Received date: 26/04/2016; Accepted date: 13/06/2016; Published date: 20/06/2016
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Hepatocellular carcinoma (HCC) is one of the major keys to cause cancer-related mortality in the whole world, with the majority of patients related to persistent infection of hepatitis B virus (HBV), especially in Asian and African, while HCV in Europe and America. HBV is the smallest DNA-containing, enveloped mammal virus known so far. High tissue and species speciÃ¯Â¬Âcity, unique genome and replication mechanism make HBV be shrouded in mystery. A high HBV load is the most significant risk element for HCC recurrence. Individuals chronically infected with HBV are 200 times more inclined to develop HCC than uninfected people. Effective antiviral treatment against HBV could alleviate HBV-associated HCC and improve survival. From several aspects including epidemiology, infection, replication of HBV, immune state, anti-HBV therapy and gut microbiota of patients, we made a summary of currently available knowledge about the epidemiology, prevention and control of HBV infection for the organism as a whole.
Hepatitis B Virus (HBV); Immunoprophylaxis; Hepatocellular Carcinoma (HCC); China
Liver disease is an important cause of illness and death, consisting of primarily viral hepatitis, alcoholic liver disease and nonalcoholic fatty liver disease. Among them viral hepatitis resulted from hepatitis B virus is predominant, especially in China. Estimated by The World Health Organization (WHO), chronic infection with HBV is a major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma, currently affects about 360 million people in worldwide [1-4]. HBV plague the people all over the whole world, especially in China, in past, at present and in the future [4-6].
In the past decade, the prevention and control of Chronic Hepatitis B (CHB) virus infection have significantly improved [4,5]. However, owing to different available anti-HBV reagents and the continuous variable guidelines, the control of HBV becomes more and more complex . Now a days, available therapies popularly used in the whole world are safe, well tolerated, and highly effective in anti-HBV therapy, both reducing HBV viremia and improving clinical course and prognosis [4,5,8-10]. However, due to antiviral resistance and HBV speciality, long-term administration remains a clinical challenge: only long-term virologic control, elimination of HBV and the recovery of CHB patients are not possible [4,11]. HBV had been devoted great deal of scientific research investment, however, understanding HBV chronicity and pathogenesis is hampered by its narrow host range, mostly restricted to human and chimpanzee [12,13]. This paper focuses on summarising the current epidemiology of HBV infection in China, giving emphasis on the use of novel treatment to deal with HBV.
Over past decade, China has made some significant efforts and progresses to shed its “leader in liver diseases” title by paying large amounts of money on funding research, vaccination and drug development, and on recruiting many Western well- trained hepatologists and experts. Different from the past two decades, liver disease research as shown by the dramatic increase in the number of publications was gradually improved and advanced both in breadth and depth. Nevertheless, many challenges troubled us and need to be tackled collaboratively. Here, we reviewed the epidemiology and characteristics of liver diseases and liver related research in China.
Among the kinds of viral causes to hepatitis, few are of greater importance than HBV, which gives rise to a major public health threat with more than 360 million humans worldwide, over 130 million in China alone . Accumulating reports have shown that HBV is accounted for about one million deaths owing to liver failure, cirrhosis, furthermore, over 75% of the hepatocellular carcinomas (HCC) develop from HBV infection every year, 0.37-0.5 million deaths occur in China , in which 65% of deaths are caused by primary hepatic carcinoma [16,17]. Liver cancer, one of the most fatal cancers, is the second most common cancer in China. In China, about 383,000 people die from liver cancer annually, which accounts for more than one half of the deaths from hepatocellular carcinoma worldwide. China was a place with most HBV carrier in the world: nearly 10% of the whole population. The medical and financial burden of HBV infection and HCC is also considered to be the world’s largest .
The HBV carrier rate varies from low (0.1-2%) in the USA and western-Europe, to in-between (2-8%) in Mediterranean littoral countries and Japan, high (8-20%) in some Asian countries and sub-Saharan Africa [4,9-21]. Based on the result of a national study in early 1979, the overall prevalence of chronic HBV was about 8.75%, and then rose to 9.75% in 1992 [22-24]. Undoubtedly, China had been classified as high HBV prevalence before 2006 . The Expanded Program on Immunization (EPI) for infants was carried out in China since 1992, what uplifting is that the EPI implement led to a significant downtrend in HBV prevalence from 9.75% to 7.18% till 2006 [19,24]. Regionally, according to the seroepidemiology study of HBV in China, the highest HBV infection rate observed in the mid-south, while lowest rate in northern China . Ominously, there are still a large population of newborns of HBsAg-positive mothers at high risk for HBV infection, and they are the main origin of HBV transmission [26,27].
