Background
Hepatitis B virus (HBV) is the cause of a spectrum of acute diseases including fatal hepatocellular necrosis. More than 350 million people have chronic HBV infection, which causes approximately one million deaths per year from the associated morbidities, liver cirrhosis and cancer [
1]. Several lines of evidence suggest that HBV infection induces suppression of the immune response to the viral components as a result of both viral tropism and antigen release.
In patients with chronic HBV infection HBV-specific T cells egress into the liver but present only a modest anti viral response [
2,
3]. By contrast, patients with an acute, self-limited HBV infection usually mount a vigorous polyclonal CTL response targeting multiple HBV epitopes [
4] and with sufficient longevity to be detectable several years after infection [
5,
6]. Here we show that this is a conclusion not always cogent in the case of patients with a resolved HBV more than two years ago.
As part of the studies to understand the immune response to HBV infection, an important part of the efforts has been focused on identifying highly immunogenic CTL epitopes. One approach involves in silico predictions on the dissociation constant (
K
D) for the binding of antigen-derived peptides to the Human Leukocyte Antigen (HLA) molecules, typically accompanied by experimental testing of CTL response to these peptides or experimentally tested ability of HLA molecules to bind these peptides. A number of studies reported that binding between HLA molecules and antigenic peptides characterized by
K
D <500 nM appears to enable CTL responses [
7]. However, other studies suggest that the effects of antigenic epitopes not always correlate with the affinity of the epitope-containing peptide to HLA [
8,
9]. Previous studies evaluated the immune response to HBV infection by testing previously confirmed HBV epitopes and assessed the T cell response by determining the frequency of reactive T cells, typically by IFN-γ enzyme-linked immunosorbent spot (ELISPOT) analysis. From such studies it was suggested that the frequencies of antigen-specific CTLs may not be the major determinant of immune-mediated protection in chronic hepatitis B, nor should immunotherapeutic approaches only aim at raising the frequency of HBV-specific T cells. Indeed, T cell functionality, such as ability to produce cytokines, may also be important parameters [
10]. However, the correlation between HLA affinity for epitope-containing peptides and T cell cytokine production as estimated by ELISPOT has not been comprehensively studied in the context of chronic HBV infection.
Objectives
Here, we combine in silico methods for predicting HBV-specific CTL epitopes and compare with the T cell response in vitro. By comparing cells from patients with chronic HBV infection (CHB) and patients with resolved HBV infection (RHB), we show that the calculations were efficient in predicting immunogenic epitopes recognized by T cells from CHB patients. Indeed, T cell functionality calculated from the amount of IFN-γ produced was increased in CHB patients with HLA-A11 genotype.
Methods
Patients
The study population consisted of patients with CHB, i.e., positive HBsAg status for more than six months, followed in Aarhus University Hospital, and former patients with RHB previously seen in Aarhus University Hospital. The diagnosis of acute HBV was based on clinical and biochemical evidence of acute liver injury according to standard diagnostic criteria of acute HBV infection, i.e. elevated liver enzymes, positive HBsAg and IgM-antibodies against HBcAg [
11] and reviewed in [
12]. Written consent was obtained from each participant, including consent to publish all personal information contained in Table
3. The study was approved by the The Central Denmark Region Committees on Health Research Ethics, ref. number M-40-12 and the National Data Protection Agency journal number 2012-41-0028.
Virological analyses
HBsAg, HBeAg, anti-HBe, anti-HBc, anti-HBs, HIV Ag/Ab, and anti-HCV were determined by commercially available chemiluminescense assays on the Architect system (Architect, Abbott Laboratories, Abbott Park, Illinois, USA). HBV DNA levels were quantified by commercial hybridization assay.
Peripheral blood mononuclear cells (PBMCs)
PBMCs were isolated from 50 mL of fresh whole blood in 6 × 8 mL cell preparation tubes prefilled with 1 mL 0.1 M sodium citrate and 3 g of polyester gel, 2.0 mL of FICOLL™ Hypaque™ solution (BD Vacutainer® CPT™). Pellet was resuspended in RPMI-1640 with 20 % (v/v) FCS and 10 % (v/v) DMSO and frozen in liquid nitrogen.
