# Circulating HBV RNA and Hepatitis B Core-Related Antigen Trajectories in Persons With HIV/HBV Coinfection and Hepatitis B Surface Antigen Loss During Tenofovir Therapy

Lorin Begré, Anders Boyd, Marie-Laure Plissonnier, Barbara Testoni, Luisa Salazar-Vizcaya, Franziska Suter-Riniker, Caroline Scholtès, Charles Béguelin,

Jürgen K Rockstroh, Huldrych F Günthard, et al.

► To cite this version:

Lorin Begré, Anders Boyd, Marie-Laure Plissonnier, Barbara Testoni, Luisa Salazar-Vizcaya, et al. Circulating HBV RNA and Hepatitis B Core-Related Antigen Trajectories in Persons With HIV/HBV Coinfection and Hepatitis B Surface Antigen Loss During Tenofovir Therapy. Journal of Infectious Diseases, 2024, 10.1093/infdis/jiae189. hal-05021442

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# Circulating HBV RNA and Hepatitis B Core-Related Antigen Trajectories in Persons With HIV/HBV Coinfection and Hepatitis B Surface Antigen Loss During Tenofovir Therapy

Lorin Begré,<fnref n="1" />,²,⁰ Anders Boyd,³,⁴,⁵,⁶,⁰ Marie-Laure Plissonnier,⁷,⁸,⁰ Barbara Testoni,⁷,⁸,⁰ Luisa Salazar-Vizcaya,<fnref n="1" />,⁰ Franziska Suter-Riniker,⁹,⁰

Caroline Scholtès,⁷,<fnref n="10" />,⁰ Charles Béguelin,<fnref n="1" />,⁰ Jürgen K. Rockstroh,¹¹,⁰ Huldrych F. Günthard,¹²,¹³,⁰ Alexandra Calmy,¹⁴,⁰ Matthias Cavassini,¹⁵,⁰ Hans H. Hirsch,¹⁶,⁰

Patrick Schmid,¹⁷,⁰ Enos Bernasconi,<fnref n="18" />,⁰ Massimo Levrero,⁷,⁸,¹⁹,²⁰,⁰ Gilles Wandeler,<fnref n="1" />,⁴,⁰ Fabien Zoulim,⁷,⁸,¹⁹,²⁰,⁴,⁰ and Andri Rauch<fnref n="1" />,⁴,⁰,⁰,⁰ on behalf of the Swiss HIV Cohort Study

**Background.** We evaluated long-term trajectories of circulating hepatitis B virus (HBV) RNA and hepatitis B core-related antigen (HBcrAg) in persons with and without hepatitis B surface antigen (HBsAg) loss during tenofovir therapy in the Swiss HIV Cohort Study.

**Methods.** We included 29 persons with HIV with HBsAg loss and 29 matched persons with HIV without HBsAg loss. We compared HBV RNA and HBcrAg decline and assessed the cumulative proportions with undetectable HBV RNA and HBcrAg levels during tenofovir therapy using Kaplan-Meier estimates.

**Results.** HBsAg loss occurred after a median of 4 years (IQR, 1–8). All participants with HBsAg loss achieved suppressed HBV DNA and undetectable HBV RNA preceding undetectable quantitative HBsAg levels, whereas 79% achieved negative HBcrAg. In comparison, 79% of participants without HBsAg loss achieved undetectable HBV-RNA and 48% negative HBcrAg. After 2 years of tenofovir therapy, an HBV RNA decline ≥1 log₁₀ copies/mL had 100% sensitivity and 36.4% specificity for HBsAg loss, whereas an HBcrAg decline ≥1 log₁₀ U/mL had 91.0% sensitivity and 64.5% specificity.

**Conclusions.** HBV RNA suppression preceded undetectable quantitative HBsAg levels and had high sensitivity but low specificity for HBsAg loss during tenofovir therapy in persons with HIV. HBcrAg remained detectable in approximately 20% of persons with HBsAg loss and 50% of persons without HBsAg loss.

**Keywords.** HBV RNA; hepatitis B core-related antigen; hepatitis B virus; HIV; kinetics.

Received 02 November 2023; editorial decision 9 April 2024; accepted 25 April 2024; published online 16 April 2024

Presented in part: 30th Conference on Retroviruses and Opportunistic Infections, Seattle, Washington, 19–22 February 2023; Global Hepatitis Summit 2023, Paris, France, 25–28 April 2023; and EASL Congress 2023, Vienna, Austria, 21–24 June 2023.

Correspondence: Lorin Begré, MD, Department of Infectious Diseases, Inselspital, Bern University Hospital, Freiburgstrasse 20, 3010 Bern, Switzerland (lorinaaron.begre@insel.ch); Andri Rauch, MD, Department of Infectious Diseases, Inselspital, Bern University Hospital, Freiburgstrasse 20, 3010 Bern, Switzerland (andri.rauch@insel.ch).

**The Journal of Infectious Diseases®** 2024;230:e954–63

© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
https://doi.org/10.1093/infdis/jiae189

With approximately 300 million people affected, hepatitis B virus (HBV) infection is a major global health problem and a frequent cause of liver cirrhosis, hepatocellular carcinoma, and death [1]. Persons with HIV and HBV are at even increased risk for liver-related complications and death [2]. Current guidelines recommend lifelong treatment with tenofovir disoproxil fumarate or tenofovir alafenamide as part of the antiretroviral therapy (ART) regimen [3, 4]. Hepatitis B surface antigen (HBsAg) loss substantially reduces the frequency of complications but occurs infrequently [5, 6]. However, some studies observed higher rates of HBsAg loss among persons with HIV as compared with persons without HIV [7–9]. In the Swiss HIV Cohort Study (SHCS; www.shcs.ch), HBsAg loss occurred in 16% of 262 persons with HIV/HBV after starting tenofovir-containing ART [8].