Currently, there are mainly four transmission manners: perinatal transmission, horizontal transmission, sexual contact and parenteral exposure to infected fluids . HBV on any surface can keep its infectivity for less than a week, such as table-tops, razor blades, blood stains and so on. The percutaneous/parenteral contact with infected body fluids, and sexual intercourse were risk factors of HBV transmission . Intrauterine infection of HBV is one of the main reasons for the failure of combined immunoprophylaxis . HBV is not able to cross the skin or the mucous membrane barrier and the placenta unless via amniocentesis which breaks in the maternal-fetal barrier. The biggest risk for babies to infect HBV from their mothers is during birth . Thus it can be seen that prevention of perinatal infection and routine infant vaccination are of the same importance with antiviral therapy.
HBV genotype and serotype
Subtypes of HBV with specific geographic distributions can serve as epidemiological markers . Due to the established serological heterogeneity, HBV isolates could be classified into nine different serological subtypes by using subtype-specific antibodies against Hepatitis B surface antigen (HBsAg) including ayw1, ayw2, ayw3, ayw4, ayr, adw2, adw4, adrq- and adrq+ [33,34]. These serological subtypes defined by two mutually exclusive determinant pairs, d/y and w/r and a common determinant a can reflect the genetic variability of HBV.
Genetic classification based on the comparison of HBV genomes has defined 10 genotypes, from A to J, exhibiting a minimum divergence of 8% in the complete genome sequences . Genotypes A and D are widely distributed in all the continents, but A is predominant in northern Europe, D prevails in the Mediterranean area. Genotypes B and C are found mainly in eastern Asia and the Far East, the Near and Middle East and south Asia, while genotype E is indigenous to sub-Saharan West Africa, genotype F is likely to be existed in populations with origins on the American continent, and genotype G is found in the USA and Mexico and Europe, genotype H is closely related to F and prevails in Central and North America . Different genotypes have shown diverse characteristics . The dominant genotypes of HBV in China are B and C. Comparing with the patients infected with HBV genotype B, people with C genotype of HBV infection showed higher HBV DNA level and activity of hepatic histology, longer immune clearance phases , more frequently or persistently fluctuating alanine transarninase (ALT), and lower response to interferon, nucleoside analog . There is also a legible and definite association between HBV genotypes and mutations of precore and basal core promoter (BCP). Genotype C has a higher frequency of substitution mutations (A1762T/G1764A) in BCP, pre-S deletion and is related with higher HBV load than B. Similarly, genotype D has a higher appearance of BCP mutations (A1762T/G1764A) than A genotype. Both Genotype C and D are correlated with severer liver disease, such as cirrhosis and HCC . Infection with genotype A and B shows better responses to IFN-based therapy than C and D, but there is no significant difference for NAs . What is more, genotype C might be a risk factor for HBV transmission from mother to child .
Both serotype and genotype can be served as epidemiological markers of HBV infection. HBV isolates of the same serotype/ genotype are believed to have evolutionary relation generally, and can be used for tracing the route of HBV transmission and geographic migration of HBV carriers. The correlation of the nine serotypes with the ten genotypes has been studied previously . Genomes encoding adw are found in genotypes A, B, C, F and G, while the genomes encoding both adr and ayr occur in genotype C, along with adw . However, these results are still incomplete and inadequate because the HBV isolates analyzed were small in number and were limited to certain geographic areas. There is no causal relationship between them, only some tiny connections.
HBV genome organization
HBV is a member of the Hepadnaviridae family and an enveloped virus with a partially double stranded DNA in 3200-bp size, which is covalently linked to the viral polymerase, it is remarkably compact and achieves its genomic economy by relying on an efficient strategy of encoding proteins from four overlapping genes, including S, C, P, and X . HBV shares similar reverse transcription with HIV, replicates through an RNA intermediate, namely, the pregenomic RNA . Despite this genetic relationship to retroviruses, there are fundamental differences between them; Hepadnavirions contain viral DNA genome instead of RNA . Due to lack of proofreading function of HBV polymerase, error-prone replication of HBV (error rate of 10−4 to 10−5) leads to an accumulation of a pool full with heterogeneous HBV genomic sequences, also called quasi species .