HLA loci genotyping
High-resolution HLA class I typing was performed using Sequence Based Typing method with PCR sequencing templates performed on both strands. Sequencing DNA templates was produced by locus- and group-specific amplifications that include exon 2 and 3, which contain the antigen recognition sites. Class I sequencing primers where the common sequences for all loci in the intron/exon boundary regions and a total of 40 locus and group-specific primers were used to amplify the target sequences (HistoGenetics LLC, Ossining, NY, USA).
Epitope selection
Published reports on acute and chronic HBV patient and response to various HBV epitopes was studied and compared to NetMHC version 3.2 predictions of 8-mer epitopes, using Artificial Neural Networks Approximation (
www.cbs.dtu.dk/services/) [
6,
8,
9,
13‐
19]. Computer predictions were made on class I genotypes HLA-A11, HLA-A24 and HLA-A2 since these genotypes covered the majority of our patientpopulation. The surface sequences vary greatly among different HBV genotypes and subtypes [
20] and we observed single and double residue variations between the published sequences and sequences found in the database of the computer algorithm. For this reason we were not able to directly compare the findings of the published reports with the computer predictions. The computer predicted epitopes were designed by cross matching the different HLA subtypes and HBV genotypes of the study population. According to classifications made earlier [
21], a dissociation constant
K
D < 50 nM predicted strong binding, 50–500 nM weak binding, and
K
D > 500 nM predicted essentially no binding. Analysis of protein parameters isoelectric point (pI) [
22], length, instability index [
23] and aliphatic index [
24] were performed on each epitope (
http://web.expasy.org/protparam/
).
Peptides mimetics of HBsAg-specific epitopes in ELISPOT assays
Twenty HBsAg and HBcAg epitopes were purchased from GL Biochem (Shanghai, China), with a purity > 95 % as estimated from mass spectometry. We used HLA class I-restricted T cell epitopes from human cytomegalovirus, Epstein Barr virus and influenza virus (CEF) for positive control (CTL-Europe GmbH, Germany).
Enzyme-linked immunospot (ELISPOT) assays were performed using the 20 different peptides seeded in separate wells. Briefly, 96-well plates (Multiscreen-IP; Millipore S.A.S., Molsheim, France) were coated overnight at 4 °C with 100 μL/well capture mouse anti-human IFN-γ monoclonal antibody (AH diagnostics, Aarhus, Denmark). Plates were washed twice with ELISPOT coating buffer and blocked with CTL serum-free media for 1–2 h at room temperature (CTL-Europe GmbH, Germany). PBMCs (3 × 105/well) were thawed and suspended in CTL serum-free media then seeded in triplicate for each individual peptide. Plates were incubated for 48 h at 37 °C and washed with PBS and 0.05 % Tween-20. 100 μL/well biotinylated secondary mouse anti-human IFN-γ monoclonal antibody was added according to manufacturer recommendations. After 2 h incubation at room temperature, plates were washed four times with PBS and 0.05 % Tween-20, 100 μl of Avidin-HRP solution was added to the wells, and the plates were incubated for further 45 min at room temperature. Plates were washed 3 times with PBS and 0.05 % Tween-20, and 2 times with PBS alone, and 100 μl of AEC substrate solution (3-amino-9-ethyl carbazole) was added. After 10–15 min, the colorimetric reaction was stopped by washing three times with distilled water. Plates were air dried, and spots were counted and analyzed using an automated ELISPOT reader (CTL-Immunospot S6 Analyzer, CTL GmbH, Germany). The T cell response was assessed as spot forming cells (SFC) and mean spot size (MSS). To exclude subjective assessment, whether the background were related to the actually response, we decided to use the stimulation index (SI) for analysis, calculated by dividing the value of the stimulated sample with the value of the unstimulated control. Four uninfected individuals (with clean HBV serology) were included to exclude unspecific findings (data not shown). All patients responded vigorously to the positive CEF control.
