HIV/HBV Coinfected Cohort: Participants were selected from a longitudinal study (ChiCTR2200064212) on HIV/AIDS conducted at Nanfang Hospital (Southern Medical University, Guangzhou, China). The inclusion criteria for the HIV/AIDS cohort included were: (1) age exceeding 18 years, (2) individuals with a confirmed HIV diagnosis according to the “Chinese guidelines for diagnosis and treatment of HIV/AIDS” (AIDS and Hepatitis C Professional Group Society of Infectious Diseases CMA and Chinese Center for Disease Control and Prevention, 2018), and (3) those who were not undergoing cART. After enrollment, patients underwent follow-up at three to six-month intervals, including routine clinical examinations and blood sample collection. Individuals with HIV/HBV coinfection, recruited from this HIV/AIDS cohort, were required to be HBsAg positive for no less than six months and initiate cART based on the guidelines’ recommendations as specified in the study’s protocol. Throughout the follow-up, antiretroviral intervention could be either halted, initiated, or modified per prevailing clinical norms, as determined by the attending physician. The enrolled individuals had undergone a minimum of one year of on-treatment follow-up. The primary endpoint of this study was HBsAg loss. Data collected from January 2019 to September 2023 were included in the analysis.

HBV Mono-infected Cohort: Participants were recruited from outpatients at the Hepatology Unit, at Nanfang Hospital, between January 2019 and September 2023. The inclusion criteria for this group were non-cirrhotic CHB patients who tested positive for HBsAg for a minimum of six months and had initiated NAs therapy (Entacavir or Tenofovir disoproxil fumarate)following international guidelines. Enrolled patients had regular evaluations of virological and serological parameters related to HBV, conducted at intervals of no more than 6 months. Data collected retrospectively from 2019 to 2023 were included in the analysis.

Individuals in both groups were excluded if they showed signs of autoimmune hepatitis, alcoholic liver disease, tested positive for hepatitis C or hepatitis D viruses, had concurrent alcohol misuse, malignancies, a history of liver transplantation, other liver diseases, or cancer. All participants provided informed consent.

The quantification of HBV DNA was carried out utilizing a domestic HBV DNA assay kit (Daan Gene Co, Ltd.; Sun Yat-sen University; Guangzhou, China) in strict accordance with the manufacturer’s instructions, with a lower limit of detection of 1000 copies/ml. Serological markers were quantitatively determined using an ARCHITECT I2000SR (Abbott Ireland Diagnostics Division). The HBsAg test featured a lower limit of detection of 0.05 IU/ml. HBsAg loss was defined as a serum HBsAg concentration of less than 0.05 IU/ml. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were determined at local laboratories, following standardized procedures. In this study, we calculated the AST-to-platelet ratio index (APRI) and the fibrosis index based on four factors (FIB-4).

HIV-1 RNA quantification was performed using the Roche RT-PCR assay, following the manufacturer’s instructions (Easy Q; Roche; lower limit of detection, 40 IU/mL). The Cells were stained with a panel of cell markers (FITC-CD4, CD8-PE, and CD3-PercP) and CountBright absolute counting beads. The cells were acquired on a BD FACS Canto II flow cytometer and the CD4+ and CD8+ lymphocyte counts were analyzed with FlowJo software. Antibodies and CountBright absolute counting beads were ordered from BD Biosciences.

Peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll-Hypaque centrifugation and cryopreserved. PBMCs were resuscitated and rested in a 37°C incubator overnight. PBMCs stimulated with Core peptide pools in RPMI medium 1640 complete medium (Gibco; Thermo Fisher Scientific) supplemented with 10% FBS (Fetal Bovine Serum). After PBMCs were added with 18-mer overlapping peptide pool covered C open reading frame (2 μg/mL, GL Biochem, Shanghai, China), CD49d (1 μg/mL; Biolengd), and CD28(1 μg/mL; Biolengd). Cells were then cultured for 10 days with recombinant IL-2 (10 ng/mL; PeproTech) added on days 3 and 7. Cells were collected, washed 3 times on day 10, and rested in a 37°C incubator for 24 hours. Following stimulation again with the Core peptide, IFN-gamma cytokines secretion of HBV-specific T cells was detected according to the manufacturer’s instructions (Human IFN-γ ELISpot PRO; MabTech). T-cell responses to DMSO were included as a negative control in each assay. Spots on the plates were quantified using ImmunoSpot (Cellular Technology Ltd., Shaker Heights, OH). Results were deemed positive if the number of spots per well exceeded twice the negative control’s background level, with a minimum of 5 spots per well.

The follow-up duration was determined by calculating the time from the initial inclusion date to either the most recent recorded follow-up or the point at which the participant was lost to follow-up. Data were presented as either mean ± standard deviation (SD) or median (interquartile range). We compared group attributes using the χ2 test for categorical variables and employed the Student t-test or Mann–Whitney test for continuous variables. To minimize potential confusion in clinical characteristics between the two groups, we utilized the PS-matching method. This involved caliper matching using the nearest available matching method in the Propensity Score Matching (PSM), considering variables such as age, sex, HBsAg, and ALT.

Follow-up data were analyzed through Kaplan–Meier analysis and the log-rank test. The relationship between variables and endpoints was investigated using Cox proportional hazards regression analysis. The statistical software used was SPSS for Windows (version 26.0; SPSS Inc., Chicago, IL, USA) for all data analysis. The analysis included the implementation of the “survival ROC” package in R to assess the performance of CD4 and HBsAg through time-dependent ROC curve estimation. Additionally, the variation in CD4 and CD8 T cell count was computed from the initial evaluation to the final follow-up using a linear mixed effects model with the “nlme” package in R. A significance level of p<0.05 was applied for all analyses.