HBV virions combined hepatocyte-surface receptors, such as Na+/taurocholate Cotransporting Polypeptide (NTCP) expressed at the basolateral membrane of hepatocytes and responsible for the uptake of Na+-dependent bile acid in liver, it was proved to be a functional receptor of HBV [44,45]. After membrane fusion, nucleocapsids are presented to the cytosol, RC DNA is releasing and then transported to the nucleus, in where with the assistance of the host DNA polymerase it is converted to a covalently closed circular DNA (cccDNA), which serves as the transcriptional template for host RNA polymerase II to generates a series of genomic and subgenomic transcripts necessary for protein production and viral replication, including 3.5 kb (over-length pregenomic RNA), 2.4 kb, 2.1 kb and 0.7 kb HBV mRNA. All viral mRNAs which are coterminal, polyadenylated, and capped are transported to the cytoplasm and translated into viral proteins, containing envelope, core, and polymerase proteins, as well as the X and pre-C polypeptides. Once the viral RNA is encapsidated, reverse transcription begins to synthetize minus DNA chain using encapsidated RNA as template, during or after the production of this strand, the RNA template is degraded and the synthesis of the complementary plus-strand DNA initiate, using the newly minus DNA strand as template to form the rcDNA. A part of nucleocapsids with mature genome are transported back to the nucleus, took part in the new production cycle to maintain a stable intranuclear pool of transcriptional templates . However, overwhelming majority of nucleocapsids bud into intracellular membranes in where they can acquire the viral envelope proteins, and then exported from the cell .
Clinical Features and Laboratory Diagnosis
The course of HBV infection may be extremely variable, and has different clinical manifestations rests with the age at infection, immune status and the disease stage of patient . Incubation phase of HBV infection varies from 6 to 24 weeks, during which patients may feel unwell, usually with gastrointestinal symptoms including nausea, vomiting, diarrhea, anorexia and headaches . Accidental patients with no obvious symptoms can be identified by detecting biochemical or HBV-specific serologic alterations. Most adult patients could recover completely from HBV infection, while about 5 to 10% of patients can’t clear HBV and will become asymptomatic carriers who constitute a reservoir for further transmission to others, or develop into CHB with high possibility of resulting in cirrhosis and/or HCC . Biochemical assessment of liver function consisting of total and direct bilirubin, ALT, AST, alkaline phosphatase, prothrombin time, total protein, albumin, serum globulin, complete blood count, and coagulation studies can be the hint and assist of HBV diagnosis . Serum microRNA profiles were proved to be novel biomarkers for HBV Infection and Hepatocarcinoma result from HBV .
The conventional HBV diagnosis is administrated based on viral specific antigens and/or antibodies . There are five clinical useful serum markers have been identified for laboratory Diagnosis of HBV, including HBsAg, anti-HBs, HBeAg, anti-HBc and anti-HBe. HBsAg can be found in the serum from 2-6 months after HBV infection . HBsAg is present in serum during acute infections and persists in chronic infections. Its presence indicates that the person is potentially infectious. In the early incubation period, pre-S1 and pre-S2 are present . HBV DNA, DNA polymerase, HBeAg could be detected in the following. HBeAg found in serum is associated with relatively high infectivity and severity of hepatitis except for the appearance of basal core promoter mutation . The first antibody of HBV appeared in serum is anti-HBc which indicates current or past viral infection. Anti-HBe follows in anti-HBc’s step, its presence correlates to a weakened infectivity. Anti-HBe replaces HBeAg in the resolution of the disease . Neutralizing anti-HBs is substituting for HBsAg as acute hepatitis is resolving and normally persists for a lifetime in most patients. If constant serum HBsAg concentration or persistent HBeAg for 8 to 10 weeks after symptoms have resolved, acute hepatitis patients possibly become HBV carriers and develop chronic liver disease . HBV DNA is the most directive, specifically and sensitive marker for HBV infection diagnosis. The higher load means more active viral replication and higher infectiousness. RT-PCR and DNA hybridization assays could be used for the detection of HBV DNA .
HBV chronicity and immunotherapy
The immunopathogenesis of HBV depends on a complex interplay of host and viral factors, such as age, gender, immune status and so on . Primary HBV infection in about 90% of perinatal and childhood, 20%-50% of children will develop into chronic infection, while 95% of immunocompetent adults with acute HBV spontaneously clear the infection . Due to unique genomic organisation and replication strategy, HBV will persist in infected hepatocytes of CHB patients. The natural procedure of CHB infection can be divided into four phases: immune tolerance, immune clearance (HBeAg-positive CHB), immune control (low or non-replicative) and immune escape . These four phases are identiÃ¯Â¬Âed based on speciÃ¯Â¬Âc biochemical, serological, virological characteristics, including serum ALT levels, HBeAg serostatus, HBsAg level and HBV DNA titre . The CHB patients in immune clearance or immune escape phases are potential candidates for antiviral and immune modulatory treatments .
