HBcAb seropositivity is correlated with poor HIV viremia control in an Italian cohort of HIV/HBV-coinfected patients on first-line therapy

The morbidity and mortality rates of human immunodeficiency virus (HIV)-hepatitis B virus (HBV) coinfection are higher than that of either infection alone. Outcomes and the virological response to antiretrovirals (combination antiretroviral therapy, cART) were explored in HIV/HBV subjects in a cohort of Italian patients treated with cART. A single-center retrospective analysis of patients enrolled from January 2007 to June 2018 was conducted by grouping patients by HBV status and recording baseline viro-immunological features, the history of virological failure, the efficacy of cART in achieving HIV viral undetectability, viral blip detection and viral rebound on follow up. Among 231 enrolled patients, 10 (4.3%) were HBV surface (s) antigen (HBsAg)-positive, 85 (36.8%) were positive for antibodies to HBV c antigen (HBcAb) and with or without antibodies to HBV s antigen (HBsAb), and 136 were (58.9%) HBV-negative. At baseline, HBcAb/HBsAb+/−-positive patients had lower CD4+ cell counts and CD4+ nadirs (188 cell/mmc, IQR 78–334, p = 0.02 and 176 cell/mmc, IQR 52–284, p = 0,001, respectively). There were significantly higher numbers of AIDS and non-AIDS events in the HBcAb+/HBsAb+/−-positive subjects than in the HBV-negative patients (41.1% vs 19.1%, p = 0.002 and 56.5% vs 28.7%, respectively, p ≤ 0.0001); additionally, HIV viremia undetectability was achieved a significantly longer time after cART was begun in the former than in the latter population (6 vs 4 months, p = 0.0001). Cox multivariable analysis confirmed that after starting cART, an HBcAb+/HBsAb+/−-positive status is a risk factor for a lower odds of achieving virological success and a higher risk of experiencing virological rebound (AHR 0.63, CI 95% 0.46–0.87, p = 0.004 and AHR 2.52, CI 95% 1.09–5.80, p = 0.030). HBcAb-positive status resulted in a delay in achieving HIV < 50 copies/mL and the appearance of viral rebound in course of cART, hence it is related to a poor control of HIV infection in a population of coinfected patients.

Based on these data, the European Clinical Practice Guidelines for the management of HBV infection 6 recommended the use of anti-HBV active drugs (NAs) containing cART in HIV-positive patients and strongly suggested avoiding the interruption of this type of treatment. Conversely, no suggestions were provided regarding the management of HIV-positive patients with HBV-resolved infections (i.e., the presence of anti-HBc and/or anti-HBs in the absence of HBsAg). Resolved HBV infection is known to be associated with HBV reactivation in immunocompromised patients subsequent to transplantation or chemotherapy for solid or hematological neoplasia 7 . As a component of resolved infection, occult hepatitis B Infection (OBI), which is defined as the absence of the HBs antigen (HBsAg) in the presence of intrahepatic or plasma HBV replication, is also known to be a risk factor for the evolution of HBV infection in cirrhosis, ESLD and HCC in immunocompromised patients 8 . Very few data are available regarding the influence of resolved HBV infection and OBI on HIV infection control 9,10 , especially in people with low-prevalence HBV infection.
The goal of this study was to explore whether having a resolved HBV infection affects overall health outcomes by comparing a group of cART-treated, HBV/HIV-coinfected Italian subjects to a group of HIV mono-infected patients. Clinical, virological and immunological data of patients seen from January 2007 until July 2018 at the Infectious Diseases Unit of the Policlinico Tor Vergata were retrospectively evaluated.

