Longitudinal Sequence and Functional Evolution within Glycoprotein E2 in Hepatitis C Virus Genotype 3a Infection

Abstract

The E2 glycoprotein of Hepatitis C virus (HCV) is a major target of the neutralizing antibody (NAb) response with the majority of epitopes located within its receptor binding domain (RBD; 384–661). Within E2 are three variable regions located at the N-terminus (HVR1; 384–411), and internally at 460–480 (HVR2) and 570–580 [intergenotypic variable region (igVR)], all of which lie outside a conserved core domain that contains the CD81 binding site, essential for attachment of virions to host cells and a major target of NAbs. In this study, we examined the evolution of the E1 and E2 region in two patients infected with genotype 3a virus. Whereas one patient was able to clear the acute infection, the other developed a chronic infection. Mutations accumulated at multiple positions within the N-terminal HVR1 as well as within the igVR in both patients over time, whereas mutations in HVR2 were observed only in the chronically infected patient. Mutations within or adjacent to the CD81 contact site were observed in both patients but were less frequent and more conservative in the patient that cleared his/her infection. The evolution of CD81 binding function and antigenicity was examined with longitudinal E2 RBD sequences. The ability of the RBD to bind CD81 was completely lost by week 108 in the patient that developed chronic HCV. In the second patient, the ability of the week 36 RBD, just prior to viral clearance, to bind CD81 was reduced ~50% relative to RBD sequences obtained earlier. The binding of a NAb specific to a conserved epitope located within E2 residues 411–428 was significantly reduced by week 108 despite complete conservation of its epitope suggesting that E2 antigenicity is allosterically modulated. The exposure of non-neutralizing antibody epitopes was similarly explored and we observed that the epitope of 3 out of 4 non-NAbs were significantly more exposed in the RBDs representing the late timepoints in the chronic patient. By contrast, the exposure of non-neutralizing epitopes was reduced in the patient that cleared his/her infection and could in part be attributed to sequence changes in the igVR. These studies reveal that during HCV infection, the exposure of the CD81 binding site on E2 becomes increasingly occluded, and the antigenicity of the E2 RBD towards both neutralizing and non-neutralizing antibodies is modulated via allosteric mechanisms.

Clearance of hepatitis C virus is associated with early and potent but narrowly-directed, Envelope-specific antibodies

“…This study confirmed two previous studies which included a total of just four subjects, that T/F variants in primary infection have a higher dependency for CD81, and CD81 binding is reduced when anti-CD81 nAbs emerge 20,33 . This is noteworthy as nAbs that block CD81 binding are prevalent in chronic progressors, and appear ineffective in clearing infection 16,19,43,44 .…”

“…reduced CD81 or SRBI binding) is an important mechanism of immune escape, as the nAbs then have no effective role in blocking ongoing replication 32 . This has also been supported by in vivo data from two small studies, each with two HCV infected patients analyzed longitudinally, which indicated that reduced CD81 receptor binding coincided with emergence of nAbs 20,33 . However, all three clearance outcomes documented in these studies were unusually delayed beyond 6 months post-infection.…”

An Optimized Hepatitis C Virus E2 Glycoprotein Core Adopts a Functional Homodimer That Efficiently Blocks Virus Entry

“…In conclusion, we propose that the Δ123 protein represents an intriguing HCV vaccine candidate, as it lacks both the HVR1 and igVR sequences which are subject to immune selection pressure during infection (23,26) yet retains key neutralization epitopes within the CD81-binding site when coupled with deletion of HVR2. The broad conservation of the Δ123 protein across diverse isolates of HCV as well as its more accessible or optimal CD81-LEL binding site configuration in the absence of the variable loops is predicted to favor a protective response against infection.…”

“…HVR1 is an immunodominant motif that acquires immune escape variants during the course of infection and is the major determinant of isolate-specific neutralizing antibody responses in vivo (21)(22)(23)(24). Hypervariable region 2 (HVR2; residues 460 to 485) and the intergenotypic variable region (igVR; residues 570 to 580) display 20% and 0% amino acid identity between HCV genotypes, respectively, yet each retains a highly conserved N-linked glycosylation site (25).We have also recently reported that the igVR is under considerable in vivo immune selection pressure, by using a longitudinal analysis of glycoprotein sequence evolution in genotype 3a-infected individuals (26). In addition to amino acid variability, a high degree of structural flexibility recently reported within the variable regions as well as the conserved CD81-binding site has been predicted to account for a significant proportion of the nonneutralizing antibody response to E2 (27).…”

Interferon lambda 4 impacts the genetic diversity of hepatitis C virus

“…Five residues 576, 576d, 577, 578 and 580 are located within the intergenotypic variable region (igVR) that has a role in virion assembly (Albecka et al, 2011; McCaffrey et al, 2011). Recent evidence suggests that the igVR is under strong selective pressure during infection and that it may function to modulate exposure of both the CD81 binding region and antibody epitopes on the surface (Alhammad et al, 2015). This is supported by a study where mutation of igVR residues to alanine reduced binding of non-neutralizing antibodies (Pierce et al, 2016).…”