In vitro assay for neutralizing antibody to hepatitis C virus: Evidence for broadly conserved neutralization epitopes

Abstract

Our understanding of the humoral immune response to hepatitis C virus (HCV) is limited because the virus can be studied only in humans and chimpanzees and because previously described neutralization assays have not been robust or simple to perform. Nevertheless, epidemiologic and laboratory studies suggested that neutralizing Ab to HCV might be important in preventing infection. We have recently described a neutralization assay based on the neutralization of pseudotyped murine retrovirus constructs bearing HCV envelope glycoproteins on their surface. We have applied the assay to well characterized clinical samples from HCV-infected patients and chimpanzees, confirmed the existence of neutralizing Ab to HCV, and validated most previously reported neutralizations of the virus. We did not find neutralizing anti-HCV in resolving infections but did find relatively high titers (>1:320) of such Ab in chronic infections. Neutralizing Ab was directed not only to epitope(s) in the hypervariable region of the E2 envelope protein but also to one or more epitopes elsewhere in the envelope of the virus. Neutralizing Ab was broadly reactive and could neutralize pseudotype particles bearing the envelope glycoproteins of two different subgenotypes (1a and 1b). The ability to assay neutralizing anti-HCV should permit an assessment of the prospects for successful Ab-mediated passive and active immunoprophylaxis against hepatitis C. H epatitis C virus (HCV) is a small enveloped virus containing single-stranded positive sense RNA. It infects up to 170 million people worldwide and is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Although HCV accounts for only Ϸ12% of acute hepatitis in the United States, its high rate of persistence (70-80%) makes it responsible for almost half of the economic burden of this disease.A better understanding of the pathogenesis of hepatitis C and its control is hampered by certain characteristics of the virus. (i) HCV is genetically and probably serologically heterogeneous.(ii) The only animal model for HCV infection is the chimpanzee. (iii) Although the virus replicates sparingly in some cell lines, the in vitro method developed for detecting neutralizing antibodies (Nt Ab) to HCV in this system is so difficult to perform that it has not been widely used. Consequently, although the two envelope proteins of HCV, E1 and E2, have been expressed individually and as heterodimers, and Ab to them has been measured, there is no confirmation that such Ab accurately reflects the response to the virus or that it is Nt Ab.Previously, we identified Nt Ab to HCV by their ability to prevent replication of the virus in a lymphoid cell line (1, 2) or to prevent hepatitis C in chimpanzees (3, 4). These and a small number of similar studies (5) have provided, for almost a decade, the only direct evidence for Ab-mediated neutralization of HCV. Consequently, although considerable new knowledge has been gained about the cellular immune response to HCV, little is known about the role of the hum...

Entry and replication of recombinant hepatitis C viruses in cell culture

“…First of all, they can be used in S2 containment facilities, an undeniable advantage in particular for antiviral screens [156]. Secondly, they are flexible tools able to incorporate a collection of patient-derived glycoproteins [157,158] (a detailed protocol for the generation of such HCVpp can be found in reference [159]), a feature particularly useful to characterize cross-neutralizing antibodies [158,160]. Thirdly, HCVpp proved to be a relevant model to study the mechanisms and steps of virus entry, with for instance the description of HCV internalization [146,147], of the cellular signalling cascades activated upon virus entry [141], or to reveal new receptors for the virus [116,119].…”

Experimental models to study the immunobiology of hepatitis C virus

“…One possible explanation is that titres of anti-hypervariable region 1 (HVR1) antibody, an antibody to the critical neutralization domain of E2 protein (Farci et al, 1996), do not correlate well with titres of HCV-specific neutralizing antibodies in vivo (Bartosch et al, 2003;Major et al, 1999). This suggests that the titre of the anti-HVR1 antibody is either not high enough to confer protection (Bartosch et al, 2003) or that anti-HVR1 antibody cannot neutralize emerged variant mutants (Farci et al, 1996).…”

“…Interestingly, humoral immunity does not seem to be associated with HCV clearance in chimpanzees (Bartosch et al, 2003;Bukh et al, 2008;Cooper et al, 1999;Major et al, 1999Major et al, , 2004Thimme et al, 2002). One possible explanation is that titres of anti-hypervariable region 1 (HVR1) antibody, an antibody to the critical neutralization domain of E2 protein (Farci et al, 1996), do not correlate well with titres of HCV-specific neutralizing antibodies in vivo (Bartosch et al, 2003;Major et al, 1999).…”

“…One possible explanation is that titres of anti-hypervariable region 1 (HVR1) antibody, an antibody to the critical neutralization domain of E2 protein (Farci et al, 1996), do not correlate well with titres of HCV-specific neutralizing antibodies in vivo (Bartosch et al, 2003;Major et al, 1999). This suggests that the titre of the anti-HVR1 antibody is either not high enough to confer protection (Bartosch et al, 2003) or that anti-HVR1 antibody cannot neutralize emerged variant mutants (Farci et al, 1996).One important issue regarding adaptive immunity is whether it can protect the resolved host against HCV reinfection. Studies in reinfected chimpanzees that previously cleared the infection have revealed the existence of a protective immunity, which may, however, not be able to consistently prevent reinfection (Bukh et al, 2008;Major et al, 2002).…”

HCV and lipoproteins: Is oxLDL an Achilles' heel of the Trojan horse?

“…However, recent results suggest that there may be more similarities than anticipated between current in vitro HCV models and virus purified from patients' plasma. HCV E1E2-targeted antibodies have been detected in patients' blood, or induced in immunized animals, that prevented contamination of patients, or infection of chimpanzees, with plasma-derived HCV 19,20 and that readily neutralized both HCVpp and/or HCVcc, 20,21 (Dreux et al, unpublished observations, 2006), indicating some shared serological and antigenic properties. Moreover, HCVcc with the highest specific infectivity was shown to have a low buoyant density, 11 similar to clinical HCV isolates with high infectivity in animals, 2 indicating that they may associate with lipoproteins.…”