Temporary HBV resolution in an HIV-coinfected patient during HBV-directed combination therapy followed by relapse of HBV

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1 Case report Antiviral Therapy 11: Temporary HBV resolution in an HIV-coinfected patient during HBV-directed combination therapy followed by relapse of HBV Karsten Wursthorn 1, Peter Buggisch 1, Marc Lutgehetmann 1, Bernhard Zollner 2 and Jorg Petersen 1,3 * 1 Department of Medicine, University of Hamburg, Germany 2 Institute for Infectious Diseases, University of Hamburg, Germany 3 Heinrich Pette Institute for Experimental Virology and Immunology, University of Hamburg, Germany Both authors contributed equally to this study *Corresponding author: Tel: ; Fax: ; joepeter@uke.uni-hamburg.de Coinfection of hepatitis B virus (HBV) and HIV is common due to overlapping routes of transmission accompanied by an increased risk for liver-related mortality. We report the case of a chronically infected hepatitis Be antigen positive patient, coinfected with HIV (CD4 + T-cell count >500 cells/µl), with histological evidence of advanced liver disease. The patient developed anti-hbs (antibody to hepatitis B surface antigen [HBsAg]) seroconversion, a strong reduction of intrahepatic covalently closed circular DNA and a marked improvement of liver histology after 24 weeks of HBVtargeted combination therapy with adefovir dipivoxil and pegylated interferon-α2b followed by another 12 weeks of adefovir dipivoxil monotherapy. Antiviral therapy was stopped after the development of stable anti-hbs titres, and anti-hbs titres remained stable for additional 9 months post-treatment. A continuous decline of anti-hbs was observed during the next 6 months until anti-hbs disappeared despite a stable HIV infection. A triple course of therapeutic vaccination failed to re-establish anti-hbs antibodies, but reappearance of HBV DNA and HBsAg was detected. By enzyme-linked immunosorbent spot analyses, HBVdirected T-cell responses clearly increased during antiviral combination therapy followed by a reduction to pre-treatment levels in association with disappearance of anti-hbs antibodies despite therapeutic vaccination. The presented case highlights the volatile nature of chronic HBV infection even after a prolonged diseasefree period in the setting of an underlying HIV coinfection in a patient with a stable and relatively high CD4 + T-cell count but nevertheless impaired immune system and calls for further investigation of probably temporary immunomodulatory effects of interferon-α and/or nucleoside analogues in immunocompromised patients. Introduction Hepatitis B virus (HBV) coinfection in HIV is frequently observed because of the shared modes of transmission, risk groups involved and chronicity of the diseases, and occurs in approximately 10% of all HIV-infected individuals in the United States and Europe [1]. Despite an effective vaccine against HBV, there is a rising incidence of HBV infection in HIVinfected individuals [2]. In patients infected with HBV, HIV can lead to higher rates of HBV chronicity, decreased rates of seroconversion to anti-hbe and anti- HBs (antibodies to hepatitis Be antigen [HBeAg] and hepatitis B surface antigen [HBsAg], respectively), and increased viral replication, probably through the impairment of innate and adaptive cellular and humoral immune responses [1]. HBV/HIV coinfection is associated with increased liver fibrosis progression and cirrhosis and rates of liver decompensation, hepatocellular carcinoma and liver-related mortality in the context of prolonged survival of HIV patients associated with highly active antiretroviral therapy (HAART) [3,4]. There is no evidence that HBV affects HIV disease progression or alters the response of HIV to antiretroviral therapy [3]. At present in Europe, only a minority of HBV/HIV-coinfected patients are treated for their viral hepatitis [1]. Here we report a case of an HBV/HIV-coinfected male patient who received HBV-targeted combination therapy with adefovir dipivoxil and pegylated interferon-α2b 2006 International Medical Press