Statistics
Statistical analysis was performed using GraphPad Prism 6 (©2014 GraphPad Software, Inc) and STATA/IC 13.1 (©2014 StataCorp, LP). Non-parametric measures were analysed by Spearman’s correlation, Mann–Whitney U test, Kruskal-Wallis test with Dunn’s correction for multiple comparison while parametric data were assessed by Student’s T-test and one-way analysis of variance (ANOVA).
Discussion
Based on findings in several past studies, we hypothesized that a stronger IFN-γ response would be produced in PBMC from resolved HBV patients compared to PBMC from chronic HBV patients when stimulating with 20 individual HBV epitopes and quantifying IFN-γ response by of number of spot forming units and the amount of IFN-γ produced. Surprisingly, T cells from CHB patients recognized a similar number of CTL epitopes compared to T cells from RHB patients. We observed a relatively unimpaired cytokine response with higher levels of IFN-γ in CHB patients, in patients without HLA-A2 genotype
, patients with HLA-A11 genotype, and we found lower levels of IFN-γ production in patients with high viral load (HBeAg positive). These results suggest that the impaired immune response in CHB is not solely due to impaired production of IFN-γ. Indeed, IFN-γ remains as an important component of the overall immune response required combating infection with HBV. Note that in our study the average stimulation index did not exceed 6, which still indicates a low IFN-γ production. Since the majority of epitopes were designed to give a strong response in patients with HLA-A2 genotype, with little difference among HLA-A*02 subtypes, and since we found a stronger response in patients with HLA-A11, this may suggest HLA-A11 is protective. No patients were both HLA-A11 and HBeAg positive and this supports HBeAg as a virulence factor and that control of HBV is more complex than T cell capability of IFN-γ production alone [
26]. Other studies report that the amount of cytokine secreted by individual antigen specific T cells rather than differences in frequencies was the factor responsible for immune deficiencies in patients with HIV infection [
27]. No studies have reported correlation between MSS and SFC in HBV infection. Most studies on HBV focus on SFC as the primary response parameter [
28‐
30]. One tuberculosis vaccination study of non-human primates, found that spot size in combination with spot number, was key in determining a protective immune response [
31]. We found no correlation between SFC and MSS respectively, but where able to describe similar results regarding SFC combined with MSS.
The three experimentally confirmed (EC) epitopes with highest SI (epitope 11 being HBcAg 18–27 which previously has been described as being highly immunogenic [
10,
32,
33]) correlated with observations from previous studies. The three new computer predicted (CP) epitopes HBsCP-7, HBsCP-9 with predicted
K
D < 50 and HBcCP-17 with predicted
K
D > 500, may be worth examining in future studies. Surprisingly we saw a high SI when stimulation with HBsCP-17 despite the high
K
D value, and this suggest that the CTL-inducing effect of an antigenic peptide not always correlate with the computer estimated
K
D value [
8,
9]. However, our results indicate that software both predict new epitopes as well as confirm previously described epitopes.
The liver cellular environment offers opportunities for immune evasion, which may partly explain why the immune response towards HBV frequently fails in the liver. Does this indicate HBV as a well-adapted pathogen - or is it the Th2 biased environment in the liver that permits the path to chronicity? The liver is an important site for T cell activation, however, the environment is biased towards induction of tolerance. This is partially due to the ongoing synthesis of IL-10 by cells constitutively exposed to traces of endotoxin and other microbial products [
34]. In addition, the structure of the liver enables open access of naïve T cells to diverse subsets of antigen presenting cells (APCs). As a result, formation of memory T cell is lacking in this tissue since CD8
+ T cell priming occurs without concomitant CD4
+ T cell activation [
35]. Furthermore, in the liver most APCs express PD-L1 providing the capacity to inactivate T cells. These mechanisms may be a key factor in the immunotolerance in chronic HBV in agreement with ours and the results of the investigations by others, this suggest that IFN-γ production is not the single major component in HBV clearance. Previous studies found a long lasting T cell response only after an acute infection [
5,
6]. Our findings suggest, however, that these observations should be interpreted with caution. Memory CD8
+ T cells can be divided into two subsets, namely central (T
CM) and effector (T
EM), which predominantly are found in the lymph node and in circulation, respectively. Nevertheless, their individual ability to confer protective immunity is not all clarified. One study found that T
CM were more efficient in mediating protective immunity resulting in antigen clearance, and that T
EM converts to T
CM in a lineage differentiation pathway. Furthermore, these results demonstrate that long-term persistence of memory T cells is primarily in the form of T
CM and that 400 days post infection, 95 % of all T cells were CCR7
hi [
36]. This may explain our low response among the resolved HBV patients, since the blood was taken from the periphery, and all donors had been infected more than 2 years ago. Recently, Loggi et al. found the total breadth and magnitude of HBV specific T cell responses in IFN-γ ELISPOT did not differ significantly between groups of CHB and RHB patients [
29] in accordance with our results.
