We have studied three groups that differed with respect to the degree of exposition to HCV: 1) chronic HCV carrier (CI); 2) individuals who remained noninfected despite repeated exposure through sexual contact contact with these carriers (EUI); 3) healthy apparently unnexposed volunteers (UI). Specific cellular immune responses against HCV Core or NS3 were assessed by Elispot or T cell proliferation assays.

The highest proportion of HCV-specific response was observed among chronically-infected subjects (Table 1). The fact that such a response was detected in less than half of the patients is in agreement with previous reports on HCV T-cell responses in chronic HCV infection 13 . Elispot responses were detected the most frequently (in 8/20 chronically-infected volunteers tested for Core, and in 2/6, tested for NS3 responses) whereas only few chronically-infected individuals presented Core or NS3-specific proliferative reactions (3/17, and 2/16, respectively).

The synthetic peptides used to screen cellular responses to HCV represent sequence of HCV genotype 1 since the majority of individuals were infected by a genotype 1 virus. Two of the three chronically-infected subjects who gave positive NS3- responses were infected with a genotype 1 virus. Seven of the eight chronically-infected subjects who were positive for Core by the Elispot assay, were also infected by HCV genotype 1. Meanwhile, chronically-infected patients carried also HCV of three other genotypes: one was infected by a genotype 2 virus, and two by each of genotypes 3 and 4 (Table 4). Very low frequency of NS3-responders amongst patients infected with HCV genotype 2, 3 or 4, may reflect a limited number of non-HCV genotype 1 infected individuals included in this study and also the genotype variation of NS3 sequence. The latter explanation is, however, hardly applicable for the core-specific responses, since HCV core is highly conserved with very few amino acid inter-genotype differences 14 .

The low frequency of proliferative responses compared to Elispot could be attributed to a higher sensitivity of the latter (assay). However, in our view, decisive is the type of the registered response. Elispot assays performed with ex vivo isolated PBMC preferentially detect effector lymphocytes that do not need to expand, while assays using in vitro expanded T lymphocytes rather detect precursors of memory T cells with a proliferative capacity 15 . The low frequency of proliferative responses among chronic HCV carriers may rather reflect a weak HCV memory response (specifically when comparing chronic hepatitis C patients to those resolving HCV infection; for review, 13). Of particular note, relatively few individuals gave a concomitant positive response in both assays. This absence of correlation between Elispot and proliferative responses in chronically-infected individuals suggests that effector and memory T cells are distinct T cell populations, probably recognizing different epitopes. Such phenomenon was described earlier 16 .

Interestingly, HCV Core-specific Elispot responses were observed in a relatively high proportion (30%) of the uninfected partners of chronically-infected individuals (Table 1). This is in agreement with previous reports on the populations of uninfected seronegative individuals exposed to HCV, including healthy relatives of HCV-infected individuals, intravenous drug users, and individuals with occupational exposure 17 18 19 20 21 .

The most striking result of the current study was that despite stringent criteria of the positive cellular response, an HCV-specific response was registered in 20% of uninfected subject tested (13/65; Tables 2 and 3). This group was split into two subgroups, depending on the possibility of exposure to HCV. Indeed, eight individuals who displayed a positive result could have been exposed to HCV (professionally), although there was no clear history of contamination (Table 3). No such risk was, however, identified to explain positive results in the remaining five individuals (Table 2).

The detection of HCV specific cellular responses in uninfected volunteers reflects the difficulty to precisely identify all (possible) risks of exposure to HCV. Furthermore, it may also reflect a past inapparent HCV infection. Clearance of HCV viremia associated with cellular immunity in the absence of seroconversion has been reported in populations at risk for HCV exposure 5 22 23 .

Other causes for detecting HCV-response in healthy risk-free individuals cannot be categorically ruled out. Two uninfected volunteers had positive proliferative response for Core: one with no risk of exposure to HCV (EFS 20) gave a response that involved both CD4 and CD8 populations, whereas the other that only implicated a CD4 population response (EFS 11) was retrospectively shown to have been exposed to HCV. For EFS 20, we could map the reactive sequence to Core amino acid residues 173-190 (not shown). An extensive sequence search using the BLAST tool 24 revealed a eight amino acid homology between HCV Core 174-185 (FSIFLLALLSCL) and HBs antigen 41-52 (FIIFLFXLLXCL). While it remains possible that the observed reactivity corresponds to a cross-reactive immunization 8 9 10 , it is noteworthy that EFS 20 was neither infected nor immunized with HBV.