<footnote n="1">¹Department of Infectious Diseases, Inselspital, Bern University Hospital, University of Bern, Switzerland; ²Graduate School for Health Sciences, University of Bern, Switzerland; ³Department of Infectious Diseases, Research and Prevention, Public Health Service of Amsterdam, The Netherlands; ⁴Stichting hiv monitoring Amsterdam, The Netherlands; ⁵Department of Infectious Diseases, Amsterdam UMC location University of Amsterdam, The Netherlands; ⁶Department of Infectious Diseases, Amsterdam Institute for Infection and Immunity, The Netherlands; ⁷Cancer Research Center of Lyon (CRCL), UMR Inserm U1052 / CNRS 5286, Lyon, France; ⁸IHU Lyon, Lyon Hepatology Institute, Lyon, France; ⁹Institute for Infectious Diseases, University of Bern, Switzerland;</footnote>

<footnote n="10">¹⁰Laboratoire de Virologie, Institut des Agents Infectieux, Hospices Civils de Lyon, France; ¹¹HIV Clinic, Department of Medicine I, University Hospital Bonn, Germany; ¹²Department of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, Switzerland; ¹³Institute of Medical Virology, University of Zurich, Switzerland; ¹⁴Division of Infectious Diseases, HIV Unit, Geneva University Hospitals, University of Geneva, Switzerland; ¹⁵Division of Infectious Diseases, Lausanne University Hospital, University of Lausanne, Switzerland; ¹⁶Transplantation and Clinical Virology, Department of Biomedicine, University of Basel, Switzerland; ¹⁷Division of Infectious Diseases, Infection Prevention and Travel Medicine, Cantonal Hospital St. Gallen, Switzerland;</footnote>

<footnote n="18">¹⁸Division of Infectious Diseases, Ente Ospedaliero Cantonale Lugano, University of Geneva and University of Southern Switzerland, Lugano, Switzerland; ¹⁹University of Lyon, University Claude Bernard Lyon 1 (UCBL1), Lyon, France; and ²⁰Department of Hepatology, Hospices Civils de Lyon, Lyon, France</footnote>

<footnote>ªG. W., F. Z., and A. R. contributed equally to this study.</footnote>

<footnote>ᵇStudy group team members are listed in the Notes.</footnote>
Novel serum markers, including hepatitis B core-related antigen (HBcrAg) and circulating HBV RNA, might improve our understanding of HBsAg loss during antiviral therapy. Moreover, previous studies identified HBcrAg and HBV RNA levels as predictors of hepatocellular carcinoma in persons without HIV [10, 11]. HBcrAg is a composite of hepatitis B core antigen, hepatitis B e antigen (HBeAg), and p22 core-related protein, which are precore/core gene products [12]. It is a surrogate of the size of the transcriptionally active pool of intrahepatic covalently closed circular DNA (cccDNA), the molecular reservoir and transcriptional template of HBV [13–15]. Circulating HBV RNA mainly consists of pregenomic RNA, the template for reverse transcription to HBV DNA, and reflects cccDNA transcriptional activity in the hepatocytes during antiviral therapy [16–19]. Thus, these 2 noninvasive biomarkers may predict functional cure of HBV infection (ie, HBsAg loss). However, data are limited on the long-term trajectories of individuals experiencing HBsAg loss during antiviral therapy, especially persons with HIV/ HBV [20, 21].

Taking advantage of a cohort of persons with HIV and HBV
with serial stored samples available during tenofovir therapy,
we compared the long-term trajectories of HBcrAg and circu-
lating HBV RNA levels in persons with and without HBsAg
loss. Moreover, we intended to assess diagnostic criteria of
these markers between persons with and without HBsAg loss.

METHODS

Study Population and Design

Our study was performed within the SHCS, an ongoing nation-
wide cohort study including >70% of all persons with HIV un-
dergoing ART in Switzerland [22]. All centers' local ethical
committees approved the cohort study, and participants provid-
ed written informed consent. We included 29 SHCS partici-
pants with chronic HBV infection (defined as 2 positive
HBsAg test results at least 6 months apart) who started
tenofovir-containing ART and later achieved HBsAg loss. A
stored plasma sample within 1 year before the start of tenofovir
and a quantitative HBsAg (qHBsAg) <0.05 IU/mL after starting
tenofovir was required for inclusion. Participants with a nega-
tive HBsAg test result before the start of tenofovir therapy
were excluded. The 29 participants with HBsAg loss were
matched 1:1 to 29 persons with HIV fulfilling the same inclusion
criteria but without HBsAg loss during tenofovir therapy.
Matching was based on age (±10 years), sex at birth, lamivudine
treatment prior to tenofovir, and CD4+ T-cell count category at
the start of tenofovir therapy (<200, 200–349, ≥350 cells/mm³).

Our primary outcomes were the cumulative proportion of
participants with negative HBV DNA, HBV RNA, and
HBcrAg levels during tenofovir-containing ART. Our second-
ary outcomes were the proportions with a decline ≥1 log₁₀ in

qHBsAg, HBcrAg, and HBV RNA levels 1 and 2 years after
starting tenofovir, as well as the sensitivity and specificity of
HBcrAg and HBV RNA declines for HBsAg loss.

