T-cells in liver-infiltrating

lymphocytes from mice with HBV persistence

Successful eradication of HBV infection requires elimination of virus-infected hepatocytes and inhibition of virus replication by the host immune system. Although the chronicity of HBV infection is the result of impaired HBV-specific immune responses that cannot efficiently eliminate or cure infected hepatocytes, many issues remain unsettled. We established a mouse model of HBV persistence in immunocompetent mice by a hydrodynamic injection of replication-competent HBV DNA. This approach generated HBV persistence in young C57BL/6 mice, but not in BALB/c mice (Huang et al., 2006; Lin et al., 2010). C57BL/6 and BALB/c mice were hydrodynamically injected with the pAAV/HBV1.2 plasmid. At the indicated time points, intrahepatic lymphocytes from mice that were HBsAg-positive (carrier) or HBsAg-negative (cleared) were isolated, and PD-1 expressions by CD8⁺ and CD4⁺ T-cells were analyzed by flow cytometry. The results in Figure 1 demonstrate that there were increased proportions of

PD1-expressing CD8⁺ and CD4⁺ T-cells in livers of C57BL/6 mice, but not in BALB/c mice. Moreover, in C57BL/6 mice, both PD-1-expressing CD8⁺ and CD4⁺ intrahepatic T-cells significantly decreased in mice which had cleared the injected pAAV/HBV1.2 plasmid (cleared mice), compared to mice with HBV persistence (carrier mice). These results indicate a significant increase in PD-1 expression in both intrahepatic CD8⁺ and CD4⁺ T-cells of mice with HBV persistence. In addition, we demonstrated that there were increased PD1-expressing CD8⁺ T-cells in liver-infiltrating lymphocytes in C57BL/6 mice, which were more susceptible to HBV persistence compared to BALB/c mice; and there were also increased PD1-expressing CD8⁺ and CD4⁺ T-cells in HBV carrier mice compared to control mice or mice which had recovered from an HBV infection (Figure 1). This is consistent with the results of recent studies of HBV-infected patients, which demonstrated that PD-1 expression can impair virus-specific CD8 T-cell responses during chronic HBV infection (Hsu et al., 2010;

Xie et al., 2009; Yang et al., 2010; Zhang et al., 2009; Zhang et al., 2008).

Furthermore, to further define the role of the HBV core in HBV persistence in this mice animal model, C57BL/6 mice were injected with wild-type (WT) pAAV/HBV1.2 or HBV core mutant DNA, including HBV-175 or HBV-38 (Lin et al., 2010), and HBV persistence and PD-1 expression in liver-infiltrating lymphocytes were analyzed by flow cytometry. Both the HBV-175 and HBV-38 core-mutant viral constructs caused

significant loss of the ability to raise the host immunity to clear the virus, with increased PD-1-expressing T-cell infiltration, leading to HBV persistence in mice, consistent with the observation that the HBV core protein plays an important role in induction of a host immune response to HBV (Figure 2)(Lin et al., 2010)

3.3 Liver-infiltrating CD8⁺ lymphocytes in carrier mice displayed the

PD-1^hiCD127^low-exhausted phenotype

Recent studies in animal models of viral infection indicated that the interaction between PD-1 on lymphocytes and its ligands plays a critical role in T-cell exhaustion by inducing T-cell inactivation. HBV-specific PD-1-positive CD8 cells of patients with chronic HBV infection also displayed lower levels of the interleukin (IL)-7 receptor, CD127, which was previously described in association with the exhausted phenotype (Boettler et al., 2006; Boni et al., 2007). To define the exhausted phenotype in the PD-1-expressing intrahepatic T-cells in mice with HBV persistence, liver-infiltrating CD8⁺ lymphocytes from mice hydrodynamically injected with the HBV construct were evaluated for the expressions of both PD-1 and CD127 by CD8⁺ T-cells by flow cytometry (Figure 3). The results in Figure 3 demonstrate that PD-1 was more highly expressed by intrahepatic CD8 cells in mice with HBsAg persistence. Also, among intrahepatic CD8 populations, CD127 expression was significantly lower in carrier mice

compared to mice which had cleared HBV. Our results indicate that liver-infiltrating CD8⁺ lymphocytes in carrier mice displayed the PD-1^hiCD127^low-exhausted phenotype.

3.4 Liver-infiltrating PD-1⁺CD4⁺ T-cells in mice with HBV persistence exhibit the

phenotype of regulatory T-cells (Tregs)

Our results indicated that there were significant increases in PD-1-expressing CD8⁺ and CD4⁺ T-cells in liver-infiltrating lymphocytes from mice with HBV persistence. We further analyzed the immune phenotype of PD-1-expressing intrahepatic CD4⁺ T-cells.

