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Centre for Sexual Health and HIV Research, Institute of Hepatology
Division of Infection and Immunity, Royal Free and University College Medical School, London, United Kingdom
Coinfection with hepatitis B virus (HBV) is a common occurrence in human immunodeficiency virus (HIV)positive patients and an increasing cause of morbidity and mortality. The CD8+ T cell response is critical for long-term control of HBV in patients resolving acute infection. Here, we examine the effect of HIV on HBV-specific CD8+ T cell responses in patients who have resolved HBV infection. A cross-sectional study showed a reduction in HBV-specific CD8+ T cell responses in HIV-positive, HBV-immune patients, compared with those in HIV-negative, HBV-immune patients. A longitudinal study of a subgroup of patients examined whether this attrition could be reversed by effective antiretroviral therapy. The introduction of highly active antiretroviral therapy (HAART) resulted in reconstitution of some HBV-specific CD4+ and CD8+ T cell responses, in association with restoration of CD4+ T cell counts. These data provide a mechanism to account for the observed impairment of control of HBV infection in the setting of HIV infection and support the ability of HAART to reconstitute functionally active T cell responses.
Coinfection with hepatitis B virus (HBV) is a frequent occurrence in HIV-positive patients and an increasing cause of morbidity and mortality in the context of the prolonged survival associated with highly active antiretroviral therapy (HAART) [14]. The proportion of HIV-positive patients with serological evidence of previous exposure to HBV ranges from 64% to 84% in published cohorts; 10%15% of such patients are chronically infected with HBV [5, 6]. HIV-related immunodepletion influences the natural history of HBV infection. Epidemiological studies have revealed that HIV-positive patients are more likely to have a prolonged duration of acute illness after HBV infection and to have lower rates of clearance of hepatitis B e antigen [7]. They have higher circulating levels of HBV DNA and higher rates of reactivation of HBV infection [8]. These findings suggest impaired immune control of HBV infection during HIV infection.
In the present study, we explore, for the first time, the effect of HIV-related immunodepletion on HBV-specific CD8+ T cell responses in patients who have resolved HBV infection. Recent data have directly demonstrated that CD8+ T cells are critical effectors in the control of acute HBV infection [9]. HBV-specific CD8+ T cell responses are difficult to detect in patients chronically infected with HBV [10], making them an unsuitable group in which to study the impact of HIV infection. We therefore studied patients who resolved HBV infection, since, in the absence of HIV, such patients maintain strong CD4+ and CD8+ T cell responses for many years after complete clinical and serological recovery [11, 12]. Patients without a history of jaundice found to have natural immunity to HBV (hepatitis B core antibody plus/minus hepatitis B surface antibody ) as part of routine screening before HBV vaccination have never previously been studied immunologically, with regard to their HBV-specific immune responses, but are common among HIV cohorts [5, 13]. HBV-specific responses were compared in HIV-positive and HIV-negative patients, with a focus on those who had resolved HBV infection without symptoms. We used the sensitive technique of intracellular cytokine staining for interferon (IFN), to explore the breadth of functionally active HBV-specific CD8+ T cell responses across a range of previously defined HLA-A2restricted HBV epitopes [1416].
To investigate to what extent any loss of HBV-specific immune responses could be reversed by antiretroviral therapy, a group of HBV-immune patients starting HAART were studied prospectively. There is a growing body of evidence suggesting that HAART may lead to successful restoration of specific immune responses to previously encountered pathogens [1719]. A previous small study of HIV patients chronically infected with HBV suggested that HAART may be associated with restoration of HBV-specific responses once HBV load is reduced [20]. Natural immunity to HBV provided a good model system in which to further explore the potential for functional restoration of HBV-specific CD4+ and CD8+ T cell responses in the setting of controlled HBV replication.
PATIENTS, MATERIALS, AND METHODS
Patients and controls.
Patients were recruited from the Mortimer Market Centre and provided written, informed consent, and the local ethics committee approved the study. All patients had HBV serologic test results confirming natural immunity to HBV (hepatitis B surface antigen negative, HBcAb positive, and HBsAb positive) and were further categorized according to whether they had a history consistent with symptomatic acute hepatitis B.
Thirty-two HIV-positive gay men with natural immunity to HBV were screened, to identify 16 HLA-A2positive patients. Two had a history of symptomatic acute hepatitis B, and 14 had no history of jaundice or other relevant symptoms. Patients had a median CD4+ T cell count of 320 cells/L and a median HIV load of 36,000 copies/mL (table 1). Fourteen patients were HAART naive, and 2 patients were already receiving antiretroviral therapy; 4 HLA-A2positive, HAART-naive patients were followed up longitudinally after starting combination therapy. Twenty-five HIV-negative patients (tested within the previous year) with natural immunity to HBV were screened, to identify 14 HLA-A2positive patients, of whom 3 had a history of symptomatic acute hepatitis B.