We defined baseline as the start date of the first tenofovir-
containing ART, and follow-up continued to the last available
stored sample before death, loss to follow-up, 6 months after
cessation of tenofovir, or database closure on 31 December
2020, whichever occurred first. Follow-up continued in case
of interruption of tenofovir therapy when participants resumed
therapy at any time later on. The time point of HBsAg loss was
defined as the first visit with a qHBsAg measurement <0.05 IU/
mL. We defined HBV DNA suppression as <20 IU/mL, an un-
detectable HBV RNA level as <10 copies/mL, and a negative
HBcrAg level as ≤3 log₁₀ U/mL.

**Laboratory Analyses**

We measured HBcrAg, HBV RNA, HBV DNA, and qHBsAg using stored plasma samples at baseline and after 6, 12, 18, and 24 months and yearly thereafter. We retrieved HBeAg status from available data. HBcrAg was quantified by the Lumipulse G HBcrAg assay on the LUMIPULSE G1200 Analyzer (Fujirebio Europe) according to the manufacturer's instructions. We used a cutoff of 3 log₁₀ U/mL to determine HBcrAg positivity, as proposed by Kimura et al [12]. As the assay has a linear range from 3 to 7 log₁₀ U/mL, samples with HBcrAg >7 log₁₀ U/mL were diluted and retested, as described previously [13]. HBV RNA levels were determined with the COBAS HBV RNA automated investigational assay on the COBAS 6800 system (Roche Molecular Diagnostics), which preferentially detects RNA expressed from cccDNA with a lower limit of detection of 3.3 copies/mL and a linear range between 10 and 10⁷ copies/mL, as described previously [19, 23]. We measured HBV DNA using a commercial quantitative nucleic acid test (COBAS HBV on the COBAS 4800 system; Roche Diagnostics) with a lower limit of detection of 4.4 IU/mL and a linear range from 10 to 1 × 10⁹ IU/mL, or we used measurements determined with accredited assays with a lower limit of detection ≤20 IU/mL during routine clinical care. We quantified qHBsAg using a commercial chemiluminescent microparticle immunoassay (ARCHITECT HBsAg; Abbott) with an initial dilution of 1:500, a sensitivity ≤0.05 IU/mL, and an upper limit of detection of 124 925 IU/mL.

**Statistical Analysis**

We modeled HBcrAg and HBV RNA levels over time using lin-
ear regression. Follow-up time was modeled by restricted cubic
splines with 5 knots located at the 5th, 27.5th, 50th, 72.5th and
95th percentiles to allow for nonlinear trajectories of HBcrAg
and HBV RNA [24]. We assessed the proportion of participants
with undetectable levels of HBV DNA, HBcrAg, and HBV RNA
at baseline and 1, 2, 3, 5, and 10 years after starting tenofovir
therapy. We determined the cumulative proportion with
negative HBV DNA, HBcrAg, and HBV RNA levels using the Kaplan-Meier method and tested differences between participants with and without HBsAg loss using log-rank tests. We calculated time-dependent area under the receiver operating characteristic curve (AUROC), sensitivity, and specificity of a decline ≥1 log₁₀ in qHBsAg, HBcrAg, and HBV RNA levels for the prediction of HBsAg loss at 2 and 5 years of follow-up using the R package *timeROC* [25]. When qHBsAg, HBcrAg, and HBV RNA decline was summarized, missing values were linearly interpolated with the closest neighboring values.

We defined statistical significance as a 2-sided *P* < .05. Statistical analyses were performed with Stata/MP (version 16.0; StataCorp) and RStudio (version 2022.7.2.576; RStudio Team) for R (version 4.2.2; R Core Team).

# RESULTS

## Patient Characteristics

The 58 participants were followed for a median of 12 years (IQR, 8–14). HBsAg loss occurred after a median of 4 years (IQR, 1–8; minimum, 0.5; maximum, 14). Prior to starting tenofovir therapy, 48 of 58 (83%) participants were treated with lamivudine-containing ART for a median of 6 years (IQR, 4–8; Table 1). Among the participants with prior lamivudine therapy, 17 of 24 (71%) with HBsAg loss and 13 of 24 (54%) without loss were treated with lamivudine at the time of tenofovir start (*P* = .37). At baseline, 8 of 29 (28%) participants with HBsAg loss and 7 of 29 (24%) without loss had a suppressed HBV DNA load. Baseline HBcrAg and HBV RNA levels were similar in participants with and without HBsAg loss (Table 1). At the start of tenofovir therapy, 13 of 27 (48%) participants with HBsAg loss and 10 of 24 (42%) without loss were HBeAg positive. During follow-up, all participants with HBsAg loss and 28 of 29 (97%) without loss achieved HBV DNA suppression (*P* > .99).

## HBV RNA and HBcrAg Trajectories During Tenofovir-Containing ART

HBV RNA and HBcrAg levels at tenofovir start could be evaluated in 26 persons with HIV with HBsAg loss and 28 without loss who had sufficient plasma volume stored for these assessments. At tenofovir start, 65% of the participants with HBsAg loss and 64% of those without loss had detectable HBV RNA levels; HBcrAg was detectable in 85% of participants with HBsAg loss and 86% without loss (Table 1). A decline ≥1 log₁₀ copies/mL in HBV RNA levels from baseline levels was observed in 88% of participants with HBsAg loss and 67% of those without loss after 2 years (*P* = .23). A decline ≥1 log₁₀ U/mL in HBcrAg levels or newly negative HBcrAg after 2 years was observed in 73% of participants with HBsAg loss and in 36% without loss (*P* = .02).