HBV whole virions and other component containing HBs/e/c/x/p protein and HBV DNA can impair the function of dendritic cells (DC), T- and B-cells, immune tolerance will take place afterwards [63-67]. HBeAg was shown to act as an immune decoy for the core antigen by depleting e-speciÃ¯Â¬Âc and c-speciÃ¯Â¬Âc T-helper cells and exerting a tolerogenic effect in utero . Furthermore, HBeAg has been proved to latently downregulate genes involved in RNA transport and processing, cell signalling, cytosol-nuclear trafÃ¯Â¬Âcking and innate immune responses . Distinct virus-mediated mechanisms may account for the limited effectiveness of the innate immune responses and subsequent adaptive immune responses in HBV infection and thereby may signiÃ¯Â¬Âcantly contribute to viral persistence .
It is very important to break the immune tolerance through immunoregulation . In vitro activation of DC and loading with HBV sub-viral particles can overcome tolerance against HBV and reactivate B- and T-cell responses in HBV transgenic mice . Till now, there are several immune-based anti-HBV drugs as follows. Zadaxin, found naturally in the circulation and synthesized in the body’s thymus gland, is a synthetic peptide in 28 amino acids size. Due to promising immunomodulatory function and related curative potential, zadaxin has been used to treat CHB through triggering lymphocyte maturation, enhancing T-cell function, and reconstituting immune defects . NOV-205 (BAM 205), a hepatoprotective agent owning well-tolerated, immunomodulating and anti-inflammatory properties , was approved to greatly reduce/eliminate viral loads of HBV and HCV, significantly improve liver function, and mainly used in the Russian Federation .
The innate immune system, invoked by the interactions between pathogen recognition receptors (PRR)-pathogen associated molecular pattern (PAMP), represents an immediate, Ã¯Â¬Ârst-line defence against foreign pathogens and plays a fundamental role in regulating the adaptive immune response . PRRs includes Toll-like receptors (TLRs), RIG-I like receptors (RLRs), NOD-like receptors (NLRs), and others triggering signals to activate intracellular pathways, production of antiviral and immune regulatory effector molecules. As the crucial parts of innate immune system, TLRs activation could develop into novel therapeutic strategy to treat HBV infection . GS-9620, an effective selective TLR-7 agonist, was designed for anti-HBV therapy . In chronically- HBV-infected chimpanzees, the pharmacokinetics of GS-9620, viral load of HBV, ISG expression, cytokine, chemokine levels, lymphocyte, NK cell activation, safety and tolerability parameters were all assessed after oral administration, it turned out that GS-9620 could suppress HBV DNA of serum and liver, serum levels of HBsAg and HBeAg, and the numbers of HBsAg-positive hepatocytes [78,79]. GI-13020 was engineered to express a chimera of HBV X, S, and C antigens based on yeast expression system. In consideration of its founction of eliciting HBV specific T cell responses, GI-13020 will possibly be used together with anti-HBV reagents to enhance HBsAg seroconversion rate of CHB patients in the future .
HBV control and current problem
The key goal for current HBV treatment is to improve and reverse the progression of liver fibrosis, to reduce the occurrence of HCC. In order to reduce the spread of HBV, prevention of transmission by immunization were carried out . Treatments for HBV infection include interferon and NAs. Interferon (IFN)-α is licensed for HBV treatment and can result in HBV clearance in a proportion of patients, but its efficacy is limited and not well-tolerated because of systemic side effects . Recombinant IFN-α has been approved and successfully used as a standard treatment for chronic HBV infection. Treatment with PEG-IFN results in a prolonged clinical remission with durable anti-HBV effect in HBeAg-positive CHB patients, enhancive rate of HBsAg seroconversion and improved liver histology . Furthermore, long-term therapy also resulted in decrease in fibrosis and regression of cirrhosis.
Until now, five kinds of NAs, powerful inhibitors of HBV replication, including lamivudine (LMV), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT) and tenofovir (TDF), have been approved for CHB treatment . They were well tolerated but the efficacy of these agents is heavily discounted by emergence of antiviral resistance. In vivo and in vitro, NAs mimic physiological nucleosides in terms of uptake and metabolism and are incorporated into newly synthesized DNA chains leading to synthesis inhibition through termination of DNA replication . In CHB patients, HBV exists in the form of quasi species due to the errorprone HBV reverse transcriptase (RT), which is responsible for the rich variety of drug-resistant mutants. Under antiviral pressure, HBV variants will contribute to the production and further selection of replication-competent resistance mutants, which will spread to other hepatocytes, eventually may replace the wild-type cccDNA molecules in the hepatocytes nucleus .
