Reduction of Hepatitis B Surface Antigen More Pronounced In Pegylated Interferon Alpha Therapy Combined With Nucleotide Analogues Than Nucleoside Analogues In Chronic Hepatitis B Patients

Background: Nucleotide analogues (NTs) monotherapy may have a greater effect on reducing hepatitis B surface antigen (HBsAg) than nucleoside analogues (NSs) due to their immunomodulatory function. However, this superiority remains unknown when combined with pegylated interferon α (PegIFNα). The study aimed to explore whether NTs have greater antiviral effects than NSs in combination therapy with PegIFNα. Methods: Chronic hepatitis B (CHB) patients treated with PegIFNα plus nucleos(t)ide analogues (NAs) were retrospectively recruited. Ecacy and the predictors of hepatitis B surface antigen (HBsAg) reduction > 1 log 10 IU/mL at 48 weeks were analyzed. Results: A total of 95 patients were investigated, including in PegIFNα plus NSs group and in PegIFNα plus NTs group. Propensity score matching (PSM) was performed. The PegIFNα + NTs group had a greater reduction of HBsAg (−3.48 vs −2.33 log 10 IU/mL, P = 0.038) and a higher proportion of patients with HBsAg reduction > 1 log 10 IU/mL (100.0% vs 72.2%, P =0.003) even after PSM. However, HBsAg and hepatitis B e-antigen (HBeAg) loss rates, HBeAg seroconversion rates, degree of HBeAg and hepatitis B virus (HBV) DNA decline, HBV DNA undetectable rates, and alanine aminotransferase (ALT) normalization rates showed no signicant differences. Higher platelet counts (OR = 1.043, 95%CI = 1.002–1.085) and PegIFNα plus NTs (OR = 77.861, 95%CI = 3.923–1545.273) were independent predictors for HBsAg reduction > 1 log 10 IU/mL at 48 weeks. Conclusion: This study suggests that PegIFNα plus NTs led to more HBsAg reduction.


Introduction
Chronic hepatitis B (CHB) is a global infectious disease. There are currently about 70 million people infected with chronic hepatitis B virus (HBV) in China, of whom more than 20 million are CHB patients. Those patients are at high risk of liver cirrhosis and hepatocellular carcinoma (HCC) especially in developing countries [1] presenting an immense medical burden [2]. The persistence of covalently closed circular DNA (cccDNA) within hepatocytes is relevant for chronic HBV infection [3]. Hepatitis B surface antigen (HBsAg) is a surrogate marker for cccDNA transcriptional activity [3][4][5]. The disappearance of HBsAg, accompanied by a sustained virological response, loss of hepatitis B e-antigen (HBeAg), recovery of alanine aminotransferase (ALT), and improvement of liver tissue lesions is de ned as functional cure. Thus, major guidelines consider sustained HBsAg disappearance after drug withdrawal an ideal treatment end point [6,7].
However, HBsAg loss is not common with current standard antiviral strategies including nucleos(t)ide analogues (NAs) and pegylated interferon-alpha (PegIFNα). Reduction of HBsAg level is often associated with better outcomes including minimizing cirrhosis and HCC and is conductive to HBsAg clearance, therefore, it is often used as an e cacy indicator. NAs are economic and convenient but cannot directly act on cccDNA.
Patients usually need to take long-term, or even life-long, medications, bringing unavoidable economic and psychological burdens, as well as drug resistance problems. In contrast, PegIFNα can reduce HBsAg more thoroughly in a subset of patients [8]. Low virologic response rate in PegIFNα monotherapy and poor reduction of HBsAg in NAs monotherapy shed light on combination strategies.
Previous studies have proven that PegIFNα combined with NAs had better clinical effects than PegIFNα or NAs monotherapy [9][10][11], particularly in reducing HBsAg level [12] and enhancing HBsAg loss rate [13]. Additionally, different NAs can vary in e cacy. Nucleotide analogues, including tenofovir disoproxil fumarate (TDF), adefovir dipivoxil (ADV), and tenofovir alafenamide (TAF), are not only structurally but also functionally different from nucleoside analogues like entecavir (ETV) and lamivudine (LAM). The reduction in HBsAg was signi cantly greater in the TDF arm than the ETV arm in NAs naïve patients according to a small randomized controlled trail [14]. Switching from ETV to TDF or TAF lead to signi cantly more decline of HBsAg [15,16]. Interestingly, nucleotide analogues have also been found with an additional immunological effect in interferon lambda 3 (IFN-λ3) induction compared to nucleoside analogues [17]. Meanwhile, TDF treatment could be associated with a signi cantly lower risk of HCC than ETV based on recent studies [18,19]. Still, the comparison remains controversial [20]. In combination strategies, PegIFNα combined with TDF can reach an HBsAg clearance rate as high as 10.4% [9], but the rate is only 0.8% when combined with ETV [11]. According to this indirect comparison, PegIFNα combined with TDF (which represents nucleotide analogues) appears to reach a better HBsAg clearance rate than PegIFNα combined with ETV (which represents nucleoside analogues) when the treatment durations are similar. However, there is currently no study directly comparing the e cacy of these two types of combination therapy. Therefore, it is useful to compare HBsAg reduction e cacy for PegIFNα therapy combined with NTs or NSs in CHB patients so that we conducted a retrospective study using the data of CHB patients treated with a combination of PegIFNα plus different NAs at Huashan Hospital of Fudan University from October 2011 to December 2018.