2 K Wursthorn et al. Figure 1. Liver biopsies before and after 24 weeks of antiviral therapy A B (A) Liver biopsy before the start of treatment: chronic active inflammation (grading 3/3) with periportal fibrosis (staging 2/4) based on the METAVIR score is shown. (B) Liver biopsy after 24 weeks of treatment with pegylated interferon a and adefovir dipivoxil. Mild hepatitis (grading 1/3) without signs of fibrosis (staging 0/4) is shown. (peg-ifn-α2b) without anti-hiv treatment since his CD4 + T-cell count remained >500 cells/µl. The patient developed HBs seroconversion during therapy and covalently closed circular DNA (cccdna) reduction to undetectability accompanied by a marked improvement in liver histology and partial temporary reconstitution of HBV immunity. However, the anti-hbs titre declined in the long-term follow-up period to an undetectable level and HBsAg reappeared along with a loss in HBVdirected T-cell responses that could not be reversed by triple therapeutic vaccination with double dosages of a commercially available HBsAg (40 µg ) despite stable HIV infection. Materials and methods A 32-year-old Caucasian male presented with a documented chronic HBeAg-positive hepatitis B virus infection that had been diagnosed 18 months previously, when the patient had presented with fatigue. Alanine aminotransferase (ALT) was increased at 5.3 upper limit of normal (ULN) and HBV DNA was detected with copies/ml by real time PCR (lower limit of detection 100 copies/ml), genotype D. Partial thromboplastin time (PTT), bilirubin and thrombocyte levels were within normal limits. The patient was diagnosed elsewhere with HIV-1 2 years before initial presentation at our clinics. HIV viral load was 49,000 copies/ml (Roche Amplicor ), CD4 + T-cell count was 605 cells/µl. The patient was treatment naive for HBV and HIV. Ultrasound results showed signs of advanced liver disease, and a liver biopsy (Figure 1A) revealed a severe inflammation (grading 3 out of 3 METAVIR) with fibrous septa (staging 2 out of 4, METAVIR score). The template of HBV replication, intrahepatocellular cccdna, was detected in liver biopsy and quantified with 2.77 cccdna copies/cell using a real-time PCR approach [5]. Details of the enzyme-linked immunospot (ELISPOT) technique: isolation of peripheral blood mononuclear cells from a total of 40 ml EDTA blood using Ficoll Hypaque gradient centrifugation; separation of CD4 + and CD8 + T cells using immunomagnetic beads (Invitrogen, Karlsruhe, Germany); splitting of separated CD4 + and CD8 + cells (2x10 6 /ml) for hil-4 and hifn-γ Elispot assays (Bionor AS, Skien, Norway), respectively; stimulation of 100 µl cell suspension per well in duplicate for 2 days at 37 C in RPMI-1640 plus 10% FCS containing either petides (5 µg/ml), PHA (positive control, 5 µg/ml) or medium (negative control). The ELISPOT assay was performed according to the manufacturer s instructions and spots were counted in a stereomicroscope. The peptides used were as follows: Core, H-Phe-Leu-Pro-Ser-Asp-Phe-Phe-Pro-Ser-Val-OH; Pol, H-Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu-OH; HBsAg, H-Trp-Leu-Ser-Val-Ile-Trp-Met-Met-Trp-OH. HBV-directed antiviral therapy was with the nucleotide analogue adefovir dipivoxil, 10 mg per day. To reduce the risk of selecting for resistance and to suppress HBV replication as fast and as much as possible, adefovir dipivoxil treatment was combined with 12 kda peg-ifn-α2b, 1.5 µg/kg body weight per week (administered subcutaneously), in accordance with suggestions of a recent first European consensus conference for the treatment of HBV/HIV-coinfected patients [1]. There was no concomitant anti-hivdirected medication due to a CD4 + T-cell count >500 cells/µl and a significantly increased level of ALT at baseline. Results After 4 weeks of HBV-directed antiviral treatment, a 5-log decrease in HBV DNA titre was observed, and International Medical Press

