Patients with chronic hepatitis B virus infection (CHB) usually mount a modest T cell response against HBV epitopes. In order to determine immunogenic epitopes of HBV recognized by HBV-specific T cells, previous studies focused on previously confirmed HBV epitopes and assessed the T cell response by the number of HBV-specific T cells by IFN-γ ELISPOT.
We studied T cell functionality by combined in silico methods predicting HBV-specific epitopes and experimental investigations on the recognition of these epitopes. 30 chronic CHB patients and 10 patients with resolved HBV (RHB) were included in the study. We identified epitopes from the literature and by in silico analysis. These were evaluated for immunogenicity by use of synthetic peptides representing the epitopes through exposure to PBMCs from patients with CHB or RHB by IFN-γ ELISPOT. The number of IFN-γ producing cells (SFC), mean spot size (MSS) and stimulation index (SI) were recorded.
The frequency of HBV-specific T cells producing IFN-γ after stimulation with HBV epitopes was similar in CHB and RHB patients. CHB patients had a higher MSS SI than RHB patients. Patients not carrying the HLA-A2 genotype had higher SFC SI and MSS SI. Patients with HLA-A11 had higher MSS SI compared to non- HLA-A11 allele patients. HBeAg-positive patients had a lower MSS SI, and none of the HBeAg positive patients had the HLA-A11 genotype. We found 3 immunogenic epitopes not described previously.
IFN-γ ELISPOT-determined MSS is an efficient marker for T cell recognition of epitopes. This experimental measure showed the in silico analysis for epitope prediction to be a valuable tool in future studies on HLA genotypes and HBV epitopes. This way our study now points to previously unappreciated consequences of carrying the HLA-A11 allele in terms of stronger immunity to HBV.
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. 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. By contrast, patients with an acute, self-limited HBV infection usually mount a vigorous polyclonal CTL response targeting multiple HBV epitopes and with sufficient longevity to be detectable several years after infection. 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. However, other studies suggest that the effects of antigenic epitopes not always correlate with the affinity of the epitope-containing peptide to HLA. 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. 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.
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.
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 and reviewed in. Written consent was obtained from each participant, including consent to publish all personal information contained in Table. 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.
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.
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.
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).
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. 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 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, 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), length, instability index and aliphatic index were performed on each epitope.
