Further research into biomarkers for HBV complications may help to predict reactivation of CHB after the endpoint of drug treatment
Hepatitis B is one of the leading causes of death in China, though it is found all over the world. Around 2 billion people worldwide have been infected by HBV (1). Because of the severe complications caused by HBV infection, it is the 10th leading cause of death in the world (2). Sub-Saharan Africa, the East Asia and North America are the endemic regions with high occurrence of HBV infections (3). Hepatitis B disease is caused by the Hepatitis B Virus (HBV), an enveloped DNA virus (4). HBV horizontal transmission is primarily through the exchange of body fluids such as saliva, semen, and blood (3). HBV primarily infects hepatocytes and HBV infection can result in either acute or chronic infection. Furthermore, asymptomatic patients of HBV can be virus carriers that allow transmission of HBV. Many HBV infections occurred via transfusion of donated blood that contains HBV. Vertical transmission can occur through the placenta blood or intake of breast milk. Before the vaccination of infants was implemented extensively around the world, many children were infected with HBV during infancy, and 90% of HBV infected infants became chronically infected with HBV (1). HBV chronic infection can cause severe complications including cirrhosis, hepatocellular carcinoma (HCC) (5). According to Dr. Richard Hunt in the University of South Carolina School of Medicine, 80% of HCC worldwide are caused by HBV infections (3).

Acute Hepatitis B versus Chronic Hepatitis B:
The infection of HBV can be classified into two types: Acute Hepatitis B (AHB), and Chronic Hepatitis B (CHB). Acute Hepatitis B (AHB) is primarily a disease of adulthood that transmitted frequently by sexual exposure or intravenous drug use. However, with the introduction of the HBV vaccine during the 1980s, reported cases of AHB have dropped from 43.4% to 28.5% in Italy and Egypt from 1991 to 2002 (5). Among all the AHB patients, two-thirds of them are asymptomatic, causing cases of HBV transmission through contact of bodily fluids. However, in endemic HBV regions around Asian-Pacific continent, perinatal transmission remains to be the top leading cause of HBV infections. The elevation of serum alanine aminotransferase (ALT) and bilirubin are typical biochemical features of AHB (6). Patients often undergo symptoms such as fever, anorexia, nausea, and jaundice. These symptoms disappear after several months. However, fatigue in patients remains for a much longer time, indicating the immunosuppressed status. During this period, patients are susceptible to the presence of pathogens.

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Worldwide, more than 350 million people have chronic Hepatitis B infections. In contrast to AHB, CHB patients can be divided into four phases with distinct symptoms, which are immune tolerance phase, immune clearance phase, immune control phase and immune escape phase. The immune clearance phase is also called the HBeAg-positive phase, showing that the e-antigen can be detected in the blood. This phase is highly relevant to cirrhosis, HCC and other complications of CHB. Similarly, the fourth phase — immune escape phase — also has the risk of complications. Therefore, wide ranges of treatments are used to reduce the risk of complications. Nevertheless, drug therapy is recommended to stop once the patients have achieved HBsAg seroclearance. After the treatment, HBV is not completely eliminated, but rather the viral replication is being suppressed to low levels by the drug, so the reactivation of HBV is still possible among CHB patients (6).

Serologic markers of HBV infection:
Through HBV serum test, several markers can show the presence and phase of infection. HBsAg, the surface antigen of HBV, is one diagnostic marker to test whether the patients are infectious. HBV DNA level is used to measure the viral replication and the disease progression. This is an important indicator to select patients for antiviral treatment and evaluate the risk of developing severe symptoms. The HBeAg level is related to both the HBV DNA level and the infectivity of patients. Anti-HBc demonstrates the persistence of cccDNA, and anti-HBs is an indicator to test the response of vaccine and monitor the treatment after liver transplantation (6).

