S抗原转阴后HBVDNA可以仍然存在(2005.4)

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HBV DNA Can Persist After HBsAg Seroconversion


  
  "ersistence of HBV-DNA in children with chronic hepatitis B who seroconverted to anti-HBs antibodies after interferon-Eo therapy: correlation with specific IgG subclass responses to HBsAg"

Journal of Hepatology
April 2005

"... it was generally believed that the disappearance of HBsAg from the serum and appearance of anti-HBs, together with normalization of liver biochemical tests, indicate complete recovery from HBV infection..

...... we analysed the presence of HBV-DNA in the sera of 37 patients with chronic hepatitis B who developed anti-HBs antibodies after IFN-Eo therapy that was given 4-10 years earlier..

...This study showed that HBV-DNA may persist for a long time after IFN-Eo therapy despite the appearance of anti-HBs antibodies..

... the study demonstrated that 13 of 37 (35.1%) patients who tested positive for anti-HBs had HBV-DNA in their sera and/or circulating immune complexes as long as 10 years after seroconversion...

.. The mechanisms responsible for HBV-DNA persistence, despite full seroconversion, are still unclear...

... Our results showed that 5.3% of patients again tested positive for HBsAg, and in one of them, HBsAg/anti-HBs were detected simultaneously..

..the authors conclude, .the results of the present study investigating the IgG subclass response to HBsAg as a biomarker of the T helper cell response provide, for the first time, evidence that the immune response in HBV-DNA-positive patients differs from that found in HBV-DNA-negative and confirm our earlier observations that immunomodulatory effect of IFN-Eo monotherapy is insufficient and non-selective. We suggest that monitoring of IgG anti-HBs profiles may be of predictive value for HBV-DNA persistence..."

Authors: Hanna Gregoreka, Katarzyna Dzier_anowska-Fangrata, Marek Woynarowskib, Paulina J—_wiaka, Ewa Witkowska-Vogtta, Jerzy Sochab, Ma_gorzata Syczewskac, Kazimierz Madali_skiad aDepartment of Microbiology and Clinical Immunology, The Children's Memorial Health Institute, 04 730 Warsaw, Poland b Department of Gastroenterology, Hepatology and Immunology, The Children's Memorial Health Institute, 04 730 Warsaw, Poland c Rehabilitation Clinic, The Children's Memorial Health Institute, 04 730 Warsaw, Poland d Department of Immunopathology, National Institute of Hygiene, 04 736 Warsaw, Poland

See Editorial

INTRODUCTION
Chronic hepatitis B virus (HBV) infection may lead to chronic hepatitis with possible progression to liver cirrhosis, or hepatocellular carcinoma, especially in hyperendemic areas 1. Thus, the search for an effective therapy against HBV infection seems to be of great clinical importance. Over the past years, the therapy of choice for most patients with chronic hepatitis B was interferon (IFN)-Eo, a cytokine which combines antiviral activity with the capacity to modulate the cellular immune response 2. Although its immunotherapeutic use as an antiviral drug has increased in recent years, a complete seroconversion to anti-HBs/anti-HBe antibodies rarely occurs.

It is well known that the humoral immune response, in particular the production of antibodies to hepatitis B surface antigen (anti-HBs), is important for successful elimination of the virus, especially by binding extracellular viral particles and therefore, limiting the spread of the infection 3. Thus, the clearance of circulating hepatitis B surface antigen (HBsAg) and the appearance of anti-HBs, accompanied by the normalization of biochemical tests of liver function, have been generally used as a criterion of hepatitis resolution in both acute and chronic hepatitis B infection 4. Recently, it has been shown that the occurrence of neutralizing antibodies to viral protein antigens of IgG1 and IgG3 isotypes is required for complete viral clearance from the host [5,6]. However, in our previous study performed in 19 children with chronic hepatitis B who seroconverted to anti-HBs after IFN-Eo therapy, we found that, in contrast to spontaneous seroconversion, anti-HBs had no characteristic selectivity of isotypes involved, and all IgG subclasses, with a wide spectrum of individual diversity in their composition, were present 7. Moreover, several recent studies have shown that circulating HBV-DNA may persist long after HBsAg has disappeared in both self-limited acute hepatitis B and after successful antiviral therapy of chronic infection [8-10]. These observations strongly suggest that serological and clinical resolution is not invariably associated with complete clearance of HBV from the host.

