New treatments to reach functional cure: Rationale and challenges for emerging immune-based therapies

Best Practice & Research Clinical Gastroenterology 31 (2017) 337e345

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New treatments to reach functional cure: Rationale and challenges for emerging immune-based therapies Adam J. Gehring, PhD, Scientist, Assistant Professor a, b, * a b

Toronto Centre for Liver Disease and Toronto General Hospital Research Institute, University Health Network, Toronto, Canada Department of Immunology, University of Toronto, Toronto, Canada

a b s t r a c t Keywords: Hepatitis B virus (HBV) Immunotherapy Vaccines Treatment

The landscape for chronic HBV therapy is rapidly evolving. The latest generation of antiviral drugs provide robust virus suppression with a high barrier to resistance that facilitates long-term treatment. However, low rates of HBsAg loss demonstrate that additional strategies are needed to consistency achieve a functional cure. The immune system can clear HBV and establish long-term control over the virus. Sufficiently boosting HBV immunity in chronic patients has been very difficult due to immune exhaustion, immune dysregulation, and inhibitory pathways suppressing the immune response. Therapeutic vaccines employing new technology, vectors and new immunomodulatory drugs that can elicit direct antiviral effects and cancel inhibitory mechanism may be able to overcome exhaustion. This review will discuss the justification for immunotherapy, lessons from previous trials and new vaccines/ drugs in early stage clinical trials. The challenges of correlating immune responses induced by these drugs to clinical efficacy will also be addressed. Crown Copyright © 2017 Published by Elsevier Ltd. All rights reserved.

Introduction The ability of Hepatitis B virus (HBV) to establish the long-lived extrachromosomal cccDNA replication template, the excessive production of viral antigens and the immune-suppressive environment of the liver have posed significant therapeutic challenges. However, it is clearly established that the immune response during acute HBV infection is able to overcome the suppressive environment and eliminate HBV from hepatocytes without complete destruction of the liver. This requires a coordinated immune response that relies on an effective CD4 T cell, CD8 T cell and B cell response resulting primarily in non-cytolytic HBV clearance and production of anti-HBV antibodies as a serological marker of longterm HBV control [1]. The contrasts between patients that resolve acute HBV infection and those with chronic infection have been extensively studied. In general, these data support the dogmatic statement that resolved patients display a broad and robust HBV-specific T cell response while T cells in chronic HBV patients display reduced frequencies and are functionally hyporesponsive. The challenge

* Toronto Medical Discovery Tower, 10-356 101 College Street, Toronto, Ontario, M5G 1L7, Canada. E-mail address: [email protected]. http://dx.doi.org/10.1016/j.bpg.2017.05.004 1521-6918/Crown Copyright © 2017 Published by Elsevier Ltd. All rights reserved.

has been to understand why the immune response has failed in chronic HBV, what maintains the hyporesponsive state, and whether it is possible to sufficiently restore HBV-specific immunity in chronic patients to achieve viral clearance and long-term control. At least some of these obstacles have been identified. We have a better understanding of inhibitory receptor expression on T cells [2], active elimination of HBV-specific T cells [3,4], potential defects in T cell priming by dendritic cells [5], metabolic suppression of immunity [6e8], dampening responses through regulatory T cells [9] and the immunosuppressive liver environment [10]. New immune-based therapies are beginning to build on this knowledge to improve potency of T cell targeted therapies such as vaccine potency and/or checkpoint blockade. Other strategies are focused on modulating the intrahepatic environment with drugs targeting innate immune cells to achieve localized production of antiviral and inflammatory cytokines. However, the history of immune-based therapies in chronic HBV infection has demonstrated the challenges that we face. In addition, immune based strategies will have to contend with the extraordinary high safety bar set by current nucleoside analogues. This review will cover the primary areas of immunomodulation currently being tested in clinical trials. I will attempt to summarize background data and previous attempts that have led us to the

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current state of HBV immunotherapy. A majority of the focus will be on human immunity but some animal models have been instrumental to our understanding of pathogenesis and resolution and will thus be referenced. The final section of the review will address the difficulty of determining if a therapy is immunologically effective and whether this correlates with clinical efficacy.

reactivation. Thus, even in chronic HBV patients, where the immune response is considered weak and dysfunctional, there is ongoing immune control. The mechanisms for this control still require better definition but it is reasonable to hypothesize that further enhancing existing immunity in chronic HBV patients holds potential for successful immunotherapy.