Patients
Between October 2011 and December 2018, a total of 159 consecutive PegIFNα-naïve CHB patients who received PegIFNα for at least 48 weeks and combined with NAs during the course were retrospectively enrolled from two clinical centers: Huashan Hospital of Fudan University (Shanghai, China). Chronic HBV infection was de ned as being HBsAg positive and/or HBV DNA positive for at least six months before enrollment. The combination therapy could be add-on (adding on NAs during the therapy of PegIFNα) and NAs experienced. NAs used were maintained consistent with the prior type. Sixty-four patients in total were excluded: four had underlying chronic hepatitis C, autoimmune hepatitis, HIV or tumor; seven had used PegIFNα for more than 48 weeks when NAs were added to the therapeutic regimen; one combined nucleoside analogues and nucleotide analogues at the same time; six used the combination therapy for less than 12 weeks; and forty-six had a PegIFNα therapy duration less than 48 weeks or incomplete data at an important time. In this study, 95 patients were ultimately included, of which one group included those who received PegIFNα combined with nucleoside analogues (ETV) (n = 18), and the other group included patients treated with PegIFNα combined with nucleotide analogues (TDF or ADV) (n = 77). This retrospective study was conducted under the approval of the Ethics Committee for Huashan Hospital of Fudan University and in accordance with the Declaration of Helsinki. Written informed consent was obtained for all patients included.
Clinical data All patients' baseline clinical data and laboratory test results were recorded. Clinical data included demographic data, previous history of hepatitis B and treatment history (name, dose, time, and complications of medication). Laboratory test results consisted of blood routine, liver and kidney function, electrolytes and hepatitis B related indicators. The baseline was de ned as the start of PegIFNα therapy. The duration of PegIFNα therapy was at least 48 weeks with a combination therapy for a minimum of 12 weeks. Laboratory examination results at 0, 12, 24, 36, and 48 weeks and the medication changes during treatment (complications, dose changes, and addition or withdrawal of NAs) were recorded in detail.

De nition of treatment response
The primary endpoint was a reduction of HBsAg levels from the baseline at 48 weeks of treatment. Serological responses: (1) Proportion of patients with HBsAg reduction > 1 log 10 IU/mL from baseline; (2) HBsAg loss rate; (3) Reduction levels of HBeAg from baseline at 48 weeks; (4) HBeAg loss rate and HBeAg seroconversion rate Serum HBsAg levels were determined by Elecsys HBsAg II assay (Roche Diagnostics GmbH, Mannheim, Germany; linear range, 0.05 to 52,000 IU/mL). HBsAg loss was de ned as HBsAg < 0.05 IU/mL. HBV DNA was measured using Taqman uorescence quanti cation, and the lower limit of detection was 500 IU/mL. Routine biochemical and hematological tests were performed locally. The upper normal limit of ALT was 40 IU/L. Data from laboratory assessments were collected at baseline, and at 12, 24, 36, and 48 weeks of treatment.