3 Temporary HBV resolution in HIV/HBV coinfection Figure 2. Summarizing the clinical course of the HBV and HIV infection during the reported history 8 ADV + IFN ADV No medication Antivirals 6 x log HBsAg vaccination LloD HBV ULN ALT Anti-HBe Loss of HBs Anti-HBs Loss of anti-hbs ALT [xuln] Anti-HBs CD4 response [in % of baseline] HIV RNA [copies/ml] Serum HBV DNA [copies/ml] CD4 + T cells [cells/µl] Shown is the course of alanine aminotransferase (ALT), serum HBV DNA, HIV RNA, CD4 + T-cell count and anti-hbs CD4/interferon response. the ALT level was 4 ULN. At week 12, loss of HBeAg and detection of anti-hbe antibodies occurred, and the ALT level was 4.2 ULN. HIV load was 6,800 copies/ml and CD4 + T-cell count had decreased to 500 cells/µl. At week 20 of combination therapy, HBsAg was lost and the ALT level was 3.1 ULN. At week 24, the ALT level was within normal limits for the first time, a low level of anti-hbs antibody titre was detectable, and HIV PCR was positive with 45,000 copies/ml. CD4 + T-cell count at week 24 was 522 cells/µl (Figure 2). A second liver biopsy performed 26 weeks after initiation of antiviral treatment showed a marked histological improvement. Hepatitis was only mild (grading 1 out of 3), and there was no fibrosis (staging 0 out of 4 METAVIR; Figure 1B). Intrahepatic nuclear total HBV DNA was reduced by 2.5 log 10 and cccdna was reduced by 2.2 log 10 to undetectable levels (detection limit 3 copies/10,000 cells, quantitative PCR, baseline 2.77 copies/cell). In addition, there was a decrease in the number of HBs- and HBc-expressing cells below the detection limit of immunostaining. The emergence of anti-hbs antibodies was accompanied by a partial restoration of HBc-directed, HBs-directed and HBV-polymerase-directed CD4 + /IFN-γ immune response, as identified by ELISPOTS at weeks 12 and 24 compared with baseline. Peg-IFN-α2b treatment was stopped after week 26 and the patient was kept on adefovir dipivoxil. After demonstration of further rising anti-hbs antibody titres, adefovir dipivoxil therapy was stopped after week 36. Genotypic resistance analysis of HIV 12 weeks after adefovir dipivoxil monotherapy (week 36) revealed the wild-type sequences of the reverse transcriptase gene (HIV RNA 24,000 copies/ml). In particular, neither mutations due to the adefovir dipivoxil medication (such as the K65R or K70E exchange) nor mutations against nucleoside reverse transcriptase inhibitors/non-nucleoside reverse transcriptase inhibitors were detected [6]. During follow up visits from end of treatment (week 36) to week 72, there were normal serum ALTs, stable anti- HBs antibody titres, and an unchanged HIV status. Follow-up visits from week 80 to week 108 showed a continuous decline of anti-hbs antibody titres, whereas CD4 + T-cell count and HIV RNA remained stable and ALT levels within normal limits. At week 108, the patient again became anti-hbs negative, CD4 + T-cell Antiviral Therapy 11:5 649

4 K Wursthorn et al. count was 512 cells/µl, and HIV PCR detected 29,000 copies/ml. In between week 108 and 120, the patient received three doses of HBsAg intramuscularly (40 µg each) in an attempt for therapeutic vaccination to reintroduce the secretion of anti-hbs antibodies. This treatment failed. At week 136 and 144, the patient started to again replicate HBV DNA with 400 and 16,000 copies/ml detected, respectively. However, ALT level remained within normal limits at week 136 and was at the ULN at week 144, but the patient became positive for HBsAg at week 144 again. ELISPOT analysis carried out at week 144 was consistent with the loss of partial restoration of HBc-directed, HBsdirected and HBV-polymerase-directed CD4 + /IFN-γ immune response. At week 144 the patient was again started on anti-hbv-directed medication with adefovir, 10 mg per day. Conclusions The most ambitious goal of anti-hbv-directed treatment in coinfected patients is to achieve anti-hbs seroconversion. This endpoint can be reached in less than 10% of HBV-monoinfected patients [1]. However, the overall response rate is much lower among HBV/HIVcoinfected patients, and anti-hbs seroconversion is very rare [3,7] and reactivation of an occult HBV infection may occur [8]. Data on the efficacy in HBV/HIV-coinfected patients are very limited, but treating HBV in HIV-coinfected patients