Justification for immune control in chronic HBV

Targeting adaptive immunity

Immune control of HBV replication has been demonstrated in every potential model of HBV infection, from in vitro co-culture with T cells to evidence from chronic HBV patients. IFN-g and TNF-a produced by HBV-specific T cells in co-culture with HBV producing hepatocyte-like cells results in a reduction in viral replication [11]. Transfer of HBV-specific T cells into HBV-transgenic mice ablates HBV replication in the liver of these mice [12]. Intrahepatic infiltration of IFN-g producing T cells in acutely infected chimpanzees, where virtually 100% of hepatocytes are infected, leads to a non-cytolytic clearance of HBV whereas depletion of T cells leads to persistent infection [13e15]. Data from chronic HBV patients undergoing bone-marrow transplant from healthy immune donors can lead to clearance of chronic HBV infection [16e18]. Case reports demonstrate that a chronically HBV infected liver, transplanted into an immune recipient, can lead to clearance of the chronic infection [19,20]. All of these examples demonstrate the immune systems' ability to control HBV replication, many of them highlighting the importance of the HBV-specific T cell response. The human data in bone marrow transplant or liver transplant patients provide evidence of the ability to clear chronic HBV infection by reconstituting the immune response but are extraordinary cases. So, what is the evidence that the immune response in chronic HBV patients is contributing to control of HBV during chronic infection, where the immune system has repeatedly been demonstrated to be weak? In fact, there are a small percentage of patients who do clear HBV, measured by a loss of HBsAg from the circulation. One study has investigated the antiviral T cell response in these patients compared to those with ongoing chronic infection and found significantly increased T cell responses in patients who lose HBsAg [21]. However, because it was cross-sectional, it is not possible to determine if the increased HBV-specific T cell frequency was the cause or result of HBsAg loss. In addition, data from liver biopsies has demonstrated that increased T cell frequency correlates with better control of viral replication and less liver inflammation [22]. Both studies provide evidence that increasing T cell immunity correlates with control of HBV replication. Additional evidence from chronic, and in some cases resolved, HBV patients supporting ongoing immune control is observed in patients receiving immune-suppressive therapy. The relative risk of these classes of drug were recently reviewed and published by the American Gastrological Association [23]. Drugs with the highest risk of HBV reactivation were Corticosteroids and B cell depleting agents. Corticosteroids, used to treat inflammatory diseases, have long been known exacerbate HBV pathology. Steroids suppress antiviral immunity and bind to responsive elements on the HBV genome, enhancing HBV transcription, leading to HBV reactivation. B cell depleting therapeutic antibodies used in lymphoma treatment carries the highest risk of HBV reactivation. Up to 60% of chronic HBV patients have been show to experience HBV reactivation following B cell depletion. Even patients with resolved HBV infection (HBsAg negative, anti-HBc positive) are at risk of reactivation when B cell immunity is ablated. Furthermore, anti-TNF-a therapies, which are increasingly used for inflammatory diseases like colitis and rheumatoid arthritis, are associated with HBV

Therapeutic vaccine trials Adaptive immunity has been the primary focus of immunebased strategies, given that T cells are the primary effectors mediating clearance of infected hepatocytes and anti-HBs antibodies define disease resolution. A majority of that effort has centered on therapeutic vaccination. Therapeutic vaccination, unlike prophylactic vaccination in healthy individuals, tries to boost HBV-specific immunity in the context of an ongoing chronic infection. The immune system in chronic HBV patients is persistently challenged with virions and HBV antigens in both the blood and liver. This persistent exposure to viral antigens is likely the primary driver of immune exhaustion, and poses a significant obstacle to overcome. The amount of antigen in the blood and liver, up to 1 mg/ml HBsAg (~200,000 IU/ml) in the serum, can easily overwhelm the amount of antigen delivered in vaccines [24,25]. Therefore, vaccination strategies have to deliver antigen in such a way as to redirect the immune system to the vaccine and associated lymphoid tissue and away from antigen in the blood and liver to facilitate priming (Fig. 1) [26]. To date, there have been numerous therapeutic vaccine attempts that have largely fallen into 2 categories 1) formulations of recombinant antigen or 2) DNA vaccination with a few additional unique approaches. A list of vaccine trials is presented in Table 1. I have tried to be exhaustive, but may have missed some studies, and this does not account for unpublished data. There have been at least 18 published clinical trials attempting a therapeutic HBV vaccine. As evidenced by a lack of a licensed therapeutic vaccine for chronic HBV infection, none of the strategies have been consistently successful. However, these trials have been important to help us understand the challenge of boosting anti-HBV immunity and provided some evidence of what may be effective. Therapeutic vaccination with recombinant antigens have tested repeated dosing with different compositions of the HBV PreS1, PreS2 and S antigens delivered with or without adjuvants [27e35]. These vaccines were relatively weak at inducing T cell immunity, which were mainly measured using low-resolution T cell proliferation assays. T cells could be detected during or shortly after vaccine regimens in some studies but rapidly waned during follow-up. It was demonstrated that adjuvanted HBsAg vaccines could induce robust anti-HBs seroconversion, without HBsAg loss, in chronic HBV patients [27]. Thus, these trials demonstrate that weak stimulation of T cell immunity, even in the presence of a significant anti-HBs response, is not sufficient to overcome the threshold needed to induce clearance of HBV. The disconnect between vaccine response and virus control is an important topic that will be discussed below. Because of the key role T cells play in HBV control, numerous studies attempted DNA vaccination, which primarily induces CD4 and CD8 T cell immunity [36e41]. Similar to trials with recombinant antigen, DNA vaccination strategies have used repeated intramuscular injections. In many of these trials, the vaccine was able to induce HBV-specific T cell immunity measured by proliferation and Elispot assays. However, the focussed effort to boost T cell immunity using DNA vaccines did not increase HBsAg loss in chronic HBV patients.

