# Targeted Removal of HCV E2 N2 N-Glycan Is Associated with Improved Immune Responses in Mice

## TL;DR

Removing a specific sugar from a hepatitis C virus protein improves immune responses in mice, suggesting a better vaccine strategy.

## Contribution

Selective removal of the N2 glycan on HCV E2 improves immune responses while preserving antigen integrity.

## Key findings

- N2 mutant (sE2-N2) showed higher anti-E2 titers and better inhibition of HCV genotype 1a.
- N2 immunization increased CD8+ T cell activity and tumor growth inhibition in mice.
- Therapeutic vaccination with sE2-N2 reduced HCV RNA and liver signals in infected mice.

## Abstract

Hepatitis C virus (HCV) still lacks a licensed vaccine. The envelope glycoprotein E2 is a key neutralizing target, but its dense N-glycan shield can hinder epitope exposure. In this study, we revisit E2 glycan editing and examine whether single-site deletion preserves antigen integrity while improving immune responses in mice under a DNA immunization setting. Using a secreted E2 ectodomain (sE2384–661), we generated five N to D mutants at conserved sites (N1, N2, N4, N6, and N11) and evaluated them in a unified DNA immunization model with identical CpG content and delivery conditions across groups. The N2 mutant (N423, sE2-N2) maintained expression, secretion, and ER localization; furthermore, in mice, it was associated with higher anti-E2 titers and greater inhibition of H77 (genotype 1a) HCVcc at the tested dilutions, with limited activity against Con1 (1b). Cellular analyses showed increased IFN-γ ELISPOT counts and higher frequencies of granzyme B+/perforin+ CD8+ T cells after N2 immunization, while IL-4 remained low. Functionally, N2 elicited stronger specific lysis of CT26-sE2 targets in vitro and slowed CT26-sE2 tumor growth in vivo. In HCV-infected ICR4R+ mice, therapeutic vaccination with sE2-N2 reduced blood HCV RNA and hepatic readouts compared with sE2. A monoclonal antibody isolated from sE2-N2-immunized mice (1C1) neutralized HCVcc in vitro and, after passive transfer, lowered viremia and liver signals in infected mice. Collectively, these findings indicate that selective removal of the N2 glycan preserves antigen properties and is associated with improved humoral and cellular immunity and measurable in vivo activity, supporting targeted glycan editing as a practical strategy to refine E2-based HCV vaccines.