The HIV/HBV coinfection rate in our cohort was 14.6% (170/2152). From the total of 170 individuals suffering from chronic hepatitis B, we eliminated the subsequent cases for analysis: 2 patients who showed positive results for anti-HCV, 1 patient diagnosed with diffuse large B-cell lymphoma, 1 patient with hepatocellular carcinoma, 10 patients with less than six months of follow-up, and 18 patients with incomplete clinical details ( Supplementary Figure 1 ). A comprehensive examination was conducted on a combined total of 138 individuals with both HIV and HBV infections, who were monitored for a median period of 2.0 years. 129 (93.5%) patients initiated tenofovir disoproxil fumarate (TDF)+lamivudine (3TC)-based HBV therapy cART schedules, 8 (5.6%) used 3TC, and one received tenofovir alafenamide fumarate/emtricitabine. The demographic and clinical characteristics of 138 patients are summarized in Table 1 . In the HBV Mono-Infected Cohort, 555 adult patients with HBV were initially screened from outpatients. Among them, 480 eligible patients were subsequently enrolled in this study ( Supplementary Figure 1 ). 326 (67.9%) patients received entecavir (ETV),128(26.7%) received tenofovir disoproxil fumarate (TDF), 25 (5.2%) received tenofovir alafenamide fumarate (TAF). The clinical characteristics of the HBV mono-infection are detailed in Table 1 .

During enrolment, individuals in HIV/HBV coinfection were found to be younger compared to HBV mono-infection (35.37 vs. 38.28 years of age, p<0.001). A total of 13 patients achieved HBsAg loss with a 3-year cumulative incidence of 11.50% in HIV/HBV coinfection. In contrast, only 1 patient in the HBV mono-infection group experienced HBsAg loss, leading to an incidence of 0.6% (p<0.001) ( Figure 1A ). At year 1, the levels of HBsAg were notably reduced in individuals with HIV/HBV coinfection compared to those with HBV mono-infection (2.61 vs. 3.16 log₁₀ IU/ml, p=0.001). This trend continued at year 2 (2.77 vs. 3.09 log₁₀ IU/ml, p=0.040) and year 3 (2.43 vs. 3.02 log10 IU/ml, p=0.035) ( Figure 1C ).

After applying the propensity score (PS)-matching technique, there were 99 individuals in the HIV-HBV coinfected category and 155 individuals in the HBV mono-infected category. At enrolment, no notable distinctions in clinical characteristics or levels of HBsAg were observed between the two groups ( Table 1 ). The 3-year cumulative occurrence of HBsAg loss was considerably greater in individuals with HIV/HBV coinfection compared to those with HBV mono-infection (12.5% vs. 1.0%, p<0.001) ( Figure 1B ). The PS-matched groups were compared over 3 years, revealing a continuous decrease in HIV/HBV coinfection. The HBsAg level in the coinfection group was significantly lower than that in the HBV-mono-infection group at year 1 (2.96 vs. 3.21 log₁₀ IU/ml, p=0.021) and year 2 (2.53 vs. 3.17 log₁₀ IU/ml, p=0.040) ( Figure 1D ).

To further assess the predictive capability of variables at enrolment for HBsAg loss in the HIV/HBV coinfected groups. Multivariable analysis showed that lower HBsAg level (HR 0.53; 95% CI 0.38-0.74. p<0.001) and CD4 cell counts <180 cells/uL (HR 0.32; 95% CI 0.10-0.96. p=0.042) at baseline was associated with an increased indicator of HBsAg loss ( Table 2 ). Furthermore, receiver-operating characteristic curve (ROC) curve analysis showed an area under the curve of 0.771 at year 1 for baseline HBsAg level and 0.758 at year 1 for baseline CD4 cell counts in predicting the incidence of HBsAg loss ( Figure 2 ). In predicting HBsAg loss at year 1, the determined threshold for CD4 cell counts was set at 180 cells/uL (with a sensitivity of 78% and specificity of 72%). The baseline of CD4 cell counts <180 cells/uL were associated with the highest indicator of HBsAg loss as compared with the level ≥180 cells/uL (15.9%&9.4% at year 3, p=0.028, Figure 3 ). At year 1, CD4 cell counts <180 cells/uL were associated with the highest indicator of HBsAg loss as compared with the level ≥180 cells/uL (19.0%&10.5% at year 3, p=0.045, Figure 3 ).

Following treatment, our study observed an increase in CD4 cell counts and a decrease in CD8 T cell count in people living with HIV/HBV coinfection. Notably, it was found that CD4 cell counts significantly increased (249.4 to 389.1, p<0.001) and CD8 T cell count significantly declined (978.1 to 733.4, p<0.001) from baseline to 1 year. Individuals with HIV/HBV coinfection experienced an annual rise of 65.9 cells/μL in CD4 cell counts. In comparison, CD8 T cell count in the same cohort declined by 110.4 cells/μL per year after cART ( Supplementary Figure 2 ). There was no significant difference observed in the CD4 cell counts and CD8 T cell count between patients with HBsAg loss and those without HBsAg loss groups after cART ( Supplementary Figure 2 ).

To further assess immunological factors in individuals with HIV/HBV coinfection, we measured IFN-γproduction by HBV-specific T cells( Figure 4 ). Due to the limited availability of PBMC samples, a total of 20 patients were tested at baseline, 4 of whom experienced HBsAg loss. After 1 year of treatment, 18 patients were tested, with 2 showing HBsAg loss. HBV-specific T-cell responses exhibited a possible increasing trend one year after cART initiation compared to baseline ( Figure 4C , p<0.05; Figure 4D , p>0.05). However, there was no significant difference in T-cell responses between the groups with and without HBsAg clearance ( Figures 4A, B ).