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Fig. 1. Boosting HBV-specific adaptive immunity to enhance intrahepatic immune responses. Strategies to boost the HBV-specific T cell, and B cell, response rely on peripheral delivery of the vaccine or checkpoint inhibitor antibody to boost or restore function to HBV-specific cells in the liver. Vaccines will have to induce T cells in the context of high antigen burden that are capable of migrating to the liver, overcome intrahepatic immune suppressive mechanisms and recognize infected hepatocytes to produce antiviral cytokines (IFN-g & TNF-a). Matching vaccines to HBV genotypes is also important to achieve sufficient population coverage and avoid escape/genotypic mutations that render antiviral T cell non-specific. To date, this data has not been demonstrated but is key to achieving an anti-HBV effect in the liver. Checkpoint blockade aims to restore function and promote expansion of HBV-specific immune cells already present in the patient. Inhibitory receptors like PD-1 are highly expressed by T cells in the liver so it is possible that blocking this interaction will have both an HBV-specific and non-specific effect that can promote and antiviral response in the liver. The amount of functional restoration achievable and the amount or restoration needed to have an antiviral effect in chronic HBV patients have yet to be quantified.

Table 1 Summary of HBV therapeutic vaccine trials. Study

Vaccine type

Composition

Nuc

Reference

Vandepapeliere Horiike Jung Dahmen Pol Couillin Xu Hoa Dikici Fontaine Yang 2012 Yang 2006 Mancini-Bourgine 2006 Mancini-Bourgine 2004 Yoon Lok Heathcote Luo

Recombinant Recombinant Recombinant Recombinant Recombinant Recombinant Recombinant Recombinant Recombinant DNA DNA DNA DNA DNA DNA Yeast Lipopeptide Cellular Therapy

HBsAg þ MPL HBsAg HBs þ PreS2 þ PreS1 þ Alum (Hepacare) HBsAg þ Alum (Engerix-B) HBsAg þ Alum (GenHevacB or Recombivax) HBsAg þ Alum (GenHevacB or Recombivax) HBsAg þ HBIG Immune complex þ Alum HBs þ PreS2 þ PreS1 (Sci-B-Vac) HBsAg þ Alum (GenHevacB) S þ PreS2 PreS2 þ IL-2 þ IFN-g S þ PreS2 þ Core þ Pol þ IL-12 S þ PreS2 S þ PreS2 HBs þ PreS1, HBc þ HBp, IL-12 Yeast expressing HBs, HBc, HBx Tetanus 830 CD4 þ HBc18-27 CD8 epitope Autologous Dendritic Cells þ CD8 epitopes

 þ  þ    þ  þ  þ   þ þ  þ/

[27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [43] [42] [44]

Despite the limited success of the DNA vaccine strategy thus far, a study by Yang et al. demonstrated that the magnitude of T cell immunity could potentially correlate with sustained viral suppression off treatment in responding patients (HBeAg seroconversion and reduced HBV replication) [38]. The T cell response in this study waned over time, eventually returning to the level of nonresponders during follow-up. This suggests that the right cocktail of plasmids or delivery may be able to overcome tolerance in chronic HBV patients but durability is an issue. It also suggests that the magnitude of the T cell response may correlate with viral control. However, functionality of DNA vaccine-induced T cells was not routinely assessed and T cell functionality may be as, or even more, important than the magnitude of the response. Other approaches to therapeutic vaccination have used HBsAgHBIG immune complexes [33], lipopeptides [42], yeast expressing HBV antigens [43] or infusion of peptide loaded dendritic cells (DC) [44]. The use of immune complexes, lipopeptides and yeast were hypothesized to activate DC more efficiently, which would lead to more effective priming of HBV-specific T cells. However, similar to above, these vaccines did not induce a significant response, either virologic or immunologic, to warrant further development. The use of peptide loaded DC circumvented the