The HIV-positive and HIV-negative groups had similar demographic characteristics, with all but 1 patient who resolved asymptomatic HBV infection being gay men, and with all being white, apart from 1 Asian patient in the HIV-positive group. The HIV-negative group had a mean age of 38 years (median, 39 years), and the HIV-positive group had a mean age of 39 years (median, 39 years).
Tissue typing.
Screening for the HLA-A2 haplotype was performed by staining peripheral blood mononuclear cells (PBMCs) with an antiHLA-A2positive monoclonal antibody (MAb) (Incstar), followed by fluorescein isothiocyanateconjugated sheep antimouse IgG second-layer MAb and flow-cytometric analysis.
Synthetic HBV peptides and antigens.
Peptides corresponding to the sequence of core 1827; envelope 18391, 33543, 33847, and 34857; and polymerase 45563, 50210, 57583, 65563, and 81624 regions of HBV genotype D were synthesized (Chiron Mimotopes). Hepatitis B core antigen (HBcAg) and HBsAg were produced in Escherichia coli strain K802 and were 90% pure. HBsAg was supplied by Rhein Biotech, and HBcAg was provided by G. Borisova (University of Latvia).
Production of T cell lines and intracellular IFN- staining.
PBMCs were seeded at a concentration of 3 × 106 cells/mL and stimulated with 1 mol/L relevant peptide (the 10 peptides listed above). Recombinant interleukin (IL)2 (10 IU/mL) (Boehringer Mannheim) was added on day 4 of cell culture. Cells were restimulated on day 10, with 1 mol/L relevant peptide (5 h), the last 4 h with 10 g/mL brefeldin A (Sigma-Aldrich). Cells were stained with anti-CD8+ (Pharmingen), permeabilized with Cytofix/Cytoperm (Pharmingen), stained with antiIFN- mAb (R&D Systems), and analyzed by use of a FACScan flow cytometer with CELLQuest software (Becton Dickinson).
For enumeration of HBV-specific CD4+ T cells, PBMCs were suspended at a concentration of 3 × 106 cells/mL in RPMI 1640 and 5% human serum and stimulated with 1 mol/L HBcAg or HBsAg for 616 h, with addition of brefeldin A after 1 h. Cells were washed, stained with anti-CD4+ phycoerythrin (Pharmingen), and subjected to intracellular cytokine staining with antiIFN- or antiIL-2 mAb (R&D Systems). For all intracellular cytokine staining experiments, responses were calculated by subtracting background production of IFN- or IL-2 in a negative control well without peptide or antigen restimulation. Stimulation with anti-CD3 and anti-CD28 MAb (1 mol/L each) or phorbol 12-myristate 13-acetate (3 ng/mL) and ionomycin (100 ng/mL), for 6 h in the presence of brefeldin A, was used as a positive control.
Statistical analysis.
In the cross-sectional study of HIV-negative and HIV-positive patients, the nonparametric Fisher's exact test was used to compare the number of patients with any HBV-specific CD8+ T cell response detectable. The nonparametric Mann-Whitney U (Wilcoxon rank sum) test was used to compare the groups, with the number and size of T cell responses for each patient taken into account.
RESULTS
Reduction of HBV-specific CD8+ T cell responses in HIV-positive, HBV-immune patients.
We initially conducted a cross-sectional study of HIV-positive and HIV-negative patients with natural immunity to HBV, either with or without a history of acute symptomatic infection. All patients were screened for CD8+ T cells producing IFN- in response to a panel of 10 peptides representing frequently recognized HLA-A2restricted HBV epitopes, and results were calculated after subtraction of background staining in wells without peptide restimulation. In 3 HLA-A2positive, HIV-negative patients who had successfully resolved acute symptomatic HBV infection 35 years before, we found peptide-specific production of IFN- for 6 or 7 of the 10 epitopes tested (figure 1 and table 2). We found that HIV-negative patients who had resolved HBV infection without prior symptoms of acute infection also had detectable CD8+ T cell responses to at least 1 HLA-A2restricted epitope in 6 of 11 cases (figure 1 and table 2). In some patients (e.g., N9, whose last risk exposure to HBV was >10 years before), responses had a level of multispecificity and expansion potential similar to those seen in patients with symptomatic acute infection, who have been the basis of all previous immunological studies of patients resolving HBV infection.