Intrahepatic lymphocytes from C57BL/6 mice that were HBsAg-positive 4 weeks after being hydrodynamically injected with WT pAAV/HBV1.2 were isolated, and the immune phenotype was analyzed. The results in Figure 4 demonstrate that there were significantly increased CD4⁺ Foxp3⁺ populations in mice with HBV persistence.

Moreover, the expression of CTLA-4 was also upregulated in PD-1⁺ CD4⁺ T-cells, indicating that these PD-1⁺ T-cells displayed the Treg phenotype. Taken together, there were increases in PD1-expressing CD8⁺ T-cells in the liver, and increased PD-1 expressing CD4⁺FoxP3⁺ T-cells and CTLA4-expressing CD4⁺ T-cells in the liver of HBV carrier mice. The results indicate that PD-1-expressing CD4⁺ T-cells in liver-infiltrating lymphocytes from mice with HBV persistence were Tregs.

We then asked whether the PD-1/PD-L1 interaction of intrahepatic T-cells was associated with the impaired immune response, resulting in a defective T-cell response to HBV in mice with HBV persistence after an infection. The HBV core-specific IFN-γ T-cell response in mice hydrodynamically injected with WT pAAV/HBV1.2 (HBV-wt), HBV-175, or HBV-38 in the presence and absence of anti-PD-1 mAb treatment were analyzed by an enzyme-linked immunosorbent spot (ELISPOT) assay. Results in Figure 5 demonstrate that the frequency of HBcAg-specific IFN-γ-secreting cells was significantly reduced in mice injected with HBV core mutant constructs; however, impairment of the HBcAg-specific IFN-γ response was restored in mice treated with the anti-PD-1 mAb. Taken together, our results indicate that the impaired T-cell response to the HBV core in the IFN-γ ELISPOT assay could be restored by in vivo treatment with an anti-PD-1 mAb, indicating that the level of PD-1 expression on intrahepatic T cells is correlated with the anti-viral T-cell response in vivo.

3.5 Blockade of the PD-1 pathway by an anti-PD-1 mAb reduced the HBV

persistence rate in this mouse animal model

To further define the role of T-cell exhaustion in the pathogenesis of persistent HBV infection in this mice animal model, and determine the effect of PD-1/PD-L1 blockade on restoring immune dysfunction and clearance of HBV, C57BL/6 mice were

intraperitoneally treated with an anti-PD-1 blocking mAb or a control isotype Ab. Ab administration was initiated every 2 days on day 6 before an injection with the HBV core-truncated (HBV-175) mutant construct. Thereafter, Abs (200 µg) were repeatedly administered every 3~4 days for a period of 8 weeks. The HBsAg level in mice serum was determined by an ELISA. The results in Figure 6 demonstrated that blocking the interaction of PD-1/PD-L1 by the anti-PD-1 mAb significantly reduced the frequency of HBV persistence in mice injected with the core-null HBV viral construct, resulting in clearance of HBV in vivo. The mice treated with anti-PD-1 mAb with higher viral clearance rate and also showed higher ALT level compared to control mice. In mice treated with anti-PD-1 mAb, the PD-1 expression level in liver infiltrating lymphocytes after hydrodynamic infection of HBV was similar to mice treated with control Ig. In contrast, among intrahepatic CD8 populations, CD127 expression was significantly higher in mice treated anti-PD-1 mAb compared to mice treated with control Ig (Figure 7), indicating PD-1 blockage reversed PD-1^hiCD127^low-exhausted phenotype in intrahepatic CD8⁺ T cells in mice treated with anti-PD-1 mAb. Taken together, these results indicate that PD-1 blockage reverses immune dysfunction as well as the PD-1^hiCD127^low-exhausted phenotype and viral persistence to HBV infection in this mouse animal model, suggesting that the anti-PD-1 mAb might be a good therapeutic candidate for chronic HBV infection.

These results are thus undertaken to further define the role of T-cell exhaustion in chronic HBV infection in a mice animal model, by comparing the phenotype and function of intrahepatic infiltrating T lymphocytes in mice with HBV persistence or HBV clearance, and the effect of PD-1/PD-L1 blockade in restoring immune dysfunction and clearance of HBV. Furthermore, PD-1/PD-L1 blockade partially restored the function of intrahepatic T-cells, leading to viral clearance. It is the first report to demonstrate PD-1/PD-L1 blockade could reverses immune dysfunction and HBV viral persistence in vivo. This observation opens new potential perspectives for the development of novel immunotherapies for chronic hepatitis B.