Materials and Methods
Study design. An observational retrospective study of 671 HIV-positive patients collected from January 2007 to July 2018 at the Infectious Diseases Unit of the Policlinico Tor Vergata in Rome, Italy was conducted. Specifically, for this study, a database was built that included all patients' data at baseline, at the start of cART treatment, and at follow-up visits. In particular, the following data were collected for all of the patients: hematochemical data and HBV and HCV serology at baseline, HIV-RNA viral load, CD4+ cell count at baseline and pre-cART, time since the HIV infection diagnosis and the start time of cART treatment. Data on flares in transaminases (defined as an increase in aspartate aminotransferase (AST) or alanine aminotransferase (ALT) > 50 IU/L), HIV viral blips (VB, defined as a single detection of HIV-RNA > 50 cp/mL), virological rebound (VR, defined as two consecutive HIV-RNA values ≥ 50 cp/ml or one > 1000 cp/mL with consequent changes in cART) and time required to achieve viral undetectability (virological success [VS], defined as two consecutive HIV-RNA < 50 cp/ mL) were collected during follow-up examinations. Moreover, data regarding the antiviral treatment for chronic hepatitis B (CHB) (LAM, TDF, Tenofovir Alafenamide [TAF] or Entecavir [ETV]) were also gathered. When available, HBV-DNA viral load data were collected.
No specific ethics committee's consent was required due to the retrospective nature of the study, which is based on information available from existing clinical documentation [Determination of the Italian Drug Agency (AIFA) of 20 March 2008]. With regard for privacy, all personal information was treated in a confidential manner, and all clinical data were anonymously analyzed. Inclusion and exclusion criteria. As shown in Fig. 1, of the 671 patients followed in our center, 401 were excluded due to a lack of virological or immunological data at the time of diagnosis (including patients who were non-adherent to follow-up visits, who transferred from other clinical centers and who voluntarily stopped first-line cART treatment), and 16 patients were excluded because of the loss of HBV serology data before the start of ART. Twenty-three patients were excluded due to the presence of transaminase flares dependent on hepatic drug toxicity (2 patients) or acute hepatitis A virus (HAV) infection (21 patients), which would invalidate the evaluation of transaminase flares. Two hundred thirty-one patients were ultimately studied. endpoints. The primary endpoint of this study was to investigate the influence of HBcAb + /HBsAb +/− status on time required to achieve an HIV viral load < 50 copies/mL after the initiation of cART and the onset of VB or VR after HIV VS. Subsequently, the effects of HBV antigen/antibody profiles (i.e., HBsAg, HBcAb and/or HBcAb/ HBsAb positivity) on overall mortality and the appearance of AIDS-related events were also evaluated.
Laboratory testing for the diagnosis of HBV and HiV infection. HBV serological markers were measured using immune-enzymatic assays (Roche/Cobas Diagnostics, Rotkreuz, Switzerland). Plasma HBV-DNA was identified using real-time polymerase chain reaction (lower limit of quantification: 20 IU/ml) (Roche/Cobas Ampliprep/Cobas Taqman, Rotkreuz, Switzerland). Plasma HIV-RNA levels were measured using a commercial test, with 20 copies/ml of HIV-RNA defined as the lower limit of quantification (COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0).

Statistical methods.
All statistical analyzes were conducted using STATA 14.2 (College Station, TX).
The study population is described using proportions and percentages for categorical values and median measurements and interquartile ranges (IQRs) for continuous values. The comparison between HBcAb + / HBsAb +/− -positive and HBV-negative patients was performed with the Kruskal-Wallis test for continuous variables and with the Chi-squared test or Fisher's exact test, when appropriate, for categorical variables.
Kaplan-Meier curves were used to estimate the time required to achieve and the probability of achieving VS after the start of cART and the probabilities of experiencing VB and VR in the HBcAb + /HBsAb +/− -positive and HBV-negative groups of patients.
Cox regression analysis was performed to evaluate the association between HBcAb and the risk of achieving VS or experiencing VR after controlling for other potential confounding factors under the assumption of proportionality of the hazards. The following variables were considered potential confounders: age, AST levels, hepatitis C virus (HCV) coinfection, pre-cART plasma HIV-RNA, pre-cART CD4 count, anti-HBV treatment, risk factor for HIV acquisition, calendar year, and type of cART.