Starting in 1988, the vaccination of HBV for newborns was implemented worldwide regardless of the HBV history of mothers. The vaccine has efficacy of 80% to 100% and duration of 20 years or more (1). The most common vaccine used is the recombinant HBsAg protein vaccine. The HBsAg gene of a CHB patient was extracted, cloned into the yeast DNA and grown in the yeast cells. The HBsAg is then collected, purified and added with adjuvants The injection of HBV vaccine usually follow the 016 rule. The second injection is followed one month after the first injection, and the third injection started six months after the second injection to booster the immune response (1).

The treatment of HBV includes pegylated interferon (PEG-IFN) and nucleotide analogues. The IFN is a treatment with limited duration because of the occurrence of side effects and high cost of treatment. By enhancing adaptive immune response and inhibit the replication process of HBV in hepatocytes, this drug can sustain the suppression of virus for relatively long periods (7). However, the drug may cause severe side effects, so the use of this treatment cannot be applied to every CHB patient.

Entecavir is one of the most common treatment for CHB infection. This treatment has few side effects and causes less drug resistance of virus. It works by inhibiting the DNA polymerase and thus suppressing the DNA replication (7). The result of treatment includes the decrease in HBV DNA level and ALT level (7). Moreover, from Tang et al., the entecavir treatment can also reduce the incidence of complications such as HCC and complications after 20 months of monitoring.
The newest tenofovir treatment still targets to suppress the viral DNA replication. From the monitoring of treatment efficacy, this treatment can also reduce the effect of complications caused by CHB infection, and no drug resistance records are reported after 6 years of treatment (7).

Current conditions:
Since humans are the only reservoir of HBV, the eradication of HBV is possible. However, due to the persistence of HBsAg and cccDNA, eliminating HBV from a CHB patient is difficult. Therefore, the suppression of virus amount and virus replication in patients is crucial for the control of HBV infections among the population (2).

Another big challenge of HBV eradication is the pantropic infection capacity in the body. Although the main target of HBV is the hepatocytes, it also infects the extrahepatic tissues. Therefore, the complete elimination of HBV is extremely difficult in CHB patients. In addition, because of the persistence of virus and the pantropic infection around organs, reactivation of HBV infection is common among cured patients. Patients who have had liver transplantation or discontinuation of drug treatment still have chances for the reactivation of infections (2).
The last difficulty is the economic cost and incomplete vaccination of infants. In order to promote the development of technology and treatment to better diagnose and treat the HBV infections, the expenditure remains to be high. Thus, some developing countries cannot afford the huge cost required for HBV breakthrough (2). Additionally, although the HBV vaccine is 80% to 100% efficacious and has already used worldwide among humans (1), the vaccination of HBV infection does not completely. In rural areas where the hospitals are not authenticated, not every newborn can receive the vaccines. Also, because the HBV vaccine requires injecting for three times, a small proportion of infants may not attend the complete vaccination series, so the memory cells for HBV antibodies may not reach the amount for long-term and effective protection.