3.6 Induction of host innate immune response by HBV

It has well established that activation of cellular immune response is required for mediating HBV clearance from the liver. In our mouse model of HBV persistence, we demonstrated that the chronicity of HBV infection is associated with PD-1 expression on intrahepatic lymphocytes (Tzeng et al., 2012). It is believed that an efficient control of HBV infection requires the coordinated actions of both innate and adaptive immune responses (Bertoletti and Ferrari, 2012; Durantel and Zoulim, 2009; Yang et al., 2010).

Innate immunity induces an antiviral state in infected cells by producing type I interferons (IFN), and supports the efficient maturation and site recruitment of adaptive

immunity through the production of pro-inflammatory cytokines, in particular, tumor necrosis factor-α (TNF-α) (Bertoletti and Ferrari, 2013). However, in chronic HBV infection, impaired HBV-specific immune responses failed to eliminate infected hepatocytes, resulting in the persistence of HBV.

Experimental viral infection in both chimpanzees and woodchucks found only limited or even non-activation of innate immunity being demonstrated in acute HBV infection (Dunn et al., 2009; Guo et al., 2009). Nevertheless, a transient though slight activation of IFN-α genes was detected in human hepatocytes infected by HBV in chimeric mice, in support of the innate immunity to sense and react to HBV (Lutgehetmann et al., 2011). However, the mechanisms responsible for sensing HBV within the infected cells have not been elucidated yet, and which molecular components of the HBV actually recognized by the pattern recognition receptors (PRR) triggering the antiviral response is still undefined. In addition, a number of recent studies have been investigated in suggesting the involvement of NK cells in chronic HBV infection and they could play a role in liver damage during reactivation (Dunn et al., 2007; Oliviero et al., 2009; Peppa et al., 2010). The role of innate immunity in viral clearance during HBV infection is still not clear.

TNF-α has long been considered as a key cytokine in HBV eradication (Larrubia et al., 2009). Higher intrahepatic levels of TNF-α have been associated with the increased

expression of HLA class I molecules and an enhanced CD8⁺ T cell response to HBV, which leads to the more effective destruction of HBV-infected hepatocytes (Hussain et al., 1994). In chronic HBV infection, CD8⁺ T cells lack the ability to secrete enough TNF-α to kill HBV-infected hepatocytes, the so-called “exhausted phenotype.” This is a functional HBV-specific CD8⁺ T cell impairment that is detectable at the peak of the disease when the majority of HBV-specific CD8⁺ T cells are activated but have little ability to proliferate and are functionally exhausted, probably due to upregulation of programmed death (PD)-1 (Fisicaro et al., 2010; Peng et al., 2008b). Studies have demonstrated that genetic polymorphisms leading to lower constitutive or inducible TNF-α expression are related to an increased risk of progression toward chronic HBV infection (Ben-Ari et al., 2003; Hohler et al., 1998). Clinically, an anti-TNF regimen also reportedly increased the number of cases of HBV reactivation (Lan et al., 2011;

Perez-Alvarez et al., 2011). However, pro-inflammatory cytokines are often undetectable during the early phases of HBV infection (Wieland et al., 2004).

3.7 The TNF-α rather than IFN-mediated pathway is critical in HBV clearance

Although the chronicity of HBV infection is the result of impaired HBV-specific immune responses that cannot efficiently eliminate or cure infected hepatocytes, however, this likely a result from the failure of immune responses at the first exposure

to HBV. Therefore, we tested a panel of KO mice with specific deficiency in the innate immune sensors or effectors for their capability in clearing HBV after hydrodynamic injection (HDI) of a replication competent HBV DNA. This approach generated HBV persistence in C57BL/6 mice but not in BALB/c mice (Huang et al., 2006; Lin et al., 2010; Tzeng et al., 2012). To identify the immune effectors of innate immunity that eliminate HBV from the liver, we monitored the persistence of HBsAg in a panel of gene knockout mice, including Nod-like receptor family protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), myeloid differentiation primary response gene 88 (MYD88), IL-1 receptor (IL-1R), IFN-α/β receptor (IFNAR), and TNF-α. The results (Figure 8) demonstrate that HBV viral clearance is not significantly different from wild type C57BL/6 mice in IFNAR, RIG-I, MDA5, MYD88, NLRP3, ASC, and IL-1R knock-out mice, indicating that these effectors are not required for HBV clearance. In contrast, only TNF-α knockout mice showed a markedly higher HBV-positive rate and prolonged HBV persistence compared to other strains, suggesting that TNF-α is an important effector cytokine that is required to clear HBV from the liver. The results (Figure 8 and Figure A1) demonstrated that the HBV persistence rate and serum HBs Ag levels was similar between IFNR knock-out mice and wild type C56BL/6 mice, indicating that IFN-mediated pathways are not essential for clearing HBV in this animal model. We