Comparison between HBcAb
HIV virological response to cART in HBcAb-positive and -negative patients. KM estimates were produced to analyze the ability to reach the VS after the start of first-line cART and the risk of incurring VR after virological success in the 2 groups of HBcAb-positive and -negative HIV patients. In Fig. 2 www.nature.com/scientificreports www.nature.com/scientificreports/ that, by 12 months after cART start, HBcAb-positive patients had a longer median time and a lower probability of achieving VS compared to HBcAb-negative patients (89.4% vs 95.6%, p < 0.001) (Panel A). Concerning the probability of experiencing a VB, Kaplan-Meier estimates showed that by the 84 th month after the achievement of VS, HBcAb-positive patients had a higher probability of experiencing a VB compared to HBcAb-negative patients (26.7% vs 89.2%, p < 0.001) (Fig. 2, Panel B).
Finally, concerning VR, in Fig. 2 panel C, it is shown that by the 84 th month after the achievement of VS, HBcAb-positive patients had a higher probability of experiencing a VR compared to HBV-negative patients (13.4% vs 27.2%, p = 0.011). Patients who experienced VR had a higher number of blips (median number of VB 2 [IQR 1-3]) compared to those who did not experience VR (median number of VB 0 [IQR 0-1]) (p < 0.001 Mann-Whitney test) (data not shown).
Therefore, Kaplan Meier estimates showed that antiHBc-positive patients exhibited less virological control from the beginning of cART, including a delayed VS and subsequently more frequent occurrence of VB and VR. Table 4 shows the adjusted hazard ratio (AHR) of achieving VS and experiencing VR after the start of cART. The following potential confounders were considered: age, AST levels, HCV coinfection, pre-cART plasma HIV-RNA, pre-cART CD4 count, anti-HBV treatment, risk factor for HIV acquisition, calendar year, and type of cART. HBcAb positivity, either with or without HBsAb, was associated with a lower AHR of achieving VS (AHR 0.63, CI 95% 0.46-0.87, p = 0.004) and an increased risk of VR after cART beginning (AHR 2.52, CI 95% 1.09-5.80, p = 0.030). After adjustment for potential confounders, HBcAb-positive status was confirmed to be a risk factor for lower HIV virological control after the initiation of first-line cART. Moreover, patients with a high pre-cART HIV viral load had a lower AHR of achieving VS (HIV-RNA > 100.000 and <500.000 cp/mL, AHR 0.68, CI 95% 0.47-0.98, p = 0.038; and HIV-RNA > 500.000 cp/mL AHR 0.40, CI 95% 0.25-0.66, p < 0.001), and patients with a pre-cART HIV-RNA > 500.000 cp/mL had a higher AHR of VR than was found in those with a lower level of viremia (AHR 4.99, CI95% 1.42-17.55, p = 0.012). The presence of integrase inhibitors (INIs) plus 2 nucleos(t)ide reverse transcriptase inhibitors (NRTIs) in the cART regimen was associated with a higher AHR of achieving VS (AHR 1.85, CI 95% 1.20-2.85, p = 0.006) as was cART initiation in a more recent calendar year (AHR 1.11, CI 95% 1.05-1.18, p < 0.001).

Discussion
In our cohort of HIV-positive patients who started cART, the presence of serological markers of a past HBV infection was significantly correlated with a delay in achieving HIV viremia undetectability and the appearance of HIV VR after the start of cART (AHR 2.52, CI 95% 1.09-5.80, p = 0.030 and AHR 0.63, CI 95% 0.46-0.87, p 0.004, respectively). Moreover, significantly higher numbers of AIDS and non-AIDS-related events were found in the group of HIV/HBcAb-positive patients than in HIV mono-infected individuals.
