Novel biomarkers for HBV including point-of-care

Despite the presence of a highly effective vaccine and potent, well-tolerated antiviral therapy, HBV infection remains an enormous global public health problem. The complicated natural history of HBV has been defined by a number of biomarkers, including serological tests, HBV DNA levels, liver biochemistry, and histology. While useful, the tests also notoriously cause great confusion among providers, particularly those with limited experience in managing HBV. In recent years, new assays have been developed with the goal of providing additional insight into what is happening inside infected hepatocytes. Hopefully, improved understanding and optimal use of new and traditional HBV biomarkers will enable simplified diagnosis and improved clinical management to reduce the global burden of HBV infection. TRADITIONAL HBV BIOMARKERS The hallmark of HBV infection is the presence of HBsAg in the blood. When HBsAg persists beyond 6 months after exposure, infection meets the definition of chronicity. Most chronic infections result from exposure during the neonatal period from mother-to-child or early horizontal transmission. Classically, the first stage of infection is deemed the immunotolerant phase, during which HBeAg is present in the blood along with very high levels (>8 log IU/mL) of HBV DNA, but normal Alanine aminotransferase (ALT) and no liver injury on liver biopsy. People may remain in the immunotolerant phase for many years, but eventually, often in the second or third decade of life, transition to the immune active phase occurs with a decline of HBV DNA associated with increases in ALT levels. During the immune active phase, repeated ALT flares or persistent ALT elevation may lead to progressive liver injury with the potential to cause fibrosis and, ultimately, cirrhosis. Eventually, HBeAg is cleared from the blood, often followed by the development of anti-HBe antibodies and a marked drop in HBV DNA levels to low or undetectable levels. After HBeAg-clearance, HBeAg-negative chronic hepatitis may develop with rises in HBV DNA, leading to ALT flares with the risk of further liver injury. Alternatively, patients may remain in an inactive phase with low or undetectable levels of HBV DNA and normal liver tests. Long-term inactive disease may eventually result in HBsAg loss, which marks the end of active HBV infection and is often referred to as a functional cure to reflect the excellent natural history of this stage of infection but also to highlight that traces of replication-competent HBV DNA remain in the liver, which canlead to HBV reactivation in the setting of potent immunosuppression.1Table 1 highlights the significance of the traditional and novel biomarkers, while Figure 1 illustrates these markers during the natural history of chronic infection. TABLE 1 - Traditional and novel biomarkers in HBV infection Meaning Point of care available Clinical utility Limitations Traditional biomarkers HBsAg Current HBV infection Yes Current infection Sensitivity (?) Anti-HBs Immunity to HBV Yes Assess immunity None HBeAg High level HBV replication Yes (limited) Phase of infection None Anti-HBe Usually follows clearance of HBeAg No Phase of infection None Anti-HBc (IgG/IgM) Exposure to HBV (IgM—acute/flare, IgG—anytime) No Identify acute/past infection False positive HBV DNA Measure of viral replication Yes (GenXpert platform) Phase of infectionTreatment indicationsTreatment response CostAvailability Novel biomarkers Quantitative HBsAg Amount of HBsAg No (unlikely) Phase of infectionResponse stopping NA therapy Source of HBsAg—cccDNA vs iDNA Hepatitis B core-related antigen (HBcrAg) Measure of cccDNA transcriptional activity No (unlikely) Phase of infectionRisk of HBV reactivationMOA novel agents Mostly HBeAg in HBeAg+Sensitivity in HBeAg- Hepatitis B core antigen (HBcAg) Measure of cccDNA transcriptional activity (P-HBcAg)Measure of HBV DNA (non-P-HBcAg) No (may be possible) Phase of infectionSerological test for HBV DNAMOA novel agents Low sensitivityLimited data HBV RNA Measure of cccDNA transcriptional activity (P-HBcAg) No (possible) Phase of infectionRisk of HBV reactivationMOA novel agents Limited sensitivityUncertain biological significance Abbreviations: cccDNA, covalently closed circular DNA; HBcrAg, Hepatitis B core-related antigen; iDNA, integrated DNA; MOA, mechanism of action; NA, nucleos(t)ide analog; P-HBcAg, phosphorylated HBcAg. FIGURE 1: Phases of chronic HBV infection with traditional and novel biomarkers. Abbreviations: ALT, alanine aminotransferase; HBcrAg, hepatitis B core-related antigen; qHBsAg, quantitative measurement of HBsAg.NOVEL HBV BIOMARKERS An important barrier to the true cure of HBV infection is the presence of the long-lasting template of HBV replication, covalently closed circular or covalently closed circular DNA (cccDNA). cccDNA is very stable episomal DNA that resides in the nucleus of infected hepatocytes and persists for the life of the cell, possibly