Abdulrahman MS, Aboelmagd O, Zhang Y, Zaky S, Johar D. Precore/core mutation relatedness to viral reactivation in patients undergoing targeted therapy for hepatitis B virus-related hepatocellular carcinoma. World J Biol Chem 2026; 17(2): 120297 [DOI: 10.4331/wjbc.v17.i2.120297]
Corresponding Author of This Article
Dina Johar, PhD, Department of Biochemistry and Nutrition, Faculty of Women for Arts, Sciences and Education, Ain Shams University, Asma Fahme Street, Cairo 11757, Egypt. dinajohar@gu.edu.eg
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Gastroenterology & Hepatology
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review-article
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Abdulrahman MS, Aboelmagd O, Zhang Y, Zaky S, Johar D. Precore/core mutation relatedness to viral reactivation in patients undergoing targeted therapy for hepatitis B virus-related hepatocellular carcinoma. World J Biol Chem 2026; 17(2): 120297 [DOI: 10.4331/wjbc.v17.i2.120297]
Author contributions: Johar D, Aboelmagd O contributed to conception, design, acquisition of data, literature analysis, discussion, writing and reviewing the manuscript; Abdulrahman MS, Zhang Y, Zaky S contributed to discussion, visualization, reviewing and editing the manuscript. All authors have read and approved the final manuscript for submission.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Corresponding author: Dina Johar, PhD, Department of Biochemistry and Nutrition, Faculty of Women for Arts, Sciences and Education, Ain Shams University, Asma Fahme Street, Cairo 11757, Egypt. dinajohar@gu.edu.eg
Received: February 25, 2026 Revised: April 20, 2026 Accepted: May 26, 2026 Published online: June 5, 2026 Processing time: 101 Days and 19.9 Hours
Abstract
Hepatitis B virus (HBV) reactivation after targeted therapy or immunomodulating therapy leads to active or fulminant hepatitis, low response to prophylactic vaccination, premature discharge from therapy and death. The hypothesis that seroreactive viral infection is caused by mutation/s in the precore/core is invaluable to elucidating the mechanisms of HBV reactivation. Precore/core mutations may correlate with, or predict susceptibility to seroreactivation in HBV-related hepatocellular carcinoma (HCC) patients receiving targeted therapy. This review’s objective is to re-analyze the relationship between the precore/core mutations of HBV-DNA and HBV reactivation in HCC patients receiving targeted therapy. Further, to re-analyze clinically significant precore/core mutations affecting pregenomic RNA initiation and synthesis, and their regulation of viral and cellular gene expressions. This review shed light on the mechanism of HBV reactivation. We analyze the effects of antivirals lamivudine, entecavir, tenofovir alafenamide, tenofovir disoproxil fumarate and immune-based strategies on reactivation after treatment for HBV-related HCC. We proposed future directions for studying mutations in the precore/core region that are likely to cause relapse. This review recommends comparing the genome/proteome of blood from overt and relapsed HCC-related chronic HBV patients. This helps identifying persistent genetic/epigenetic profiles of HBV resistant variants, thus accurately selecting the appropriate antiviral therapy and eliminating the risk of viral reactivation.
Core Tip: Current antiviral treatments can slow down hepatitis B virus (HBV) replication and help improve liver damage. However, they rarely fully clear chronic HBV infections. There is an urgent need for new drugs and better strategies to combat the virus. The hypothesis that seroreactive viral infection is caused by mutation/s in the precore/core is invaluable to elucidating the mechanisms of HBV reactivation. This review re-analyzes clinically significant HBV precore/core mutations affecting pregenomic RNA initiation and synthesis, and their regulation of viral and cellular gene expressions. Further, it analyzes the effects of antivirals lamivudine, entecavir, tenofovir alafenamide, and immune-based strategies on viral reactivation after treatment.
Citation: Abdulrahman MS, Aboelmagd O, Zhang Y, Zaky S, Johar D. Precore/core mutation relatedness to viral reactivation in patients undergoing targeted therapy for hepatitis B virus-related hepatocellular carcinoma. World J Biol Chem 2026; 17(2): 120297
Hepatitis B virus (HBV) infection is epidemic where 350 million infections have been reported worldwide[1]. Egypt has had a very high prevalence of chronic liver diseases[2,3] and inaccurate statistics about hepatocellular carcinoma (HCC)[4]. Globally, an estimated 254 million people are living with hepatitis B; 20%-30% of patients with chronic HBV infection will develop cirrhosis and/or HCC; 2% of all deaths are due to liver cirrhosis and HCC[1]. HBV infection caused about half of the mortality from HCC, 62% of such mortality was associated with cirrhosis, which increased by 29% between the 1990s and 2010[5]. Vaccines and antiviral medications are available[6], but yet about 30% of people worldwide have signs of current or past infection with HBV[7].
HBV
Chronic active HBV patients have HBV DNA in their blood or liver tissue, along with detectable hepatitis B surface antigen (HBsAg) in the serum. Usually, the amount of DNA in the serum is < 104 copies/mL. A highly sensitive polymerase chain reaction (PCR) test identifies this level. Hepatitis B e antigen (HBeAg) is the HBV envelope protein; its presence indicates active viral replication and thus greater infectivity. Anti-hepatitis B core (anti-HBc) antibodies indicate current or former HBV infection[8].
HBV DNA lacks proofreading activity and exhibits a high replication rate, with genomic variations throughout reverse transcription. Accumulating evidence has been published on the viral genes pre s/s, x, and p, whereas little research has been conducted on the precore/core region. The latter region plays a key role in upregulating viral replication, liver pathogenesis, and immune evasion. The precore/core open reading frame (ORF) of HBV DNA encodes both the HBeAg and the hepatitis B core antigen (HBcAg). The core protein forms a 28 nm core particle within the viral envelope. The basic core promoter (BCP), located upstream of the core region, regulates transcription of both the precore/core mRNA and the pregenomic RNA (pgRNA) during viral replication. The BCP ORF overlaps with the x ORF. The core contains a partially double-stranded DNA genome and a viral enzyme with DNA polymerase, reverse transcriptase (RT), and RNase H activities. Mutations in the BCP link to accelerated progression to cirrhosis and HCC, suppression of HBeAg expression, emergence of drug-resistant mutants, and ultimately, treatment failure and mortality.
Serological markers for HBV infection
The HBsAg, anti-HBs, HBeAg, anti-HBe, and anti-HBc immunoglobulin M (IgM) and IgG are typical markers of HBV infection. The presence of HBsAg signifies an active infection. IgG remains in the body during long-term or chronic infection. IgM anti-HBc appear during severe flare-ups of chronic hepatitis B, but their level is lower compared to acute cases. Anti-HBs shows protection or immunity against the virus. It is the primary marker in vaccinated individuals and appears alongside anti-HBc IgG in those who recovered from a past infection. If both IgG anti-HBc and HBsAg are positive, it indicates an ongoing infection. The presence of IgM and IgG anti-HBc suggests a past infection. The presence of HbeAg indicates high viral activity and infectivity, whereas the absence of HBeAg indicates low viral activity and infectivity, as reflected by HBeAb. Some individuals test negative for HBsAg but positive for anti-HBc IgG without anti-HBs. This pattern is called isolated anti-HBc. People with no HBsAg but positive anti-HBc can still have the virus reactivates during treatments like chemotherapy or immunosuppressive therapies. In such cases, HBsAg may reappear in the blood[9]. Table 1 shows the HBV serologic profiles.
Table 1 Hepatitis B serologic testing involves measurement of several hepatitis B virus-specific antigens and antibodies. Different serologic markers or combinations of markers are used to identify different phases of hepatitis B virus infection and to determine whether a patient has acute or chronic hepatitis B virus infection, is immune to hepatitis B virus as a result of prior infection or vaccination, or is susceptible to infection.