In our population, 41% of the 231 patients included in this study had an ongoing or resolved HBV infection. Slightly lower rates of HBV-resolved infection or CHB were found in two American cohorts, which reported values of 23% and 35% in HIV seroconverters and HIV patients on cART, respectively 11,12 .   In our study cohort, HBcAb + /HBsAb +/− -positive subjects presented flares of transaminases more frequently than was found in HBV-negative patients (65.9% vs 36%, p < 0.0001), suggesting the potentially transient reactivation of HBV. Hepatitis flares have been widely documented in HBcAb-positive patients during immunosuppressive therapy 7 , whereas the association of liver diseases with signs of resolved HBV infection in coinfected HIV people is difficult to ascertain as few studies have assessed the association between liver damage and both isolated anti-HBc patterns and occult HBV infections 14 . In the Veterans Aging Cohort Study 15 , the presence of HBcAb was associated with more advanced hepatic fibrosis in HIV-HCV coinfected patients, while Morsica and his colleagues 9 showed that 21% of HBcAb-positive HIV-coinfected patients in the Italian national cohort (ICONA) presented detectable HBV-DNA associated with an increase in transaminase levels.
An increased number of AIDS and non-AIDS events were documented in the follow-up of our study cohort, which is associated with ongoing or resolved HBV infections. A similar result was reported by Chun HM and his collaborators 12 , who observed that the risk of AIDS and death was high in both CHB and resolved HBV subjects from a cohort of 2536 HIV-positive cART recipients. Similarly, HBV coinfection negatively impacted patient survival in a group of HIV seroconverters, among whom the rates of AIDS and death were significantly higher in CHB patients and increased, although not significantly, in patients with resolved HBV infections 11 .
A series of potential factors have been shown to form the basis of the HBV and HIV interaction and to result in the acceleration of both infection courses 16 . HIV exerts direct activity on hepatocytes and Kupffer cells, thereby contributing to liver inflammation. Moreover, the rate of microbial translocation was higher in HIV/ HBV-coinfected individuals than in HBV mono-infected individuals 17 . Microbial translocation has been associated with ongoing immune dysfunction and contributes to the persistence of chronic immune activation and inflammation, which have been associated with the development of comorbidities in HIV-infected persons, even those receiving effective cART 18,19 .
In our study cohort, virological control was worse in HIV/HBcAb/HBsAb +/− patients than in HIV mono-infected subjects in a multivariate analysis, and the status of the resolved HBV coinfection was strongly correlated with HIV VB onset in HIV monoinfected people (89.2% vs 26.7%, p < 0.001).
The appearance of VB is commonly described in cART-treated HIV patients 20 , and some authors have found a correlation between VB and subsequent VR [21][22][23] . In our study, we found that the median VB was higher in patients with VR than in those without VR. The origin of VB remains uncertain, but an increased HIV replication rate has been correlated with the presence of other infections and with certain vaccinations 24,25 . A number of viral coinfections, when present in HIV-positive subjects, seem to contribute to unfavorable outcomes. Cytomegalovirus coinfection was associated with HIV disease progression 26,27 , and hepatitis C virus is a leading cause of non-AIDS-related mortality among HIV-coinfected patients 28 .
HBV DNA is less rarely detected among patients with HIV and those with isolated anti-HBc (OBI patients), with its rate reported to vary from 0.6% to 89% in several studies 29 , and is related to a poor immune status 8 . Therefore, it is possible to argue that occasional, although limited, HBV replications may also contribute to the appearance of HIV viremia during the course of cART.
Before any conclusions can be drawn, some of the limitations of our study need to be discussed. First, the lack of HBV-DNA data in our population was a major limitation. Currently, no shared indications are used in the monitoring of HBV viremia in coinfected patients with resolved HBV infections, but it may be interesting to ascertain whether poor control of HIV viremia during cART is correlated with the simultaneous presence of HBV-DNA in patients with resolved HBV infections. Second, all of the evaluated patients were included from a   Table 4. Cox regression models for estimated factors with predictive impact on VS and VR. a Adjusted for age, AST levels, HCV coinfection, pre-ART plasma HIV-RNA, pre-ART CD4 count, anti-HBV treatment, risk factor, calendar year, and type of ART. b Reference (dummy). Bold variables were significantly associated with virological response (p < 0.05). VS: virological success; VR: virological rebound; 3TC: lamivudine; AST: alanine aminotransferase; ART: antiretroviral therapy; INI: integrase inhibitor; NRTI: nucleos(t)ide reverse transcriptase inhibitor; NNRTI: Non-NRTI; PIb: ritonavir/cobicistat boosted protease inhibitor.