Inserm U370, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris Cédex 15, France1
Inserm U25; Hôpital Necker; 75015 Paris, France2
Innogenetics, Gent, Belgium3
Author for correspondence: Christian Bréchot. Fax +33 1 40615581. e-mail Brechot{at}necker.fr
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Abstract |
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The overall profile of the immune response to HCV is now emerging: recovery from acute HCV infection is clearly associated with a strong cellular immune response to the virus, i.e. the proliferation of CD4+ and cytotoxic T cells against several HCV proteins (Cerny & Chisari, 1999 ). The persistence of HCV develops despite humoral and cellular polyclonal and polyspecific immune responses to viral proteins (Baumert et al., 1998
; Cerny & Chisari, 1999
; Farci et al., 2000
; Klenerman et al., 2000
). However, the vigorous proliferation of HCV-specific CD4+ and CD8+ lymphocytes correlates well with the clearance of the virus under therapy. Under these conditions, the mechanisms underlying the ineffectiveness of the immune response in clearing the virus during establishment of a chronic carrier state are largely unknown.
There is some evidence for the appearance of neutralizing antibodies to the virus during the natural course of HCV infection (Shimizu et al., 1994 ). In vivo, infection of a naive chimpanzee can be prevented by preincubating the challenging virus with serum from an infected animal (Farci et al., 1994
). In humans, co-infected with HCV and hepatitis B virus (HBV) and transplanted for end-stage cirrhosis, reinfection of liver grafts by HCV can be prevented partially by administrating anti-HBV immunoglobulins, this observation being consistent with the presence in such immunoglobulin preparations of neutralizing antibodies to HCV (Feray et al., 1998
).
A major problem concerning studies on this issue is the lack of appropriate and reproducible in vitro experimental systems. Attempts have been made to identify neutralizing antibodies to HCV by testing for the inhibition of HCV envelope protein E2 binding to target cells such as MOLT-4 cells (Rosa et al., 1996 ). The relevance of this assay has been established though the correlation of their detection and the resolution of HCV infection in humans (Abrignani & Rosa, 1998
), as well as by the partial protection of chimpanzees from a challenge virus after immunization with HCV envelope proteins. Neutralizing antibodies have also been tested for by inhibiting HCV in vitro infection of mononuclear cell lines (Azzari et al., 2000
; Shimizu et al., 1994
) or HCV binding to fibroblasts (Zibert et al., 1995
). These experimental systems have provided significant information, demonstrating both the appearance of neutralizing anti-HCV antibodies during the course of chronic HCV infection in chimpanzees and humans (Ishii et al., 1998
; van Doorn et al., 1995
) and the importance of antibodies directed against epitopes encoded by the hypervariable region of the envelope protein E2 for virus clearance. However, it is difficult to achieve reproducible infection of these cells and fibroblasts are not infected in vivo. Thus, it is still necessary to develop reproducible assays to precisely define the patterns of neutralizing anti-HCV antibodies and their actual relevance to HCV pathogenesis.
We reported previously the permissivity of human biliary cells for in vitro HCV infection (Loriot et al., 1999 ). Although HCV replicates at a very low level in biliary cells, we were able to show that in vitro HCV infection is highly reproducible. In the present report, we demonstrate the ability of this experimental system to detect neutralizing antibodies.
As described previously (Loriot et al., 1999 ), biliary cells could be maintained in culture with a well-differentiated status for at least 2 months (Fig. 1A
, left panel). The expression of cytokeratin 7 was detected, indicating the purity and differentiation of these cells (Fig. 1A
, right panel).
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Having validated HCV infection in our experimental model, we went on to test the ability of selected serum samples to neutralize HCV infection. The neutralization assay was performed using sera from patients who, after therapy, had recovered from infection (Table 1). We investigated ten patients with HCV-related chronic active hepatitis (CAH). All ten patients were long-term responders after interferon (IFN) or IFN+ribavirin therapy (normal liver tests and negative serum HCV RNA assay at least 1 year after the end of therapy). The HCV genotypes involved were: 1b (two patients), 1a (two patients), 2a (three patients) and 3a (three patients). Serum samples were obtained 6 to 84 months after the end of therapy. Fig. 2(A
, C
) shows representative results obtained with serum samples from patients 2 and 6; these patients had recovered from HCV infection with HCV genotypes 3a (patient 2) and 1b (patient 6). HCV RNA was undetectable when infectious reference serum 1b was incubated with undiluted sera or with 1:25 dilutions of sera from both patients. In contrast, HCV RNA was detected in the positive control of cells infected with HCV reference serum 1b only. HCV RNA was also observed when 1b reference serum was mixed with a 1:100 dilution of sera from patients 2 and 6. These results demonstrated neutralization activity in the sera from both patients.
