Epigenetics and Immunotherapy: New Perspective for Breaking Chronic Viral Infection

Chronic or persistent viral infections, like HBV, HCV and HIV, are still a challenge for human health. More importantly, most human chronic viral infection will result in cancer, the top leading cause of death. So far antiviral drugs and immunotherapy have limited therapeutic effects on patients. New potential mechanisms of chronic viral infections are needed to explore before new effective therapeutic strategies can be put into use. Epigenetic alterations are an important hallmark of cancer. Recent studies indicate that chronic viral infections also induce epigenetic changes in the host. This review will try to summarize some of the most recent studies in these fields and explore a new strategy to break chronic viral infection by combining epigenetic therapy and immunotherapy.


Introduction
Epigenetics refers to heritable and potentially reversible changes in gene expression that do not change the primary sequence of genomic DNA [1]. The most extensively studied epigenetic modifications are DNA methylation and histone posttranslational modifications [2]. It is well-known that there are eight hallmarks of cancer [3]. Recently, epigenetic alterations are considered as a new hallmark of cancer [4][5][6]. However, more and more studies have indicated that epigenetic modifications are also associated with chronic viral infections, including HIV [7], HBV [8] and HCV [9]. In the present review, I will give a brief summary of chronic viral infection and further discuss a new strategy to break chronic viral infection by combining immunotherapy and epigenetic modifications.

Chronic Viral Infection
Most viral infections are resolved due to clearance of viruses by the host immune system. However, some viruses can establish persistent infections (in many cases initiated by an acute infection) through adoption of sophisticated relationships with their hosts and manipulation of a wide array of cellular mechanisms for their own advantage [10]. The so-called chronic viral infections are largely due to the failure of immediate viral clearance, where the host's immune system is not appropriately induced or infected viruses modify the relevant immune signaling and cellular apoptotic pathways [10].
Both DNA viruses (like HBV and HPV) and RNA viruses (like HCV and HIV) can induce chronic infections in humans [11]. Persistent viral infections are also found in nonhuman primates and mice, like simian immunodeficiency virus (SIV) and lymphocytic choriomeningitis virus (LCMV), respectively. Both SIV and LCMV infections are good models to study the mechanisms of chronic viral infection. Several mechanisms have been reported to regulate immune responses during chronic viral infection. Upregulation of inhibitory receptors on exhausted CD8 T cells is an important one. These inhibitory receptors include PD-, CTLA, LAG, etc [11].
Chronic viral infection is a challenging health problem for humans. Now it is clear that persistent viral infection will result in cancer development, like HPV and HBV [12]. It is estimated that chronic HBV infection accounts for more than 80% of HCC [13]. As for RNA viruses, HIV predisposes to the development of non-Hodgkin's lymphoma, squamous cell carcinomas, and Kaposi's sarcoma while the human T-cell lymphoma virus causes adult T-cell leukemia [14]. Viruses that induce cancer are also called tumor viruses. Tumor viruses induce malignant transformation in host through several strategies [15], including apoptosis inhibition, cell cycle deregulation, blocking of senescence, induction of genetic instability and promotion of angiogenesis.

Epigenetics in Viral Infection
Epigenetic regulation in viral infection includes two different meanings. One is that tumor viruses themselves undergo epigenetic changes; the other is that viruses cause epigenetic changes in host. Epigenetics play an important role in control of tumor viruses' life cycle and the transformation of a normal cell to a cancer cell [16]. One example is silenced HIV genomes, which is a major obstacle for a curative treatment of AIDS patients [17]. The two extensively studied epigenetics are DNA methylation and histone modification. Okamoto et al., [18] reported that HBV and HCV infection induced aberrant DNA methylation in mice with humanized livers. Lusic et al., [19] found that histone acetylation was involved in regulating of HIV-1 gene expression and factor recruitment at the LTR promoter. In addition, DNA methylation is involved in controlling viral genome of HPV [20]. Repression of tumor suppressor genes by inducing promoter hypermethylation is an important reason for carcinogenesis [,22]. More interestingly, it is found that HPV16 can epigenetically silence the interferon-κ gene [23], which indicates that chronic viral infection is able to suppress the host's innate immunity. Actually chronic viral infections also influence adaptive immune responses by epigenetic strategy. Youngblood et al., [2,25] reported that decreased DNA methylation of the PD1 locus in CD8 + T cells happened in both chronic LCMV and HIV infections. Taken together, epigenetic control of tumor viruses' life cycle is popular [26,27]. In addition, chronic viral infections are able to modify both hosts' innate and adaptive immunity by epigenetic strategy. These indicate that epigenetic therapy may be useful for interrupting and breaking chronic viral infection.

