Protective effect of human leukocyte antigen B27 in hepatitis C virus infection requires the presence of a genotype-specific immunodominant CD8+ T-cell epitope

Abstract

Human leukocyte antigen B27 (HLA-B27) is associated with protection in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. This protective role is linked to single immunodominant HLA-B27-restricted CD8+ T-cell epitopes in both infections. In order to define the relative contribution of a specific HLA-B27-restricted epitope to the natural course of HCV infection, we compared the biological impact of the highly conserved HCV genotype 1 epitope, for which the protective role has been described, with the corresponding region in genotype 3 that differs in its sequence by three amino acid residues. The genotype 3a peptide was not recognized by CD8+ T cells specific for the genotype 1 peptide. Furthermore, patients with acute or chronic infection with HCV genotype 3a did not mount T-cell responses to this epitope region, and their autologous viral sequences showed no evidence of T-cell pressure. Finally, we found a significantly higher frequency of HLA-B27 positivity in patients with chronic HCV genotype 3a infection compared to genotype 1 infection, indicating that there is no protection by HLA-B27 in HCV genotype 3 infection.

Conclusion: Our data indicate that the protective effect of HLA-B27 is limited to HCV genotype 1 infection and does not expand to other genotypes such as genotype 3a. This can most likely be explained by intergenotype sequence diversity leading to the loss of the immunodominant HLA-B27 epitope in viral strains other than genotype 1. Our results underline the central role of a single HLA-B27-restricted epitope-specific CD8+ T-cell response in mediating protection in HCV genotype 1 infection.

Fig. 1

The HLA-B27 NS5B_2841-2849 epitope region is highly conserved within but not between different HCV genotypes. (A) Genotype-specific consensus sequences corresponding to the HLA-B27 NS5B_2841-2849 epitope are displayed. The numbers present sequences identical to consensus/total sequences in the Los Alamos HCV Sequence Database. (B) Genotype 1a, 1b, and 3a sequences available at the Los Alamos HCV Sequence Database have been aligned to the respective genotypespecific consensus sequence. Amino acids differing between genotype 1a/1b and genotype 3a are underlined.

Fig. 2

CD8+ T cells specific for the genotype 1 HLA-B27 NS5B_2841-2849 epitope do not cross-recognize the corresponding genotype 3a peptide. PBMC from an HLA-B27+ patient chronically infected with HCV genotype 1 were stimulated for 2 weeks with the genotype 1 HLA-B27 NS5B_2841-2849 epitope peptide (ARMILMTHF). The resulting CTL line was restimulated for 5 hours prior to intracellular INF-γ staining either with the genotype 1 peptide at different concentrations as indicated (upper panel) or with the corresponding genotype 3a peptide (V RM VM MTHF) (lower panel). Percent values indicate the subset of IFN-γ+ CD8+ T cells after subtracting the background in the absence of peptide.

Fig. 3

HLA-B27+ individuals with acute or chronic HCV genotype 3a infection do not target the HLA-B27 NS5B_2841-2849 epitope region and show no sequence variation within the epitope region. CD8+ T cells from HLA-B27+ patients with acute (3/A1 and 3/A2) or chronic (3/C1-C9) HCV genotype 3a infection were tested for INF-γ production after 5 hours of stimulation with the genotype-specific HLA-B27 NS5B_2841-2849 peptide. In addition, PBMC were stimulated for 2 weeks with the genotype-specific peptide, and the resulting CTL lines were tested for INF-γ production after 5 hours of peptide restimulation. Autologous viral sequences corresponding to the epitope region were determined and are aligned to genotypespecific consensus. For comparison, data from genotype 1-infected patients that have in part been published^, are also shown. Percent values are percent INF-γ+ per CD8+ T cells. “−” indicates below the cutoff of 0.02%. “·” indicates homologous to consensus. ND: not done. ¹Infecting virus is known to harbor the genotype 1 consensus sequence within the epitope region.

Fig. 4

Genotype 1 epitope-specific CD8+ T cell response suggest a previous genotype 1 infection. (A) PBMC of patient 3/C3 were stimulated for 2 weeks with the genotype 1 HLA-B27 NS5B_2841-2849 epitope peptide and then tested for INF-γ production after 5 hours of restimulation with the genotype 1 peptide (ARMILMTHF) (upper panel) or the corresponding genotype 3a peptide (V RM VM MTHF) (lower panel) in the indicated concentration. (B) PBMCs of the same patient were stimulated for 2 weeks with three additional genotype 1a-specific epitope peptides and then tested for IFN-γ production after 5 hours of restimulation with the genotype 1a-specific peptide as well as the corresponding genotype 3a specific peptide at a concentration of 10⁻⁵ M. (C) For the HLA-B35 restricted epitope, restimulation was also performed with autologous B-cell lines that had been loaded with peptide overnight in the indicated concentrations and washed extensively prior to the 5-hour coculture with the CTL line.

Fig. 5

The frequency of HLA-B27 is significantly higher in patients chronically infected with genotype 3a compared to patients infected with genotype 1. The frequency of HLA-B27 was determined in a cohort of 265 patients chronically infected with HCV genotype 1 and 98 patients chronically infected with HCV genotype 3a. The frequency of HLA-B27 in the general population includes 11,407 individuals from the German bone marrow registry. P-value was calculated by two-tailed Fisher's exact test.