HBV DNA testing directly measures the amount of virus present by assessing the viral load, which indicates the virus replication activity. Most current HBV DNA tests use real-time PCR, which can detect levels as low as 10-20 IU/mL within its broad range. During chronic HBV infection, serum HBV DNA levels change widely, from undetectable to over 109 IU/mL. Studies have found a link between serum HBsAg levels and the activity of covalently closed circular DNA (cccDNA) in the liver, specially in patients who are HBeAg-positive. Monitoring HBsAg helps predict how patients will respond to interferon (IFN) treatment. It can also show which HBeAg-negative patients with normal alanine aminotransferase (ALT) levels might develop ongoing liver problems[10]. A significant obstacle to advancing these studies is the lack of cell lines permissive to infection. To date, all tested cell lines have demonstrated resistance to known HBV strains, including duck HBV. While primary human hepatocyte cultures are susceptible to infection, their viability is limited to five to seven days post-plating. The capacity of in vivo models to sustain infection for longer durations remains untested. HBV replication initiates with the formation of cccDNA from incoming virion DNA. The virion DNA adopts a relaxed circular conformation, comprising a complete minus strand and a partial plus strand. The 5’ end of the minus strand is protein-linked, whereas the 5’ end of the plus strand contains RNA modifications. Modifications to the minus strand remove the viral RT from its 5’ end and eliminate one set of terminal repeats. Plus strand modifications complete DNA synthesis and remove an RNA oligomer from its 5’ end. Subsequently, both strands are ligated at their 5’ and 3’ termini to generate cccDNA. Critical questions remain regarding the timing and mechanism by which the viral polymerase dissociates from the 5’ end of the minus strand DNA, and whether this process is associated with minus strand end joining. This mechanism may resemble the cleavage and ligation events observed during retroviral DNA integration. Rogler[11] analyzed nucleotide sequences at the termini of minus strand DNA and proposed that topoisomerase I mediates the conversion of relaxed DNA to cccDNA. Yang et al[12] provided evidence for an alternative cccDNA synthesis pathway involving recombination of terminally redundant double-stranded linear DNA. Plus strand DNA synthesis generates these molecules by extending beyond the direct repeat 2 to the 5’ end of the minus strand. The cccDNA persists as a mini-chromosome and serves as the template for viral RNA transcription from multiple promoters. Its chromatin structure likely contributes to its stability within the nucleus of infected cells. This stability determines the half-life of cccDNA and influences responses to antiviral therapy. Nucleocapsids must either bind the core protein or translocate to the nucleus, and partial dephosphorylation of the core protein is essential for both regulatory steps[13]. Antiviral drugs targeting viral DNA synthesis must overcome the persistence of cccDNA in hepatocytes and the extended lifespan of infected cells, particularly in asymptomatic carriers. Although short-term administration of these drugs may not result in adverse effects, prolonged use can lead to liver failure and mortality. Both human and animal models have been used to assess the adverse effects of long-term administration of HBV antivirals[14,15]. These studies consistently report that extended nucleoside therapy alone does not eradicate infection. While treatment suppresses viral replication, viral rebound typically occurs upon drug withdrawal, except in certain human patients receiving IFN therapy[15]. The observed success rates are comparable to those achieved with IFN, indicating that antiviral therapy may enhance the host immune response against infection. Remission of infection in human HBV carriers is further promoted by IFN therapy. In practice, a study[16] on 55 HBV-related HCC patients receiving curative therapy, measured blood HBcAg and liver cccDNA levels that reveal virus states and associate with HCC recurrence. Cancer recurred in 38% of patients over 2.2 years (range 0.2-7.4). Multivariate analysis revealed blood HBcAg at or above 4.8 log U/mL raised risk (hazard ratio: 8.96; 95% confidence interval: 1.94-41.4). Patients with high liver cccDNA had worse recurrence-free survival than low cccDNA group after resection (P = 0.0438). Liver cccDNA levels associated with blood HBcAg at HCC onset (P = 0.028; r = 0.479). HBcAg predicted HCC return post treatment with antivirals.
Immunopathogenesis of HBV-related HCC
The development of HCC after HBV infection clears is immune-related. Cirrhosis is itself a step before cancer, making it a warning sign. Checking the host’s immune status is crucial for monitoring the disease progression. People with chronic HBV infections often have weak T-cell responses, and their immune cells target only a few regions of the virus’s genome[17]. In fulminant hepatitis (FH), flare-ups that associate chronic hepatitis often result from a strong immune activity that raises ALT levels. Some flares are associated with a drop in HBV DNA and a switch from HBeAg to anti-HBe, indicating immune clearance of infected liver cells. Repeated ALT flares can raise the risk of cirrhosis and liver cancer. Extrahepatic problems can also occur if humoral responses form circulating immune complexes, disrupting the normal balance[18]. HBcAg is an important marker that triggers the immune system. Changes in its mRNA sequence can help the virus evade immune responses, making the infection harder to fight and leading to a more stubborn disease[19,20].
HBV genotypes
Two major determinants of chronic HBV infection outcome are HBV genotypes and subgenotypes. HBV has at least 10 confirmed genotypes, designated A through J. The impact of these various HBV genotypes and subgenotypes on the likelihood of reactivation in individuals with HBV-related HCC remains poorly understood. Longitudinal, population-based studies offer the most effective approach to elucidating these genotypic relationships. Such studies should involve comparative analyses of cohorts with differing genotypes, followed prospectively over extended periods. Egypt demonstrates considerable uniformity in HBV subtypes, predominantly genotype D, specifically subgenotype D3, and HBsAg subtype ayw2. Furthermore, mutations within the major hydrophilic region are prevalent in this population[21]. Genotype D has been associated with a higher incidence of advanced liver disease, including HCC, compared to other genotypes, and constitutes an independent risk factor for FH[22]. The objectives of this review are to: (1) Re-analyze profiles of mutations in the precore/core region of HBV DNA published to date, in serum of chronic HBV-related HCC patients who undergo targeted therapy; and (2) To assess the impact of such mutations on viral and cellular gene expressions that may predispose for HBV reactivation.
IDENTIFIED PROFILES OF MUTATIONS IN PRECORE/CORE REGIONS OF HBV DNA IN THE SERUM OF CHRONIC OVERT HCC PATIENTS
Research has focused on identifying specific mutations associated with the development of HCC and severe liver disease. Table 2 shows a summary of the identified mutations to date. One of the HBV ORFs encodes for HBeAg and HBcAg that share the same 149 aa sequence. The precore/core regions are controlled by an alternative upstream start codon, which influences the production of these proteins[23]. The popularity of mutations in the HBV four ORFs is due to their ability to enable the virus to evade the immune system and relapse, and to stabilize its secondary structure. In liver tissue specimens, the BCP region may downregulate HBeAg expression (negative chronic). i.e., the precore mutation G1896A and the A1762T/G1764A double mutations are known to affect early prognosis and severity of HCC[24,25], and are independent predictors of worse survival after surgery for HCC[26]. The emergence of the G1896A mutation in the precore region is known to be associated with HBeAg seroconversion[27]. Rastegarvand et al[28] found an unexpected stop codon at position 1896 in patients on hemodialysis with precore- or core-positive occult hepatitis B. A research group analyzed how mutations in the HBV precore/core region affect HBV DNA levels associated with HCC survival. The multivariate survival analysis used a Cox proportional hazards model and identified 5 mutational sites 1915, 2134, 2221, 2245, 2288 as independent predictors of HCC survival, after adjusting for clinical characteristics. Two of such mutations express aa variants. The association of site 1896 with survival was close to statistical significance[29].
Table 2 A summary of the identified and published hepatitis B virus mutations.
