HBV pgRNA profiles in Chinese HIV/HBV coinfected patients under pre‐ and posttreatment: a multicentre observational cohort study

Abstract Data on hepatitis B virus (HBV) pregenomic (pgRNA) levels in HIV/HBV coinfected patients pre‐ and post‐combined antiretroviral therapy (cART) are limited. This study aimed to evaluate the distribution of HBV pgRNA levels in treatment‐naive coinfected patients and explore the changes that occur after the initiation of cART by examining patients from multicentre cohort studies performed in China. We included HIV/HBV coinfected subjects from the China AIDS Clinical Trial cohorts established from 2008 to 2014. Clinical and serological markers of HIV and HBV infection and biochemical data were acquired at baseline and after 96 and 240–480 weeks of cART. The correlations between HBV pgRNA and HBV DNA levels as well as HBsAg levels were calculated using Spearman's bivariate correlation analysis, and multivariate regression analysis was performed to determine factors associated with undetectable HBV pgRNA levels before cART and HBeAg loss after cART. A total of 132 HIV/HBV coinfected patients were enrolled, and 100 individuals were HBeAg‐negative. A total of 34.4% (32/93) of patients were positive for HBV pgRNA, and the median HBV pgRNA level was 4.92 (IQR: 4.21–6.12) log10 copies/mL before cART. The median HBV pgRNA level was significantly lower in HBeAg‐negative individuals than in HBeAg‐positive individuals (4.22 (IQR: 2.70–4.84) log10 copies/mL vs. 5.77 (IQR: 4.63–6.55) log10 copies/mL, p = 0.002). HBV pgRNA was moderately correlated with HBsAg (r = 0.594, p = 0.001), and positively associated with HBV DNA (r = 0.445, p = 0.011). The factors independently associated with undetectable HBV pgRNA level before cART were HBV DNA (OR: 5.61, 95% CI: 1.50–20.96, p = 0.01) and HBeAg status (OR: 5.95, 95% CI: 1.52–23.25, p = 0.01). A total of 87.5% (28/32) of patients were followed for a median duration of 138 (IQR: 54–240) weeks, and the HBV pgRNA levels became undetectable in seven patients. The 132 patients were observed for 695.5 person‐years, and no HBsAg loss occurred. Thirteen individuals achieved HBeAg loss, four patients had undetectable levels of HBV pgRNA pre‐cART, and the level of six individuals became undetectable during the 48‐week (IQR: 48–264) follow‐up period. HBeAg status was significantly associated with HBV pgRNA level in HIV/HBV coinfected patients pre‐ and post‐cART. Additionally, undetectable HBV pgRNA level may be associated with HBeAg loss after cART.


| INTRODUC TI ON
HIV/HBV coinfection is fairly common in clinical practice due to shared transmission routes. The prevalence of coinfection is 8.4% worldwide 1 and 9.5% in China. 2 It has been reported that the coinfected prevalence is higher in populations where vaccination is not available such as sub-Saharan. 1 HIV/HBV coinfected patients are characterized by accelerated progression of liver disease and increased liver-associated mortality. 3 Although anti-HBV regimencontaining combined antiretroviral therapy (cART) has effectively suppressed HIV and HBV replication, morbidity and mortality remain significantly higher in coinfected patients than in those infected with HIV alone. 4 Previous studies indicated that the HBV DNA suppression rate after 48 weeks of cART was 78.8% in coinfected patients, 5 and 98.5% had undetectable levels of HBV DNA by year 5 of TDF-inclusive cART. 6 Unfortunately, cART is not able to eliminate covalently closed circular DNA (cccDNA), which is the main transcriptional template of HBV. Consequently, the level of HBV DNA rebounds once the treatment is discontinued in many chronic hepatitis B (CHB) patients.
Therefore, monitoring the cccDNA levels plays an important role in evaluating the therapeutic efficacy and estimating the treatment endpoint. Intrahepatic cccDNA monitoring is conducted by liver biopsy, which is not widely used in clinical practice due to certain disadvantages, such as its invasiveness, an inadequate specimen size, interobserver variability and potential complications. 7 Hepatitis B virus pgRNA is thought to be a transcription product of HBV and used as a plasma marker for infection outcome and for hepatic cell carcinoma (HCC). [8][9][10][11][12] Patients who remain HBV pgRNApositive after treatment have an increased risk of viral rebound when the treatment is discontinued. 12 Detectable HBV pgRNA is associated with HCC development in CHB patients receivingantiviral therapy. 11 Unfortunately, the HBV pgRNA distribution in HIV/HBV coinfected patients has not been well elucidated. These HIV-associated immunodeficiency patients are known to exhibit depletion of HBV-specific cytotoxic T lymphocytes (CTLs) pre-and post-cART, which may impact the control of HBV replication. 13,14 This implies that coinfected patients may display different characteristics of viral markers from HBV monoinfection patients and further study is necessary to explore the levels of potential serological markers, such as HBV pgRNA, in coinfected patients. To date, we found that only a cross-sectional study demonstrated that there was no significant difference in the levels of HBV pgRNA between treatment-naive individuals with HBV monoinfection and those with HIV/HBV coinfection. HBV pgRNA levels were strongly correlated with HBV DNA levels in coinfected patients. 15 However, the small sample size and some limitations inherent from the single-centre nature of that study make it difficult to fully understand the HBV pgRNA levels in treatment-naive HIV/HBV coinfected patients.
Moreover, no study has been conducted to demonstrate the changes in HBV pgRNA levels after cART in coinfected patients.
Additionally, highly potent tenofovir disoproxil fumarate (TDF)containing cART is able to effectively suppress HBV replication in either HBV-infected or HIV/HBV coinfected patients. However, long-acting drugs such as cabotegravir and rilpivirine, which do not contain any active anti-HBV agents, are noninferior to standard TDF combined with 3TC-containing therapy for maintaining the HIV RNA suppression rate. 16,17 It is worth considering whether HIV/HBV coinfected patients who have been treated with anti-HBV regimensincluding cART for a long time could discontinue 3TC and TDF and commence on cabotegravir and rilpivirine therapies. Based on this fact, it is essential to understand the effect of long-term TDF with and without 3TC-containing treatment in HIV/HBV coinfected patients, which may provide important clues for the further clinical management of HIV/HBV coinfected patients. Hence, the primary aims of this multicentre cohort study were to evaluate the levels of HBV pgRNA pre-and post-cART and explore their association with the long-term prognoses in HIV/HBV coinfected patients.