For the reasons above, HBV reactivation is still prevalent. Due to the drug resistance and high cost of antiviral drugs, the therapy is recommended to stop once the endpoint has reached. Ideally, the endpoint of HBV treatment is the seroclearance of HBsAg, the surface antigen of Hepatitis B that can be found in serum in 30 to 60 days after HBV exposure, and studies have shown that it is correlated with the amount of cccDNA in the liver (8). However, the relapse rate of HBV infection remains to be as high as 25% to 50% among patients who have discontinued the treatments. In He et al., the researchers investigated the outcomes for the discontinuation of treatment and the appropriate endpoint of treatment. The result showed that HBeAg seroconversion can result in a more durable suppression of HBV. Although the risk of relapse of infection still exits, the relapse rate in the sample has decreased by 10% to 15% (9). In addition, 48 weeks is a marker for long-term suppression of HBV since all the relapses in this case happened in 48 weeks after discontinuation of Nucleotide Analogue treatment (9).
Notably, other factors can also influence HBV activation after stopping the treatment, including age and gender. One study showed that patients with age >50 are recommended to continue their treatment irrespective of their HBsAg levels because they have very high recurrence rates for 84.6% (10). Some studies suggested that patients with age <36 years old have more sustained responses to the drug treatment, resulting in lower rate of recurrences. Also, male HBV infection patients have a higher rate of relapse comparing to female patients (8).
Current Research:
Due to the poor prognosis of HBV infection reactivation, the prognosis of HBV recurrence can provide patients and physicians with prepared treatment to prevent the occurrence or exacerbation of reactivation, and hopefully adjust the treatment for each patient according to the probability of relapse. The prediction includes the recurrence of infections and the possible complications accompanying with reactivations. Therefore, in order to predict the recurrence of HBV infections in patients who have ceased the treatments, several ways and also combined methods are used to obtain an accurate prediction of relapse and complication occurrence in individual CHB patients. The prognosis methods include HBsAg level, HBV DNA level, prevalence of deletions in Pre-S region, and combined measuring method. However, so far, none of these methods can fully and accurately predict the long-term clinical outcome of HBV drug treatments.
The quantification of HBsAg level after HBeAg seroconversion is one method that can be a predictive marker for the recurrence of HBV infections. The study recruited 112 patients with CHB who have stopped the entecavir treatment following the guideline published by Asian Pacific Association (10). According to the study, the HBsAg level shows the amount of cccDNA remained in the liver tissue, which can be used to predict the relapse of HBV infections. The researchers observed the relapse condition for 52 weeks (10). The data indicated that a decrease in HBsAg level at the first 12 weeks after discontinuation of entecavir treatment can result in a low relapse rate with 17% (10). On the contrary, the patients who have increases in their HBsAg level by 1.0 log10 IU/ml after stopping the treatment for 12 weeks have a relapse rate of 81.3%, higher than the patients with slowly increased or decreased HBsAg levels (10). Liang et al. showed that patients with HBsAg level lower than 2.0 log10 IU/ml had only 9% rate of reactivation, highlighting the importance of HBsAg level at the end of the treatment (11). The use of HBsAg level is plausible for predicting the relapse rate of patients with different age groups. However, the accuracy of this method needs to be further investigated. Additionally, HBsAg level is not the only factor that affects the relapse rate. Due to the mixing of different factors in the HBV recurrence prognosis, a more complete and detailed test should be done to estimate the chance of reactivation in HBV infections.

A combined measurement composing of HBsAg level and HBV DNA level was examined to predict the risk of disease progression in HBeAg-negative patients. The study aimed to find the inactive carriers with low risk of disease CHB progression after drug treatments. 1529 patients with CHB were measured with HBsAg level, HBeAg level, HBV DNA level and ALT level (12). An 18-month follow-up was conducted to distinguish the inactive carriers and the feasibility of using single-point measurement for prediction. Other studies have found that inactive carriers have better prognosis and low risk of relapse and complication occurrence (13). Thus, identifying inactive carriers can in turn predict the clinical outcome and future disease progression. In this study, researchers used single-point measurement to test for the accuracy of HBsAg level and HBV DNA level as markers for inactive carriers and active CHB classification. The result indicated that the baseline of HBsAg level for <1000 IU/mL and HBV DNA level for <2000 IU/mL had 71% sensitivity and 85% specificity can be used to classify inactive carriers and predict clinical outcomes (12). Although the accuracy of inactive carrier identification can be further improved, it provides insights for the possibility of using combined measurement as markers for disease progression prediction of CHB patients.

