Early therapy for hepatitis C virus (HCV) is associated with a high rate of viral clearance, but the long-term effects on immune responses remain controversial. The role of antibodies, both acutely and in the long term, is not clearly defined. We investigated these issues in a unique cohort of 7 individuals with primary antibody failure, who had received early interferon therapy after infection through contaminated immunoglobulin therapy a decade previously.

In 1994, an outbreak of hepatitis C virus infection, genotype 1a, occurred in 30 hypogammaglobulinemic patients in the United Kingdom from 1 batch of contaminated immunoglobulin. Treatment with interferon (IFN)–α was initiated within 6 months of inoculation, as reported previously.1,2  We were able to study 7 patients who had survived for 10 years, 5 remaining polymerase chain reaction–negative (PCR) many years after treatment (or spontaneous resolution in 2 cases) and 2 who were still PCR+.

T-cell responses in peripheral blood were first studied using enzyme-linked immunospot assays with peptide pools spanning the whole HCV genome arranged in a matrix (described as matrix ELISpot), as well as specific human leukocyte antigen (HLA)–A2– and HLA-A1–restricted peptides.3  Incorporation of 3H-thymidine and CFSE (5,6-carboxylfuorescein diacetate succinimidyl ester; Molecular Probes, Eugene, OR) proliferation assays were performed, using recombinant HCV proteins NS3, NS4, and NS5 (Chiron, Emeryville, CA); HCV core peptide pools; tetanus toxoid (TT); or cytomegalovirus (CMV) lysate as previously described.4,5 

Figure 1.

Analysis of HCV-specific and other T-cell responses in hypogammaglobulinemic cohort. (A) Percentage of positive responses to different antigens tested using a range of techniques. Numbers on top of each bar show absolute numbers of responding individuals tested for the various antigens. The numbers include those tested from the whole cohort, both PCR+ and PCR. Matrix ELISpot indicates antigens tested were 58 pools containing 301 overlapping peptides (P1-P58) spanning the whole HCV genome; TT, tetanus toxoid, CMV-L, cytomegalovirus lysate; A2-EBV, Epstein Barr virus peptide HLA-A2–GLCTLVAML, A2-HCV, HCV peptide HLA-A2–CINGVCWTV; and A1 HCV, HCV peptide HLA-A1–ATDALMTGY. (B) Mean magnitude of antigen-specific T-cell responses in ELISpot. Magnitude is shown as IFN-γ spot-forming cells (SFCs)/106 peripheral blood mononuclear cells (PBMCs). For matrix ELISpot, mean number of reactive pools is 11 (range, 4-26 pools); mean magnitude is 137 SFC/106 PBMCs (range, 45-360 SFC/106 PBMCs). (A-B) The vertical line divides each graph into control antigens (left) and HCV antigens (right). (C) HCV-specific CD4+ proliferative responses as determined by the CFSE assay. Results are shown for 2 PCR hypogammaglobulinemic individuals previously exposed to HCV (first patient in top panels; second patient in middle and bottom control panels). PBMCs were stimulated for 6 days using HCV core peptide pools and HCV nonstructural proteins NS3/4 and NS5 as described. Responses represent the percentage of proliferating CD4+ T cells after subtraction of the background (negative). Undivided CD4+ T cells are detected in the top right quadrants of each FACS plot, and the CFSE signal is diluted with each cell division as the dye is distributed to the daughter cells. Numbers in the top left quadrants of each plot represent the percentage of HCV peptide– or protein-specific CD4+ T cells that have proliferated during the 6-day culture. One negative and 1 positive (phytohemagglutinin [PHA]) control are shown in the bottom panel. Cells are gated on CD4+ and Viaprobe-negative (live) cells (Viaprobe; Becton Dickinson, San Diego, CA).

Figure 1.