We found evidence that HBeAg status and HLA-type affected the amount of IFN-γ production. IL-7 signaling is essential to CD8
+T cell proliferation and function [
37] and persistent viral antigen load suppresses CD127 expression, i.e., the α-chain on the IL-7 receptor on primed T cells. Studies have found that CD127 negatively correlates with serum HBV DNA and HBeAg levels in chronic HBV [
38]. In addition to the impaired CD127 expression, exhausted HBV-specific T cells in the liver display increased PD-1 expression [
39]. In several studies the PD-1/PD-L1 pathway has been shown to contribute to the suppression of HBV-specific T cell function and IFN-γ secretion is rapidly suppressed despite a continued presence of antigens. This loss of function coincides with the up-regulation of PD-1 [
40]. We now report that a higher viral load in HBeAg positive patients influenced the number of HBV-specific T cells. Despite the higher number of SFC, however, the amount of IFN-γ produced was impaired compared to patients negative for HBeAg. This suggests that the IFN-γ producing subset of T cells may possibly be inactivated or exhausted by PD-1 linkage and CD127 down regulation while T cell ability to register and bind to the antigen is partly maintained.
To date, no HLA class I allele has been confirmed to be a protective or risk factor of CHB. However, many proposals have been made [
41,
42] and, indeed, our findings are in support of some of these previous studies. The IFN-γ production was significantly higher in CHB patients with HLA-A11 genotype compared to the other groups, and with the highest SI levels observed among all groups tested (Fig.
3a). It has been reported previously that HLA-A11 may contribute in the development of natural immunity against HBV [
43] and HLA-A11 were among the most common genotypes of responders to an HBsAg vaccine [
44].
Competing interest
The authors declare that they have no competing interests.
Authors’ contributions
Nanna-Sophie Brinck-Jensen (NSBJ), Thomas Vorup-Jensen (TVJ), Peter Christian Derek Leutscher (PCDL), Christian Eristrup (CE), Eskild Petersen (EP). NSBJ, TVJ and EP were heads of the study concept and design. NSBJ and PCDL acquired all patient data and blood samples. CE was responsible for the HLA acquisition and interpretation. NSBJ carried out all immunoassays. NSBJ TVJ and CE carried out all analysis and interpretation of data. NSBJ and TVJ drafted the manuscript. TVJ, CE and EP made critical revision of the manuscript for important intellectual content. NSBJ, TVJ and CE carried out statistical analysis. PCDL and CE were in charge of administrative, technical, and material supports. TVJ and EP were the overall study supervisors. All authors read and approved the final manuscript.
NSBJ is the PhD student enrolled at Aarhus University. She has been part of the Hepatitis B research group for more than 5 years and has been doing HBV related research in Aarhus, Herlev, Beijing and Liverpool, learning different techniques and skills.
TVJ is professor at Department of Biomedicine, Aarhus University, with special responsibilities in biophysical immunology. He is head of the PhD graduate program for biomedicine at Aarhus University.
PCDL is a medical doctor, PhD, at Department of Infectious Diseases, Aarhus University. He is head of the InfCare Hepatitis DK project, a real-time quality database for patients with chronic HBV.
CE is chief physician and associated professor at Department of Clinical Immunology, Aarhus University Hospital. He is head of HIV and hepatitis testing in the Central Denmark Region, Denmark.
EP is professor at Department of Infectious Diseases, Aarhus University, with many years of experience in the field of infectious diseases, tropical diseases and immunological research. He is chief editor of the international peer review journal International Journal of Infectious Diseases.