Characterization of secreted and intracellular forms of a truncated hepatitis C virus E2 protein expressed by a recombinant herpes simplex virus

Abstract

A replication-defective herpes simplex virus type 1 (HSV-1) recombinant lacking the glycoprotein H (gH)-encoding gene and expressing a truncated form of the hepatitis C (HCV) E2 glycoprotein (E2-661) was constructed and characterized. We show here that cells infected with the HSV/HCV recombinant virus efficiently express the HCV E2-661 protein. Most importantly, cellular and secreted E2-661 protein were both readily detected by the E2-conformational mAb H53 and despite the high expression levels, only limited amounts of misfolded aggregates were detected in either the cellular or secreted fractions. Furthermore, cell-associated and secreted E2-661 protein bound to the major extracellular loop (MEL) of CD81 in a concentration-dependent manner and both were highly reactive with sera from HCV-infected patients. Finally, BALB/c mice immunized intraperitoneally with the recombinant HSV/HCV virus induced high levels of anti-E2 antibodies. Analysis of the induced immunoglobulin G (IgG) isotypes showed high levels of IgG2a while the levels of the IgG1 isotype were significantly lower, suggesting a Th1-type of response. We conclude that the HSV-1 recombinant virus represents a promising tool for production of nonaggregated, immunologically active forms of the E2-661 protein and might have potential applications in vaccine development.

Three Different Functional Microdomains in the Hepatitis C Virus Hypervariable Region 1 (HVR1) Mediate Entry and Immune Evasion

“…The cell lysates were centrifuged, and the supernatants were concentrated 5-fold using a Centricon ultrafiltration tube (Millipore, Bedford, MA). E2 protein in crude cell extract was normalized using Galanthus nivalis agglutinin (GNA) capture ELISA with conformation-dependent anti-E2 mAb H53 or H48 as described (50,51).…”

“…Envelope Protein Binding to HVR1 Antibodies-The binding of envelope proteins to HVR1 antibodies was assayed by GNA capture ELISA as described previously (50,51). In brief, ELISA plates were coated with GNA (Sigma) and incubated overnight at 4°C.…”

CD81-Dependent Binding of Hepatitis C Virus E1E2 Heterodimers

“…Several putative cell surface receptors of HCV or recombinant E2 proteins have been identified (1,25,34,45,46,50,51). Among these receptors, human CD81 has been repeatedly shown to interact with recombinant soluble E2, the E1E2 complex, HCV-like particles, and HCV particles from infectious plasma (3,8,17,22,27,30,31,35,42,45,48,55,59). …”

Amplicons as Vaccine Vectors

“…Protein expression was then evaluated in a panel of cultured cell lines; results showed that most of the expressed HCV E1 and E2 glycoproteins formed misfolded aggregates, although substantial amounts of non-aggregated products were also detected, which exhibited the size, glycosylation pattern and subcellular localization expected for authentic HCV E1 and E2 [Tsitoura et al, 2002]. Subsequent studies by this group used a replication-defective HSV-1 vector deleted in gH (a disabled infectious single-cycle [DISC] vector) to express HCV E2, from the CMV immediate-early promoter [Lucas et al, 2003]. This resulted in the generation of glycoprotein products that were mostly non-aggregated, and able to bind to CD81 (as expected for authentic HCV E2).…”

“…This resulted in the generation of glycoprotein products that were mostly non-aggregated, and able to bind to CD81 (as expected for authentic HCV E2). Intraperitoneal delivery of this vector also elicited a humoral immune response to HCV E2 in mice, which was largely of the IgG2a isotype -consistent with a Th1-type response [Lucas et al, 2003]. No direct comparison of E2 expression by amplicon-versus DISC-vectors containing matched inserts was performed, and immune responses to amplicon-vectored HCV glycoproteins were not analyzed.…”