In a similar group of 12 HAART-naive, HIV-positive patients who also had serological evidence of past HBV infection without a history of symptoms, HBV-specific CD8+ T cell responses were markedly diminished. This group was matched with the HIV-negative, HBV-immune group for sex, age, ethnic origin, and likely route of acquisition of HBV infection, and all were antiretroviral naive. HBV-specific CD8+ T cell responses were undetectable after 10 days of specific peptide stimulation, by both intracellular cytokine staining (figure 1) and tetramer staining (data not shown), apart from 1 low-level core 1827 response in P16. In an HIV-positive patient with a history of acute symptomatic hepatitis B (P10; 8 years before), intracellular cytokine staining for IFN- after 10 days of in vitro stimulation showed recognition of 4 of 10 epitopes tested. The proportion of patients with any HBV-specific CD8+ T cell response and the total number and magnitude of responses was significantly greater for the whole HIV-negative group than for the whole HIV-positive group (P = .018, Fischer's exact test, and P = .006, Mann-Whitney U test, respectively). These differences remained significant in a comparison of the closely matched subsets in the HIV-negative and HIV-positive groups who resolved HBV infection without symptoms (P = .027, for the proportion of patients with at least 1 response, Fisher's exact test; P = .01, for the number and size of responses, Mann-Whitney U test) (figure 1).
Intracellular cytokine staining directly ex vivo, in a subgroup of 2 HBV-immune patients who resolved symptomatic infection and 2 HBV-immune patients who resolved asymptomatic infection, from both the HIV-negative and HIV-positive groups (table 3), confirmed that responses were detectable in all of the HIV-negative patients tested. In the HIV-positive group, responses were detected directly ex vivo in only 1 of the HIV-positive patients (in P10, who had a history of acute symptomatic HBV infection 8 years before, but not in P11, who had acute infection >15 years before, or in the patients with asymptomatic infection). In all patients studied by both methods, the breadth of the HBV-specific immune responses seen after direct ex vivo expansion was similar to that seen after in vitro expansion.
HBV-specific CD8+ T cell responses in HIV-positive patients receiving antiretroviral therapy.
To investigate any potential for reconstitution of HBV-specific CD8+ T cell responses, we initially examined responses in 2 patients who had resolved HBV infection without symptoms and were already receiving treatment for HIV infection. P12 started zidovudine and lamivudine at a CD4+ T cell count nadir of 240 cells/L; at the time that samples were obtained, he had been maintained on this regimen for 3 years (at which time his CD4+ T cell count was 350 cells/L and his HIV load was 500 copies/mL). P2 had been receiving HAART for 6 months, but lamivudine was substituted for didanosine 1 month before samples were obtained. His CD4+ T cell count increased from a nadir of 160 cells/L to 700 cells/L, and his HIV load was suppressed to <50 copies/mL. Both of these patients had CD8+ T cell responses to 3 of the 10 HBV-specific, HLA-A2restricted epitopes tested (figure 2), which, after 10 days of in vitro expansion, were present at a magnitude similar to that seen in the HIV-negative, HBV-immune patients. This contrasted with the lack of responses detectable in 11 of the 12 untreated HIV-positive patients who had an equivalent pattern of HBV immunity and no history of symptomatic acute infection (figure 2). HBV-specific CD8+ T cell responses were detectable in the treated HIV-positive patients but not in the untreated HIV-positive, HBV-immune patients tested directly ex vivo (figure 2).
Direct ex vivo analysis of P7 revealed reconstitution of HBV-specific CD8+ T cell responses at week 24 (figure 3B), which is consistent in specificity and timing with the in vitro data (figure 3A), and a simultaneous decrease in levels of CD8+ specific for an HIV-1specific, HLA-A2restricted epitope (gag 7785; data not shown). This patient was also tested directly ex vivo for HBcAg-specific CD4+ T cell responses, by intracellular IFN- staining, which revealed a parallel increase in HBV-specific CD4+ and CD8+ T cell responsiveness (figure 3B).
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DISCUSSION
In the present study, we explored the effect of HIV immunodepletion and HAART-related immune restoration on reshaping immune responses to HBV infection. We observed a decrease in functionally active HBV-specific CD8+ T cell responses in association with HIV infection in HBV-immune patients. Accumulating data suggest that residual virus is kept under tight control by an ongoing immune response in such patients [11, 12, 22]. Thus, the reduction in HBV-specific T cell responses observed in the present study provides a mechanism that could contribute to the increased risk of reactivation of hepatitis B surface antigenemia seen in HIV infection and other situations of clinical immunodepletion [23, 24].
To date, immunological studies of HBV immunity after acute infection have only included patients presenting with symptomatic infection. However, a large proportion of patients are found to have immunity to HBV without having had any prior symptoms of acute infection. Although the duration since the initial exposure to HBV was not always known, a number of HIV-negative patients in the potentially heterogeneous group in the present study had preserved HBV-specific responses over the course of many years. Although we cannot exclude the possibility that HIV-positive patients had a longer interval between resolution of HBV infection and the time that samples were obtained, the efficient long-term preservation of HBV-specific CD8+ T cell responses makes this unlikely to account for their lack of responsiveness.