In another study, the researchers used the Pre-S2 mutant protein as a biomarker to predict the occurrence of tumor formation caused by HBV infection. Since HCC is one of the most severe complications of CHB, the prediction of HCC is indispensable when predicting reactivation of HBV infections. Qiu et al. has found that the deletion sites at Pre-S2 region matched were corresponding with the epitopes of CTLs and B-cells epitopes for neutralizing responses. Therefore, the presence of this deletion can indicate the immune evasion of HBV, which further predicts the disease progression. Pre-S region also worked as a biomarker for the presence of hepatocellular carcinoma (13). One study investigated the correlation between prevalence of Pre-S deletions and development of hepatocellular carcinoma. The HBV DNA is extracted from 115 patients with CHB, and then amplified using PCR. The PCR products were sequenced and analyzed to detect the deletions in Pre-S region. The result showed that the prevalence of Pre-S region deletions is much higher in HCC patients compared to non-HCC patients, with an average percentage prevalence 29.2% in HCC patients and 14.6% in non-HCC patients (14). Furthermore, in the study, most patients identified with HCC were males with HBV genotype C and deletions in Pre-S2 region (14). According to another research, the epitopes in Pre-S2 region are highly related to the binding of T cells and B cells, showing the significance of the deletion of this region on host immune response (15).
Reactivation of HBV infection is an intractable problem for CHB patients. After the HBsAg or HBeAg seroclearance has achieved, no more drug treatments are suggested to continue for the sake of drug resistance prevention. However, due to the persistence of HBV, relapse of HBV infection and its complications still occur. A regular check for HBV is required to monitor the disease progression after the drug treatment. However, patients who do not follow the regular check may have exacerbation in HBV reactivation and cause even severe complications. Therefore, in order to solve this issue, a detailed and precise prognosis of disease progression and prediction of HBV reactivation need to be developed to predict the risk of disease relapse using a single-point measurement right after the endpoint of drug treatment. The development of this prognosis technique allows the pre-treatment for HBV reactivation and customization of follow-up monitoring, preventing the recurrence of severe infections and complications.

In this proposal, I suggest that the sequencing of Pre-S region and a detailed HBV reactivation assessment should be done in patients to determine the long-term clinical outcome and risk of HBV reactivation after the discontinuation of drug treatments. Previous research found that the Pre-S region sequence is potentially related to the development of HCC. Therefore, Pre-S region sequences may help predict serious HBV relapse symptoms. The deletions in Pre-S region are indicators for immune evasion, showing the possibility of viral mutation and disease progression. Thus, due to the significance of Pre-S region deletion on HCC and liver cirrhosis development, further research should be done to investigate its relationship with HBV related liver decompensation as another HBV complication. If Pre-S region genetic sequence has correlates with disease markers such as HBsAg level or level of HBV DNA level, then the Pre-S region could be used to predict disease outcome. At the same time, an evaluation test of pre-existing factors including age, gender, HBV genotype should be made as a HBV general reactivation risk assessment.

Previous research showed that deletions in Pre-S region are correlated with the development of HBV induced HCC and liver cirrhosis. Therefore, a similar experiment can be done to investigate the potential correlation between Pre-S region deletions and occurrence of liver decompensation. CHB patients can be recruited and tested for the regular HBV serum test. Then, the HBV DNA should be extracted and amplified with PCR. The precise baselines of disease markers that correlate with deletions in Pre-S region need to be determined through experiments with a large number of samples by examining the boundary of disease markers that separate patients with high relapse risk from low risk ones. In this way, the baseline of every disease marker used to evaluate the potential risk of complications development after endpoint of drug point can be determined.

Thus, by integrating the general reactivation risk assessment, the existing combined measurement of HBsAg level and DNA, and the disease markers that related to the Pre-S region deletion, the risk of HBV reactivation and HBV complication development can be tested by using single-point measurement right after the endpoint of drug treatment. This test has an overall evaluation of potential risk of disease recurrence, enabling a customized checking schedule after drug treatment for individual patients. Patients with high risk of relapse need to be constantly monitored and probably instructed to prevent or reduce the relapse symptoms, and patients with a lower risk can have less frequent check for HBV serum test.
The sequencing of Pre-S region is a back-up measurement to help the prediction. Therefore, even though the sequences in Pre-S region do not have correlations with other disease markers, the sequencing of Pre-S region can still be applied to patients who are predicted to have high risk of relapse by the general risk assessment and combined measurement of HBV reactivation.

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