NS3-specific proliferative responses were observed in four uninfected volunteers (COC 13, and EFS 14, 21 and 24). The PBMC of these individuals were also reactive to the CEF peptides including 12 influenza epitopes. As immunization against Influenza virus neuraminidase was reported to generate immune responses crossreactive with HCV NS3 10 , we cannot formally exclude that T-cell proliferation in response to NS3 resulted from cross-reactivity.

In all three groups, a much higher proportion of individuals tested positive for CEF-specific response registered by Elispot and proliferation tests. The proportion of responders (number of positive/total number tested) varied between groups, but was within the limits of stochastic variations: between 56% (9/16) for the chronically-infected subjects and 29% (5/17) for their uninfected partners (in proliferation). These figures matched the range of proportions seen in CEF-positive Elispots: 60% (18/30) for at risk uninfected volunteers and 46% (13/28) for uninfected volunteers with no known risk of HCV-exposure (Table 1). This was somewhat lower that the data reported by Currier et al. 12 , but similar to that reported by Horton et al. 25 possibly reflecting the heterogeneity of the HLA alleles in the studied groups. All CEF-specific proliferative responses involved the CD8 subpopulation, and in 1 of 95 individuals, both the CD8 and CD4 compartments. This is not surprising since most of the CEF peptides were 8 to 9 mers representing CD8 class I-restricted epitopes, although CD4-specific cytotoxic responses have also been reported in human viral infections 26 27 28 .

As there were no statistical difference between the groups in the frequencies of proliferative or Elispot responses to the control (CEF) antigens (Table 1), exposure to or infection by HCV did not seem to have any major impact on the frequency of cellular responses to unrelated viruses. Hence, it is unlikely that the number of positive cellular responses to HCV antigens could be explained by antigen stimulation(s) specific to other viral antigens. In addition, pair-wise comparisons revealed no difference in the occurrence of cellular immune response against HCV core and/or NS3 among CEF-negative versus CEF-positive individuals in any of the groups (UI with known risk, UI at risk, EUI, or CI; all p values > 0.3). Thus, there is no evidence demonstrating that anti-CEF cellular reactivity interfere with the detection of anti-HCV cellular responses.

Alternatively, atypical HCV-specific immune responses may be generated by the occult HCV infections of the liver 29 . Such infections have been described for patients with abnormal liver function of unknown origin, who present negative HCV PCR and Elisa results in the serum but where HCV RNA is detected in the liver 30 . However, in our study, all uninfected CIC volunteers had normal liver biology. For the twenty COC individuals, liver function was investigated using the Fibrotest 31 , and all gave a normal value (not shown). Thus, it is likely that, in this study, the detection of a positive HCV-specific cellular response did not reflect an occult HCV infection.

The polymorphism of the IL28B gene has been recently associated with both spontaneous resolution of HCV infection and sustained virologic response in pegylated interferon/ribavirin treated patients 32 33 34 ; we can speculate that such a polymorphism may explain partially our results but this study was initiated before the first report and we are unauthorized to make a retrospective genetic study.

In summary, the detection of HCV-specific immune responses in uninfected volunteers may reflect an under-estimated prevalence of inapparent and resolving acute HCV infections. This changes our understanding of the epidemiology and the physiopathology of HCV infection. An alternative, not mutually exclusive, hypothesis is the existence of cross-reactivity between HCV antigens and other viral or common antigens present in the general population, as previously suggested by other researchers.

EUI: Exposed Uninfected Individuals; UI: Uninfected Individuals; CI: Chronically Infected Individuals.

The authors have no competing interests with any commercial or other association in conjunction with the research presented herein.

TM, GJ, CM, LD carried out the elispot and proliferation assays. MLC carried out the virological assays. MLC, OL, JLB, SP participated in the design of the study and were responsible for the clinical data from volunteers. MI, TM ad JLB participated in the statistical analysis. YR was responsible for the design and coordination of the study, and for the writting of the manuscript. All authors read and approved the final manuscript.

Present address for L. Durrieu: Centre de recherche CHU Ste-Justine, Département de Microbiologie et Immunologie, Université de Montréal, 3175, Chemin de la Côte-Sainte-Catherine, Montréal H3T 1C5 Québec, Canada.

Corresponding author contact information: Yves Rivière, Viral Immunopathology URA CNRS 3015, Department of Virology, Lwoff Building, Institut Pasteur, 28 rue du Dr Roux, 75015 Paris. tel 331 4568 8778; fax 331 4061 3298; yves.riviere@pasteur.fr or yves.l.riviere@gmail.com.