Statistical analysis
Continuous variables are represented by the mean ± standard deviation (SD) and median (interquartile range [IQR]). Independent t tests were used to compare continuous variables with normally distributed data (Z-score between ± 1.96, which was calculated by skewness and kurtosis), while Mann-Whitney U tests were used to compare continuous variables with a skewed distribution. Categorical data were presented as n (%) and analyzed by the chi-squared test. Differences among groups were evaluated using one-way analysis of variance (ANOVA), if the variances were homogeneous and LSD-T test was used for intergroup comparison. Otherwise, the Kruskal-Wallis test (K-W test) for nonparametric statistics was conducted. Multivariate logistic regression analysis was applied to determine the predictors that affected HBsAg reduction > 1 log 10 IU/mL from baseline at 48 weeks of treatment. To adjust for potential bias that could in uence the results, including sample size with excessive deviation, we applied a balanced study on the basis of the propensity score-matching (PSM) technique at a 1:1 ratio with a caliper of 0.2 separately between PegIFNα + ETV group and PegIFNα + ADV group or PegIFNα + ETV group and PegIFNα + TDF group. Age, HBsAg, and prior treatment duration of NAs before combined with PegIFNα were imputed for PSM. When the absolute value of the standard difference was less than 10%, the balance of the variables between the groups was considered acceptable. Differences were considered signi cant at a two-tailed P < 0.05. All statistical analyses were carried out using SPSS statistical software version 24.0 (IBM, Armonk, NY, USA).

Ethical approval
This study was approved by the Institutional Ethics Committee of Huashan Hospital, Fudan University, China (KY2018-251). Informed consent was obtained from all patients.

Baseline characteristics
A total of 95 cases were selected for effective analysis, including 18 patients who received a therapy combining PegIFNα with nucleoside analogues (PegIFNα + NSs) and 77 patients who received PegIFNα combined with nucleotide analogues (PegIFNα + NTs) (Fig. 1). Subgroups of different drugs combined were PegIFNα + ETV, PegIFNα + ADV and PegIFNα + TDF. Before PSM, there was no signi cant difference in baseline information between the two groups or among different drugs (Table 1). PSM was performed, yielding 18 patients matched in each group. After PSM, relative multivariate imbalance L1 waslower than the imbalance before PSM, indicating a better balance. No covariate exhibited a large imbalance, and all of the covariates reached a balance within 10%. There were no statistically signi cant differences among patients in each group after PSM (Table 1).  (Table 2). Both PegIFNα + ADV group (−3.47 vs −2.33 log 10 IU/mL, P = 0.029) and PegIFNα + TDF group (−3.44 vs −2.33 log 10 IU/mL, P = 0.046) reduced signi cantly more HBsAg levels than PegIFNα + ETV group. After PSM, the change in HBsAg from baseline was −3.52 log 10 IU/mL in the PegIFNα + NTs group and −2.33 log 10 IU/mL (P = 0.032) in the PegIFNα+NSs group (Table 3). HBsAg declined signi cantly more in the PegIFNα + NTs group (Fig.2 A, D). In subgroup comparison, both PegIFNα + ADV group (−3.55 vs −2.33 log 10 IU/mL, P = 0.035) and PegIFNα + TDF group (−3.49 vs −2.33 log 10 IU/mL, P = 0.039) reduced HBsAg more than PegIFNα + ETV group (Table 3).
We further analyzed patients with HBsAg loss after receiving different treatments. Before PSM, four patients (22.2%) achieved HBsAg loss in the PegIFNα + NSs group, while only ve patients (6.5%) in the PegIFNα + NTs group achieved the same, but the difference was not statistically signi cant (P = 0.109) ( Table 3). After PSM, patients achieving HBsAg loss in the PegIFNα + NTs and PegIFNα + NSs group were three (8.3%) and four (22.2%), respectively, without signi cant statistical difference (P = 0.205) (Fig. 3). Subgroup analysis did not show a statistically signi cant difference ( Table 2, 3).