further investigated the roles of innate cytokines TNF-α during HBV infection (Figure 9). In contrast, significantly impaired HBV clearance and enhanced HBV persistence was observed when TNF-α was neutralized with the soluble TNF receptor Etanercept in HBV-cleared mouse strain BALB/C, suggesting that TNF-α is required for HBV clearance (Figure 9A). Similarly, the HBV persistence rate and serum HBs Ag levels was significantly enhanced in TNF-α knockout mice compared to wild-type C57BL/6 mice (Figure 9B). Taken together, these results indicate that TNF rather than the IFN-mediated pathway is required for HBV clearance, and TNF blockage enhances HBV persistence in vivo.

3.8 TNF-α deficiency leads to cytotoxic T lymphocyte dysfunction against HBV

We then asked whether TNF-α deficiency is associated with an impaired T cell response to HBV in mice with HBV persistence. Recent studies in viral infection indicate that the interaction between the PD-1 on lymphocytes and its ligands plays a critical role in T-cell exhaustion by inducing T-cell inactivation and displayed lower levels of interleukin (IL)-7 receptor CD127, which had previously been described in association with the exhausted phenotype (Boettler et al., 2006; Boni et al., 2007). The results in Figure 10A demonstrate that PD-1 is more highly expressed by intrahepatic CD8 cells in BALB/c mice hydrodynamically injected with HBV constructs and treated with

Etanercept. Also, among intrahepatic PD-1 -expressing CD8 populations, CD127 expression was significantly reduced in mice treated with Etanercept. Interestingly, there are no such differences noted for the spleen. Our results indicate that there are significantly increased liver-infiltrating PD-1^hiCD127^low-exhausted CD8⁺ lymphocytes in Etancercept-treated HBV infected mice. Similar results were observed in TNF-α knockout mice (Figure 10B). Both results indicate that liver-infiltrating CD8⁺ lymphocytes in response to HBV in mice with TNF-α deficiency displayed the PD-1^hiCD127^low-exhausted phenotype.

We then evaluated T cell response to HBV in mice with HBV persistence after an infection. The HBV core-specific IFN-γ T-cell response in mice hydrodynamically injected with HBV DNA in the presence and absence of TNF-α were analyzed by an ELISPOT assay. Results in Figure 10C demonstrate that the frequency of HBcAg-specific IFN-γ-secreting cells was significantly reduced in TNF-α knockout C57BL/6 mice or Etanercept-treated BALB/c mice. Taken together, our results indicate TNF-α is correlated with the anti-HBV T cell response in vivo.

3.9 TNF-α blockage-induced elevation of serum HBV viral loads and maintained

HBV viral gene expression within the liver

Our results indicate that TNF-α deficiency is associated with the impaired T-cell response to HBV. We then investigated the effects of TNF blockage on HBV viral load and viral replication. The results (Figure 11A) show the control group BALB/c mice were almost cleared of HBV viral loads in the initial five weeks after hydrodynamic transfection of HBV. In contrast, persistently elevated HBV viral loads in serum were observed in BALB/c mice treated with Etanercept (Figure 11A). TNF-α knockout mice showed similar results with persistently elevated HBV viral loads in serum compared to the wild type C57BL/6 mice (Figure 11B). We then analyzed the HBV transcripts in the livers of mice transfected with /HBV by Northern blotting after hydrodynamic injection (Figure 12). The HBV transcripts remained detectable in the liver of TNF-α knockout and Etanercept-treated BALB/c mice up to day 42 post-transfection. In contrast, the HBV transcripts were almost undetectable in wild type C57BL/6 mice on day 42 post-transfection. Similarly, the results from an immunohistochemistry analysis also revealed that the staining for HBcAg and HBsAg remained detectable in the livers of TNF-α knockout and Etanercept-treated BALB/c mice on day 42 post-transfection.

However, HBcAg and HBsAg staining was much lower in wild type C57BL/6 mice on day 42 post-transfection, which is correlated with serum viral loads and HBV transcripts in the liver (Figure 12B). These results indicate that a TNF-α deficiency impairs viral clearance and increases HBV viral load and viral replication in vivo.

3.10 Lack of TNF-α eliminates HBcAg-induced HBsAg clearance

Previous studies showed that the immune response triggered in mice by HBcAg during exposure to HBV is important in determining HBV clearance (Lin et al., 2010; Tzeng et al., 2012; Yang et al., 2013). We then test whether HBcAg can induce cytokines

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