| Study population
We reviewed cART-naive HIV/HBV coinfected patients from the following prospective, multicentre cohort studies: the China AIDS Clinical Trial (CACT) 0810 and CACT1215 (ClinicalTrials. gov identifiers: NCT00872417 and NCT01844297). As previously described, 2 HIV-infected individuals in CACT0810 were enrolled between November 2008 and January 2010. Eligibility criteria included 1 age between 18 and 65 years, 2 CD4 + cell count lower than 350 cells/μL, and 3 cART naivety. Participants in CACT1215 were enrolled between August 2012 and September 2014. CACT1215 had the same inclusion criteria as CACT0810, except that the CD4 + cell count threshold was 500 cells/μL. Subjects received zidovudine or stavudine in combination with lamivudine and nevirapine four patients had undetectable levels of HBV pgRNA pre-cART, and the level of six individuals became undetectable during the 48-week (IQR: 48-264) follow-up period.
HBeAg status was significantly associated with HBV pgRNA level in HIV/HBV coinfected patients pre-and post-cART. Additionally, undetectable HBV pgRNA level may be associated with HBeAg loss after cART.

K E Y W O R D S
cART, HBeAg loss, HBV DNA, HBV pgRNA, HIV/HBV coinfected patients or efavirenz in the CACT0810 cohort and tenofovir combined with lamivudine and efavirenz or nevirapine in the CACT1215 cohort.
The CACT1315 was an extension of the above two studies beyond the initial 96-week follow-up period to the time of the present analysis, so these data in this study were collected from 2008 to 2020.
Subjects visited local medical centres for clinical evaluation and blood collection before cART (baseline) and at the following weeks after cART initiation at 4, 8, and 12 weeks and then every 12 weeks.
In this study, we retrieved demographic and clinical data including Participants were defined as having HIV/HBV coinfection if they were HBsAg-positive and anti-HCV-negative. In this study, subjects who were both HBsAg and anti-HCV-positive were excluded (n = 6).

| Measurement of serum HBV pgRNA
The extraction of HBV pgRNA from 200 μl serum of HIV/HBV coinfected individuals and DNase I treatment were performed using commercial kits (Beijing Hotgen Biotech. Co. Ltd) following the manufacturer's instructions. The levels of HBV pgRNA were detected with the HBV pgRNA One-Step RT-qPCR Kit (Beijing Hotgen Biotech.Co. Ltd) as previously described. 12,18 During quantification, five gradients of quantification standards were used, with 10 3 , 10 4 , 10 5 , 10 6 and 10 7 copies/mL, respectively. The minimum and maximum detection limits of this method were 300 copies/mL and 10 8 copies/mL, respectively.

| Ethics statement
The Institutional Review Board of Peking Union Medical College Hospital (PUMCH) approved the parent studies and each participant provided written informed consent.

| Statistical analysis
Analyses were performed using SPSS 23.0 (IBM Corp, Armonk, NY, United States). Descriptive statistics are presented as the mean with standard deviation (SD) or median (M) with interquartile range (IQR).
Student's t-test was used for comparisons of parametric data, and the Mann-Whitney U test was used for comparisons of noncategorical variables. Categorical variables were analysed by the chi-squared test or Fisher's exact test. Associations between two variables were calculated using Spearman's bivariate correlation analysis. A logistic regression model and Cox regression model were applied to identify factors closely related to undetectable HBV pgRNA levels and HBeAg seroconversion, respectively. Statistical significance was defined as a p value less than 0.05. Notably, 100 patients were HBeAg-negative, and 32 were HBeAg-positive. The HBV DNA, HBsAg and ALT levels in HBeAgnegative individuals were significantly lower than those in HBeAgpositive patients. HIV RNA and CD4 + cell counts were comparable in the two groups ( Table 1).    However, the HIV RNA levels, CD4 + cell count and CD4 + /CD8 + ratio was comparable between the two groups ( Table 2).