Data describing the impact of HIV on responses to other viruses suggest that the persistence of detectable responses is related to the magnitude of antigen-specific response generated by each virus. For example, Epstein-Barr virus (EBV) and cytomegalovirus (CMV) are both associated with stable high-frequency memory cytotoxic T lymphocyte (CTL) populations, which, although reduced by HIV infection, remain easily detectable [18, 25]. Since the responses generated in association with control of HBV infection are typically lower in frequency than those to viruses such as EBV and CMV, it is not surprising that few remain detectable in HIV-positive patients. A recent study [26] including long-term follow-up of HIV-positive patients revealed maintenance of numbers but functional impairment of the EBV-specific CD8+ T cell response associated with subtle increases in EBV load and progression to non-Hodgkin lymphoma. Thus, careful long-term assessment of HIV-positive, HBV-immune patients would be required to examine the virological impact of the impaired HBV-specific CD8+ T cell response in this group of patients. A reduction of HBV-specific CD8+ T cell responses after HIV infection would be consistent with the demonstrated ability of HIV to induce apoptosis of CTLs of unrelated specificities through FasL-mediated [27] and tumor necrosis factormediated [28] counterattack. Similarly, data from murine models show that heterologous viral infections can quantitatively and qualitatively alter the memory pool of existing antiviral CD8+ [29, 30].
Our previous study of HIV-positive patients with ongoing HBV infection revealed the potential to recover some HBV-specific T cell responses in patients receiving HAART, in association with the reduction in HBV load induced by anti-HBV agents [20]. By contrast, in the present study, we investigated a group of patients in whom HBV load was already efficiently suppressed, to dissect the potential contribution of HAART-mediated immune reconstitution to the restoration of HBV-specific T cell responses. There is evidence from studies of the herpes viridae (EBV, CMV, and Kaposi sarcomaassociated herpesvirus) that HAART can restore specific T cell frequencies [17, 18, 31, 32], and this correlates with a decrease in end-organ disease [4, 33, 34]. The 2 patients studied cross-sectionally and 2 of the 4 patients studied longitudinally restored functionally active HBV-specific CD8+ T cell responses that had expansion potential after in vitro stimulation. That CD8+ T cell responses were reconstituted only in a proportion of these HBV-immune patients is in accordance with our findings in HIV-negative patients without a history of symptomatic HBV infection and are in line with those of a study of the impact of HIV infection and antiretroviral therapy on responses to Mycobacterium avium complex [35].
An important contribution to the reconstitution of HBV-specific CD8+ T cell responses is likely to be a restoration of CD4+ T cell help associated with the increasing CD4+ T cell counts. T cell help is known to be important for maintaining functionally active CD8+ T cell responses [36], and recent data suggest that IL-2producing CD4+ T cells play a key role in antiviral immunity [21, 37]. In both of the patients with restoration of HBV-specific CD8+ T cell responses, we observed a concomitant reconstitution of IFN-positive and IL-2positive CD4+ T cell responses to HBcAg and HBsAg after the start of HAART. These HBV-specific CD4+ T cell responses were similarly observed in a patient receiving an antiretroviral combination that did not include lamivudine or other drugs with anti-HBV activity. Importantly, we demonstrated the potential for long-term maintenance of both CD4+ and CD8+ T cells during prolonged follow-up of 2 patients, in contrast to the rapid but short-lived effects of lamivudine on HBV-specific T cell function reported in patients with ongoing HBV infection [20, 38]. There is a temporal correlation between the reconstitution of T cell responses observed in the present study and that seen in other studies of HAART-mediated reconstitution of T cell function (reviewed in [39]), with initial reconstitution at 3 months and subsequent reconstitution continuing gradually for at least 2 years [40]. These findings should be extended to a larger group of patients, since the present study was not powered to detect an effect of CD4+ T cell count nadir, magnitude of changes in CD4+ T cell count, or HIV load on the extent of HBV-specific T cell reconstitution. However, it is worth noting that the patient with the most effective reconstitution of HBV-specific CD4+ and CD8+ T cell responses was the one who started HAART with the highest CD4+ T cell count nadir and the lowest HIV load and who achieved the most efficient containment of HIV to undetectable levels.
The findings of the present study could be extrapolated to suggest that, in patients treated for HBV/HIV coinfection, reconstitution of HBV-specific T cell responses may involve 2 distinct components: the reconstitution of responses associated with reduction in HBV load [20], as seen in treatment of HBV monoinfection [41, 42], and the more gradual, sustained reconstitution associated with the prolonged suppression of HIV viremia and reconstitution of HBV-specific CD4+ T cell responses demonstrated here. Thus, antiretroviral therapy can lead to an increase in functional CD4+ and CD8+ T cell responses to HBV infection, supporting the potential of HAART to reconstitute immune responses to clinically important pathogens.
Acknowledgments
We thank the patients and staff of the Mortimer Market Centre who were involved in this study.
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