Mutation
Effect
Mechanism
G1896A in precore
Affects early prognosis and severity of HCC
The study compared results between ultra-deep pyrosequencing and cloning based sequencing using HBeAg-positive and negative sera infected, with either genotype D or E
A1762T/G1764A double mutation
HBcAg mutations might increase the risk for HCC; affect early prognosis and severity of HCC; a factor that independently predicts worse survival rates after surgery
When combined with HBx mutants, they upregulate SKP2, which then down-regulates the cyclin kinase inhibitor p21 via ubiquitin-mediated proteasomal degradation. Eventually mRNA precore inhibition; upregulation of pgRNA transcription, and ultimately HCC
A1762T; 1753-1757 jointly with A1762T/G1764A; 1766T jointly with 1768A
Mutation in the precore/core and s regions results in undetectable s region
1915, 2134, 2221, 2245, 2288 in precore/core
Independent predictors of HCC survival
-
S87 and P156
Cancer recurrence
-
E77, P79, E83, L84, and S87 in core
Shorter survival times and increased viral activity. The S87 mutation could interfere with the assembly of the core
-
x/precore of HBV genotype D1, T1673/G1679, G1727, C1741, C1761, A1757/T1764/G1766, T1773, T1773/G1775 and C1909
A marker of HCC
-
G1896A; G1899A, T1846A, G1862C, G1888A, T1821C, C1826T, A1827C, A1850T precore start codon Kozak and G1951T, G1957A, genotype A1 and D
HBeAg-negative chronic; HBV infection and a more severe outcome
-
A2051C
Associated with increased viral replication both in vivo and in vitro
-
P5H/L/T, E83D, I97F/L, L100I, and Q182K/stop
More prevalent in chronic HBV and cirrhotic patients
Evading the host-immune system
The stop codon of W28*(G1896A), precore; S21, E40 and 1105; the epitope substitutions (117-131)
Identified to correlate with the development of the inactive carrier state. Higher in the cirrhotic/HCC group than in the inactive and the chronic active HBV groups
Evading the host-immune system
T1938C (V13A) with A2051C (N51H) in core
Those substitutions in the core correlated with HBV-related HCC and disease progression in Alaskan natives
-
YMDD mutation at codons 203-206 of the HBV RT
It targets the catalytic site of the HBV polymerase, specially at rtM204V/I
Multiple studies have elucidated mechanisms by which the HBcAg localizes to the cytoplasm during active hepatic necroinflammation. These mechanisms include alterations in the aa sequence of the HBV x gene, upregulation of S-phase kinase-associated protein 2 by the HBV core, and suppression of the cyclin-dependent kinase inhibitor p21. These processes collectively result in inhibition of mRNA precore expression, increased transcription of pgRNA, and ultimately contribute to the development of HCC[25] (Figure 1).
Figure 1 Hepatitis B e antigen and hepatitis B core antigen share the same 149 aa sequence and are produced by one of the four open reading frames of the hepatitis B virus.
The popularity of mutations in the hepatitis B virus (HBV) four open reading frames is a variation that enables the virus to evade the immune system and relapse. In liver tissue specimens, the basic core promoter region may downregulate hepatitis B e antigen expression (negative chronic). i.e., the precore mutation G1896A and the A1762T/G1764A double mutation are known to affect early prognosis and severity of hepatocellular carcinoma (HCC), and were identified as a factor that independently predicts worse survival rates after surgery for HCC. Versatile studies provided mechanisms that position HBV core antigen into cytoplasm with active hepatic necroinflammation; HBV core upregulation of the S-phase kinase-associated protein 2; and suppression of its target’s activity, the cyclin kinase inhibitor p21, and eventually mRNA precore inhibition, upregulation of pregenomic RNA transcription, and ultimately HCC. The mutations S87 and P156 were identified as independent risk factors for cancer recurrence. The bottom capture is an autopsy case of metastatic HCC from our laboratory. HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; HBeAg: Hepatitis B e antigen; HBcAg: Hepatitis B core antigen; SKP2: S-phase kinase-associated protein 2; DR: Direct repeat; pgRNA: Pregenomic RNA.
A study in China examined the precore/core regions of tumor and non-tumor tissues from 98 patients with HBV-related HCC after surgery. The mutations S87 and P156 were independent risk factors for cancer recurrence. There was no link between serum samples and liver tissues in patients with the S87 mutation. Mutations in the core region at E77, P79, E83, L84, and S87 were associated with shorter survival and increased viral activity. The research indicated that the S87 mutation could interfere with core assembly[20].
Further, the 1762T variant is associated with HBeAg seroconversion and liver histopathological inflammation. The 1753-1757 mutations, jointly with the 1762T and 1764A mutations, are common in FH, HCC and ALT[30]. Whereas the 1766T, jointly with 1768A, are common in FH, and jointly with A1762T and G1764A are common in recurrent hepatitis B post liver transplantation. Furthermore, 1766T jointly with G1764A, exacerbate HBV infection in HBeAg negative asymptomatic carriers. The x/precore region of HBV genotype D1, i.e. T1673/G1679, G1727, C1741, C1761, A1757/T1764/G1766, T1773, T1773/G1775 and C1909 have been identified as a marker of HCC[31].
To date, research on HBV genotypes and subgenotypes, such as A1 and D, has primarily focused on their sequences. These types link to higher risks of cirrhosis and liver cancer in short-term studies. For example, Yousif et al[24] utilized a well-characterized HBV BCP, precore, and core sequences as a bioinformatics model. The study compared ultra-deep pyrosequencing with standard cloning-based sequencing by analyzing sera that were HBeAg positive or negative from genotype D or E human infections. Ultra-deep pyrosequencing identified significantly more substitutions than cloning-based sequencing; however, many of these were not authentic. While deep sequencing offers advantages, the absence of validation reduces its reliability. It is essential to identify variants relative to a known reference or consensus sequence, as a mutation under investigation may represent a normal trait within the appropriate genotype or subgenotype. Maasoumy et al’s study[32] examined HBcAg in a large European cohort with HBV genotypes A and D throughout all phases of infection. Groups included 30 people with HBeAg-positive immune tolerance, immune clearance (60), HBeAg-negative (50), inactive carriers (109), and acute cases (8). The tolerance and clearance groups had the highest HBcAg medians (8.41 log U/mL), (8.11 log U/mL) respectively. Whereas hepatitis cases had low core antigen levels (4.82 log U/mL) and the lowest in quiescent carriers (2 log U/mL). HBcAg shifted a lot by infection phase, although it linked well to HBsAg across all phases. HBcAg tracks virus replication and transcriptional activity of liver cccDNA. For HBeAg negative patients, it discriminates carriers from active disease. To date, no long-term or cross-sectional studies have looked at these specific genotypes. No data are available for genotypes and subgenotypes A3, E, F4, and H regarding disease outcomes. Future research should analyze the full genomes of these virus types from patients with and without serious health issues.
Since the immune response influences the extent of liver damage, studies should include peptides unique to each genotype and subgenotype. It is essential to investigate how immune cells, such as T-cells and regulatory cells, respond to these specific peptides. This approach will help us understand how different virus types affect disease severity. The latter genotyping model was conducted by a group from Nigeria in 2021[33]. They genotyped the precore/core mutations in 72 patients with chronic HBV infection. The G1896A mutation was predominant. Other sites included G1899A, T1846A, G1862C, G1888A, T1821C, C1826T, A1827C, A1850T precore start codon Kozak and G1951T, G1957A mutations. Phylogenetic analysis revealed HBV genotypes A1 and D. Further, the occurrence of precore/core mutations in patients with HBV vs those with HBV/hepatitis C virus (HCV) dual infections was similar. They concluded that this variation in the precore/core region was causing HBeAg negative chronic HBV infection and a more severe outcome. Furthermore, a population of Alaskan natives with HBV genotype F1b showed correlations between the T1938C (V13A) with A2051C (N51H) substitutions in the core with HBV-related HCC and disease progression. In particular, A2051C associates with increased viral replication both in vivo and in vitro[34]. Another Korean study[35] identified five HBcAg mutations: P5H/L/T, E83D, I97F/L, L100I/ and Q182K/Stop - as being more prevalent in chronic HBV and cirrhotic patients. Such accumulation of mutants, along with viral persistence, provided sufficient evidence of the mutants’ capability to escape the host immune system[20]. For this reason, a group from Iran identified precore/core mutations at different stages of HBV-infected patients. They found the most prevalent mutation in the stop codon of W28*(G1896A) in the precore, whereas, among group comparisons, S21, E40 and 1105 were identified as correlating with the development of the inactive carrier state. Further, the epitope substitutions (117-131) were higher in the cirrhotic/HCC group than in the inactive and the chronic active HBV groups (P = 0.001)[36]. In summary, establishing correlations between specific mutations in the HBV genome and the clinical progression of HBV infection is challenging. This complexity results from the presence of diverse viral variants within infected individuals and the continuous generation, accumulation, and elimination of these variants. Additionally, multiple mutations often coexist within a single viral mutant, which complicates the assignment of clinical significance to individual mutations[37]. Coinfections with other viruses may also affect the natural course of HBV infection through mechanisms such as viral interference or immune modulation.