Therefore, the aim of our study was to investigate the persistence of serum HBV-DNA in a larger series of patients with chronic hepatitis B who seroconverted to anti-HBs after interferon-Eo therapy completed 4-10 years earlier. We also wanted to assess whether any association exists between the presence of HBV-DNA in serum and the specific humoral response to HBsAg measured at the IgG subclass level.

ABSTRACT
Background/Aims;
We examined the persistence of HBV-DNA in sera collected 4-10 years after IFN-Eo therapy from patients with chronic hepatitis B who had seroconverted to anti-HBs antibodies. We also wanted to assess whether any association exists between HBV-DNA status and the IgG anti-HBs subclass responses.

Methods:
Sera were obtained from 38 patients and the following parameters were determined in each of them: (1) serological markers of HBV; (2) concentrations of IgM, IgG, IgA; (3) total IgG subclasses. HBV-DNA and IgG anti-HBs subclasses were determined in anti-HBs positive sera.

Results:
Four to 10 years after therapy, anti-HBs were found in 37 of 38 patients (GMT: 775IU/L). In 13 of them (35.1%) free and/or bound HBV-DNA was present in sera. Significant differences in the profiles of IgG anti-HBs were observed when the HBV-DNA status was considered. Patients with undetectable HBV-DNA responded mainly with IgG1 and/or IgG3, while in the HBV-DNA-positive group, a high contribution of IgG4 was found.

Conclusions:
Our study showed that HBV-DNA may persist for a long time after IFN-Eo therapy despite the appearance of anti-HBs antibodies. The monitoring of specific IgG subclasses may be of predictive value for HBV-DNA persistence.

AUTHOR DISCUSSION
Discussion
return to Article Outline

In this study, we analysed the presence of HBV-DNA in the sera of 37 patients with chronic hepatitis B who developed anti-HBs antibodies after IFN-Eo therapy that was given 4-10 years earlier. We also analysed the distribution of anti-HBs antibodies among IgG subclasses and correlated their profiles with HBV-DNA status. Our results showed that 5.3% of patients again tested positive for HBsAg, and in one of them, HBsAg/anti-HBs were detected simultaneously. These observations confirm earlier findings that seroconversion to anti-HBs in chronic hepatitis B may not be a stable phenomenon 12. The remaining 94.7% of patients studied remained HBsAg negative and anti-HBs positive. Because it was generally believed that the disappearance of HBsAg from the serum and appearance of anti-HBs, together with normalization of liver biochemical tests, indicate complete recovery from HBV infection 13, serum titres of anti-HBs antibodies were used as a criterion of successful immune response to HBsAg. Recently, however, the development of sensitive molecular biology methods for the determination of trace amounts of HBV-DNA in the serum and liver tissues has allowed to detect HBV-DNA long after serological recovery [14-16]. Therefore, we wished to verify if HBV-DNA might persist in the sera of children with chronic hepatitis B who responded to IFN-Eo therapy with seroconversion to anti-HBs many years earlier. Our present study demonstrated that 13 of 37 (35.1%) patients who tested positive for anti-HBs had HBV-DNA in their sera and/or circulating immune complexes as long as 10 years after seroconversion. In addition to original serum samples, we tested the possibility that serum proteins or non-specific circulating immune complexes may interfere with the determination of low levels of HBV-DNA, or that viral particles may circulate as specific immune complexes. In fact, removal of CIC from the serum allowed detection of free HBV-DNA in five patients (13.5%) in whom it was previously undetectable. Moreover, in four patients HBV-DNA was found in the form of CIC and in three of them, free HBV-DNA was not detected. These results were significantly different from those observed in the group who spontaneously seroconverted, where only free form of HBV-DNA was found, in 15.8% of cases. Our results are in contrast to the observations reported by Yotsuyanagi et al. 14 who found only Ig-bound HBV in anti-HBs positive patients, and postulated that after seroconversion to anti-HBs, HBV remaining in the circulation is not infectious.