Analysis of HCV-specific and other T-cell responses in hypogammaglobulinemic cohort. (A) Percentage of positive responses to different antigens tested using a range of techniques. Numbers on top of each bar show absolute numbers of responding individuals tested for the various antigens. The numbers include those tested from the whole cohort, both PCR+ and PCR. Matrix ELISpot indicates antigens tested were 58 pools containing 301 overlapping peptides (P1-P58) spanning the whole HCV genome; TT, tetanus toxoid, CMV-L, cytomegalovirus lysate; A2-EBV, Epstein Barr virus peptide HLA-A2–GLCTLVAML, A2-HCV, HCV peptide HLA-A2–CINGVCWTV; and A1 HCV, HCV peptide HLA-A1–ATDALMTGY. (B) Mean magnitude of antigen-specific T-cell responses in ELISpot. Magnitude is shown as IFN-γ spot-forming cells (SFCs)/106 peripheral blood mononuclear cells (PBMCs). For matrix ELISpot, mean number of reactive pools is 11 (range, 4-26 pools); mean magnitude is 137 SFC/106 PBMCs (range, 45-360 SFC/106 PBMCs). (A-B) The vertical line divides each graph into control antigens (left) and HCV antigens (right). (C) HCV-specific CD4+ proliferative responses as determined by the CFSE assay. Results are shown for 2 PCR hypogammaglobulinemic individuals previously exposed to HCV (first patient in top panels; second patient in middle and bottom control panels). PBMCs were stimulated for 6 days using HCV core peptide pools and HCV nonstructural proteins NS3/4 and NS5 as described. Responses represent the percentage of proliferating CD4+ T cells after subtraction of the background (negative). Undivided CD4+ T cells are detected in the top right quadrants of each FACS plot, and the CFSE signal is diluted with each cell division as the dye is distributed to the daughter cells. Numbers in the top left quadrants of each plot represent the percentage of HCV peptide– or protein-specific CD4+ T cells that have proliferated during the 6-day culture. One negative and 1 positive (phytohemagglutinin [PHA]) control are shown in the bottom panel. Cells are gated on CD4+ and Viaprobe-negative (live) cells (Viaprobe; Becton Dickinson, San Diego, CA).

Matrix ELISpot analysis revealed responses in 6 of 7 HCV-exposed patients (Figure 1A), but 0 of 5 hypogammaglobulinemic HCV-PCR controls (P = .015). Strong HCV-specific responses were detected in all 5 HCV-exposed PCR patients (Figure 1A-B) and 1 of 2 PCR+ patients.

CD8+ T-cell responses were examined using class I–restricted peptides in IFN-γ ELISpot. These were demonstrated in all patients tested (2 HLA-A2 and 1 HLA-A1; Figure 1A-B). We also analyzed HCV-specific CD4+ T-cell populations using a fluorescence-activated cell sorting (FACS)–based assay (Figure 1A,C), revealing substantial proliferation in 5 of 7 individuals. Responses to NS3-5, characteristic of immunocompetent spontaneous resolvers, were observed in 4 of 5 PCR patients, but 0 of 2 PCR+ patients, and confirmed using conventional tritium assays.

Here we have shown a sustained response to antiviral therapy in acute HCV in the absence of specific antibody, associated with T-cell responsiveness maintained over a decade. Such activity was specific, was detected using a variety of methods, and included CD8+ and CD4+ T-cell responses. Two patients in the group did not receive therapy because they controlled the infection spontaneously, and these showed comparable responses in ELISpot and proliferation assays. Thus, the responses seen in these antibody-deficient patients who were IFN-α treated are not substantially different from untreated, spontaneously resolved, antibody-deficient individuals, or other spontaneous resolver cohorts,3,4  although a much larger cohort would be needed to assess this definitively.

The role of antibodies in long-term antiviral T-cell memory in humans is not known, although it plays a critical role in murine models. Single-source outbreaks of HCV have been highly informative as to the natural history of HCV infection and long-term outcomes of early treatment. Here, such an outbreak provides a unique insight into the longevity of T-cell responses in the absence of antibodies.

This work was supported by grants from the Fritz Thyssen Foundation and the Wellcome Trust.

We thank all patients for their participation; Suranjith Seneviratne, Janet Burton, Nicola Salome-Bentley, and Carol Ross for assistance with samples and patients; and Rodney Phillips for continued support in The Peter Medawar Building.

1
Chapel HM, Christie JM, Peach V, Chapman RW. Five-year follow-up of patients with primary antibody deficiencies following an outbreak of acute hepatitis C.
Clin Immunol
.
2001
;
99
:
320
-324.
2
Christie JM, Healey CJ, Watson J, et al. Clinical outcome of hypogammaglobulinaemic patients following outbreak of acute hepatitis C: 2 year follow up.
Clin Exp Immunol
.
1997
;
110
:
4
-8.
3
Lauer G, Barnes E, Lucas M, et al. High resolution analysis of cellular immune responses in resolved and persistent hepatitis C virus infection.
Gastroenterology
.
2004
;
127
:
924
-936.
4
Semmo N, Day CL, Ward SM, et al. Preferential loss of IL-2-secreting CD4+ T helper cells in chronic HCV infection.
Hepatology
.
2005
;
41
:
1019
-1028.
5
Mannering SI, Morris JS, Jensen KP, et al. A sensitive method for detecting proliferation of rare autoantigen-specific human T cells.
J Immunol Methods
.
2003
;
283
:
173
-183.