The individual long-term trajectories of HBV RNA and HBcrAg levels are depicted in Figure 1. All participants with HBsAg loss reached undetectable HBV RNA levels during

<table><caption>**Table 1.** Baseline Characteristics of Participants With and Without HBsAg Loss During Tenofovir-Containing Antiretroviral Therapy</caption><thead><tr><th></th><th colspan="2">HBsAg Loss<sup>a</sup></th></tr><tr><th></th><th>Participants<br>Without (n = 29)</th><th>Participants<br>With (n = 29)</th></tr></thead><tbody><tr><td>Female sex at birth</td><td>6 (21)</td><td>6 (21)</td></tr><tr><td>Age, y</td><td>39 (36–46)</td><td>42 (38–46)</td></tr><tr><td>Calendar year of tenofovir start</td><td>2005 (2003–2007)</td><td>2005 (2003–2007)</td></tr><tr><td>Follow-up duration, y</td><td>11.1 (7.9–14.1)</td><td>12.3 (10.4–14.1)</td></tr><tr><td>European origin</td><td>14 (48)</td><td>22 (76)</td></tr><tr><td>Ethnicity</td><td></td><td></td></tr><tr><td>White</td><td>16 (55)</td><td>22 (76)</td></tr><tr><td>Black</td><td>10 (34)</td><td>4 (14)</td></tr><tr><td>Asian</td><td>3 (10)</td><td>1 (3)</td></tr><tr><td>Other or unknown</td><td>0 (0)</td><td>2 (7)</td></tr><tr><td>Body mass index, kg/m<sup>2</sup></td><td>22.7 (19.2–26.9)</td><td>22.9 (21.0–25.5)</td></tr><tr><td>Lamivudine pretreatment</td><td></td><td></td></tr><tr><td>Duration, y</td><td>6.3 (3.9–7.3)</td><td>6.2 (4.8–7.8)</td></tr><tr><td>CD4+ T-cell count &lt;200 cells/µL</td><td>4 (14)</td><td>4 (14)</td></tr><tr><td>CD4/CD8 ratio</td><td>0.4 (0.3–0.6)</td><td>0.5 (0.3–0.7)</td></tr><tr><td>HIV viral load ≥50 copies/mL</td><td>15 (52)</td><td>13 (45)</td></tr><tr><td>Stage C<sup>b</sup></td><td>10 (34)</td><td>8 (28)</td></tr><tr><td>HBV genotype</td><td></td><td></td></tr><tr><td>A</td><td>11 (38)</td><td>8 (28)</td></tr><tr><td>A + G</td><td>0 (0)</td><td>3 (10)</td></tr><tr><td>C</td><td>1 (3)</td><td>0 (0)</td></tr><tr><td>D</td><td>2 (7)</td><td>4 (14)</td></tr><tr><td>Not available</td><td>15 (52)</td><td>14 (48)</td></tr><tr><td>HBV DNA, log₁₀ IU/mL</td><td>4.0 (1.5–7.9)</td><td>3.0 (1.2–7.5)</td></tr><tr><td>Quantitative HBsAg</td><td></td><td></td></tr><tr><td>log₁₀ IU/mL</td><td>4.0 (3.5–4.2)</td><td>3.4 (2.1–4.5)</td></tr><tr><td>&lt;1 log₁₀ IU/mL</td><td>1 (3)</td><td>5 (17)</td></tr><tr><td>HBcrAg<sup>c</sup></td><td></td><td></td></tr><tr><td>log₁₀ U/mL</td><td>5.6 (3.7–7.9)</td><td>6.9 (3.8–8.6)</td></tr><tr><td>≤3 log₁₀ U/mL</td><td>4/28 (14)</td><td>4/26 (15)</td></tr><tr><td>HBV RNA<sup>c</sup></td><td></td><td></td></tr><tr><td>log₁₀ copies/mL</td><td>3.3 (&lt;1.0–5.7)</td><td>4.7 (&lt;1.0–5.6)</td></tr><tr><td>&lt;10 copies/mL</td><td>10/28 (36)</td><td>9/26 (35)</td></tr><tr><td>HBeAg positive</td><td>10/24 (42)</td><td>13/27 (48)</td></tr><tr><td>Alanine aminotransferase<br>elevation</td><td>15 (52)</td><td>16 (55)</td></tr></tbody></table>

Data are presented as No. (%) or median (IQR).

Abbreviations: HBcrAg, hepatitis B core-related antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus.

<sup>a</sup>Defined as quantitative HBsAg <0.05 IU/mL.

<sup>b</sup>According to the clinical classification of HIV disease by the US Centers for Disease Control and Prevention.