Interestingly, we could not find any correlation between HBV-DNA status, the titres of anti-HBs and time of sampling after the last dose of IFN-Eo or spontaneous loss of HBsAg.

It has been postulated that the antibody response to viral envelope antigens, in addition to cellular immunity, plays a critical role in removing free viral particles from circulation and in limiting virus spread in the host 17. Moreover, it has been shown that strong activation of the Th1 response by viruses with production of Th1 type cytokines, mainly IFN-ƒÁ, and induction of specific antibodies of the IgG1 and IgG3 isotypes is most important for effective virus neutralization [5,6,18]. In humans, it has been demonstrated that Th1 cytokines skew antibody responses toward the IgG1 isotype which strongly activates complement system, enhances opsonization and phagocytosis, and antibody-dependent cellular cytotoxicity. In contrast, Th2 cytokines, mainly IL-4, direct antibody synthesis toward the allergic/blocking IgG4 subclass. Thus, it has been postulated that the IgG subclass response to infection is a biomarker of the T helper cell response. In this study we wanted to assess if any correlation exists between persistence of HBV-DNA in the serum long after HBsAg disappearance and the distribution of anti-HBs among IgG subclasses. In contrast to HBV-DNA-negative patients and controls, in whom the response was strongly restricted to neutralizing antibodies, in HBV-DNA-positive group the most interesting finding was a high frequency and contribution to the response of IgG4 anti-HBs. Although the real biological role of this IgG isotype in viral and bacterial infections is still unresolved, the high prevalence of IgG4 anti-HBs in samples with detectable HBV-DNA indicates that in these patients, the response to HBV induced in vivo by IFN-Eo was moved toward the Th2 type. IgG4 subclass antibodies do not activate complement, while in some circumstances may inhibit its activation, have a low affinity to macrophage Fc receptors, and are up-regulated by IL-4, which in turn inhibits IFN-ƒÁ synthesis, thereby reducing the Th1 cell-mediated response. Thus, we suggest that the production of high titres of specific IgG4 antibodies at the early stage of humoral response to HBsAg may be responsible, at least in part, for unsuccessful clearance of virus from the host. On the other hand, paradoxically, prolonged stimulation of IgG4 anti-HBs during years after seroconversion may protect, in addition to other factors, against severe inflammatory reactions mediated by persistent HBV infection, which were also not observed in our HBV-DNA/anti-HBs-positive patients at the time of follow-up.

The mechanisms responsible for HBV-DNA persistence, despite full seroconversion, are still unclear. However, the interaction between viral load and individual status of immune system at the time of infection as well as the strength of the immune response against HBV may be a crucial factors in the effects of immunomodulatory therapy [19].

In conclusion, the results of the present study investigating the IgG subclass response to HBsAg as a biomarker of the T helper cell response provide, for the first time, evidence that the immune response in HBV-DNA-positive patients differs from that found in HBV-DNA-negative and confirm our earlier observations that immunomodulatory effect of IFN-Eo monotherapy is insufficient and non-selective. We suggest that monitoring of IgG anti-HBs profiles may be of predictive value for HBV-DNA persistence.

Materials and methods
2.1. Patients

Between 1993 and 1997 in a multicentre open, uncontrolled trial we collected data on interferon-Eo (rIFN-Eo; Intron A, Schering-Plough, Kenilworth, NJ; Roferon, Hoffman La Roche, Switzerland) treatment of 1689 children with chronic active hepatitis B. Serological markers of HBV were assessed 12 months after completion of therapy in 910 children, and 91 of them (10%) were HBsAg and HBeAg-negative, but anti-HBs-, anti-HBc- and/or anti-HBe- positive. All these patients were recalled at a mean of 8 years (range: 4-10 years) after the last dose of rIFN-Eo for the follow-up.