need to activate efficient antigen presenting cells in vivo by producing these cells in vitro. The T cell response was not quantified in the DC vaccine trial by Luo et al. so it is difficult to determine how efficient the vaccine was but virologic responses in the study did not surpass what can be achieved with IFN-a. A DC-based vaccine also represents a complicated and costly strategy for HBV immunotherapy, given that there are 250 million people infected worldwide [45]. New therapeutic vaccines in development While the data may be largely negative results, trials thus far have been hugely helpful in defining the challenges faced by therapeutic vaccination. The use of recombinant antigen appears least effective. As mentioned above, these antigens are introduced on top of the massive antigen burden present within chronic patients. Plus, the adjuvants favored the production of antibodies and were noticeably weak at inducing antiviral T cell responses. DNA vaccines were more effective at inducing T cells but responses waned and there was no evaluation of their functionality to determine if responding cells had the capacity of mediate and antiHBV effect. New strategies are building on these trials and advances

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in our understanding of the immune response in chronic HBV infection. They employ new vectors and technology to provide maximum stimulation to the T cell compartment that will hopefully improve magnitude, durability and functionality. New DNA vaccines Two companies, Inovio (INO-1800) and Ichor Medical Systems (HB-110) currently have a new generation of DNA vaccines in phase I clinical trials. This new generation of vaccines have improved on delivery, adjuvant and patient selection compared to previous DNA vaccine attempts. Inovio's INO-1800 is composed of plasmids encoding HBV genotypes A & C HBsAg and a consensus HBcAg. Ichor's HB-110 is composed of plasmids encoding HBs þ PreS1 and HBc þ HBp. Both companies use electroporation to deliver the DNA vaccine. The use of proprietary in vivo electroporation devices increases delivery efficiency and expression of the vaccine antigens in vivo. Both formulations include a plasmid encoding IL-12. IL-12 serves as an adjuvant to polarize T cell responses and has been shown to increase HBV-specific T cell frequency and effector function in chronic HBV patient PBMC [46]. Furthermore, a previous attempt to use IL-12 in combination with DNA vaccination was the only demonstration that increased T cell responses correlated with sustained suppression of viral load [38]. Patient selection/conditioning is also an important component of new vaccine trials. Previous studies mainly used short courses (6e12 weeks) of lamivudine or adefovir whereas long-term treatment may allow better recovery of HBV-specific T cell immunity prior to vaccination [21]. Thus, in an effort to maximize T cell responses, chronic HBV patients will be on potent long-term antiviral therapy. Ichor Medical Systems has published pilot clinical data from patients treated for 8 weeks with adefovir and given escalating doses of the vaccine [41]. In this study, the DNA vaccine was administered by injection, not electroporation. They observed a dose dependent induction of T cell immunity but no HBsAg clearance. The Phase I study for Ichor's HB-110 should build on the immunogenicity of their vaccine using in vivo electroporation. No pilot data is published for INO-1800 but pre-clinical data with Inovio's vaccine showed robust immunity in mice and non-human primates [47]. Phase I trials and data analysis are ongoing for both companies. Given that the T cell response is the primary mode of action in DNA vaccination, careful analysis of T cell frequency and function, and how this correlates with viral and HBsAg load, will be important to evaluate the effectiveness of the vaccine. Synthetic epitope arrays Altimmune is developing a multi-epitope peptide vaccine linked to a fluorocarbon tail that stabilizes the peptide and creates a depot upon injection (HepTcell). The vaccine is composed of 9 multi-epitope peptides that include CD4 and CD8 T cell epitopes targeting multiple HBV genotypes. This differs from the previous lipopetide vaccination strategy because it incorporates larger number of both CD8 and CD4 T cell epitopes. The depot forming nature of the vaccine is also hypothesized to increase its immunogenicity by increasing antigen duration and bioavailability. The vaccine started Phase 1 trials in July, 2015 enrolling chronic HBV patients on antiviral therapy. Given that the vaccine is composed of HLA-restricted epitopes, the HBV-specific T cell response will be the primary surrogate for vaccine immunogenicity, which should be predictably monitored based on the composition of the included epitopes. Thus, similar to above, antiviral efficacy should be compared to the magnitude and functionality of T cell immunity.

