Combination therapy including CpG oligodeoxynucleotides and entecavir induces early viral response and enhanced inhibition of viral replication in a woodchuck model of chronic hepadnaviral infection
Introduction
According to World Health Organization estimates, approximately 240 million people worldwide are chronically infected with the hepatitis B virus (HBV), a hepadnavirus that is one of the major causes of liver cirrhosis and hepatocellular carcinoma (http://www.who.int/hiv/pub/hepatitis/hepatitis-b-guidelines/en/). Over the past two decades, major advances have been made in the treatment of chronic HBV infection, but complete and sustained viral eradication is difficult to achieve with the currently available treatments (Tang et al., 2014, Wong and Chan, 2013). Preclinical and clinical studies have indicated the necessity of stimulating HBV-specific B cell and T cell responses to bring about long-term viral control in chronically infected patients (Bertoletti and Ferrari, 2012, Dandri and Locarnini, 2012, Grimm et al., 2013, Kosinska et al., 2015), and several reviews have summarized recent therapeutic approaches that have been attempted to achieve this control (Kosinska et al., 2015, Liu et al., 2014a, Liu et al., 2014b, Marimani et al., 2013, Michel et al., 2015, Michel et al., 2011, Wang et al., 2014, Zhang et al., 2013). Traditional subunit vaccines have failed to treat chronic HBV infection, but newer approaches, such as DNA vaccines and anti-HBs antibody-HBsAg immune complexes, are being tested in clinical trails (Godon et al., 2014, Xu et al., 2013, Yang et al., 2012). Pre-clinically, the woodchuck model of hepadnaviral infection is well established as a suitable model of chronic HBV infection (Kosinska et al., 2012, 2013; Lu et al., 2007; Roggendorf et al., 2010; Wang et al., 2014). In the woodchuck model, combinations of antiviral agents and immunomodulation have been shown to be capable of inducing virus-specific T and B cells and sustained suppression of viral replication (Kosinska et al., 2013, Liu et al., 2014a, Liu et al., 2014b), but the overall efficacy has been low, with only some of the animals exhibiting responses.
Toll-like receptors (TLRs) play critical roles in the recognition of pathogen-associated molecular patterns and in the activation of innate immune responses to infectious agents (Lester and Li, 2014, Takeuchi and Akira, 2010). While TLR agonists have not been tested on chronic HBV infection in humans, pre-clinical data suggest that they may be useful for this indication (Ma et al., 2015, Zhang et al., 2012). For example, various TLR agonists have been shown to suppress HBV replication in HBV transgenic mice (Isogawa et al., 2005). We and others have shown that TLR agonists stimulate antiviral cytokines, leading to suppression of hepadnaviral replication in HBV-met cells and primary woodchuck hepatocytes (Wu et al., 2007, Zhang et al., 2009). Finally, a TLR7 ligand (GS-9620) has been demonstrated to induce marked viral suppression in both woodchuck (WHV) and chimpanzee (HBV) models (Lanford et al., 2013, Menne et al., 2015).
CpG oligodeoxynucleotides (ODNs) are TLR9 agonists that stimulate B cells and plasmacytoid dendritic cells (pDCs) in humans (Jurk and Vollmer, 2007). Four main CpG classes have been described that differ structurally by their abilities to form higher-ordered structures due to palindromic sequences and by their backbones, namely the native phosphodiester (PO) backbone or nuclease-resistant phosphorothioate (PS) backbone (Vollmer et al., 2004). Class B CpG ODNs, which are monomeric and have a PS backbone, strongly activate B cells, but they only induce a low level of IFN-α secretion in pDCs. Class A CpG ODNs, which form higher-ordered structures and have a chimeric PO/PS backbone, strongly induce IFN-α secretion in pDCs but have a limited capacity for B cell activation. Class C CpG ODNs, which form dimers and have a PS backbone, induce B cell activation similar to class B CpG ODNs, in addition to IFN-α secretion at a level that is intermediate to that induced by the B and A classes. Class P CpG ODNs, which form concatemers due to a double palindrome and have a PS backbone, stimulate B cells, similar to class B, and they are more potent than class C in terms of IFN-α induction (Samulowitz et al., 2010). CpG ODNs, especially those classes that induce type I IFN, may be suitable immunomodulators for the treatment of chronic viral infections (Bodera et al., 2012, Hanagata, 2012, Krieg, 2007). A class C CpG ODN (CpG 10101) has been administered weekly to chronically infected hepatitis C virus (HCV) patients, resulting in dose-dependent increases in the levels of serum cytokines, including IFN-α, and a decrease in HCV viremia, although long-term viral control was not achieved (McHutchison et al., 2007). In the present study, we evaluated CpG, alone or in combination with an antiviral agent, in WHV-infected woodchucks.
Section snippets
CpG ODNs and entecavir (ETV)
Seven CpG ODNs (R2 to R8) and one non-CpG control ODN (R1) were provided by the Pfizer Oligonucleotides Therapeutics Unit (Düsseldorf, Germany) (Table 1). ETV was purchased from Bristol-Myers Squibb (Munich, Germany).
Woodchucks
Woodchucks were purchased from North Eastern Wildlife (Ithaca, NY, USA) and were kept at the Animal Laboratory of the University of Essen. The experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the
Response of woodchuck PBMCs to stimulation with CpG ODNs
Among the seven CpG ODNs, all but one class B CpG ODN (R2) induced some IFN production in PBMCs from at least two of the three animals tested, although the effects were strongest with class P (R7 and R8) and weakest with classes B (R2 and R3) and A (R6), with class C (R4 and R5) producing intermediate results (Fig. S1A). Statistical analysis was hindered by the small number of animals, but combining all of the data for the same class revealed that class P induced a significantly increased level
Discussion
The present study demonstrates that CpG ODNs are able to stimulate woodchuck PBMCs to produce IFN in vitro, and furthermore, that immunotherapy with a class P CpG ODN combined with a conventional nucleoside analogue (ETV) results in the profound suppression of WHV DNA and WHsAg loads in vivo.
Various classes of CpG ODNs have been described that have different immunostimulatory effects on mouse or human cells (Hanagata, 2012, Jurk and Vollmer, 2007, Vollmer et al., 2004). This study, which is the
Acknowledgments
This work was partly supported by grants from Deutsche Forschungsgemeinschaft (TRR60 und GK1494 to M.L.), Wilhelm Sander-Stiftung (No. 2011.023.1 to M.L.), and the Chinese Research Foundation (Key Discipline Project of Hubei Province (2014XKJSSJ00 and 2014CFB645).
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