Biochemical response
For patients with elevated baseline ALT, the proportion of those who returned to normal levels at 48 weeks also differed between the two groups, although the difference was not statistically signi cant. In all, 33 patients (43.4%) in the PegIFNα + NTs group and nine patients (52.9%) in the PegIFNα + NSs group achieved a biochemical response of serum ALT level < 40 IU/L at the end of therapy before PSM (P = 0.476) ( Table 2). After matching, 15 patients (42.9%) and nine patients (52.9%) in the PegIFNα + NTs and PegIFNα + NSs groups had biochemical responses, respectively (P = 0.494) (Fig. 3). Biochemical responses did not vary substantially by subgroups ( Table 2, 3).   inhibiting HBV DNA polymerase and reverse transcriptase. Whereas, they cannot directly inhibit the transcriptional activity of cccDNA. Therefore, it is di cult to obtain durable immunological control so that clearance and seroconversion of HBsAg and HBeAg are not easily achievable. As a result, a long-term medication is often required. PegIFNα can enhance innate immunity, trigger T cell-mediated immune responses, and prevent HBV protein formation and a depleted cccDNA pool [21], resulting in superior effectiveness to NAs in reducing HBsAg [8]. Nearly one-third of PegIFNα responders achieve HBsAg clearance. Strong inhibition of viral replication by NAs can assist PegIFNα's immunomodulatory effect [22]. Hence, a combination strategy with PegIFNα plus NAs is not only theoretically feasible, but also an inevitable trend for future development. Before a new generation of effective drugs is introduced and popularized, exploration of the combination treatment has become a major focus of current research.
There have been a number of studies on the e cacy of combination therapy, among which many have shown combination therapy to be superior to monotherapy in reducing HBsAg levels [9,23,24] and found that combination therapy could even signi cantly increase HBsAg loss rate (9.1% vs 2.8%) [24]. Compared with NAs monotherapy, combination therapy resulted in a higher percentage of HBeAg loss (26% vs 13%, at 96 weeks) [21] and a higher HBeAg seroconversion rate (15% vs 5%, at 48 weeks) [25] as well. Therefore, it is obvious that combination therapy has prominent advantages over monotherapy, but the baseline conditions, optimal treatment duration, and sustained response rate of combination therapy require further exploration.
At the same time, it remains controversial whether e cacy differs between nucleotide analogues and nucleoside analogues when combined with PegIFNα. The two types of oral drugs have been found to be functionally different especially in HBsAg reduction. Koike et al. found that TDF reduced signi cantly more HBsAg levels at week 24 (-0.147 vs -0.027 log 10 IU/mL, P < 0.05) and 48 (-0.208 vs -0.051 log 10 IU/mL, P < 0.05) in NAs naïve patients [14]. Furthermore, HBeAg negative patients whose HBsAg had not been reduced in 48 weeks during ETV treatment had a signi cantly higher HBsAg reduction after switching to TDF or TAF than in the ETV continuation group [15]. HBV infection is a risk factor for hepatocarcinogenesis. Nevertheless controversial, previous researches have proven that TDF treatment was associated with lower risk of HCC than ETV therapy. A large retrospective analysis in China found that over a median follow-up time of 3.6 years, 4.9% ETV-treated patients developed HCC while it occurred in only 0.6% TDF-treated patients [19]. Similarly, a research in Korea had a consistent nding that the annual incidence rate of HCC was signi cantly lower in the TDF group than ETV group (0.64 vs 1.06 per 100 person-year) [18]. Notably, studies have indicated that patients treated with nucleotide analogues, especially ADV, have higher serum IFN-λ3 levels than those treated with nucleoside analogues [26,27]. The ability of IFN-λ3 to induce interferon-stimulated genes (ISGs) in Huh7 cell lines is stronger than that of interferon lambda 1/2 (IFN-λ1/λ2), and this ability is weaker but longer-lasting monotherapy reduced HBsAg signi cantly more than addition of ETV to Peg-IFNα-2b (−1.799 log 10 IU/mL vs −1.078 log 10 IU/mL, P = 0.0491) [30]. It was an important result as it compared the addition of TDF or ETV to Peg-IFNα-2b directly. However, considering the small sample size and the restrictive conditions for the selected population, it slightly lack universality and a larger sample size study is required to verify the results. Therefore, it is presently still no so clear whether PegIFNα combined with different NAs in uences HBsAg reduction and clearance. The loss rate of HBeAg after 48 weeks was similar between PegIFNα + TDF and PegIFNα + ETV (29.0% vs 31.0%) [31]. Recent data from another study pointed out that PegIFNα combined with TDF could improve HBeAg responses in a short time. No advantages were found when PegIFNα was combined with LAM or ETV [32]. But Lin et al. showed than the HBeAg loss rate was signi cantly higher in TDF add-on group than that in ETV add-on group at week 48 (40% vs 10%, P = 0.028) [30]. Interestingly, these studies suggested a possibility that PegIFNα combined with different NAs could have different e cacies, but direct evidence was demanded and mechanism behind the differences need to be discussed. Based on these ndings, we conducted this retrospective study to provide this evidence. TAF has only been launched in recent years, and with insu cient studies discussing the e cacy of PegIFNα plus TAF, we therefore did not include patients who received TAF in the current study. Meanwhile, no patients in our cohort used LAM, so the only nucleoside analogue analyzed was ETV. To our knowledge, our study was the rst to retrospectively compare HBsAg level reduction e cacy for CHB patients treated with different NAs in PegIFNα combination therapy no matter which combination strategy was adopted. This could be helpful to prove that the difference in reduction was due to the types of NAs.