even surviving cell division.2 The goal of HBV treatment is to clear or at least transcriptionally silence all cccDNA in the infected liver. Traditional biomarkers provide very limited information about the presence, quantity, and/or transcriptional activity of cccDNA. Novel biomarkers attempt to fill this gap (Table 2). TABLE 2 - Novel HBV biomarkers qHBsAg HBcrAg HBcAg HBV RNA Identifying inactive HBV <1000 IU/mL useful Negative may be useful (conflicting data) Unknown Negative may be useful (conflicting data) Treatment response IFNNA Stopping RulesRarely helpful Limited utilityNot useful UnknownUnknown Marginally usefulNot useful Stopping NA therapy Asian <100 IU/mLCaucasian <1000 IU/mL Helpful if qHBsAg 10–100 IU/mL (small niche) Unknown Negative may be helpful (not critical) New agents Challenging—source (cccDNA vs. iDNA) unclear Potentially useful Potentially useful Potentially useful Pros Standard serology (low cost) Reflects cccDNA activitySerological assay (low cost) Reflects cccDNA activity (P-HBcAg)Reflects HBV DNA (non-P0-HbcAg) Reflects cccDNA activity Cons Does not distinguish cccDNA vs iDNARequires central lab SensitivityRequires central lab Sensitivity SensitivityStandardizationNucleic acid testing—cost and stability? Abbreviations: cccDNA, covalently closed circular DNA; iDNA, integrated DNA; IFN, interferon; NA, nucleos(t)ide analog; P-HBcAg, phosphorylated HBcAg; qHBsAg, quantitative measurement of HBsAg. QUANTITATIVE HBsAg AND POINT-OF-CARE QUALITATIVE HBsAg The first advance was the development of a quantitative measurement of HBsAg (qHBsAg). qHBsAg levels have been shown to be associated with the different phases of chronic HBV infection, as well as with both spontaneous and treatment-induced HBsAg loss.3 The major limitation of qHBsAg is that the level of HBsAg does not reflect the source of HBsAg. In addition to cccDNA, viral proteins may be derived from HBV DNA that has been integrated into the host genome through the incorporation of double-stranded linear that forms due to an error in strand-switching during the complicated replication cycle.4 Integration events are random in size and location. Only the HBsAg (and HBV X protein) genes are likely to integrate in the correct orientation with their native primers.4 The consequence is that circulating HBsAg in the blood may come from cccDNA and/or integrated DNA (iDNA). Early in infection, with high replication levels, most HBsAg likely comes from cccDNA, whereas in older people with HBeAg-negative HBV, HBsAg often comes predominantly from iDNA.5 Unfortunately, current HBsAg assays—both qualitative and quantitative—do not distinguish the source of HBsAg. This has very important implications for evaluating new therapies. Although it would be ideal to eliminate all traces of HBV in the liver, including iDNA, most therapeutic strategies aim to eliminate or silence all cccDNA. However, even if this ambitious goal were achieved, a person could remain HBsAg positive due to HBsAg derived entirely from iDNA and thus would not be considered to have achieved a functional cure of infection—effectively a false negative. Multiple groups are working on assays to distinguish the source of qHBsAg; however, to date, none is available. qHBsAg has still proven useful as a clinical tool. Not surprisingly, lower qHBsAg levels are associated with future HBsAg loss. Perhaps the greatest utility of qHBsAg to date has been the stratification of patients interested in stopping long-term nucleos(t)ide analog therapy as a therapeutic strategy. HBsAg loss after stopping is strongly associated with qHBsAg level and ethnicity. For Caucasian patients, qHBsAg levels below 1000 IU/mL are associated with HBsAg loss rates of ~40% over 4 years compared to 5% above this level. In contrast, Asian patients with qHBsAg levels >100 IU/mL have only a 2% chance of HBsAg loss, which increases to 33% if qHBsAg is below 100 IU/mL.6 qHBsAg is also useful to assess novel therapeutic strategies and to stratify patients likely to respond to specific therapies. qHBsAg is likely underutilized in clinical practice, in large part due to limited availability in the United States. Furthermore, point-of-care (POC) tests for HBsAg, which are widely available in many HBV-endemic regions, are not available in North America. African studies have shown that rapid diagnosis improves linkage to care and ultimately treatment uptake. There is interest in simplifying treatment guidelines and expanding treatment eligibility, including the notion of treating all HBsAg-positive individuals. Particularly in regions of the world where access to HBV DNA is limited, decisions could be made on HBsAg +/− HBeAg and ALT results alone. POC tests generally have very high specificity but somewhat reduced sensitivity at the low end of the qHBsAg range. Since many/most patients with very low levels of qHBsAg will have inactive HBV, the assays, even in their current form, may be adequate to identify those likely to benefit from