CURRENT HBV ANTIVIRAL THERAPIES AND RISK OF HBV REACTIVATION
Pegylated IFN-alpha or one of five Nucleos(t)ide Analogues (NAs) that block the HBV DNA polymerase and are known as direct-acting antivirals (DAAs) can treat chronic HBV infection. Pegylated IFN-alpha helps about one in three patients and can boost the immune response, but it often does not fully clear the virus. The polymerase inhibitors quickly lower viral load in most patients, but rarely lead to a complete cure. People who clear the infection on their own without treatment usually live normal lives and are considered to have a “functional cure”. Their viral load and antigen levels remain very low without medication, and their laboratory tests, such as ALT levels, return to normal. Still, a “true cure”, where the virus is completely gone, is rarely achieved with current medicines.
The popular use of infant hepatitis B vaccines has lowered chronic HBV infections by over 90%. Still, some people who do not respond to the vaccine show anti-HBs levels below 10 IU/L. Over time, these levels in vaccinated individuals tend to drop below 10 IU/L. Vaccinating HBV negative patients is strongly recommended. Individuals with weakened immune systems often require higher vaccine doses or additional booster shots to elicit a robust anti-HBs response.
The 2025 European Association for the Study of the Liver consensus guidelines regarding HBV reactivation prevention have been released[38]. HBV reactivation in patients undergoing immunosuppressive or cytotoxic therapy, with reports of fulminant liver failure occurs following cessation of these treatments[39-41]. Individuals who are HBsAg positive or HBsAg negative but anti-HBc positive and receiving chemotherapy or immunosuppressive therapy are at increased risk for HBV reactivation. This risk is particularly elevated when agents such as rituximab are used alone or in combination with steroids. The probability of reactivation is classified as high (10%), moderate (1%-10%), or low (< 1%). Consequently, all patients commencing chemotherapy or immunosuppressive therapy should undergo screening for HBsAg, anti-HBs, and anti-HBc prior to treatment initiation. The use of hepatitis B Ig (HBIG) in combination with NAs prevents HBV recurrence following liver transplantation. Nevertheless, the optimal prophylactic strategy remains a subject of ongoing debate among experts. A major randomized, placebo-controlled trial could clarify the role of antiviral therapy after curative treatment for HCC. Still, such a trial might seem unethical as antiviral therapy is frequently recommended for these patients by various international guidelines[42,43].
The core pregenomic promoter regulates the expression of multiple RNAs with 5’ heterogeneity, including those encoding core antigen, e antigen, polymerase mRNAs, and pgRNA. pgRNA acts as the template for reverse transcription during HBV replication. Lamivudine (LAM), or 2’,3’-dideoxy-3’-thiacytidine, has been the principal antiviral agent used to treat HBV infection. Cellular kinases convert this synthetic nucleoside analogue into its active 5’-triphosphate form, LAM-triphosphate. LAM-triphosphate competes with endogenous deoxycytidine triphosphate to inhibit HBV DNA polymerase, acting as a chain terminator. This inhibition blocks the reverse transcription of pregenomic mRNA into DNA, as well as plus strand and double stranded DNA synthesis. LAM has no 3’-OH group, which prevents the formation of the 5’-to-3’ phosphodiester bond, thereby arresting HBV DNA replication and reducing viral load by 3 logarithmic units to 4 logarithmic units, which subsequently decreases liver disease activity.
The preventive use of LAM lowers the risk of HBV reactivation and reduces related health problems and death. However, about 10% of patients with chronic HBV and low viral levels (HBV DNA around 2000 IU/mL) still face reactivation risks. This risk is higher in patients with elevated viremia. Those who test positive for HBsAg and are resistant to LAM must jointly receive HBV RT inhibitors entecavir (ETV), tenofovir alafenamide, or tenofovir disoproxil fumarate (TDF) as treatments or prevention. TDF, an acyclic nucleoside phosphonate with simpler stereochemistry than ETV, is often considered superior for manufacturing purposes. Its straightforward chemical synthesis enables high-yield, large-scale production and significantly reduces manufacturing costs. ETV is a carbocyclic 2’-deoxyguanosine analogue. Its synthesis is complex because it requires the formation of a specific exocyclic double bond and multiple chiral centers. This process requires advanced reagents and rigorous purification steps, thereby increasing raw material production costs per kg. The rules for monitoring and stopping these medications are the same as HBIG. Patients who are HBsAg-negative but anti-HBc positive should get anti-HBV prophylaxis if they face a high risk of HBV reactivation. Therefore, using prophylaxis with ETV, TDF, or tenofovir alafenamide is recommended. The treatment should last at least 12 months after stopping immunosuppressive therapy, and 18 months if rituximab is involved. It should only be stopped when the underlying disease is in remission. During prophylaxis, liver tests and HBV DNA levels should be checked every 3 months to 6 months. Monitoring should continue for at least 12 months after stopping NAs, as many reactivations occur after withdrawal.
Investigators tried to address what predicts HBV reactivation after a liver transplant, and assessed post-transplant outcomes. Trials compared placebo, HBIG alone, or NAs alone as interventions. The joint use of HBIG and NAs lowers the rate of HBV recurrence. Two clinical trials and systematic reviews on this topic have been published until 2014[44]. One trial examined 209 consecutive patients who all tested positive for HBsAg and received liver transplants. Most of these patients (89%) received a combination of HBIG and LAM to prevent recurrence. A smaller group (11%) received HBIG alone. The study concluded that high viremia at orthotopic liver transplantation and longitudinal intervention with LAM should be given further attention as a preferential predictor of HBV reactivation[45].
Many studies propose that antiviral therapy can reduce incidence of HCC, although they require specifying the type of drug used and the stage of the disease. We reviewed studies published from 2005 to 2025 on LAM as a prophylactic therapy, a summary is shown in Supplementary Table 1. We looked at patients with positive HBsAg who have been undergoing HCC-targeted chemotherapy and no HBV reactivation after chemotherapy. The included articles focused on outcomes such as HBV-related hepatitis (HBV-DNA levels + liver function test); HBV-related death (secondary to liver failure induced by HBV reactivation); all causes of hepatitis; all causes of death. We found 159 results: 3 meta-analyses, 11 systematic reviews, and 3 clinical trials that tested different approaches to treating HCC. One trial enrolled 73 consecutive patients with HCC who received transarterial chemolipiodolization. They were treated with epirubicin 50 mg/m2 and cisplatin 60 mg/m2 every month. Participants were randomly split into two groups: One started taking 100 mg of LAM daily from the start of transarterial chemolipiodolization (preemptive), while the other did not (control)[46]. In another trial conducted from January 2000 to December 2002, 33 patients with HCC and active hepatitis B infection participated. These patients were divided into two groups. One group of 17 patients had surgery alone, while 16 received surgery plus LAM and thymosin α1 after the operation. The study tracked changes in HBV-DNA levels, HBeAg seroconversion, tumor recurrence, and survival time[47]. A third trial from January 2000 to December 2002, enrolled a group of 33 patients with HCC and active hepatitis B infection. They were split into two groups. The control group, with 17 patients, only had surgery to remove the tumor. The treatment group, with 16 patients, had surgery plus additional medicine. They received LAM and thymosin α1 after surgery. Researchers monitored the effectiveness of the virus control, tracked change in virus markers, recorded tumor recurrence, and measured patient survival in both groups. The study found that LAM and thymosin α1 after surgery can help reduce HBV viral load. This treatment may also slow down the return of the disease and help patients with HCC live longer[48].
From January 2004 to June 2007, a randomized trial compared different treatments after surgery for advanced HCC[49]. The study looked at patients who had a complete removal of liver cancer. The treatment group included 43 patients who received LAM, with some also receiving adefovir dipivoxil (ADV). The control group had 36 patients who did not receive antivirals. While NAs did not lower the short-term recurrence rate, they helped clear the virus after surgery. They also increased the amount of leftover liver tissue, making it easier for patients to tolerate future treatments if the disease recurs. In another study[50], 18 patients received liver transplants due to HBV-related liver disease. Before surgery, all patients tested positive for HBsAg, and 3 also had HBV DNA. HBV recurrence was identified when HBsAg appeared again after the transplant. Two patients (15%) were treated with HBIG alone, 4 (31%) with LAM alone, and 7 (54%) with a combination of LAM and HBIG. One patient had HCC. Their ongoing immunosuppressive treatment involved cyclosporine or tacrolimus. Liver transplants for HBV patients using combination antiviral drugs have good survival rates. A positive outcome is achievable when HBV-infected livers are transplanted with HBIG, administered in a dose based on its pharmacokinetics. This, along with LAM, protected against the virus. Using preventive treatment against the virus has greatly lowered HBV recurrence and helped patients live longer after transplantation.