<sup>c</sup>Measurements not available for 1 participant without HBsAg loss and 3 with HBsAg loss due to limited plasma sample volume.

tenofovir-containing ART, as did 79% of those without loss (*P* = .02). All participants with HBsAg loss achieved undetectable HBV RNA before or at the time of HBsAg loss. In contrast, 19 (66%) participants had negative HBcrAg levels at the time of HBsAg loss, whereas 14% reached negative HBcrAg levels after HBsAg clearance and 21% had HBcrAg levels >3 log₁₀ U/mL until the end of follow-up (Supplementary Figure 1). In comparison, 52% of the participants without HBsAg loss remained HBcrAg
A

Participants with HBsAg loss

<figure><img src="image_2.png" /></figure>

C

Participants with HBsAg loss

<figure><img src="image_3.png" /></figure>

B

Participants without HBsAg loss

<figure><img src="image_4.png" /></figure>

D

Participants without HBsAg loss

<figure><img src="image_5.png" /></figure>

**Figure 1.** Circulating HBV RNA levels and HBcrAg levels in participants (A, C) with and (B, D) without HBsAg loss (defined as quantitative HBsAg <0.05 IU/mL) during tenofovir-containing antiretroviral therapy. HBV RNA and HBcrAg levels over time were modeled using linear regression while incorporating follow-up time as restricted cubic splines with 5 knots located at the 5th, 27.5th, 50th, 72.5th, and 95th percentiles. Follow-up time refers to time since start of tenofovir therapy. Circles with connecting solid lines, individual trajectories; dashed line, modeled HBV RNA and HBcrAg levels; shaded area, 95% confidence intervals. cp, copies; HBcrAg, hepatitis B core-related antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus.

positive (*P* = .03). Combined HBV DNA suppression, undetectable HBV RNA, and negative HBcrAg during follow-up were achieved in 13 of 29 (45%) participants without HBsAg loss and was more likely in individuals who were HBeAg negative (9/14, 64%) than HBeAg positive (1/10, 10%; *P* = .01). Among participants with HBsAg loss, 23 of 29 (79%) reached combined HBV DNA suppression, undetectable HBV RNA, and negative HBcrAg, without significant differences between individuals who were HBeAg negative (12/14, 89%) and HBeAg positive (9/13, 69%; *P* = .38). Among the participants with HBsAg loss, 28 of 29 (97%) had sustained qHBsAg <0.05 IU/mL (ie, at least 2 consecutive samples with qHBsAg <0.05 IU/mL), whereas those without HBsAg loss all remained at qHBsAg ≥0.05 IU/mL throughout follow-up. One participant with HBsAg loss had a transient detectable level of qHBsAg coinciding with ART interruption. This participant was able to reach qHBsAg <0.05 IU/mL 4 years after this event, while HBcrAg remained negative and HBV RNA undetectable during this period. **Supplementary Table 1** depicts the number of participants as well as the number of qHBsAg, HBV DNA, HBcrAg, and HBV RNA measurements at each follow-up time point.

## Cumulative Proportions With HBV DNA Suppression, Negative HBcrAg, and Undetectable HBV RNA Levels

Median time to HBV DNA suppression was similar in participants with and without HBsAg loss (12 months [95% CI, 6–18] vs 12 months [95% CI, 6–24], *P* = .35; **Figure 2A**). Among those with detectable levels at baseline, the Kaplan-Meier cumulative probabilities for HBcrAg ≤3 log₁₀ U/mL (*P* = .001) and HBV RNA <10 copies/mL (*P* = .03) were significantly higher for participants with HBsAg loss as compared with those without loss (**Figure 2D** and **2G**). The cumulative probabilities for HBcrAg ≤3 log₁₀ U/mL for persons with and without HBsAg loss, stratified by HBeAg status, are shown in **Figure 2E** and **2F**. The respective estimates for undetectable HBV RNA levels are presented in **Figure 2H** and **2I**. Crude proportions of participants with HBV DNA suppression, negative HBcrAg, and undetectable HBV RNA levels at baseline and after 1, 2, 3, 5, and 10 years after tenofovir start are shown in **Supplementary Figure 2**.

## HBcrAg and HBV RNA as Predictive Markers for HBsAg Loss

In comparison with qHBsAg decline ≥1 log₁₀ IU/mL, HBcrAg decline ≥1 log₁₀ U/mL after 1 and 2 years had higher sensitivity
<figure><img src="image_6.png" /><figcaption>**Figure 2.** Kaplan-Meier curves for cumulative proportions with HBV DNA <20 IU/mL, HBcrAg level ≤3 log10 U/mL, and HBV RNA <10 copies/mL after starting tenofovir-containing antiretroviral therapy, stratified by participants with and without HBsAg loss. Cumulative proportions: A, D, G, all participants; B, E, H, participants who were HBeAg positive; C, F, I, participants who were HBeAg negative. Only participants with detectable levels at the start of tenofovir-containing antiretroviral therapy were included in the analysis; HBsAg loss was defined as quantitative HBsAg <0.05 IU/mL. HBcrAg, hepatitis B core-related antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus.</figcaption></figure>

but lower specificity in predicting HBsAg loss at 2 or 5 years. HBV RNA decline ≥1 log₁₀ copies/mL after 1 and 2 years had 100% sensitivity in predicting HBsAg loss at 2 and 5 years but only 40.0% and 36.4% specificity (Table 2). A combination of HBcrAg or HBV RNA decline with qHBsAg decline did not improve sensitivity and showed a similar AUROC than qHBsAg decline alone. Supplementary Table 2 shows detailed reports of the performance of qHBsAg, HBcrAg, and HBV RNA as markers for HBsAg loss at 2 and 5 years based on time-dependent receiver operating characteristic curves. A combination of undetectable HBV RNA levels and HBcrAg decline ≥1 log₁₀ U/mL after 1 year of tenofovir therapy revealed the highest AUROC (0.831) for the prediction of HBsAg loss after 2 years. For the prediction of HBsAg loss after 5 years, the highest AUROC values were identified with the combination of qHBsAg and HBcrAg decline ≥1 log₁₀ (AUROC, 0.814) and in HBcrAg decline ≥1 log₁₀ U/mL (AUROC, 0.810) 1 year after starting tenofovir.