should not diminish future therapeutic options for the treatment of either HIV or HBV. IFN-α therapy is an option for coinfected patients who do not need to start HAART (CD4 + T-cell count >500 cells/mm 3 ) [1], although IFN has been reported to be little effective in HIV-positive patients and CD4 + T- cell drops may be of particular concern [3,7]. Lamivudine was reported to induce HBV-escape mutants with a 5-year actuarial rate close to 100% in HIV-coinfected patients [9]. Adefovir dipivoxil at a dose of 10 mg daily is thought to have no activity against HIV and the application of the HBV-directed 10 mg dosage in HIV-1-coinfected patients in vivo does not select resistance-associated mutations in the HIV-1 reverse transcriptase gene [6,10]. It is the preferred approved drug besides IFN in coinfected patients not requiring HAART [3]. Current research in HBV suggests that, as in HIV, combination therapy reduces the risk of selecting for resistance [1,11] and diminishes the rate of clinical decompensation in cirrhotic patients, which is even more important in coinfected patients. Analysis of total intrahepatic HBV DNA revealed a sharp decline of 2.5 log 10 copies/cell in the second liver biopsy consistent with the inhibition of viral DNA synthesis during therapy. A significant decline of viral intrahepatic cccdna by 2.2 log 10 copies/cell was also observed, which was greater than the cccdna decline observed in another recent study [5]. In that study, monoinfected patients received adefovir dipivoxil only, and the decline of cccdna was 0.8 log 10 copies/cell [5]. However, cccdna clearance may take several years and it remains questionable if cccdna clearance is an option at all [5]. Due to its stability and longevity in the nuclei of hepatocytes, cccdna is believed to be responsible for viral rebound and for the seroreversion of anti-hbc/e and/or anti-hbs-positive chronic hepatitis B. Its distinct reduction on the other hand is believed to contribute to a long-lasting and stable anti-hbe and/or anti-hbs seroconversion. Currently it is unknown if there exists a certain threshold for cccdna below which the host immune response will control HBV infection. Although cccdna was reduced to undetectability by quantitative PCR reaction in this patient, the continuous decline of anti-hbs antibody titres starting at week 80 followed by reappearance of viral replication at week 136 is consistent with occult infection or residual intact cccdna copies within the liver and insufficient immune control of remaining HBV DNA despite stable HIV disease [11]. Sampling errors in paired liver biopsies is always a concern that cannot be ruled out entirely, and the marked improvement with regression of fibrosis within 6 months of antiviral treatment is drawing a very optimistic picture. On the other hand, cccdna reduction was associated with a significant decline of HBV antigen-expressing cells below the detection limit of immunostaining assays and was positively correlated with serum markers such as HBsAg seroconversion [5], confirming that antiviral therapy indeed reduced the reservoir of transcriptionally active viral cccdna. The spontaneous loss of HBsAg is extremely rare in HIV-infected patients [7,13]. However, this patient had a fairly high CD4 + T-cell count (>500 cells/µl), which may have favoured HBsAG seroconversion. Furthermore, anti-hbe and anti-hbs seroconversion was not associated with a distinctive ALT flare, the patient showed a more stable and continuous elevation pattern of ALT from baseline to week 20 with 3 5 ULN and a peak within this range between week 8 and week 12 of antiviral therapy. The emergence of anti-hbe and anti-hbs antibodies during antiviral treatment was accompanied by a significant increase in HBV-specific CD4 + T-cell response after 12 and 24 weeks of antiviral combination therapy. In HIV-negative patients, HBV chronicity is associated with a reduction in HBV-specific CD4 + and CD8 + T-cell responses that can be partially restored by antiviral treatment [12,14]. In this coinfected patient it seemed that reconstitution of some HBV-specific T-cell responses after reduction of HBV load occurred in the context of ongoing HIV viraemia, but only International Medical Press