A group of 48 patients with HBV-related HCC received 3D conformal radiation therapy (3D-CRT) to the liver[51]. Among them, 16 patients started LAM treatment before and during the therapy (group 1). The remaining 32 patients did not receive antiviral treatment before 3D-CRT (group 2). To study spontaneous reactivation of HBV, researchers included a control group of 43 HCC patients who had no specific treatment for either HCC or chronic HBV. The study concluded that patients with HBV-related HCC treated with 3D-CRT should be watched carefully for HBV reactivation. Reactivation can lead to a flare-up of chronic HBV, which might be mistaken for radiation-induced liver damage. Using antiviral therapy could help prevent the worsening of liver function after radiation therapy.
Another study[52] aimed to assess how 30 patients with controlled HCC respond to LAM treatment, comparing them with a clinically matched 40 HCC patients who did not receive LAM. The study then examined differences in liver function, tumor recurrence, survival rates, and causes of death between the two groups. LAM therapy resulted in a significant improvement in liver function, even in patients with HCC. LAM could help reduce death caused by liver failure.
A review was conducted on 17 patients with HBV-related HCC who underwent transhepatic arterial infusion chemotherapy[6]. Eight of these patients received LAM, while 9 did not. All patients tested positive for HBeAg. Various tests were performed, including liver function, liver enzymes, HBV-DNA levels, HBeAg, HBeAb, and mutations in the precore/core promoter regions of HBV DNA. The use of LAM before treatment lowered HBV-DNA levels and helped prevent worsening liver damage during chemotherapy for HBeAg-positive patients with HCC.
TDF is a nucleotide analogue, whereas ETV is a nucleoside analogue. This structural distinction leads to different immunological effects. Recent studies demonstrate that TDF selectively enhances IFN-lambda 3 production, an effect not observed with ETV. Enhanced IFN-lambda 3 production activates IFN-stimulated genes with anti-tumor properties and contributes to the suppression of HBsAg. This mechanism may account for the more rapid and significant reduction of HBV DNA by TDF compared to ETV in specific patient populations. Rapid viral suppression is particularly critical when vascular endothelial growth factor receptor inhibitors are administered, as these agents modulate immune responses and increase the risk of abrupt viral reactivation. Consequently, the additional anti-cancer and antiviral properties of TDF are beneficial in contexts where viral mutations enable evasion of standard immune surveillance[53]. Retrospective and prospective studies examined serum HBV DNA levels, liver function, complication rates, and hospital stay duration. They compared two groups: One that had only liver surgery and another that received ETV therapy before and after surgery. Each group had 44 patients in the study. Patients with HCC and low HBV DNA levels were at risk of HBV reactivation after surgery, and that preoperative ETV reduced such risk. ETV improved liver function and shortened hospitalization[54]. Another retrospective study[55] followed a group of 108 patients with HBV-related HCC who received TACE from January 2007 to January 2013. The study examined sudden liver problems that occurred after the procedure. Preemptive antiviral therapy targets only high-risk chronic hepatitis B patients to prevent worsening liver damage. They defined at least 6 months of antiviral treatment as necessary before a patient was diagnosed with HCC. A third study[56] looked at whether high-dose TDF therapy can stop HBV from causing HCC to recure. They designed a study where everyone received the same treatment, with no comparison group. The goal was to determine if using high-dose TDF is practical in real life. They enrolled 10 patients and monitored their progress for 3 months or until they discontinued treatment early. They found that high doses of tumor necrosis factor (TNF), up to five times the recommended amount, are poorly tolerated by many patients. These doses also do not effectively stop HBV from replicating as HCC progresses. Another retrospective study[57] examined the effectiveness of prophylactic ETV in HCC patients undergoing TACE. The study included 191 consecutive patients with HBV-related HCC, of whom 44 received ETV before treatment. The main focus was on virologic changes, which meant an increase in serum HBV DNA levels to more than 1 log10 copies/mL from the lowest point (higher than nadir). They also looked at hepatitis flares caused by reactivation of HBV. Prophylactic ETV achieved efficacy in HBV-related HCC patients receiving TACE. In 2018, a study[58] examined 607 patients with HBV-related HCC who underwent surgery or radiofrequency ablation (RFA). They divided them into three groups based on the antiviral drugs they administered. The first group, with 261 patients, did not get any antiviral treatment. The second group, with 90 patients, received low-strength NAs. The last group, with 256 patients, was treated with high-strength NAs. The main goal was to see how long patients stayed free of cancer recurring. Patients on ETV and TDF had fewer recurrences than those on other antivirals. Another study[59] followed up 1695 patients who had surgery for HBV-related HCC at Korea’s Barcelona Clinic Liver Cancer stage 0 or A between 2010 and 2018. Of those, 813 patients received ETV while 882 took TDF. The study compared cancer recurrence and overall survival between the two groups, using statistical methods to match patients’ backgrounds and adjust for predisposing factors. The analysis started from the day of their liver surgery. Patients on TDF had a notably lower chance of their cancer recurred and survived longer overall than those on ETV.
A retrospective study[60] examined 87 patients who had a curative liver surgery for HCC. The goal was to determine if antiviral therapy administered after surgery could improve outcomes for patients who had not received antiviral treatment before surgery. The study examined clinical signs, tissue pathologies, and the duration patients remained free of cancer. Patients were followed up for a median of 31 months to track HCC recurrence. Results showed that antiviral therapy improved survival rates for HBV-related HCC tumors up to 3 cm in diameter. The findings suggest that antiviral drugs should be considered a standard part of post-surgery treatment for HBV-related HCC.
Regarding viral suppression kinetics, several studies suggest that TDF achieves more rapid or complete viral suppression in certain subgroups, such as HBeAg positive patients with high baseline HBV DNA, a key factor in oncogenesis[53]. A meta-analysis[61] was conducted across EMBASE, the Cochrane Library, and the Science Citation Index, expanded to include relevant studies investigating NAs in HBV-related HCC patients after curative resection. Relative parametric data, including 1-, 3-, and 5-year overall survival rates and 1-, 3-, and 5-year recurrence-free survival rates, were quantitatively pooled and estimated. The inconsistency factor, the cumulative ranking curve, and the publication bias were evaluated. Patients with HBV-related HCC who received NAs antiviral therapy after surgery had better survival rates than those who did not receive antivirals. Drugs like ADV and TDF showed better efficacy in preventing early and late cancer recurrence than other NAs. Drugs like ADV and TDF showed better efficacy in preventing early and late cancer recurrence than other NAs. Making antiviral treatment an important part of post-surgery care for these patients.
Between 2013 and 2017, three hospitals enrolled patients with HBV-related HCC who had surgery or ablation as their first treatment. A total of 421 patients had part of their liver removed, and 305 received RFA. All of these patients started antiviral medication using either ETV or TDF. The study examined how often the cancer reoccurred and how many patients died over time. Researchers adjusted for factors including HBV DNA levels, tumor characteristics, and patient demographics. The results showed no significant difference in cancer recurrence or mortality rates between those treated with ETV and those treated with TDF[62]. In another study[63] patients with HCC who went beyond the Milan criteria tended to have a high chance of the cancer recurring after surgery. When comparing treatments, TDF significantly reduced the risk of HCC recurrence more than ETV therapy. Using propensity score matching (PSM) from the date of liver resection for HCC, TDF showed better overall survival. It also offered stronger protection of liver function. However, there was no difference in the rate of HCC recurrence between TDF and ETV treatments[64]. TDF works better than ETV for eliminating hepatitis B symptoms after RFA treatment. It helps improve the albumin-bilirubin grade more effectively[65]. A research group[66] performed a thorough search across multiple electronic databases from 2000 to January 2022. The goal was to find studies comparing ETV and TDF in patients with HBV-related liver cancer who had received curative treatment. The data on adjusted hazard ratios were combined using a random-effects model. Nine studies involving 5298 patients were included in the final analysis. TDF was superior than ETV in reducing the risk of recurrence and mortality following resection or ablation of HBV-related HCC.