DISCUSSION

In our study, we described the trajectories of HBcrAg and HBV RNA during a median follow-up of 12 years on tenofovir-containing ART in persons with and without HBsAg loss. Of the participants with HBsAg loss, all achieved undetectable circulating HBV RNA levels, and approximately 80% reached negative HBcrAg levels during follow-up. In persons without HBsAg loss, the probability of achieving undetectable HBV RNA and HBcrAg levels was significantly lower than in persons with HBsAg loss. A decline ≥1 log₁₀ in HBV RNA or in HBcrAg levels after 2 years of tenofovir therapy had high sensitivity but low specificity for predicting HBsAg loss.

Undetectable HBV RNA levels preceded the first occurrence of undetectable qHBsAg levels in all participants with HBsAg loss. Similar findings were reported in a retrospective analysis including participants with HIV and HBV from 2 randomized controlled ART trials, where all but 1 participant with HBsAg loss had undetectable HBV RNA levels [21]. In line with these
<table><caption>**Table 2.** qHBsAg, HBcrAg, and HBV RNA Decline After 1 and 2 Years of Tenofovir-Containing ART as Predicting Markers for HBsAg Loss Within 2 and 5 Years Based on Time-Dependent AUROCs</caption><thead><tr><th rowspan="2"></th><th colspan="3">HBsAg Loss<sup>a</sup> Within 2 y</th></tr><tr><th>Sensitivity, %</th><th>Specificity, %</th><th>AUROC</th></tr></thead><tbody><tr><td colspan="4"><strong>Decline ≥1 log<sub>10</sub> after 1 y of tenofovir-containing ART<sup>b</sup></strong></td></tr><tr><td>qHBsAg, IU/mL</td><td>70.0</td><td>88.1</td><td>0.791</td></tr><tr><td>HBcrAg, U/mL</td><td>87.5</td><td>64.7</td><td>0.761</td></tr><tr><td>HBV RNA, copies/mL</td><td>100.0</td><td>40.0</td><td>0.700</td></tr><tr><td>HBV RNA and HBcrAg</td><td>85.7</td><td>64.7</td><td>0.752</td></tr><tr><td>HBcrAg and qHBsAg</td><td>70.0</td><td>88.1</td><td>0.791</td></tr><tr><td>HBV RNA and qHBsAg</td><td>66.7</td><td>88.1</td><td>0.774</td></tr></tbody><tbody><tr><td colspan="4"><strong>Decline ≥1 log<sub>10</sub> after 2 y of tenofovir-containing ART<sup>b</sup></strong></td></tr><tr><td>qHBsAg, IU/mL</td><td>73.6</td><td>81.6</td><td>0.776</td></tr><tr><td>HBcrAg, U/mL</td><td>91.0</td><td>64.5</td><td>0.778</td></tr><tr><td>HBV RNA, copies/mL</td><td>100.0</td><td>36.4</td><td>0.682</td></tr><tr><td>HBV RNA and HBcrAg</td><td>89.0</td><td>67.7</td><td>0.784</td></tr><tr><td>HBcrAg and qHBsAg</td><td>69.4</td><td>89.2</td><td>0.793</td></tr><tr><td>HBV RNA and qHBsAg</td><td>66.9</td><td>88.6</td><td>0.777</td></tr></tbody></table>

Abbreviations: ART, antiretroviral therapy; AUROC, area under the receiver operating characteristic curve; HBcrAg, hepatitis B core-related antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; qHBsAg, quantitative hepatitis B surface antigen.

results, a study from Beijing in persons without HIV reported undetectable HBV RNA preceding HBsAg loss during nucleos(t)ide analogue therapy [26]. In contrast, a study from Taiwan found detectable HBV RNA levels at the time of HBsAg clearance in the majority of participants, but undetectable levels were achieved in all participants within the following 3 years [27]. Differences in the population characteristics, inclusion criteria, and technical characteristics of the assays used might explain these discrepancies. A recent study in 5 French centers reported undetectable circulating HBV RNA among all 27 persons with HBV 12 months after liver transplantation, whereas HBcrAg remained detectable in 30% of the transplant recipients [28]. Our results are in line with these findings, although the setting and population differ considerably between the studies. Similarly, a study from Hong Kong observed detectable HBcrAg levels in 12 of 55 persons without HIV who experienced spontaneous HBsAg loss [29]. Detectable HBcrAg despite undetectable HBV DNA and HBsAg may reflect ongoing low-level cccDNA transcriptional activity, while qHBsAg and HBV DNA may still be present in the serum below detection levels of the assays used in our study [28, 29]. Whether low-level transcriptional activity also has clinical implications with regard to the development of novel HBV drugs or for predicting the risk of hepatocellular carcinoma is currently uncertain.

A decline ≥ 1 log₁₀ in HBV RNA or HBcrAg levels after 2 years of tenofovir-containing ART had higher sensitivity but low specificity for HBsAg loss than a decline in qHBsAg levels. A recent systematic review of 6 studies including 1257 persons

reported the predictive value of HBcrAg levels for HBsAg loss with a median AUROC of 0.645 [30]. However, only 1 of these studies investigated the change in HBcrAg levels for predicting HBsAg loss during HBV therapy in persons without HIV with an AUROC of 0.521 [31]. A recent study including persons without HIV who were HBeAg positive and negative reported that a decline >2 log U/mL in HBcrAg after 4 weeks of antiviral therapy had a sensitivity and specificity for HBsAg loss of 75% and 62.5%, respectively [32]. In our study, baseline levels in HBcrAg and HBV RNA were not significantly different in both groups, consistent with previous reports on the limited use of baseline markers for predicting HBsAg loss [30]. As shown in Supplementary Table 2, a combination of negative HBV RNA and HBcrAg decline after 1 year of tenofovir therapy showed good performance in predicting HBsAg loss after 2 years and could serve as an alternative end point in the development of new HBV treatment strategies [33]. Adding qHBsAg decline to these 2 markers did not lead to further improvement in sensitivity and specificity. As these assays can be used on widely available diagnostic platforms, integrating these biomarkers into clinical care would be realistic in high-income countries. Yet, this would not be the case in low- and middle-income countries, where even HBV DNA measurements are often inaccessible due to high costs.