Thirty-eight subjects (24 males and 14 females; median age: 9.7 years; range 5-22 years) responded to our invitation. All of them showed biochemical recovery (normal levels of ALT and total bilirubin) and were in good clinical condition without evidence of hepatosplenomegaly or other clinical symptoms of chronic liver disease. At the time of study the patients were not treated with antibiotics, antiviral or immunomodulatory drugs.

The control group consisted of 19 patients (eight males and 11 females; median age: 10 years; range 6-45 years) who had lost HBsAg spontaneously 5-13 years earlier (mean 9.5 years) and were HBsAg, HBeAg-negative and anti-HBs, anti-HBc and/or anti-HBe-positive with normal level of ALT and bilirubin.

Serum samples obtained from all patients and controls were divided into small portions and kept at -70¡C until analysis.

2.2. Serological markers of hepatitis B virus

HBsAg, HBeAg, anti-HBs, anti-HBe and anti-HBc were determined using Abbott AXSYM system and commercially available kits (Abbott Laboratories, Chicago, IL). The geometric means of anti-HBs antibody titres (GMT's) were calculated.

2.3. HBV-DNA detection

Serum samples stored at -70¡C were thawed immediately before testing, centrifuged at 15,000rpm for 1h and subjected to HBV-DNA analysis by use of the semi-automated HBV-DNA PCR assay (Cobas Amplicor HBV Monitor, Roche Diagnostic System, Branchburg, NJ, USA), according to the manufacturer's instructions. The primers used in the test were HBV-104UB and HBV-104D amplifying a 104-bp fragment within the highly conserved pre-core/core region of the HBV genome. The limit of detection was 200 HBV-DNA copies per ml, and each run included appropriate positive and negative controls, according to the test requirements. 2.4. Isolation of circulating immune complexes

To assess whether the absence of detectable serum HBV-DNA is because it is protected within a layer of serum proteins or masked in the form of immune complexes (CIC), serum samples were precipitated by the addition of polyethylene glycol solution (PEG 6000) to a final concentration of 3.5%. The mixtures were kept at 4¡C overnight, centrifuged at 3000~g at 4¡C for 20min, and separated into supernatants and pellets. The pellets were resuspended to their original serum volume in glycin-HCL buffer, pH 2.8 and subjected to acid dissociation according to the procedure previously described 11. The supernatants and pellets were concentrated by centrifugation at 15,000rpm for 1h and were tested for the presence of HBV-DNA using the method described above.

2.5. Serum immunoglobulins and IgG subclasses

To exclude possible deficiencies of total serum immunoglobulins, IgM, IgA and IgG subclasses [1-4] were determined by nephelometry (BN ProSpec, Dade Behring Marburg GmbH, Germany).

2.6. IgG anti-HBs subclasses

IgG anti-HBs subclasses were determined by an ELISA standardized according to the general principles of solid phase immunoassay; the detailed protocol has been described elsewhere 5.

2.7. Statistical analysis

All results were analysed by applying the Kolmogorow-Smirnov, Shapiro-Wilk and E2tests to determine the normality of the data distribution. Then, the data were summarised either by mean values and SD (normal distribution) or by geometric mean accompanied by minimum and maximum value (log-normal distribution). The differences in the frequency and activity of specific subclasses were analysed by the E2test and individual results were compared by non-parametric sign tests for dependent samples. The correlation between two variables was analysed by the Spearman rank correlation coefficient test. GMT was accompanied by min.-max. values. A level of P<0.05 was considered significant.

Results

In all patients studied, the concentrations of total serum immunoglobulins and IgG subclasses were within the normal range for their ages.

3.1. Hepatitis B virus markers

Four to 10 years after IFN-Eo therapy, 37 (97.4%) of 38 patients studied had detectable levels of anti-HBs antibodies with a GMT of 775IU/L (min.-max., 16-28,500IU/L), which was 6-fold higher than this found in sera 5-13 years after natural seroconversion (GMT, 127IU/L; min.-max. 9-2105IU/L) (P<0.01). In one child, an 8.7-year-old boy who was examined 6 years after treatment, the simultaneous presence of HBsAg and anti-HBs (217IU/L) was found. Moreover, a 12-year-old girl, who tested positive for anti-HBs 12 months after therapy, was again positive for HBsAg 10 years after the last dose of IFN-Eo. This child was excluded from further study. All patients tested positive for anti-HBc and 55% of them had anti-HBe. Serological and clinical characteristics of the patients and controls are presented in Table 1.