treatment.7 Increased availability of both qHBsAg and POC HBsAg (qualitative) could have a significant impact on improving the overall cascade of care and prioritizing optimal patients for newer curative therapies. HEPATITIS B CORE-RELATED ANTIGEN AND HBCAG Because of the limitations of HBsAg, other biomarkers for predicting natural history and treatment response have been sought. Hepatitis B core-related antigen (HBcrAg) was developed using monoclonal antibodies to a 149 amino acid sequence, which is found in the HBV core protein but is also present in HBeAg and the truncated p22 component of the core protein.8 Because all 3 products recognized by HBcrAg are derived exclusively from cccDNA (not from iDNA), the presence of HBcrAg in the blood is thought to represent transcriptionally active cccDNA in the liver. Indeed, liver biopsy studies have shown that HBcrAg levels correlate with the ratio of HBV pregenomic RNA to cccDNA, a measure of cccDNA transcription.9 The clinical utility of HBcrAg has been explored in different settings. Like qHBsAg, HBcrAg levels are higher in the immunotolerant and HBeAg-positive immune active phases of chronic HBV, and it may be useful to help distinguish inactive carriers from those likely to develop HBeAg-negative chronic HBV.10 HBcrAg has also been evaluated prior to stopping nucleos(t)ide analog therapy. Detectable HBcrAg in patients with qHBsAg levels between 10 and 100 IU/mL are predictive of worse outcome than those with undetectable HBcrAg; however, this is a relatively small group of patients.11 Detectable HBcrAg is also associated with a higher risk of HBV reactivation with immunosuppression.12 The major limitations of HBcrAg are that in HBeAg-positive disease, HBcrAg levels largely reflect HBeAg levels, and in HBeAg-negative disease, the sensitivity of HBcrAg is limited, with many patients having undetectable levels.8 The iTACT-HBcrAg is a more sensitive version of the assay with somewhat improved clinical utility. To avoid the overlap with HBeAg, a novel HBcAg assay is specific for the core protein. The HBcAg assay detects phosphorylated HBcAg that is present in empty viral particles and unphosphorylated HBcAg that is found in association with HBV DNA inside virions.13 In preliminary studies, phosphorylated HBcAg correlates very well with HBcrAg and HBV RNA levels, while nonphosphorylated HBcAg correlates strongly with HBV DNA levels. Like with HBcrAg, sensitivity is still an issue but can likely be improved. The main potential of this assay may be its utility to effectively detect HBV DNA using a serological (and thus potentially inexpensive) assay. This could have a major impact in resource-limited settings where HBV DNA availability is a major impediment to HBV care. Unfortunately, it will likely be difficult to make HBcAg (or HBcrAg) into POC assays due to the need to dissociate the antigens from tightly bound circulating antibodies. HBV RNA An alternative approach to assessing cccDNA transcriptional activity is measurement of HBV RNA in the blood. HBV is a DNA virus but uses an RNA intermediate in its lifecycle (DNA to RNA back to DNA). For reasons that are not well understood, many viral capsids contain HBV RNA with no HBV DNA.14 Most circulating HBV RNA is the pregenomic RNA, which only comes from cccDNA and is the template for HBV DNA production.14 As with HBcrAg, HBV RNA has been shown to correlate well with pregenomic RNA:cccDNA levels on liver biopsy indicating that it reflects transcriptionally active cccDNA.15 However, as with HBcrAg, the clinical benefit over qHBsAg remains somewhat uncertain because many/most HBeAg-negative patients have undetectable HBV RNA levels. Standardization of assays between labs will be important to ensure all are measuring the same thing. HBV RNA has been useful in assessing new curative approaches to HBV, helping to clarify the mechanism(s) of action and potentially to predict response. Although HBV RNA could be developed as a POC assay similar to HCV RNA or HBV DNA, this is unlikely to happen until the clinical utility is more clearly defined. SUMMARY Biomarkers play an essential role in understanding the biology and guiding clinical management of chronic HBV infection. Guidelines have been slow to incorporate new biomarkers into clinical practice, limiting their uptake to date. qHBsAg levels are very useful for predicting response to stopping nucleos(t)ide analog therapy but are confounded by the challenge of distinguishing HBsAg from cccDNA or iDNA. HBcrAg and HBV RNA reflect cccDNA transcriptional activity that may prove very useful to select and optimize new HBV curative regimens; however, clinical utility is limited currently due to the low sensitivity of the assays. HBcAg may hold promise as a serological test for HBV DNA. POC tests for HBsAg have been underutilized, but as guidelines move closer to a treat-all strategy, rapid diagnosis may be a key to improving the global cascade of care.

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