Additional study[67] compared how TDF and ETV affect patients with HBV-related HCC who were treated with FOLFOX-hepatic arterial infusion chemotherapy. The study involved 683 patients treated between January 2016 and December 2021. Some patients received TDF, while others took ETV. Researchers analyzed their outcomes, focusing on survival rates, disease progression, HBV reactivation, and liver health. To obtain accurate results, they employed PSM to compare the two groups fairly. The findings indicated that TDF led to better outcomes in patients with advanced HBV-HCC who were treated with FOLFOX-hepatic arterial infusion chemotherapy. These patients experienced fewer cases of HBV reactivation and maintained better liver function for a longer period. Notably, these enhancements were particularly evident in patients who underwent a minimum of four treatment cycles. A meta-analysis[68] examined the comparative efficacy of TDF and ETV in patients with HBV-related HCC. The analysis encompassed detailed survival data extracted from six sources, including some gray literature, up to August 30, 2023. The researchers utilized Kaplan-Meier survival curves, stratified Cox models, and shared frailty models to analyze outcomes and address inter-study variability. The study employed restricted mean survival time analysis to assess the duration patients remained cancer-free or survived overall. The analysis also explored recurrence patterns by categorizing recurrences as early (less than 2 years) or late (2 years or more) for both treatment groups. The results revealed no significant differences in the timing of tumor recurrence or mortality between patients administered TDF and those receiving ETV.
A 2024 study[69] compared the effects of TDF and ETV on long-term health in patients with HCC, fatty liver, and hepatitis B. The researchers analyzed patient data using a multivariate Cox proportional hazards model and applied a PSM method. They then compared survival outcomes with Kaplan-Meier survival curves. The results showed that TDF helped improve long-term prognosis for patients. The latter finding was confirmed in 2025 in patients with high HBsAg levels after liver resection[70]. A study[71] looked back over 10 years, finishing in 2025. It included 1396 patients with HBV-related cirrhotic HCC who had curative surgery. The patients were divided into two groups: Those who took antiviral medicine and those who did not. The research focused on how often the cancer recurs, considering when the antiviral treatment was started, how well the virus was kept under control, and the levels of HBV. Recurrences were labeled early if they occurred within 2 years and late if they occurred after. The study found that long-term antiviral therapy helped prevent late recurrence after surgery, regardless of whether it was started before or after the operation. Patients who responded well to the virus treatment saw the biggest benefit.
A key goal is to understand how early suppression of HBV-DNA with antiviral drugs impacts long-term survival after liver surgery for patients with HBV-related HCC. A retrospective study[72] examined patients with baseline HBV-DNA levels > 2000 IU/mL. The study examined the number of patients who reached undetectable HBV-DNA at weeks 24 and 48. It also examined the rates of tumor recurrence and overall survival over time. There was little difference in disease-free survival between those who received treatment and those who did not. The analysis identified several factors that increased the risk of HCC recurrence. These included tumor size over 5 cm, blood transfusions, surgical margins less than 1 cm, satellite nodules, portal vein tumor thrombus, and a high Ishak inflammation score. The same factors also predicted worse overall survival after surgery. On the other hand, having undetectable HBV-DNA before 24 weeks was a strong factor in reducing the risk of both recurrence and death.
IMMUNE CELL THERAPY AND VACCINATION IN THE TREATMENT OF HBV
Key elements of the host immune response, particularly the human leukocyte antigen system, are critical in determining clinical outcomes. Human leukocyte antigen typing analyses have linked the major histocompatibility complex class II allele DRB1*1302 to a robust immune response and clearance of HBV, i.e. FH[73-75]. Viral mutations can generate variants with cytotoxic epitopes, which may alter T-cell recognition[76], antagonize T-cell receptors for antiviral cytotoxic T lymphocytes (CTLs)[77], or enable evasion of established immune surveillance[27]. Binding sites within the enhancer core domain are essential for virion enhancer 1 activity. A binding motif at the 3’ end of the enhancer core interacts with the consensus factor EF-C, which is identical to the transcription factor regulatory factor X-1 (RFX-1)[78]. RFX-1 regulates major histocompatibility complex class II gene expression[79]. Alterations in the EF-C/RFX-1 sequence significantly reduce enhancer 1 activity. Garcia et al[80] demonstrated that this site cooperates with the HBV retinoic acid response element. All binding sites in the entire enhancer core domain coordinate, i.e., a single site cannot compensate for the functional loss caused by a mutation in another region of the enhancer core. This coordination linking binding factors to enhancers 1 and 2, is regulated by protein-protein interactions[81], and may need exact DNA conformation[82].
Transgenic mice that express partial or full copies of the HBV genome[83] and even replicate the virus were generated using constructs containing only HBV-derived regulatory sequences[84]. The use of transgenic mice in producing HBV products has been their primary benefit in immunopathogenic research. Except for the large HBs proteins, overexpression of every viral protein was not cytopathic in vivo. In chronic HBV infection, the CD8+ CTLs are the site of necrosis[85]. Liver cells that make IFN-γ trigger chronic inflammation in a transgenic mice model[86]. CTLs produce IFN-γ and TNF-α to eliminate HBV replication non-cytolytically[87]. The majority of the damage observed was due to inflammatory cells recruited by the CTL rather than the CTL itself[86]. These factors trigger 2 virus-killing mechanisms in such mice. One clears HBV nucleocapsids. The other breaks down viral RNA[87,88]. Additional research showed that CTLs attach to HBsAg positive liver cells in transgenic mice and induce apoptosis[89].
Blocking the pathways that cause T-cell exhaustion has shown promise in treating cancer. When these pathways are blocked, T-cells can regain some of their function. In experimental studies, this approach helps recover HBV-specific T-cell activity in patients with chronic hepatitis B. However, data from real patients to know how well it works in living bodies are still lacking. Vaccine therapy aims to boost HBV-specific T-cells that are weakened by chronic viral exposure. Several trials have tested different approaches, but most have failed. Often, the vaccines did not trigger a strong T-cell response. When they did, the response did not yield significant health benefits. It has been demonstrated that new, highly immunogenic vaccines, when combined with antiviral drugs, can be effective. This combination produced better immune responses and improved outcomes in a chronic HBV infection model using woodchucks.
Natural killer cells (NKs) are a special type of lymphocytes known for their ability to kill infected or cancerous cells. They are important in fighting tumors and infections. Scientists have paid more attention to how these cells affect HBV infection. NK’s-induced immunogenic injury can lead to serious problems like cirrhosis or liver cancer. Numerous studies demonstrate that NKs combat HBV infection at early stages and during its long-term progression. They do this by killing infected cells, releasing harmful substances, and signaling via cytokines. However, NKs have two sides. They help control the virus and regulate the immune system, but they can also cause damage to the liver. This dual role makes their impact complex and important to understand. Studies confirm that HBV genotypes/subgenotypes have important influences on the outcome of chronic HBV infection.
Dendritic cells (DCs) are versatile cells that trigger the body’s adaptive immune response. In people with chronic HBV infection, the number of DCs is often low, and these cells may not work properly. This defect might help the virus stay in the body longer. Using a person’s own DCs for immunotherapy is seen as a possible treatment for chronic HBV. We looked at patients with chronic HBV who received a treatment involving HBV-loaded DCs along with ETV. This group compares to those who only received ETV. The main goal was to determine if viral DNA levels decreased in blood. One clinical trial[90] tested the efficacy of HBV-pulsed DCs in combination with ETV for treating patients with chronic HBV. A total of 80 patients were divided into four groups: Those receiving only HBV-pulsed DCs, those receiving both HBV-pulsed DCs and ETV, those receiving only ETV, and a group that received no treatment. The results showed that patients who received joint therapies had stronger antiviral responses than those who received only a single therapy. When combined, HBV-pulsed DCs and ETV caused the biggest drops in serum viral DNA levels and the highest rate of viral response. This combination also led to HBeAg loss and its seroconversion. These findings suggest that using both autologous HBV-pulsed DCs and ETV could be a more effective treatment for people with chronic HBV.