HBV RT is subdivided into 7 domains (A-E, G and F), in which nearly all resistance mutations are located . NA-resistant mutations inhibit the anti-HBV effects of NAs and induce virological advancement and hepatopathological progression. A number of mutations have been reported to account for drug resistance. Mutations of rtM204I or rtM204V in the YMDD motif within the RT domain of the HBV polymerase lead to LMV resistance (LMVr) and LdT resistance (LdTr). ETV resistance (ETVr) has also been observed in patients with LMVr . Under the background of rtM204I/V, a combination of mutations in the B, C or D domains of RT may result in ETVr, such as rtI169T, rtL180M, rtS184G, rtA186T, rtS202I, rtM204V, rtM250V [5,6]. The substitution mutations rtA181V/T, rtN236T, rtN238R, rtT240Y and rtN248H can reduce the anti-HBV effects of ADV, while rtP177G and rtF249A have been reported to induce TDF resistance, apart from rtV214A, rtQ215S and rtA194T which all need further confirmation . Usually, when LMVr occurs, ADV remains effective. Due to a higher genetic barrier to HBV resistance than LMV and ADV, ETV and TDF are high potency drugs for the therapy of CHB patients, even those with LMVr and ADVr . Compensatory mutations that increase viral replication levels can be found in other domains of the HBV Polymerase, such as rtL80V/I, rtI163V, rtI169T, rtV173L, rtT184S/G, rtS202I, rtQ215S and rtQ267H .
The HBV transmission from mother to child is another serious problem for HBV prevention and control. There is no doubt that HBV plague the people all over the whole world, especially in China, the greatest suffered country with at least 130 million CHB carriers, 30%-50% of whom are considered to infect HBV from mother [86,87]. About 90% of infants infected during 1-year-old and 30% to 50% of children infected between 1 to 5 years old likely develop into chronic infection . The virus with mutation on “a” region of surface protein located from 121 to 149 amino acids may escape vaccine-elicited antibodies or hepatitis B immune globulin (HBIG) and may be transmitted from mother to infant . Therefore effective cutting off the mother-to-child transmission route is the only way to decrease HBV infection and relieve the HBV disease burden . About 90% of HBV mother-to-child transmission can be blocked by the administration of immunoprophylaxis with the HB vaccine and HBIG in China. It has been confirmed that the immunoprophylaxis failure of a child is mainly associated with his mother with seropositivity for HBeAg and high HBV DNA viral load. Hence the pregnant women with a high HBV DNA viral load were recommended to take anti-HBV therapy in late pregnancy to inhibit virema .
Prophylactic and therapeutic vaccine for HBV
HBV was discovered for about half a century ago, since 1966, but finding a cure for CHB remains a challenging task. Thankfully, hepatitis B is a vaccine-preventable disease, till now the vaccine is the first and the only one towards a major human cancer . HBV vaccine which is a most effective tool in preventing HBV and HDV transmission is composed of HBsAg (plasma derived or recombinant DNA expression). A safe and effective HBV vaccine has been used for more than 20 years, and 95% of recipients were protected after being administered properly . Due to the contribution of preventive vaccine, the incidence of HBV carriers worldwide was induced signiÃ¯Â¬Âcantly . Nonetheless, a great population is still HBV-suffered. Besides currently approved antiviral treatment with NAs and INF, therapeutic vaccine is a hopeful new strategy for the control of CHB, including the peptide/protein-based, DNA-based, cell-based and antigen-antibody-based therapeutic vaccines . There are three groups of people should receive HBV vaccination: newborns with incomplete immunity and no incomplete against HBV, preschoolers who are also susceptible for HBV, people with HBV-infected spouse or who have jobs in risk of HBV infection, people exposed to HBV . In China, the whole course of HBV vaccination includes three injections with one and six months interval .