| HBV DNA response during the followup period
We further studied the factors associated with HBV pgRNA levels  (Table 3).

| HBV pgRNA response during the followup period
Among the 32 subjects with detectable HBV pgRNA at baseline,  Table 4).
The transaminase and glycemic level in HIV/HBV coinfected patients were comparable during follow-up period (

| HIV RNA suppression and CD4 + cell count improvements
We also evaluated the HIV RNA suppression and recovery of CD4

| DISCUSS ION
This is the largest multicentre cohort study to evaluate the levels of HBV pgRNA changes in HIV/HBV coinfected subjects pre-and post-cART in China. We demonstrated that 34.4% of cART-naive HIV/HBV coinfected patients were positive for HBV pgRNA. HBeAg status and HBV DNA levels are two significant factors associated with undetectable HBV pgRNA levels. Additionally, the decrease in the HBV pgRNA level was more attenuated than the decrease in the HBV DNA level following cART, and HBV pgRNA is frequently undetectable in HBeAg-negative patients after long-term treatment.
Notably, patients with undetectable HBV pgRNA levels are more likely to achieve HBeAg seroconversion.
HBeAg status is a marker of infectivity and of transcriptional activity of HBV. 19 Therefore, HBV DNA and HBV pgRNA levels in HBeAg-negative individuals are different from those in HBeAgpositive subjects. Other studies reported that the HBV pgRNA levels in HBeAg-negative patients were lower than those in HBeAgpositive patients. 8,20,21 In this study, the proportion of patients with undetectable levels of HBV pgRNA was significantly higher and the HBV pgRNA levels were lower in HBeAg-negative patients than in HBeAg-positive patients. Our multivariable analyses also demonstrate that HBeAg status is an independent factor associated with the proportion of patients with undetectable levels of HBV pgRNA.
These results are in line with the results of a previous study. 20 A possible explanation for these findings may be the lower transcriptional F I G U R E 5 Correlations between HBV pgRNA and HBsAg. The association between HBV pgRNA and HBsAg in enrolled HIV/HBV coinfected patients (A), HBeAg-positive coinfected patients (B) and HBeAg-negative coinfected patients (C) before treatment activity of cccDNA in HBeAg-negative patients than that in HBeAgpositive patients. This explanation is supported by the findings of the study conducted by Goncalves and his colleagues. 22 Their study used a multiscale viral dynamic model to show that HBeAg-negative patients had a lower rate of production of encapsidated pgRNA and a smaller basic reproduction number. Based on these results, we recommend dual HBV therapy for HBeAg-positive coinfected patients when they initiate cART to effectively suppress HBV DNA and decrease HBV pgRNA levels.
It is worth mentioning that the proportion of patients with undetectable HBV pgRNA levels in our study was higher than that in previous study. 23 This difference may be due to the normal or slightly elevated transaminase levels in the enrolled patients, in addition to the fact that most of the patients in this study were HBeAg-negative.
A study reported that ALT levels were positively associated with HBV pgRNA levels 24 and other studies included mostly patients with elevated ALT levels, 23 which may lead to higher HBV pgRNA levels than our results.   15 The data from our study show that the HBV pgRNA level was 4.87 (4.19-6.11) log 10 copies/mL in treatment-naive HIV/HBV coinfected patients, which is similar to a previous report. We also show that HBV pgRNA levels are modestly correlated with HBV DNA and moderately correlated with HBsAg levels before treatment.
However, these associations were not observed when patients were stratified by HBeAg status, which is consistent with the results of previous studies. 10,15 Notably, a few studies found that the positive correlation between HBV pgRNA and HBsAg remained in HBeAgpositive patients but was absent in HBeAg-negative patients, 9,24

| CON CLUS IONS
In conclusion, our study reveals the HBV pgRNA distribution in HIV/ HBV coinfected Chinese patients from a multicentre cohort pre-and post-cART. After cART initiation, HBV pgRNA is still detectable even if HBV DNA is suppressed in most HBeAg-positive patients and undetectable HBV pgRNA levels may be associated with HBeAg seroconversion. Based on these results, we suggest that anti-HBV treatment is still needed for HIV/HBV coinfected patients with detectable HBV pgRNA even after long-term cART.

AUTH O R CO NTR I B UTI O N S
LX performed laboratory experiments, acquired, analysed all the data and drafted the manuscript; XDL, LFL, XSL, YH and TZ performed laboratory experiments; XJS, YLL and WC collected the clinical data; TSL designed the study, evaluated and interpreted data, obtained funding; All authors participated in the manuscript review and approved the final version of the text.

ACK N OWLED G EM ENT
We thank all participants for their contributions to this study and the PUMCH team who established the original cohort.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

F I G U R E 7
Recovery of T-cell counts after the initiation of cART. The dynamics of CD4 + cell counts (A) and the CD4/CD8 ratio (B) after treatment in coinfected patients