Thymosin α1 is effective in treating chronic HBV infection. In another population of chronic HBV patients, a clinical trial focused on the immune response after using Thymosin α1 alone in 25 patients who tested positive for HBeAg. These patients were split into three groups: One received 1.6 mg of active thymosin α1 (group A), another 1.6 mg of recombinant thymosin α1 (group B), and the last group 3.2 mg of recombinant thymosin α1 (group C). The treatment lasted for 52 weeks. Researchers measured the levels of cytokine-producing T-cells, including T-helper 1 cytokines such as interlukin-2 (IL-2), IFN-γ, and TNF-α, as well as the T-helper 2 cytokine IL-4, using flow cytometry. In all patients given thymosin α1, cytokine levels and the percentage of blood cells producing these cytokines increased significantly compared to their baseline levels and healthy controls. Over time, the number of cytokine-producing cells rose gradually and returned to normal levels. The levels of IFN-γ and IL-4-producing cells even exceeded those observed in healthy individuals. The findings show that thymosin α1 boosts cytokine production, especially IFN-γ. Higher doses of thymosin α1 were more effective against HBV than lower doses or other treatments tested[91].
There have been no previous systematic reviews or meta-analysis on the role of toll-like receptor 9 (TLR9) in HBV treatment. We demonstrated a primary search on a population of patients with active HBV infection. The search yielded 4 clinical trials that compared patients who received a TLR9 agonist as adjuvant therapy with placebo or conventional therapy. The HBsAg/anti-HBs/anti-HBc were the outcome. Those trials are analysed in the following section. In the first trial, oligodeoxynucleotides were examined for their immunogenic effects. Oligodeoxynucleotides with immunostimulatory cytosine-phosphate-guanine (CpG) motifs are potent T helper 1-like immune boosters, shown to improve responses to many antigens in animal studies. CPG 7909 is a small DNA piece with immune-boosting CpG motifs. It triggers human B-cells and plasmacytoid DCs by activating TLR9. Previous studies showed that adding CPG 7909 to a standard HBV vaccine improves the speed, strength, and duration of protective antibody responses for up to 48 weeks. These findings were extended to human studies with the first clinical trial in 2004[92], which assessed the safety, tolerability, and immunogenicity of CPG 7909 when added to a commercial hepatitis B vaccine. In a randomized, double-blind phase I study, healthy volunteers aged 18-35 received Engerix-B (produced by GlaxoSmithKline) via intramuscular injection at weeks 0, 4, and 24. The standard adult dose of 20 μg of recombinant HBsAg, formulated with alum, was administered either alone with saline (control) or combined with CPG 7909 at one of three doses (0.125 mgs, 0.5 mgs, or 1.0 mgs). Those receiving CPG 7909 showed earlier and stronger antibody responses (anti-HBs) compared to controls at all time points up to 24 weeks (P ≤ 0.001). Remarkably, most participants in the CPG 7909 groups reached protective anti-HBs IgG levels within 2 weeks of the first dose. There was also a pattern suggesting higher CTL responses in the groups receiving the two higher doses of CPG 7909. Common side effects included reactions at the injection site, flu-like symptoms, and headaches. These were more common in the CPG 7909 groups than in controls (P < 0.0001), but most were mild or moderate across groups. Overall, CPG 7909 added to Engerix-B was safe and improved the immune response. It may enable a two-dose hepatitis B vaccine that works well. The second trial[93] assessed adults over 40. They often have weaker immune responses to hepatitis B vaccines. An experimental vaccine combining three doses of a TLR9 stimulator, termed 1018 immunostimulatory sequence, with HBsAg showed better results. This vaccine led to faster and longer-lasting protection than the standard Engerix-B vaccine. It was well tolerated and had a safety profile similar to the existing shot. These findings suggest that HBsAg-1018 immunostimulatory sequence may be more effective in older adults with weaker immune responses.
Hemodialysis patients are at higher risk of HBV infection. Many patients with chronic kidney disease (CKD) respond poorly to HBV vaccines. Guidelines recommend administering 4 double doses of a licensed hepatitis B vaccine (2 μg × 20 μg HBsAg) to CKD patients. The third clinical trial[94] was a large, multicenter study involving 521 patients with CKD aged 18 to 75. The study compared three doses of an experimental hepatitis B vaccine, called HBsAg-1018 (20 μg rHBsAg plus 3000 μg 1018, a TLR9 agonist), administered at 0, 4, and 24 weeks. This was tested against 4 double doses of HBsAg-Eng (2 μg × 20 μg rHBsAg plus 500 μg alum) given at 0, 4, 8, and 24 weeks, for a total of eight injections. Patients were followed for a year. In the final analysis with 467 patients, the primary goal at week 28 was to determine how many had protective antibody levels. The HBsAg-1018 group had a seroprotection rate of 89.9%, which was as good as or better than the 81.8% seen in the HBsAg-Eng group. More patients in the HBsAg-1018 group had higher anti-HBs levels-73.6% with anti-HBs above 100 mIU/mL, compared with 63.2% in the other group. The average antibody level was also higher in the HBsAg-1018 group, at 587.1 mIU/mL, compared with 156.5 mIU/mL. Two months after the first shot, the HBsAg-1018 group continued to show higher protection. After a year, their immune response remained stronger. The new vaccine was well-tolerated and had a safety profile comparable to that of the standard vaccine. For CKD patients, three doses of HBsAg-1018 gave better, earlier, and longer-lasting protection than 4 double doses of HBsAg-Eng.
Patients with HIV tend to respond poorly to hepatitis B vaccines. A fourth clinical trial[95] provided data covering five years after vaccination. The latter was a double-blind, controlled trial that was performed to test the safety and effectiveness of the HBV vaccine in HIV-positive adults who were on effective intervention. Participants were tested for susceptibility to HBV, with half having failed previous vaccinations. They received three doses at 0, 1, and 2 months, either with or without 1 mg of CPG 7909 (19 patients in each group). Anti-HBs antibody levels were monitored every six months for up to 5 years. Those given CPG 7909 showed a higher rate of sustained protection, with more maintaining anti-HBs levels of 10 mIU/mL or higher. The average anti-HBs levels were also higher than those of the control group that did not get CPG 7909. The immune-boosting power of CPG 7909 appeared to be a key approach for longer-lasting protection, especially for HIV-infected patients and others who do not respond well to HBV vaccines.
HBV REACTIVATION IN PATIENTS COINFECTED WITH HCV WHILE RECEIVING DAAS
Anti-HBc seropositivity had little effect on the success and safety of HCV treatment. For patients with chronic HCV who comorbid with active HBV infection, there is risk of HBV reactivation. Monitoring HBV DNA levels during treatment is recommended. We searched whether HCV DAAs could trigger reactivation in patients with inactive or resolved HBV infections. A systematic review[96] included a group of chronic HCV patients with different genotypes who were treated with DAAs. These patients had their HBV DNA, HBsAg, and anti-HBc tested before and after therapy. During treatment and follow up, ALT levels were examined regularly. Such studies suggested that HBV reactivation may occur after HCV DAAs are administered. A 2017 study[97] found no evidence of HBV reactivation in patients with resolved infection treated with DAAs for HCV in a large real-world cohort. Another study[98] revealed IFN-based therapy was not a risk factor for HBV reactivation. Most studies were conducted on a large number of populations. No clinical trials exist. In summary, HBV DNA remains in cells outside the liver after recovery from HBV infection. Recovery may come from IFN treatment or occur on its own. Peripheral blood mononuclear cells (PBMCs) store the HBV outside the liver. Studies show that PBMC-associated HBV DNA comes from virus particles that stuck to cell surfaces. It does not stem from PBMC infection or virus growth inside. The role of HBV in extrahepatic cells remains unclear. HBV outside the liver may trigger key events. These include reactivation in long-term infections, setbacks after IFN-α treatment for chronic hepatitis B, and new liver infections in transplant patients with liver failure from severe or ongoing HBV infection.