Immunotherapy for Chronic Viral Infection
Immunotherapy is a therapeutic method to boost the host's innate and adaptive immune responses to fight infectious diseases and cancer. As antiviral drugs are expensive and showing viral resistance and drug toxicity, immunotherapy is becoming a promising strategy to conquer chronic viral infection [28]. One strong evidence of HBV immunotherapy is that resolution of chronic HBV infection can be achieved by bone marrow transplantation from an immune donor [29]. Using a dually functional vector containing both an immunostimulating single-stranded RNA (ssRNA) and an HBxsilencing short hairpin RNA (shRNA), Lan et al., [30] reported that HBV-impaired hepatocyte-intrinsic innate immunity could be recovered in a mouse model. They further showed that the recovered innate immunity could overcome systemic adaptive immunotolerance in an IFN-α-and TLR7-dependent manner. Moody et al., [31] found that combination of TLR7/8 and TLR9 agonists was able to elicit the highest titers of binding, neutralizing, and antibody-dependent cellular cytotoxicity-mediating antibodies against HIV-1 envelope gp140. As the enhanced immune response was associated with the release of CXCL10 (IP-10), it indicated that the TLR adjuvant formulation may have optimally stimulated innate and adaptive immunity to elicit high titers of antibodies.
There are different types of immunotherapy for chronic viral infection, including cytokine delivery [32], therapeutic vaccines and blocking antibodies targeting co-inhibitory receptors such as PD-1 [33]. Therapeutic vaccines are one major immunotherapy strategy, where a variety of platforms and approaches including DNA, viral vectors, dendritic cells and peptides are tested, together with some adjuvants and immune modulators [34]. It is well-accepted that combination therapy will have better effect. One example is that combining antagonists of IL-10R or PD-1 with a therapeutic vaccine strengthened the effects in a murine model of persistent lymphocytic choriomeningitis virus (LCMV) infection [35].
So far most epigenetic therapies are studied for cancer. Besides the modifying effect on gene expression, recent studies also show that epigenetic drugs are good immunomodulators. They influence both innate and adaptive immune responses [4]. Many reports have shown that epigenetic drugs are able to improve the costimulatory properties of cancer cells by up regulating surface expression of CD40, CD80, CD86, and ICAM1 and restore their sensitivity to immune cell triggered apoptosis by enhancing the expression of death-inducing receptors [40][41][42] . Zhang et al., [43] reported that HDAC inhibitors restored function of defective CD8 + T cells from chronic LCMV infection. Archin et al., [44] reported that selective HDAC inhibitors could activate latency HIV infection, which suggests a clinical strategy to target persistent HIV infection. The same group [45] further showed that administration of vorinostat disrupted HIV-1 latency in patients on antiretroviral therapy. This study is of great significance as it indicates that eradication of latent HIV infection is possible. Wei et al. [46] reported that romidepsin could induce HIV expression in CD4 T cells from HIV patients. Recently Tian et al., [47] showed that Telbivudine treatment corrected HBV-induced epigenetic alterations in liver cells of patients with chronic hepatitis B. HBV nuclear cccDNA accumulates in hepatocyte nuclei as a stable minichromosome organized by histone and non-histone viral and cellular proteins [48]. Identification of the molecular mechanisms regulating cccDNA stability and its transcriptional activity at the RNA, DNA and epigenetic levels may reveal new potential therapeutic targets. Taken together, considering the epigenetic regulation of latent HIV and HBV cccDNA, epigenetic therapy may be a promising strategy to conquer chronic/persistent viral infection. Table 2 lists some of the representative studies for epigenetic therapy on chronic viral infections.

Breaking Chronic Viral Infection by Combining Epigenetic Drugs and Immunotherapy
Many studies have shown that either antiviral drugs or immunotherapy alone have limited effects on patients with chronic viral infection. Kelly et al., [52] reported that immunotherapy with therapeutic vaccine alone failed to restore T cell immunity in HCV infected patients. Kang et al., [53] showed that combination of EGCG and DNA vaccination led to an enhanced tumor-specific T-cell immune response and enhanced antitumor effects, resulting in a higher cure rate than either immunotherapy or EGCG alone. These studies suggest that combining epigenetic therapy and immunotherapy have better therapeutic effects on tumor. It is promising to try the same strategy on patients with chronic viral infection. Tropberger et al., [8] found that HBV cccDNA chromatin was modulated by innate immunity and manipulated with an epigenetic agent. Their findings provide rational evidence for treating chronic HBV patients by combining immunotherapy and epigenetic therapy.
It should be noted that, epigenetic reagents may have more effects on viral control besides modulating gene expression. One recent paper [54] showed that an epigenetic chemical epigallocatechin-3-gallate (EGCG), the main polyphenol compound of green tea, was able to inhibit HCV entry.
One specific goal of epigenetic therapy is to restore normal DNA methylation patterns and to prevent the cells from acquiring further methylation in DNA that could lead to silencing of genes crucial for normal cell function. Concerns appear immediately about epigenetic therapy with DNMT inhibitors and HDAC inhibitors: Is that effect malignance-directed in vivo or universal? In other words, the use of demethylating agents may aggravate the situation by further decreasing the level of methylation and activation of genes that are potentially deleterious, like oncogenes. One recent report [55] showed that 5-Aza-CdR was able to modulate immune-related genes in mouse tumor while normal tissues were substantially unaffected. Cheng et al [50] reported that zebularine could be selective toward cancer cells rather than normal cells [56]. Although safety concerns still exist [57], epigenetic therapy combined with current immunotherapy methods (including some antiviral drugs) may be promising to break chronic viral infection and eradicate viruses from host completely.

Conclusion
Epigenetics provide new strategy to study chronic viral infection, especially those caused by tumor viruses. It is necessary to explore as many as possible molecular mechanisms of epigenetic drugs on cancer and virus infection treatment. As most current epigenetic therapies are carried out on cancer patients, it is interesting to check whether the similar recipe will work for patients with chronic viral infection.
Finally and most importantly, combining epigenetic therapy together with immunotherapy may provide a promising strategy to break chronic viral infection and induce appropriate immune responses to eradicate viruses from the host.

Conflicts of Interest
The author declares no conflict of interest.