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Attempts were then made to determine whether neutralization activity might differ before and after the end of HCV multiplication in given patients. Fig. 2(E) shows representative results obtained with serum from patient 2, before anti-viral therapy and after a long-term response to treatment. Using serum obtained before therapy, HCV RNA was detected when infectious serum was incubated with undiluted serum, suggesting an absence of detectable neutralizing anti-HCV antibodies before therapy; in contrast, when using serum samples obtained after therapy, neutralizing anti-HCV antibodies were shown at a serum dilution of 1:25 but not 1:100. These results indicated that neutralizing antibodies were identified after a long-term response but not before therapy.
Globally, this report demonstrates that in vitro infection of primary biliary cells may be used efficiently for studies aimed at detecting and characterizing neutralizing antibodies to HCV. Biliary cells can easily be obtained during gallbladder surgery and primary biliary cells retain a well-differentiated status for up to 2 months. In vitro infection of biliary cells can be achieved reproducibly and we were able to confirm our initial observations by combining the immunohistochemical detection of HCV antigens with HCV RNA detection. No robust experimental system currently exists for the culture of HCV. Although interesting data have been reported in primary human hepatocytes and the levels of HCV multiplication reported with that system are clearly higher than those obtained in our model, it nonetheless remains difficult to obtain this material on a sustained basis and, furthermore, the differentiation status of donor hepatocytes markedly influences the quality of results.
Using this material, we were able to detect antibodies capable of neutralizing in vitro infection in the serum of patients who had recovered from HCV infection after IFN or IFN+ribavirin therapy. The fact that neutralizing antibodies were indeed present in these serum samples was shown by inhibition of neutralization upon serum dilution and by the neutralization achieved with purified IgG. Several interesting features are suggested by this study: firstly, amongst the few subjects tested, neutralizing anti-HCV antibodies were detected after the end of HCV multiplication but not before therapy, thus suggesting an association between their detection and the efficacy of therapy. Some previous reports had suggested that in chronically infected chimpanzees neutralizing anti-HCV could be detected at some but not all time-points, several years after contamination (Farci et al., 1994 ). On the other hand, anti-HCV antibodies detected using baculovirus-engineered virus particles (HCV-LPs) (Baumert et al., 2000
) have been transiently detected in patients with self-limited acute hepatitis. Studies based on a binding neutralization assay have also revealed an association between the detection of anti-NOB (neutralization of binding) antibodies and the resolution of HCV infection (Ishii et al., 1998
). Thus, although our experimental system now needs to be tested in a large series of patients, our findings were consistent with an association between the detection of anti-HCV antibodies with neutralizing activity and the inhibition of HCV multiplication. Nevertheless, an intriguing point remains: the long-term persistence of neutralizing anti-HCV antibodies that we identified during this study (antibodies identified up to 84 months after the end of therapy). Our result contrasts with those of other studies, during which it was not possible to detect such neutralizing activity or where there was a marked decline in the anti-HCV-LP antibody titres in sera obtained long after the end of HCV multiplication (Baumert et al., 2000
). In long-term responders to IFN or IFN+ribavirin, there is evidence for eradication of the virus, as shown by negative PCR tests for HCV RNA in serum, PBMCs and liver (Romeo et al., 1993
; Zeuzem et al., 1998
). Further studies will be needed to determine the kinetics of the anti-HCV antibodies detected in our assay and to correlate these findings with virus elimination patterns. Secondly, our findings demonstrate that infection with our reference HCV 1b inoculum could be neutralized by sera obtained from patients infected with different HCV genotypes. Antibodies to HCV-LPs detected with HCV 1b proteins expressed in baculovirus have been detected in patients infected by different HCV genotypes. Consistent with these observations, conserved domains have been identified in the hypervariable region 1 sequence of the envelope protein E2. Thus, taken together, our data support the possibility of inducing neutralizing anti-HCV antibodies with cross-reactivity between different genotypes, which may have major implications in the prevention of an HCV infection. Thirdly, in most, if not all, sera with neutralizing activity, our results demonstrated serological reactivity against different peptide-derived E1 and E2 envelope proteins (Table 1
) (Lechner et al., 1998
; Rosa et al., 1996
; Weiner et al., 1992
; Zhou et al., 1999
; Zibert et al., 1995
). Several reports have, however, shown that antibodies to E2, as determined using the tests currently available, can be detected throughout the course of HCV infection, whether it has resolved or is progressing to chronicity, and their detection is not correlated with HCV clearance (Cerny & Chisari, 1999
; Hassoba et al., 1997
; Tanaka et al., 1999
). During the present study, one patient did not exhibit any detectable anti-E2 and anti-E1 reactivity, although this patients serum showed neutralizing activity. Our data suggest the importance of conformational antibodies and, perhaps, that of other virus epitopes in eliciting neutralizing antibodies.
Taken together, the present study provides a valuable in vitro infection model to test the potential neutralizing activity of various anti-HCV antibodies.
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Acknowledgments |
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References |
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Received 17 October 2001;
accepted 28 February 2002.