FUTURE RESEARCH ON THE RELATIONSHIP BETWEEN HBV GENOTYPES AND SUBGENOTYPES WITH DISEASE OUTCOME
Whether new classes of drugs are needed to manage chronic HBV, whether a complete cure is possible, or even necessary, has not been fully addressed. The goal of new therapies for chronic hepatitis B should be to achieve a “virological cure”. Current antiviral treatments can slow down HBV replication and help improve liver damage. However, they rarely fully clear chronic HBV infections. There is an urgent need for new drugs and better strategies to combat the virus. Several promising new medicines are in development, but further research is needed before they can be used in clinics.
Understanding how HBV genotypes and subgenotypes affect chronic HBV infection is crucial. Still, more research is needed to find clear links between specific genotypes and risks like cirrhosis or liver cancer. The best studies follow large groups of people over many years, comparing different genotypes. Some research has been conducted in Asia and Alaska, but many genotypes, including A1 and D, have not been studied in longer-term, prospective research. Data is missing for some genotypes and subgenotypes, such as A3, E, F4, and H, on how they impact health. Collaboration between institutions from different countries enhances the power of these studies.
Comprehensive genetic analysis of virus sequences should also be carried out. Researchers need to examine full genome data from patients with and without liver problems. These studies should identify mutations that increase the risk of HCC and severe damage. Finding such mutations can help pinpoint patients who might benefit from early treatment or closer watch. More research should align the sequences of such mutants in relapsed HBV patients vs patients with overt infection, to compare the genome and proteome profile in their blood. Accurate methods to study HBV reactivation in HCC patients through aligning the sequence of such mutants in relapsed HBV patients vs patients with overt infection are required. In the following section, we briefly propose how to study HBV reactivation in HCC patients. Such approach helps investigate the specific aims of this review. Prospective longitudinal cohorts should enroll patients who meet the clinical case definition of chronic active hepatitis B, i.e. group 1: Patients with laboratory-confirmed viral hepatitis B who have HBsAg positive, HBcAg IgG positive. Group 2: Hepatitis B patients who had resolved infection, for at least 12 months after the end of therapy (DNA < 2000 IU/mL, ALT normal, anti-HBe negative). The latter are relapsed after the targeted therapy and seroconverted to chronically active disease, thus their current status presents HBsAg positive; DNA > 2000; liver enzymes above normal levels. For assessing HCC status, it is necessary to include a control group of patients with chronic HBV infection without HCC to assess viral kinetics and mutations, in addition to assessing host immune status. Diagnosing HCC relies on finding two of these signs: Liver lesions seen with two different imaging methods, such as ultrasound and triple-phase computed tomography scan, along with a serum alpha-fetoprotein level over 200 ng/mL. Patients who test positive for HCV or have anti-HCV antibodies are excluded from this diagnosis. The laboratory investigations should include collecting blood samples from chronic HBV patients belonging to the following categories: Group 1 (positive control): HCC patients presenting with chronic active HBV status. Group 2: HBV-HCC patients who had targeted therapy and seroconverted from chronic inactive to chronic active disease. In addition to alpha-fetoprotein levels in serum, tri-phasic computed tomography, and serum creatinine should be examined. Serology testing by enzyme-linked immunosorbent assay is performed to detect HBc IgG, HBsAg, HBeAg and anti-HBe in plasma. Plasma from patients who tested positive for HBsAg is used to isolate HBV DNA. This is done using reverse transcription-quantitative PCR, focusing on the s and precore/core regions of the virus. After amplification, the PCR products are sequenced and compared to the HBV reference sequence. Mutations in the s and precore/core regions are identified by deletions in aa or changes in nucleotides. Multiple sequence alignment of both overt chronic HBV isolates and the positive control isolates, along with proteomics analysis of data are informative at the genomic and proteomic levels. Statistical analysis of results should consider one way analysis of variance, and the t test. HBV transgenic mice models for studying HBV oncogenic potential often utilize mice expressing the viral x protein, large surface antigens, or pre-s mutants. The murine fibroblast line NIH3T3 is commonly employed as an immortalized cell line for oncogene characterization assays, but yet, no innovative therapeutic strategies have been developed to study HBV infection using them[99]. Cellular transformation has also been studied using the immortalized murine hepatocyte line FMH202[100,101]. Further research is necessary, as the Simian Virus 40 polyomavirus immortalizes FMH202 cells independently of HBV.
RECOMMENDATIONS FOR ANTIVIRAL THERAPY IN PATIENTS WITH VIRAL HEPATITIS AND HCC
Patients with chronic hepatitis B or C who do not have HCC should be checked for antiviral treatment because these medicines can lower the chance of developing HCC. Those with advanced fibrosis or cirrhosis from viral hepatitis face a high risk of HCC. Regular ultrasound scans, performed every 6 months, are essential for early detection. HBV patients with cirrhosis should start antiviral drugs like ETV or TNF, while HCV patients with cirrhosis should be quickly treated with IFN-free options. Antiviral treatment should be offered to patients with HBV who have HCC, especially to help prevent the tumor relapse after treatment aimed at a cure. It can also protect against worsening liver function. For patients with HCV and HCC, antiviral therapy free of IFN should be considered, based on how advanced the tumor is and the patient’s overall lifespan.
ANTIVIRAL DRUG RESISTANCE
Antiviral drug resistance plays a key role in how successful long-term treatment for chronic hepatitis B will be. When patients develop resistance to NAs, their livers’ health can worsen. Using different drugs one after another raises the chance of resistance affecting multiple medications. Starting treatment with a strong antiviral that can challenge resistance usually offers the best chance for long-term control. This approach has led to lower resistance rates in countries where it is affordable. The ability of an antiviral to overcome resistance depends on factors like the genetic barrier, drug potency, patient adherence, drug mechanism of action, viral fitness, and cross-resistance. It is critical to keep researching new methods to target the virus and the immune system. Developing new medicines can help control the virus, prevent resistance, and reduce related health problems over time.
To understand resistance to LAM in chronic hepatitis B, we reviewed published studies in PubMed using keywords such as LAM, resistant HBV, chronic HBV, incidence, and associated factors. Our focus was on patients with resistant HBV. We examined studies of LAM without a specific comparison group to determine how often resistance occurs and which factors influence it. The initial search yielded 60 articles and one meta-analysis from March 2016, with no subsequent studies identified, see Supplementary Table 1. We also searched the Cochrane database using similar terms and identified 14 studies; however, only two were relevant to our question, as aforementioned. The YMDD mutation at codons 203-206 of the RT targets the catalytic site of the HBV polymerase, specifically at rtM204V/I, thereby reducing both the susceptibility and functional capacity of the viral polymerase to LAM, ultimately leading to LAM resistance. This resistance is frequently observed in clinical trials and is associated with increased risks of death, hepatic decompensation, hepatitis flare-ups, and fatal liver failure[102]. In the YMDD motif of the RNA-dependent DNA polymerase, resistant HBV strains display isoleucine (I) or valine (V) substitutions for methionine (M). The M204S substitution in the YMDD motif has also been documented[103]. These mutations are often accompanied by a substitution at position 180, where leucine (L) is replaced by M. Consequently, HBV DNA accumulates, and relapse occurs. Persistent viremia independently elevates the risk of HCC recurrence following surgery. In such cases, close monitoring of HBV DNA levels to detect antiviral resistance, together with the use of potent oral agents, is recommended.
CONCLUSION
Accurate methods through aligning the sequence of HBV mutants in relapsed HBV patients vs patients with overt infection are required. More research is needed to find clear links between specific HBV genotypes and risks like cirrhosis or HCC. Comparing the genome/proteome of blood from overt and relapsed HCC-related chronic HBV patients helps identifying persistent genetic/epigenetic profiles of HBV resistant variants, thus accurately selecting the appropriate antiviral therapy and eliminating the risk of viral reactivation.
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Specialty type: Biochemistry and molecular biology
Country of origin: Egypt
Peer-review report’s classification
Scientific quality: Grade B, Grade B, Grade B, Grade E
Novelty: Grade B, Grade B, Grade B, Grade B
Creativity or innovation: Grade A, Grade B, Grade B, Grade C
Scientific significance: Grade B, Grade B, Grade B, Grade C
P-Reviewer: Liu HY, China; Wen DG, PhD, Academic Fellow, Professor, China; You R, Associate Chief Physician, China S-Editor: Hu XY L-Editor: A P-Editor: Zhao YQ