The majority of participants experienced a decline ≥ 1 log₁₀ in HBcrAg and HBV RNA levels within 2 years of tenofovir therapy, even in the absence of HBsAg loss. Among participants without HBsAg loss, the combined suppression of HBV DNA, HBV RNA, and HBcrAg was more likely in

<footnote><sup>a</sup>Defined as qHBsAg <0.05 IU/mL.</footnote>

<footnote><sup>b</sup>Details of this analysis are shown in Supplementary Table 2.</footnote>
participants who were HBeAg negative than HBeAg positive. Integrated DNA has been identified as the main source of HBsAg production in individuals who are HBeAg negative, which could explain the persistently detectable qHBsAg levels despite serologic evidence of cccDNA silencing as reflected by undetectable HBV RNA and HBcrAg levels [34, 35]. Previous studies found an association between (1) undetectable HBcrAg levels and HBV RNA levels and (2) favorable outcomes after cessation of nucleos(t)ide analogue therapy in persons without HIV who were HBeAg negative, but the generalizability of these findings to persons with HIV is limited as lifelong HBV-active therapy is recommended by current guidelines [3, 4, 36, 37].

Our study provides detailed insights into the kinetics of HBcrAg and HBV RNA in persons with HIV/HBV undergoing tenofovir-containing ART. We were able to compare the trajectories of both markers in individuals experiencing HBsAg loss with the trajectories in similar individuals not experiencing HBsAg loss using stringent inclusion and matching criteria. Despite the relatively small sample size due to the low frequency of HBsAg loss, our study design allowed us to involve one of the largest number of persons with HIV experiencing HBsAg loss during tenofovir therapy followed longitudinally to date. Several reports have highlighted the potential of circulating HBV RNA quantification to serve as a surrogate marker for intrahepatic cccDNA transcriptional activity and assessment of antiviral efficacy [17, 18, 38, 39]. The majority of currently available tests have a lower limit of quantification of around 1000 copies/mL, although in-house reverse transcription drop-let digital polymerase chain reaction assays and the Abbott serum HBV RNA assay have lower limits of quantification of approximately 100 copies/mL [16, 40, 41]. In our study, we used a recently developed, highly sensitive investigational assay to quantify serum HBV RNA preferentially expressed from cccDNA with a lower limit of detection <5 copies/mL across a broad range of HBV genotypes, which ascertained robust results and potentially improves the diagnostic value of circulating HBV RNA in the prediction of HBsAg loss [19, 23]. However, currently available HBV RNA assays are not yet standardized, and detectable HBV RNA may not exclusively consist of pregenomic RNA. The applicability of HBcrAg is currently limited by the lower limit of sensitivity of the assay. To avoid false-positive results, we used a stringent cutoff of 1000 U/mL, as recommended by the manufacturer. An assay with a lower limit of sensitivity has recently been developed [42]. In addition, we were unable to correlate directly circulating HBV RNA and HBcrAg levels with intrahepatic cccDNA due to the absence of liver biopsies in this cohort. Moreover, HBeAg status at the start of tenofovir therapy was available in only 51 of 58 participants. Yet, the equal distribution of HBeAg status among participants with and without HBsAg loss should have limited any nondifferential bias with respect to this factor.

In conclusion, our findings indicate that in persons with HIV receiving tenofovir-containing ART, HBV RNA suppression precedes HBsAg loss. However, as HBV RNA clearance occurs in the majority of persons without HBsAg loss, its predictive value is limited. HBV RNA and HBcrAg declines during the first 2 years of tenofovir therapy have a high sensitivity for HBsAg loss, emphasizing the potential of these markers for the identification of individuals who will not experience HBsAg loss on tenofovir-containing ART. A combination of HBV RNA and HBcrAg levels over time could improve predictions of HBsAg loss in clinical trials of HBV drugs with novel modes of action.

## Supplementary Data

Supplementary materials are available at *The Journal of Infectious Diseases* online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

## Notes

**Acknowledgments.** We thank all patients, physicians, and nurses associated with the participating cohorts. The data are gathered by the 5 Swiss university hospitals, 2 cantonal hospitals, 15 affiliated hospitals, and 36 private physicians (listed in http://www.shcs.ch/180-health-care-providers).

