Immunologic evidence for lack of heterologous protection following resolution of HCV in patients with non-genotype 1 infection

Abstract

Chronic hepatitis C virus (HCV) infection is typically characterized by a lack of virus-specific CD4(+) T-cell-proliferative responses, but strong responses have been described in a subset of persons with persistent viremia. One possible explanation for these responses is that they were primed by an earlier resolved infection and do not recognize the current circulating virus. We defined all targeted epitopes using overlapping peptides corresponding to a genotype 1a strain in 44 patients chronically infected with different HCV genotypes (GT). Surprisingly, more HCV-specific CD4(+) T-cell responses were detected in patients with chronic non-GT1 infection compared with patients with chronic GT1 infection (P = .017). Notably, we found serologic evidence of a previous exposure to GT1 in 4 patients with non-GT1 infection, and these persons also demonstrated significantly more responses than non-GT1 patients in whom genotype and HCV serotype were identical (P < .001). Comparison of recognition of GT1-specific peptides to peptides representing autologous virus revealed the absence of cross-recognition of the autologous circulating virus. These data indicate that persistent HCV infection can occur in the presence of an HCV-specific T-cell response primed against a heterologous HCV strain, and suggest that clearance of 1 GT does not necessarily protect against subsequent exposure to a second GT.

Figure 1

Strong lymphoproliferative responses to recombinant HCV antigens are detectable in patient C35 despite high GT2b viremia. Lymphocyte proliferation assays (LPAs) with recombinant HCV antigens based on GT1 sequence: core (c22.3), NS4 (c100.3), NS3 + NS4 (c200), NS5, and tetanus toxoid (TT) and PHA as control were performed on fresh PBMCs of patient C35 (“Patients, materials, and methods”) and demonstrate (A) strong responses to multiple antigens despite high untreated viremia. These responses stayed stable over time (data not shown), and did not change (B) after successful antiviral treatment (sustained virologic response) with Peg-interferon and ribavirin for 24 weeks. Patient C35 showed serologic evidence of prior exposure to GT1 virus. Serotyping was performed with the Murex HCV serotyping 1-6 assay (Abbott; “Patients, materials, and methods”).

Figure 2

Comparison of the breath of the HCV-specific CD4⁺ T-cell response in patients with chronic GT1 or non-GT1 infection or spontaneously resolved HCV. (A) PBMCs from 44 patients with chronic HCV infection were CD8⁺ depleted and stimulated with recombinant HCV proteins (or overlapping peptides) covering the entire polyprotein in the presence of recombinant IL-2. Expanded cell lines were tested for IFN-γ production upon stimulation with pools of 10 to 20 overlapping peptides by ELISPOT. All peptide pools eliciting a positive response by IFN-γ production were further deconvoluted to identify specific 20mer peptides targeted by the CD4⁺ T-cell response. Each single response detected in the ELISPOT assay was confirmed by ICS for IFN-γ production as well. Horizontal bars indicate the mean numbers of targeted HCV CD4⁺ T-cell responses against single peptides for each patient as tested by ICS. The number of responses is significantly higher (P = .017) in chronically infected patients with non-GT1 virus than in patients infected with GT1 virus when comparing the number of CD4⁺ T-cell responses detected with GT1-specific peptides in short-term lines across the entire polyprotein. (B) Genotype-specific subanalysis of previously published data reveals that within the group of patients with spontaneously resolved HCV, more responses are detected with GT1 peptide set in patients who are serologically GT1 than patients who are tested non-GT1 (P < .05). Serotyping was performed with the Murex HCV serotyping 1-6 assay (Abbott; “Patients, materials, and methods”).

Figure 3

Summary and location of all the specificities recognized by each individual with chronic infection. Shaded boxes indicate a positive CD4⁺ response. Peptides: p1 = aa11-30, p298 = aa2981-3000.

Figure 4

Comparison of GT1a peptide sequence with autologous sequences from chronic subjects.

Figure 5

Autologous sequence of GT1a-infected chronic patients in the region of three frequently detected epitopes in resolved HCV infection.

Figure 6

Patients with chronic non-GT1 infection who have serologic evidence of previous exposure to GT1 show significantly more responses than patients with non-GT1 infection in whom genotype and serotype correspond. Horizontal bars indicate the mean numbers of targeted HCV CD4⁺ T-cell responses against single peptides for each patient as tested by ICS. The number of responses is significantly higher (P < .001) in chronically infected patients with non-GT1 virus that serologically showed evidence for exposure to GT1 than patients chronically infected with a non-GT1 virus in whom genotype and serotype corresponded non-GT1. Serotyping test was performed with the Murex HCV serotyping 1-6 assay (Abbott; “Patients, materials, and methods”).

Figure 7

HCV GT1a-based peptides, but not peptides representing autologous sequence, are recognized in patients infected with non-GT1 strains. Strong and durable responses against 5 different GT1a-based HCV peptides were detected in patient C29 (Figure 3). This patient was infected with GT4 virus and showed a serologic response to GT1 virus. We were able to cultivate peptide-specific lines (A) by stimulating with GT1-based peptides, but not with autologous peptides (B) (“Patients, materials, and methods”). (C) dilution ICS assays performed on GT1-specific CD4⁺ T-cell lines for all specificities confirm the lack of recognition of peptides representing the autologous sequence of patient C29 (“Patients, materials, and methods”) (D) Ex vivo dilution ELISPOT data for 2 strong responses against p158 (1581-1600) and p180 (1801-1820) confirm that peptides based on GT1a sequence (■) elicited much higher responses than peptides based on autologous GT2b sequence (▲) in patient non-GT1 C35 (“Patients, materials, and methods”).

Figure 8

GT4 and GT2-specific peptide variants representing autologous viral sequence of patients C29 and C35 are not cross-recognized in patients with resolved GT1 infection. HCV-specific cell lines of 2 patients with spontaneously resolved HCV infection (serologic GT1 according to Murex serotyping test: “Patients, materials, and methods”) were tested by dilution ICS for IFN-γ production for their responses against GT1 peptides (■) and autologous peptides (▲) of non-GT1 patients C29 and C35. (A) spontaneous resolver patient R23 shared the restricting HLA DR1*1 alleles for epitope p158 (aa1581-1600) that was detected in patient C35. The GT1-specific peptdie sequence but not the GT2b-specific autologous variant of the non-GT1 patient was recognized in dilution ICS experiments (“Patients, materials, and methods”). (B) Spontaneous resolver patient R1 shared the restricting HLA allele DRB1*11 for the previously described epitope P227 (aa2271-2290) that was detected in non-GT1 patient C29. Here also, the GT1 peptide sequence, but not the GT4-specific autologous variant of non-GT1 patient C29 was recognized.