3.2. Detection of HBV-DNA in sera and circulating immune complexes

In total, free and/or bound HBV-DNA was present in sera or immune complexes in 13 of 37 (35.1%) patients.

We first, tested for the presence of HBV-DNA in the sera collected from all anti-HBs positive subjects. HBV-DNA was detected in 5 of 37 (13.5%) children who completed their therapy 4-10 years earlier. After precipitation of circulating immune complexes by 3.5% PEG (non-specific and/or specific CIC) from the sera, a free form of HBV-DNA was detected in 7 of 37 (18.9%) supernatants and five of these patients showed no evidence of HBV-DNA in the original serum, including an HBsAg/anti-HBs-positive case. In addition, HBV-DNA was detected in 4 of 37 (10.8%) pellets, and in three of these subjects a free form of HBV was not found. In contrast, in the control group only a free form of HBV-DNA was detected in serum samples obtained from three of the 19 (15.8%) subjects at a period of 5-8 years after spontaneous seroconversion to anti-HBs. In both groups no significant correlations between HBV-DNA status and anti-HBs titres or time of blood sampling were observed.

3.3. IgG subclass profiles of anti-HBs

The analysis of anti-HBs response performed 4-10 years after IFN-Eo therapy showed high individual variability in the IgG subclass composition, engaging three IgG isotypes: IgG1 was present in 95% of serum samples and comprised, on average, 56% of the total IgG response. Specific IgG3 was found in 49% and IgG4 in 41% of serum samples, and their percentage contribution to the total IgG response was similar (20 vs. 24%, respectively). Together, in the group as a whole, specific IgG1 and IgG3 subclasses with strong opsonic and complement-fixing activities comprised, on average, 76% of total anti-HBs response. (Fig. 1a). After natural seroconversion, the response was highly restricted to the IgG1 subclass, which was present in 18 of 19 serum samples (95%) with 84% contribution to the total anti-HBs response. Specific IgG3 and/or IgG4 were present in four sera (21% for each), and their mean percentage contribution, to the IgG response was low (9 and 7%, respectively), (Fig. 1b).

3.4. HBV-DNA status and specific IgG subclass profiles

Statistically significant differences with respect to frequency and proportions of IgG isotypes involved in the response to HBsAg were found when HBV-DNA status was considered. Thirteen of 37 patients treated with IFN-Eo had detectable levels of HBV-DNA in their sera and/or immune complexes (group I), and the remaining 24 patients were HBV-DNA-negative (group II). In both groups, IgG1 was still the predominant subclass with respect to frequency (85 and 100% positive sera, respectively), with similar contribution to the total IgG anti-HBs response (62 vs. 76%; P=ns). IgG3 was present in 38% of sera from group I and in 54% of sera in group II (P=ns). However, in HBV-DNA-positive patients this subclass comprised, on average, 9% of the total IgG anti-HBs response, as compared with 18% in the HBV-DNA-negative group (P<0.05). The frequency and percentage contribution of IgG4 anti-HBs (77 and 29%, respectively), significantly dominated in HBV-DNA-positive patients when compared with those observed in the HBV-DNA-negative group (29 and 6%, respectively; P<0.001). Individual analysis showed that in 43% of HBV-DNA-positive patients, IgG4 anti-HBs became the most represented isotype with ca. 54-100% contribution to the total anti-HBs response. Similar statistical analysis of anti-HBs profiles in the control group was not possible because of low HBV-DNA frequency (3/19). Nevertheless, IgG4 anti-HBs were found in four cases (54-100% of total response) and three of them were HBV-DNA-positive (data not shown). In one HBV-DNA-positive child only IgG4 anti-HBs was detected.

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