The hepatitis C virus (HCV) genome encodes two membrane-associated envelope glycoproteins (E1 and E2), which are released from the viral polyprotein precursor by host signal peptidase cleavages. These glycoproteins interact to form a noncovalent heterodimeric complex, which is retained in the endoplasmic reticulum. HCV glycoproteins, E1 and E2, are heavily modified by Nlinked glycosylation. A recent study has revealed that upon partial deglycosylation with endoglycosidase H only four of the five potential glycosylation sites of HCV glycoprotein E1 are utilized. In this work, the unused glycosylation site on the E1 glycoprotein was identified and the influence of N-linked glycosylation on the formation of the HCV glycoprotein complex was studied by expressing a panel of E1 glycosylation mutants in HepG2 cells. Each of the five potential Nlinked glycosylation sites, located at amino acid positions 196, 209, 234, 305 and 325, respectively, on the HCV polyprotein, was mutated separately as well as in combination with the other sites. Expression of the mutated E1 proteins in HepG2 cells indicated that the fifth glycosylation site is not used for the addition of N-linked oligosaccharides and the Pro immediately following the sequon (Asn-Trp-Ser) precludes core glycosylation. The effect of each mutation on the formation of noncovalent E1E2 complexes was also analysed. As determined with the use of a conformation-sensitive monoclonal antibody, mutations at positions N2 and N3 had no, or only minor, effects on the assembly of the E1E2 complex, whereas a mutation at position N1 and predominantly at position N4 dramatically reduced the efficiency of the formation of noncovalent E1E2 complexes.

The hepatitis C virus (HCV) is the etiologic agent of non-A non-B hepatitis, the leading cause of chronic liver infections. Chronic HCV infection is correlated with the risk of developing liver cirrhosis and hepatocellular carcinoma (1). It is estimated that there are more than 170 million chronic carriers worldwide (44). To date, there is neither a prophylactic vaccine nor a satisfactory therapeutic treatment (27), and although routine testing of blood products for HCV has reduced posttransfusion infection in the Western world, not all of the routes of transmission are known, and new cases are still accumulating.The development of an HCV vaccine has been problematic due to the logistics and the ethical restrictions in the numbers and use of chimpanzees, the only species other than Homo sapiens that is susceptible to chronic HCV infection. Furthermore, essential information regarding the immune correlates of protective immunity is still lacking. Although important proof-of-principle ex vivo neutralization studies have been undertaken with chimpanzees (14), neutralizing antibodies are not easily attained and are considered insufficient for viral clearance or the prevention of reinfection (9, 13). The study of immune responses induced in individuals and chimpanzees with acute resolved versus chronic infections has so far revealed only an emerging picture of potential protective immune responses following the establishment of active viral infection. In addition to innate host immunity (38), the importance of cytotoxic T lymphocytes (CTLs) (9,12,18,43,46), gamma interferon (IFN-␥)-producing T cells homing into the liver (36), and T-helper (Th) responses (4,10,20,26) in the control of HCV infection has been demonstrated. However, the correlates of prophylactic vaccine protection following parenteral immunization are expected to be largely extrahepatic prior to HCV exposure. We set out to correlate such peripheral immune responses with vaccine efficacy in order to identify peripheral immune readouts that are likely to be indicative of a desirable vaccine-induced response. Such end points should be readily measurable in human volunteers without necessitating invasive liver biopsies.A limited number of vaccine efficacy studies have been performed with chimpanzees. Immunization with E1 and E2 proteins protected five out of seven animals from infection after a low-dose homologous challenge (with exactly the same clone being used for immunization and challenge) (8) but not against heterologous challenge. In another study, DNA expressing E2 was used to immunize two chimpanzees. Following homologous challenge, both vaccinees became infected but resolved the infection (15). However, none of these studies provided insights into the nature of the vaccine-induced immune correlates of clearance. In addition, HCV presents a high degree of variability (35). Multiple genotypes coexist worldwide, and in * Corresponding author. Mailing address: UMR 2142 CNRS/BioMerieux, 46 allee d'Italie,

Deglycosylation of viral glycoproteins has been shown to influence the number of available epitopes and to modulate immune recognition of antigens. We investigated the role played by N-glycans in the immunogenicity of hepatitis C virus (HCV) E1 envelope glycoprotein, a naturally poor immunogen. Eight plasmids were engineered, encoding E1 protein mutants in which the four N-linked glycosylation sites of the protein were mutated separately or in combination. In vitro expression studies showed an influence of N-linked glycosylation on expression efficiency, instability, and/or secretion of the mutated proteins. Immunogenicity of the E1 mutants was studied in BALB/c mice following intramuscular and intraepidermal injection of the plasmids. Whereas some mutations had no or only minor effects on the antibody titers induced, mutation of the fourth glycosylation site (N4) significantly enhanced the anti-E1 humoral response in terms of both seroconversion rates and antibody titers. Moreover, antibody induced by the N4 mutant was able to recognize HCV-like particles with higher titers than those induced by the wild-type construct. Epitope mapping indicated that the E1 mutant antigens induced antibody directed at two major domains: one, located at amino acids (aa) 313 to 332, which is known to be reactive with sera from HCV patients, and a second one, located in the N-terminal domain of E1 (aa 192 to 226). Analysis of the induced immune cellular response confirmed the induction of gamma interferon-producing cells by all mutants, albeit to different levels. These results show that N-linked glycosylation can limit the antibody response to the HCV E1 protein and reveal a potential vaccine candidate with enhanced immunogenicity.