In order to minimize the impact of bias, PSM was performed to eliminate the inequality caused by excessive deviation of the general data and sample size. After PSM, the results showed that the HBsAg of the PegIFNα + NSs group decreased by an average of −2.33 log 10 IU/mL from baseline at 48 weeks, while it decreased signi cantly more in the PegIFNα + NTs group, by an average of −3.52 log 10 IU/mL (P = 0.032). The reductions of HBsAg in both groups were more than the reductions in Lin's study (Lin et al. 2020). This might be because our study had a longer combination course and some patients had a prior treatment of NAs. The proportion of patients achieving HBsAg reduction > 1 log 10 IU/mL was signi cantly higher at 48 weeks in the PegIFNα + NTs group compared to the PegIFNα + NSs group (100% vs 72.2%, P = 0.003). However, even after PSM adjustment, no signi cant differences between the two groups were found in the following indicators: HBsAg loss rate, HBV DNA reduction, HBeAg reduction, HBeAg loss rate, HBeAg seroconversion rate, HBV DNA undetectable rate, and ALT normalization rate. The observation end point of this study was the 48th week of treatment, and subsequent follow-up had not yet been carried out, resulting in di culty achieving HBsAg clearance especially for antiviral treatment-naïve patients. The ability to maintain HBsAg clearance steadily after combination therapy also cannot be con rmed. Another reason for the signi cant differences in decline levels, but not in HBsAg loss rates, may be the small sample size. Based on the results of our study, we believe that nucleotide analogues can signi cantly reduce more HBsAg than nucleoside analogues when combined with PegIFNα. This reduction will contribute to achieving HBsAg clearance and even functional cure. In our study, the proportion of patients who simultaneously reached HBV DNA below the lower detection limit and HBsAg reduction > 1 log 10 IU/mL from baseline at 48 weeks differed between PegIFNα + ETV group and PegIFNα + TDF group after PSM (100.0% vs 72.2%, P = 0.045). This result exempli es the dual effectiveness of combination therapy with TDF over combination therapy with ETV in inhibiting viral replication and reducing HBsAg levels simultaneously.
Furthermore, multivariate logistic regression showed that treatment with PegIFNα plus nucleotide analogues was an independent predictor for HBsAg decline > 1 log 10 IU/mL at 48 weeks, suggesting that the combination of PegIFNα and nucleotide analogues can increase HBsAg decline. Higher platelet count was also an independent predictor for HBsAg reduction > 1 log 10 IU/mL.
Combination strategies been studied include "De novo", "NA-experienced", "add-on", and "switch-to". Several studies have shown that the "NA-experienced" strategy seemed to be the best. The "switch-to" strategy was particularly effective and improved HBsAg clearance [13,29,33]. This may be because the direct antiviral activity of NAs can lead to virological suppression, which can further improve the immunomodulatory effect of PegIFNα, thereby maximizing the advantages of combination therapy. Among the patients included in this study, the number of NA-experienced patients was relatively small and was prone to bias, so no statistical analysis of this sub-population was conducted.
Limitations of our study include that it is a retrospective study with a small sample size and short therapy duration without a long-term follow-up. Furthermore, the combination strategy was not precisely uniform although the duration of combination therapy had been guaranteed to be at least 24 weeks. Even though, the prior treatment duration and drugs before combination for NAs-experienced patients, the weeks of adding-on NAs for "add-on" patients and the total weeks of combination at baseline before and after PSM were not statistically different so the following analysis was considered reliable. Further randomized controlled trials are required for veri cation, and patients who are NAs-experienced for at least 48 weeks before the initiation of PegIFNα add-on need to be particularly examined.

Conclusion
In conclusion, reduction of HBsAg was more pronounced in PegIFNα therapy combined with nucleotide analogues than nucleoside analogues, a nding that will be bene cial for promoting further HBsAg clearance and functional cure. This result can provide a basis for clinical decision-making. Similar results and related mechanisms need to be further con rmed.