**Members of the Swiss HIV Cohort Study.** I. Abela, K. Aebi-Popp, A. Anagnostopoulos, M. Battegay, E. Bernasconi, D. L. Braun, H. C. Bucher, A. Calmy, M. Cavassini, A. Ciuffi, G. Dollenmaier, M. Egger, L. Elzi, J. Fehr, J. Fellay, H. Furrer, C. A. Fux, H. F. Günthard (president of the SHCS), A. Hachfeld, D. Haerry (deputy of Positive Council), B. Hasse, H. H. Hirsch, M. Hoffmann, I. Hösl, M. Huber, D. Jackson-Perry (patient representatives), C. R. Kahlert (chair of the Mother & Child Substudy), O. Keiser, T. Klimkait, R. D. Kouyos, H. Kovari, K. Kusejko (head of data center), N. Labhardt, K. Leuzinger, B. Martinez de Tejada, C. Marzolini, K. J. Metzner, N. Müller, J. Nemeth, D. Nicca, J. Notter, P. Paioni, G. Pantaleo, M. Perreau, A. Rauch (chair of the Scientific Board), L. Salazar-Vizcaya, P. Schmid, R. Speck, M. Stöckle (chair of the Clinical and Laboratory Committee), P. Tarr, A. Trkola, G. Wandeler, M. Weisser, and S. Yerly.

**Author contributions.** L. B., A. B., G. W., A. R., M. L., and F. Z. designed the study. M.-L. P., B. T., F. S.-R., and C. S. performed the serologic and virologic analyses. L. B. and A. B. analyzed the data. L. B., G. W., and A. R. prepared the first draft, which was revised by all coauthors. C. B., H. F. G., A. C., M. C., H. H. H., P. S., E. B., G. W., and A. R. contributed to data acquisition. All authors contributed to the interpretation of the analyses.
**Data sharing.** Data are not publicly available.

**Disclaimer.** The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Financial support.** This work was supported by an investigator-initiated trial grant from Gilead Sciences (CO-SW-985-5602), by the NEAT-ID Foundation, by the Department of Teaching and Research, Inselspital, Bern University Hospital, and by the Liquid Biobank Inselspital Bern. This study has been financed within the framework of the SHCS, supported by the Swiss National Science Foundation (grant 201369), by SHCS projects 809 and 868, and by the SHCS research foundation. The SHCS biobank was supported by the Liquid Biobank Inselspital Bern and by the Department of Teaching and Research, Inselspital, Bern University Hospital. L. B. was supported by the Young Talents in Clinical Research program of the Swiss Academy of Medical Sciences and Gottfried and Julia Bangerter Rhyner Foundation (grant YTCR 13/19). G. W. was supported by a professorship from the Swiss National Science Foundation (PP00P3_211025). F. Z. and M. L. received public grants overseen by the French National Research Agency as part of the second Investissements d'Avenir Program (reference ANR-17-RHUS-0003) and by the European Union (grant EU H2020-847939-IP-cure-B).

**Potential conflicts of interest.** L. B. reports support for travel and conference participation from the CROI Foundation and the SAFE-ID Foundation. A. B. has received speaker honoraria from Gilead unrelated to this work. M.-L. P. reported support for the present article from the CirB-RNA-RHU program paid to her institution. B. T. has received honoraria for lectures from Gilead Sciences France, Hopital Vall D'Hebron, and the Belgian Association for the Study of the Liver and payment for expert testimony and travel support from the International Hepatology Education Program. L. S.-V. has received honoraria for consulting from Gilead paid to her institution and unrelated to this work. F. S.-R. reports no potential conflicts. C. S. received speaker honoraria and travel support from Gilead and a grant from Roche Diagnostics paid to her institution and unrelated to this work. C. B. has received advisory board membership fees from Gilead paid to his institution. J. K. R. has received honoraria for consulting or speaking at educational events from Boehringer, Gilead, Merck, Janssen, and ViiV. H. F. G. has received unrestricted research grants from Gilead Sciences; fees for data and safety monitoring board membership from Merck; consulting/advisory board membership fees from Gilead Sciences, Merck, Johnson & Johnson, Janssen, GSK, Novartis, and ViiV Healthcare; and grants from the Swiss National Science Foundation, the Bill & Melinda Gates Foundation, the Yvonne Jacob Foundation, Gilead, ViiV, and the National Institutes of Health. A. C. reported unrestricted educational grants from ViiV, Gilead, and MSD and industry-

sponsored clinical trials at her HIV unit. M. C.'s institution received research grants from Gilead, MSD, and ViiV and financial compensation for expert opinion given to Gilead, MSD, and ViiV. H. H. H. received grant support from Moderna paid to his institution and honoraria for consulting or speaking at educational events from AICuris, Allovir, Moderna, VeraTx, Roche, Takeda, Biotest, and Gilead. P.S.'s institution has received travel grants and congress/advisory fees from Gilead and ViiV unrelated to this work. The institution of E. B. received grants from MSD; consulting fees from Moderna; speaker fees from Pfizer AG Switzerland; and payments for the participation of E. B. to advisory boards or travel grants from Gilead Sciences, ViiV Healthcare, MSD, Pfizer AG Switzerland, Moderna, Astra Zeneca, Eli Lilly, and Abbvie. M. L. has received lecture and presentation fees from Abbvie and Gilead and coverage and reimbursement of travel expenses from Abbvie, Gilead, Inventiva, Madrigal, and MSD. G. W. received unrestricted research grants from Gilead Sciences and Roche Diagnostics and lecture/advisory board membership fees from Gilead Sciences, MSD, and ViiV Healthcare, all paid to his institution. F. Z. has received speaker fees from Gilead; consulting fees from Aligos, Assembly, Blue Jay, and GSK; and research grants through INSERM from Assembly, Beam, Blue Jay, and Janssen. A. R. reports support to his institution for advisory boards and/or travel grants from MSD, Gilead Sciences, Pfizer, and Moderna and an investigator-initiated trial grant from Gilead Sciences. All remuneration went to his home institution and not to A. R. personally, and all remuneration was provided outside the submitted work.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

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