5 Temporary HBV resolution in HIV/HBV coinfection temporarily. The potential to recover T-cell responses, which has been thought to be crucial for HBV control, would provide support for an anti-hbv therapy in the early stage of HIV in HBV/HIV-coinfected patients [15]. However, one of the most interesting aspects in this case report is the loss of the temporarily improved T-cell responses associated with relapse of HBV replication despite an HIV infection with stable CD4 and HIV RNA after stopping antiviral treatment. Seroreversion of HBV in this patient was not associated with chemotherapy or other immunosuppressing medication, which has been described in HBV monoinfected patients before [1]. It has been shown that IFN treatment or nucleoside treatment alone can lead to improvement of immunological responses [12] and it is difficult to assess which medication was responsible for the temporarily improved immunological response in this patient, but the results are consistent with a longterm sustained antiviral response of only about 20% after IFN treatment of HBV-monoinfected patients and should reassure us that counting of CD4 + T cells or determining ratios of CD4/CD8 cells in coinfected patients is not sufficient to predict the course of HBV infection despite temporary resolution of HBV. The role of IFN treatment especially in this patient group needs to be further determined. The histological pattern with loss of HBsAg- and HBcAg-expressing hepatocytes during therapy in this patient is consistent with a higher level of cytolytic disappearance of HBV [16], in contrast to adefovir dipivoxil monotherapy trials, in which 48 weeks of treatment did not alter significantly the number of infected cells, suggesting a more non-cytolytic immune response [5]. Therefore, it is possible that the main antiviral effect was due to the peg-ifn-α treatment and we cannot distinguish to what extent adefovir dipivoxil contributed to the temporary resolution of HBV. Recent reports have highlighted the potential benefits of additional anti-hbv drugs, such as tenofovir, entecavir, telbivudine, clevudine and others for HBV/HIVcoinfected patients. Whether a mostly immunomodulatory drug like IFN-α or combination of IFN with nucleos(t)ide analogues or combination of nucleos(t)ide analogues will ultimately be able to further enhance reconstitution of HBV-specific T-cell responses and resolution of HBV in the setting of HIV remains to be established [14]. The continuation of adefovir dipivoxil for another 24 weeks after stopping IFN-α in this patient might have helped to achieve anti- HBs seroconversion. The course of the HIV infection in this patient remained basically unaffected; the low, non-mutagenic dosage of adefovir dipivoxil together with the IFN-α lacked a resistance-inducing activity. In summary, HIV coinfection accelerates the natural course of hepatitis B, but at present only a minority of HBV/HIV-coinfected patients are treated for their hepatitis [1]. This patient had a relatively high CD4 + T- cell count and showed a remarkable course of his HBV infection, strongly supporting the idea of early HBV treatment in coinfected patients. Particularly high CD4 + T-cell counts may improve the possibility of driving a cellular-mediated immune response to HBV epitopes during antiviral therapy. The relapse of HBV even after long-term anti-hbs seroconversion in the absence of further immunosuppression in this coinfected patient reminds us that we need to dissect further the effects of immunomodulatory and direct antiviral drugs for a better understanding of immune regulations in chronic hepatitis B. Effective treatment strategies including the role of combination therapy for this special difficult-to-treat group of coinfected patients are urgently needed. Acknowledgements The authors like to thank Gilead Sc., Foster Lake, CA, USA and essex pharma GmbH, Martinsried, Germany, for providing medication. 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