Unfortunately, conventional antiviral therapy directly target HBV to inhibit its replication and protein expression, the virus and virus-infected cells may not be eliminated . Therapeutic vaccines are considered to be able to reverse the dysfunctional immune state of chronic hepatitis B and thus hold the promise for HBV cure. Unlike antiviral treatment, immunotherapy shows indirect therapy through triggering host immune responses to repair the immunologic defects and supplement necessary immune factors to inhibit or clear the persisting virus and virus-infected cells . Currently, several therapeutic vaccines for CHB are in experimental or clinical studies, including autologous monocyte-derived DCs, hepatitis B DNA vaccine, HBsAg-HBIG immunogenic complex and so on [90,94-97]. Wen et al. have developed an HBsAg-HBIG immunogenic complex therapeutic vaccine candidate, aimed at breaking immune tolerance to HBV through modulating HBV antigen processing and presentation, it shows a satisfying effect and needs in-depth test in further clinical trials [98,99]. Sung etc. evaluated the safety and immunogenicity of HB-110, one kind of HBV DNA vaccine, in mice and CHB patients, the result showed that HB-110 was safe and tolerable in CHB patients . Xu et al. developed a novel therapeutic vaccine of HBV consisting of HBs and c antigen, using CpG as adjuvant, which elicits forceful humoral responses directed against s and c antigen, promotes a Th1/Th2 balance response against HBs and a Th1- biased response against HBc in mice model . Another novel adenovirus-based therapeutic vaccine named TG1050, induced long-lasting anti-HBV CD8+ T-cell immunity in mouse models .
Novel strategies to achieve sustained virologic remission of HBV
Potential anti-HBV drug targeting viral entry, as described above, Na+/taurocholate Cotransporting Polypeptide (NTCP) is a functional receptor of HBV, and opens the door for HBV infection. HBV has the tendency to bind and infect hepatocytes and the following HBV entry is mediated by the specific interactions between HBV membrane proteins and cellular receptors . There are two kinds of agents which can specifically target the NTCP, including Myrcludex-B (a synthetic lipopeptide derived from pre-S1 of HBV) and Vanitaracin A (a novel tricyclic polyketide), they all can efficiently inhibit the entry of HBV [103-105]. NTCP antagonists targeting viral entry will be new strategy to treat HBV . Furthermore, HBV infection could only infect freshly isolated primary hepatocytes of humans, chimpanzees or northern treeshrews in the past, but recently some NTCP-expressing cells could be infected by HBV, it will be a powerful platform for anti-HBV drug screening .
Potential anti-HBV strategy targeting viral assembly and/or encapsidation
The HBV capsid composed of capsid protein (Cp), polymerase, and pregenomic RNA plays an important role in HBV life cycle, is involved in genome packaging, reverse transcription, intracellular trafficking, and maintenance of HBV stable infection . Assembly is a key step of HBV replication and dominates HBV persistence and transmission. To perturb HBV assembly and thereby alter either the timing or the geometry of capsid formation will be a promising antiviral strategy . Multiple classes of compounds could inhibit HBV assembly and encapsidation, such as Heteroaryldihydropyrimidines (HAPs) which could inhibit capsid formation through triggering assembly inappropriately, misleading assembly, reducing the stability of normal HBV capsids. The most important is that they are effective to both wild-type and NAs-resistance HBV mutants . In the HAPs family, Bay 41- 4109 can induce inappropriate assembly and yield an aberrant virus particle which is incompetent for HBV whole life cycle. GLS4, another member of HAP family, is a small molecular compound with the effect to inhibit the replication of both wild-type and ADVr HBV mutants in vitro .
There is another phenylpropenamide family w shown to suppress HBV replication in vitro including AT-61 and AT-130 . These two compounds have specific antiviral effect only to HBV (both wild-type and LMVr), but no activity against other hepadnaviruses, such as DHBV, WHBV and HIV-1 . Based on previous studies, they inhibit HBV replication through interfering with pregenomic RNA encapsidation, yielding pseudo HBV particles which are in normal appearance, but lack genetic material .
Potential anti-HBV policy targeting HBsAg secretion
Due to ineffective anti-HBV immune response towards HBV, the infection will progress and persist. High level of HBsAg is considered to play a crucial role in impairing the HBV-specific immunity . The impaired innate immunity can be regulated by direct interaction between HBV antigens and host. Therefore, control of HBsAg secretion could be a potential anti-HBV method . Several classes of drugs containing Nitazoxanide (NTZ) and tizoxanide (TIZ) have been studied to reduce HBsAg secretion and HBV replication . Phosphorothioated oligonucleotides (PS-ONs) with sequence-independent antiviral activity is a novel class of compounds with promising microbicide pathway, and is described as amphipathic DNA polymers (APs) with broad spectrum antiviral activity, against a wide range of viruses, including HBV, HIV-1, HCV, herpes simplex virus, arenavirus and lymphocytic choriomeningitis virus (LCMV) . REP 9 AC which is currently in phaseÃ¢Â Â /Ã¢Â Â¡ clinical trial is a polycytidine amphipathic DNA polymer in 40 nucleotides with the effect to inhibit HBsAg release from HBV-infected hepatocytes and allows CHB patients to reestablish durable immunity through eliminating the immunosuppression mediated by HBsAg . Clinical trial results suggest that REP 9AC possesses promising effect of HBsAg clearance and HBV DNA inhibition with no pro-inflammatory side effects and may develop into an effective new tool to treat HBV .