Adenovirus Adenovirus has emerged as a highly immunogenic vector inducing both innate and adaptive immunity. Transgene is currently testing a non-replicative adenovirus 5 vector encoding HBV Core, Polymerase and selected domains of Envelop proteins (TG1050). Preclinical data for TG1050 demonstrated that the vaccine can induce T cell immunity in a persistent HBV mouse model [48]. Although, T cell induction was weak in HBV mice compared to non-HBV animals, similar to what has been observed between chronic HBV patients and healthy donors [42]. Decreases in viral load and HBsAg levels were observed with the vaccine but no animals cleared HBV infection. The TG1050 vaccine is currently in Phase I dose escalation studies in chronic HBV patients treated with tenofovir or entecavir. Checkpoint inhibitors Checkpoint inhibitors have been attracting significant discussion in terms of chronic HBV treatment, particularly the PD-1-PDL1 pathway. PD-1 is the primary inhibitory receptor expressed by HBV-specific T cells [2]. Blocking the PD-1-PD-L1 interaction can increase the frequency and restore functionality to HBV-specific T cells in vitro [49e51]. The woodchuck model of chronic HBV infection has also provided positive data showing that a combination of antiviral therapy, DNA vaccination and anti-PD-L1 can lead to HBsAg loss [52]. Thus, there is a substantial amount of evidence to argue that blocking this inhibitory pathway could benefit the induction of T cell immunity in chronic HBV patients (Fig. 1). Many companies are developing PD-1/PD-L1 drugs, which are now clinically used with success in cancer therapy for solid tumors. There is hesitation regarding the used of checkpoint inhibitors due to the potential toxicity profile of anti-PD-1/PD-L1 drugs [53]. They have the potential of inducing hepatitis, colitis, or pneumonitis. For oncology, the occurrence rates of these adverse events are equal to or better than previous/current chemotherapy regimens. This contrasts with the current level of safety and tolerability seen with antiviral drugs for chronic HBV treatment. However, ongoing clinical trials testing PD-1 blockade in hepatocellular carcinoma patients with chronic viral hepatitis may be able to provide insight into both safety and efficacy of the drug. Interim reports from a Phase I/II study suggest that the anti-PD-1 drug, Nivolumab, which was administered to over 100 patients with chronic HBV and HCV related liver cancer, did not induce severe hepatic toxicity [54]. Because PD-1/PD-L1 pathway is involved with suppressing the immune response in chronic HBV, careful investigation as monotherapy or in combination with vaccines is warranted. Targeting innate immunity Evidence for innate immune control from HBV models Our understanding of innate immunity in chronic HBV infection has trailed behind that of T cell research because early studies and clinical data suggested that HBV does not trigger an inflammatory response [55]. There is now evidence that HBV and HBV antigens are recognized by multiple pattern recognition receptors (TLR-2, TLR-4, Mannose receptor, RIG-I) [56e61]. However, HBV activation of innate immunity is still debatable and is likely context dependent e related to viral replication and the intrahepatic environment. There is also evidence that HBV employs mechanisms to inhibit signaling through pattern recognition receptor pathways, which could counter innate targeted therapies

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[62e71]. Many of the studies investigating HBV inhibition of innate signaling pathways employed overexpression in tumor cell lines or exposure to recombinant antigens in vitro and require confirmation in vivo. Even though Innate recognition of HBV is still an issue of debate, it has become increasingly clear that cells of the innate immune system play a key role in HBV pathogenesis and can serve as potential targets for immune based therapies [72]. Current strategies for targeting the innate immune system use synthetic drugs that activate pathogen recognition receptors such as Toll-like receptors (TLRs) or cytoplasmic nucleic acid sensors. There is significant evidence that therapeutically manipulating innate immunity through these receptors can have a strong antiviral effect [73]. Injection of TLR agonists into HBV-transgenic mice clears HBV RNA from the liver [74,75]. This was identified as an IFNa mediated effect but there are multiple points within the HBV life cycle where cytokines elicited from innate immune activation disrupt HBV replication [76]. Furthermore, it was shown that direct injection of IL-12, a Th1 polarizing cytokine could induce clearance of HBV in transgenic mice [77]. While the data in animal models is compelling and encouraging, challenges will no doubt arise in the translation to chronic HBV patients. This is evident in the use of IFN-a for treatment, which is highly effective at clearing HBV in mice but much less effective in patients [78]. In addition, IL-12 cytokine has been administered to chronic HBV patients with no significant virologic response [79]. Targeting innate immunity is likely to have an impact beyond simply the production of cytokines with direct antiviral activity. Innate immune cells, in particular Kupffer cells, act as gatekeepers in the liver by regulating the inflammatory/antiinflammatory intrahepatic environment. Therefore, providing the correct stimulus can promote an environment that facilitates an antiviral response [80]. It was also demonstrated in mouse models that TLR-9-mediated production of TNF-a promotes the intrahepatic expansion of activated T cells [81]. When combined with therapeutic vaccination in a chronic HBV mouse model, there was HBV-specific T cell expansion in the liver and a significant reduction in viral antigens. Thus, targeting innate immunity has the potential to elicit cytokines with direct antiviral activity and an adjuvant effect that modulates the intrahepatic environment, making it more permissible for an effective immune response (Fig. 2).

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TLR-7 agonists Toll-like receptors are responsible for recognizing conserved molecular patterns associated with invading pathogens and are highly expressed on myeloid cells (DC, monocytes, macrophages). They are expressed either at the cell surface or endosomally in the case of TLR-3, -7, -8, and -9 [82]. Exploiting the inflammatory potential of TLRs has been the basis of new vaccine adjuvants and can dramatically boost immune responses [83,84]. The most advanced are Monophosphorylated lipid A (MPLA), a derivative of LPS that triggers TLR-4, and CpG oligos that activate TLR-9 in B cells and plasmacytoid DC (pDC). However, it was only recently that the inflammatory and antiviral properties of TLR agonists were developed for oral HBV therapy. In an attempt minimize systemic side effects and concentrate the antiviral activity of IFN-a production in the liver, Gilead (GS9620) and Roche (RO6864018) have developed synthetic TLR-7 agonists. These are orally delivered drugs that are absorbed in the intestine and activate TLR-7 expressed on pDC. This activation elicits the production of IFN-a and additional inflammatory cytokines. Gilead has published results from Phase I studies in treatment naïve and virologically suppressed patients showing dose dependent induction of biomarkers of the IFN-a pathway [85]. Interferon stimulated gene (ISG) induction was detectable in the cells from the blood within 24h. IFN-a protein was rarely detected in the serum, suggesting localized production of IFN-a in the intestine/liver. The published Phase I study showed activity, safety and tolerability but because responses were measured within a 2 week window, there was no significant effect on HBV DNA or HBsAg. Data from the Phase 2 study will provide more information on efficacy. Data for the Roche drug, RO6864018, are not available. This is currently in Phase II for chronic HBV patients. RIG-I agonists RIG-I is a cytosolic RNA sensor that can trigger an inflammatory response through activation of IRF-3 and NFkB. RIG-I has been shown to recognize and bind the epsilon stem loop of the HBV pregenomic RNA and stimulate the production of IFN-b and type III interferon (IFN-lambda) [59]. Springbank Pharmaceuticals has developed a RIG-I/NOD2 based drug for chronic HBV (SB-9200). SB9200 is a small molecule nucleic acid hybrid in Phase 2 clinical

Fig. 2. Innate immunomodulatory drugs stimulate production of antiviral and environment modifying cytokines in the liver. New drugs that target pattern recognition receptors like TLRs, RIG-I and STING are being developed for oral administration. These drugs are absorbed through the intestine and delivered to the liver via the portal vein. The target of these drugs is likely to be broad. They will activate immune and parenchymal cells in the intestinal track as well as in the liver. Target cells are dictated by the expression profile of pattern recognition receptor such as TLR-7 being highly restricted to plasmacytoid DC and RIG-I being expressed in myeloid cells and hepatocytes. These drugs will stimulate production of IFN-a, IFN-l, TNF-a, IL-6 and IL-12 that have antiviral effects and alter the liver inflammatory environment. The profile of cytokine produced is likely to be influenced by the target cell population, immune vs. parenchymal.