Potential new NAs targeting HBV replication
Besides these five inhibitors of HBV DNA polymerase, Emtricitabine was originally approved for anti-HIV therapy and found to be also powerful anti-HBV effect in both HBeAg positive and negative patients, and was applied in combination with TDF in HIV/HBV coinfected patients . Clevudine [1-(2-deoxy-2-f luoro-β-L-arabinofuranosyl) thymine, L-FMAU] has effective antiviral activities against both HBV and Epstein-Barr virus (EBV) but not HIV, approved in South Korea and Philippines . MIV-210 (Lagociclovir valactate) is a prodrug of 3’-fluoro-2’, 3’-dideoxyguanosine (FLG) with high oral bioavailability and potent activity against HBV will be a good candidate for anti-HIV/HBV. Besifovir (LB80380), with chemical similarity to adefovir and tenofovir, is an acyclic nucleotide phosphonate and the prodrug of LB80331, and will be metabolized to the active metabolite with anti- HBV effect after intracellular phosphorylation to triphosphate form . Tenofovir Alafenamide (TAF; GS-7340),a prodrug of TDF, achieves higher metabolite concentrations in peripheral blood mononuclear lymphocytes (PBMCs) and lymphatic tissues . TAF will behydrolyzed to tenofovir-alanine conjugate (TFV-Ala) which is the intermediate and then converted into the parent tenofovir, which is phosphorylated in the following to yield the active tenofovir diphosphate (TFV-DP) metabolite . CMX157 is a hexadecyloxypropyl conjugate of TDF with anti-HIV and HBV, 267-fold more active than TDF against HIV-1 and 4.5-fold more active against HBV in vitro. CMX157 is currently in phaseÃ¢Â Â clinical study . AGX-1009 is another novel patented prodrug of TDF and exibits ideal efficacy in inhibiting HBV replication, phaseÃ¢Â Â trials were carried out in 2013 in China . A class of simple unnatural L-nucleosides which have in common a hydroxyl group in the 3'-position (3'-OH) of the beta-L-2'-deoxyribose sugar were found to own specific anti-HBV effect. The more interesting is that the replacement of the 3'-OH will broaden their activity to other viruses. What’s important is that human DNA polymerases and mitochondrial function are not hurt. They can be used alone or in combination to treat CHB .
Gene silence targeting HBV antigen
RNA interference (RNAi) induces a sequence-speciÃ¯Â¬Âc degradation of homologous mRNA. This process can be mediated by MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) which are two popular used classes of small RNAs with different origin and early processing pathways [125,126]. HBV is an approving target for an RNAi approach because its high-efficiency, compact and overlapping genome that lacks significant redundancy . There was a breakthrough platform developed for disease treatment named ddRNAi which was designed to specifically treat human diseases through inserting a DNA construct into host cells, triggering the synthesis of dsRNA, subsequently, the dsRNA will be cleaved into siRNA which will play an efficient role in destructing target mRNA and knocking-down or silencing the expression of target gene . Unlike other RNAi-based technologies, treatments of ddRNAi leads to long-term benefit and potentially cure. ARC-520, a siRNA-based therapeutic agent which is targeting HBV sequences, resulted in long-term suppression in serum levels of HBsAg, HBeAg and HBV DNA both in transient and transgenic mouse, and chimpanzee chronically infected with HBV model by intravenous injection . PhaseÃ¢Â Â study of ARC-520 in healthy adult volunteers implemented in Melbourne, Australia had been finished in the fourth quarter of 2013, phase II trial in chronic HBV patients began in 2014. The satisfactory anti-HBV efficacy of ARC-520 lets it be a new promising anti-HBV drug [98,128]. In future, the nucleic acid-based therapeutics will be improved gradually and put into use increasingly.