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trials. This will be the first use of the drug for HBV therapy in humans. Phase I studies were performed in chronic HCV patients. SB-9200 is hypothesized to have a direct antiviral effect on the HBV polymerase and stimulate localized production of interferons and inflammatory cytokines in the liver. Preclinical data in the woodchuck model of chronic HBV infection has been published. In this model, SB-9200 showed a dose dependent reduction in HBV DNA and HBsAg during the treatment window that rebounded following treatment withdraw [86,87]. The Phase II study currently underway with SB-9200 will incorporate 12 weeks of treatment followed by Tenofovir, with the presumed goal of maintaining/enhancing viral suppression after stopping immunomodulatory treatment. STING agonists STING agonists are still in preclinical development but represent another potentially effective innate pathway for therapy. STING is the primary transducer of intracellular DNA pathogen recognition receptors such as cGAS [88]. It was shown in a mouse hepatocyte cell line that cGAS-STING could recognize HBV DNA and suppress viral replication [89]. A role for cGAS-STING in chronic HBV infection is debated because the expression of these proteins in human hepatocytes is still being investigated [90,91]. However, cGASSTING is expressed in Kupffer cells and intrahepatic DC. Activation of cGAS-STING using synthetic agonists has been shown to induce interferon production in persistent HBV mouse models and inhibit HBV replication [92,93]. Given the interest in innate immunotherapy, we are likely to see further development of these agonists as drugs for chronic HBV therapy. Effect, efficacy and the difficulty of linking the two together The primary assessment of efficacy will remain a virological readout, HBV DNA, HBsAg, ALT. These tests are established and clearly track the improvement or resolution of disease. Thus, keeping these at the center of all trials is accepted practice. However, measuring the effectiveness of new immune-based therapies will pose specific challenges based on the mechanism of action for each drug. Clinical trials may use multiple, specifically adapted assays to test drug function in vivo. The goal being to confirm it is working through the hypothesized mechanism of action and correlating immune stimulation with virological response. Effect vs. Efficacy Effect, as referred to here, is the ability of the drug or vaccine to activate the desired immune response. Many of the previous therapeutic vaccine trials did not include immunological assessment of the vaccine, or used low resolution assays such as proliferation. Without specifically measuring the targeted immune response, it is virtually impossible to determine why an immunebased therapy failed and hinders the ability to correlate immunological response with virological outcome. Current and future therapeutic vaccines will require thorough assessment of antibody production and induction, phenotype and function of T cell immunity. Immunomodulators activating innate cellular pathways will have to expand beyond the production of interferons and upregulation of interferon stimulated genes (ISGs) to include inflammatory cytokines and assessment of phenotypic markers on innate and adaptive effectors. The approach to measuring the immunological effect also presents issues. Immunogenicity is often monitored longitudinally in treated patients. Positive responses are judged as change from baseline. While this is not wrong, the immune response in

chronic HBV patients, in particular the T cell response, is exhausted and weak. Thus, a change from undetectable to barely detectable after therapy may indicate a positive response to therapy but is ineffective in terms of virus control. Many vaccines were capable or inducing some level of T cell immunity but none to date have increased the rate of HBsAg seroconversion [27,29,30,32,38e42]. The use of adjuvants was shown to boost antibody responses, in some cases to levels considered protective in healthy vaccinated individuals [27]. Thus, simply inducing a T cell or antibody response e immunogenicity e is not sufficient to achieve efficacy. Based on limited therapeutic vaccine-related data, but substantial in vitro data, T cell induction in chronic HBV patients is only a fraction of that induced in healthy donors [42,43,94]. Similar trends have been observed in preclinical mouse models [48]. Therefore, immunogenicity in chronic HBV patients compared to immunogenicity data obtained in healthy individuals may be a better measure of drug effectiveness. Hopefully, with the new generation of vaccines, which are likely to be significantly more immunogenic, careful monitoring of the T cell response may help establish a standardized cut-off for the magnitude of T cell induction associated with an effective virologic response in patients. We also have to consider that the magnitude of a peripheral T cell response is not the only factor correlating with viral control. The discrepancy between T cell induction and lack of virological response may partially be due to a misdirected immune response. Vaccines use a defined antigen where the amino acid sequence may not match that of the virus infecting the patient. Therefore, immunity induced by the vaccine may not effectively target the virus present in the patient. In addition, a vaccine may induce robust immunity in the blood but if these cells do not traffic to the liver, or are suppressed in liver microenvironment, then the virological response will be minimal. To date, this issue has not been investigated. Understanding this fundamental biological question could argue in favor of combination therapy with an orally delivered immunomodulatory drug that functions similar to a “prime and pull” vaccine strategy, driving T cells to the liver and altering the liver environment [95]. In addition to quantification and localization, the quality of therapy-induced, HBV-specific T cells is an important parameter that has not been investigated. Data from patients that cleared HBsAg suggest clearance is associated with a greater breadth of T cell function [21]. The ability to proliferate, produce multiple cytokines, lyse infected targets are all factors that have been demonstrated to be important for control of chronic infections yet are rarely investigated in early vaccine trials [96]. How to assess the function of innate immunity with clinical efficacy is less clear. The hypothesized mechanism of action from new immunomodulatory drugs is localized intrahepatic production of interferons. Thus, assessment of ISG seems logical and ISG induction can be detected in cells from the blood [85]. However, induction of ISGs does not correlate with viral control and triggering the pattern recognition receptors will stimulate the production of antiviral/inflammatory other than interferons [97]. TNF-a [11], IL-6 [98], IL-12 [46,77], all potentially induced by this class of drug, have anti-HBV activity and maybe be more important for viral control because of their impact on the environment and adaptive immunity. If these cytokines are not detectable in the serum then its unknown if the drug is achieving optimal stimulation. In addition, the role of innate immunity is to coordinate the adaptive immune response. Thus, monitoring T cell immunity could prove a useful correlate to virological response using innate immunomodulators.

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Obstacles to immune monitoring Practice points While easy to list ideal assay scenarios for monitoring immunotherapy, practical issues create significant obstacles to careful evaluation of the immune response. Monitoring a vaccine induced T cell response is probably the most challenging assay for large-scale Phase II/III clinical trials but the most informative in terms of vaccine immunogenicity. Elispot is currently the most sensitive and effective assay to measure the magnitude, polyclonality and potentially function of the total T cell response. However, a single assay requires a significant amount of blood and capacity in clinical labs to consistently handle, cryopreserve, store and ship samples. If PBMCs can be obtained, there are currently no guidelines for optimal time points to measure T cell responses nor a consistent set of reagents and protocols used across multiple clinical trials that would facilitate direct comparisons. Being able to reasonably compare data across clinical trials may help establish a threshold for a therapeutically effective immune response. In addition, evaluation of the PBMCs does not address the potential issue of whether vaccine-induced T cells traffic to the liver. Fine needle aspirate biopsies in subsets of patients should be considered to investigate this question. The relatively short dosing regimens of new immunomodulatory drugs will limit the amount of blood drawn at each time point. This will limit the depth of immunological studies that can be performed, making it difficult to identify biomarkers or determine when the drug has reached its maximum immunological effectiveness. Cytokines can be measured in the serum and RNA can be extracted from relatively few total cells for ISG expression. However, as mentioned above, analysis should not be limited to ISG expression because other cytokines produced upon pattern recognition receptor activation are likely to play a significant role in reducing HBV replication. In addition, the target of these orally administered drugs is localized intrahepatic production of antiviral and inflammatory cytokines, which is rapidly detectable e within 24 h [85]. Thus, assays to assess activation of innate immunity should be performed both in the liver and blood, including early time points (1-3d). These issues should be carefully considered at the beginning of clinical trials and incorporated into study designs. Without this information, immunomodulatory/vaccine clinical trials become empirical e dose escalation vs. virological response. This model works if the drug displays some efficacy because additional trials will be performed and, theoretically, immunological mechanisms can be investigated at later stages of development. However, if a drug fails to show efficacy, the data is lost. There will be no motivation to perform a study to evaluate the immune mechanism of a drug that failed. However, if we have the immunological data, even if the drug fails, we learn something about what didn't work and can build on that data to improve the next generation of immunotherapy drugs. Concluding remarks Boosting HBV-specific immunity is widely accepted to be important to obtain a functional cure for chronic HBV infection. There have been important, informative, efforts in the past that have led to the development of new highly immunogenic vaccines and new classes of drugs in the innate immunomodulators. It is too early to determine the efficacy of the different strategies, or how new immunological knowledge might shape the evolution of therapy, but the importance of immune monitoring during these clinical trials is critical. Practical issues will present challenges for thorough immunological evaluation, particularly in sample requirements, standardized assays and reagents, but efforts to obtain this data will likely provide return on investment by understanding the in vivo mechanism of action.

 Therapeutic vaccines have not been able to sufficiently induce HBV-specific immunity to achieve consistent virological responses.  Innate immunomodulatory drugs are in the first generation of development. They have the potential to induce cytokine with direct antiviral activity and cytokines that modulate the inflammatory environment of the liver. This functional combination could help promote adaptive immune responses and virological response.  Vaccines with new adjuvants and delivery methods may still require combination with checkpoint blockade for maximum T cell stimulation or innate immunomodulators to drive vaccine-induced T cells to the liver.

Research agenda  Detailed monitoring of the T cell response has to be performed to determine if there are correlates between magnitude, breadth or function of vaccine-induced T cells and reduced viral replication.  Innate immunomodulators will have immunological effects beyond interferon production. Their impact on effector cells in both the innate and adaptive immune response needs to be carefully defined.  The immune response in the liver during any immunotherapy needs to be investigated to ensure T cells are trafficking to the liver and that innate efforts are responding as predicted to help identify biomarkers of an effective immune response.

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