Targeting covalently closed circular DNA (cccDNA) for “cure”
Current antivirals aimed towards inhibition of viral replication can inhibit but not eradicate HBV, because of cccDNA, it steadily exists in hepatocyte nuclear and association with histones and DNA chaperone proteins as a minichromosome that serves as template to generate all RNAs necessary for protein production and viral replication, and also secures HBV persistence avoiding host innate immune responses . As we know, the failure of HBV clearance through anti-HBV therapy with NAs is mostly due to the persistence of the cccDNA in CHB patients. Furthermore, long half-life of HBV-infected hepatocytes allows the maintenance of cccDNA in the hepatocytes nuclei which acts as reservoir for HBV reactivation . CHB infection persistence will permit the virus to manipulate the cellular machinery to meet its replication and propagation requirements, and to evade the antiviral response of the host. So to help host being superior in the war of virus-host interactions is the priority among priorities. Efficient and nontoxic clearance of cccDNA in nuclear is a final goal of anti-HBV treatment. Interferon-α (IFN-α) and lymphotoxin-β-receptor activation were reported to respectively up-regulate APOBEC3A and 3B cytidine-deaminases both in HBV-infected cells and human liverneedle biopsies. It is gratifying that this up-regulation can inhibit HBV replication and lead to cccDNA degradation .
The way to cure CHB
In cancer, proteins called the inhibitor of apoptosis proteins (IAPs) are over-expressed and block the apoptosis pathways, causing cancer cells to survive and become resistant to chemotherapies. IAPs impair HBV clearance through preventing TNFmediated killing of HBV-infected cells. The second mitochondrial-derived activator of caspases (SMAC) is a natural antagonist of IAPs and enables apoptotic cell death [132,133]. Birinapant is a member of SMAC mimetics and can be used as antagonists of IAP. It can specifically target and inhibit IAPs, thus naturally drive tumor cells to apoptosis. The latest studies show that birinapant and other SMAC mimetics are able to rapidly reduce HBsAg and HBV DNA in serum, to promote the clearance of hepatocytes with HBcAg, to enhance the anti-HBV ability of ETV in CHB mouse model. It follows that combination of ETV and birinapant or other SMAC mimetics is a promising weapon to cure CHB patients .
Relationship between HBV, innate immunity and gut microbiota
The liver connects and interacts directly with the gut through the hepatic portal and bile secretion. This is the reason why enteric dysbiosis, bacteria translocation and their products are involved in the progression of liver cirrhosis . Nan Qin et al. utilized a unusual system sequencing 16S ribosomal RNA genes of gut microbiota from patients with liver cirrhosis, the change of the gutmicrobiota, invasion of the gut from oral bacterial species, biomarkers specific to liver cirrhosis, genes that differ in abundance between the patients and healthy individuals were analyzed in details, something interesting and important was found and concluded. Their study paves the way to the development of novel probiotics to help combat the aggravation of liver cirrhosis, also to the modulation of microbiota to correct the major dysbioses systems .
HBV clearance in patients heavily depends on the age of exposure, but the reason for this remains obscure . More recently, in hydrodynamic transfection mouse model, gut microbiota was reported to correlate with the age dependence of HBV immunity. In young mice, the gut bacteria was not established, immuno-tolerating pathway to HBV which depend on TLR4 was prevailed, while the maturation of gut microbiota could significantly stimulate liver immunity in adult mice, leading to rapid HBV clearance. The above study reveals the close relationship between gut microbiota and immune tolerance, paves the vital way to control HBV infection, transmission .
To sum up, the causes and treaments of some clinical diseases including child obesity, intestinal inflammatory diseases, allergic dermatitises, hepatic cirrhosis, tumor and so on, are closely related with intestine mucosal organism barrier and intestinal probiotics [138,139]. The intestinal microbiota plays a key role in maintaining the normal function of the human gastrointestinal tract. Many probiotics are derived from human gut flora, and have been conÃ¯Â¬Ârmed to be valuable in the management of gastrointestinal diseases . HBV is a key cause to liver cirrhosis, chronic HBV infection must be characterized by similar changes in the gut of CHB patients as reported. To regulate and improve gut microbiota of host may be a hopeful adjuvant therapy for conventional anti-HBV strategy.
For a long period of time in the future, due to the large population base with CHB infection, the persistent of HBV will sustain in the whole world, especially in China. But be assured: the affected population will become smaller and smaller in view of successful available vaccines popular used. Neonatal inoculation and appropriate antiviral therapy for CHB patients is the key to control HBV.
It is critical to understand the basis and mechanism of HBV persistence, and design more suitable therapeutic strategies to eradicate HBV. Combination approaches enhancing host immune responses, degrading cccDNA which is a key viral target, improving gut microbiota along with conventional antiviral drugs will offer the most appropriate, pragmatic and effective technique to achieve sustained HBV remission of most CHB patients.
This work was supported by Medical Scientific Research Foundation of Zhejiang Province, China (2013KYA208) and Shaoxing, Zhejiang province of China (2013B70063 and 2012B70063).