Prevalence of hepatitis C virus hypervariable region 1 insertions and their role in antibody evasion

Background and Aims: Chronic hepatitis C virus (HCV) infection afflicts around 50 million people globally, causing 250,000 deaths yearly. An effective vaccine needs to overcome high viral diversity and HCV's ability to evade neutralizing antibodies (NAbs). Rapid antigenic drift in the N-terminal motif of envelope protein E2, named hypervariable region 1 (HVR1), is critically involved in NAb evasion via an incompletely understood mechanism involving viral entry factors. The canonical length of HVR1 is 27 amino acids, but insertions of 2-4 amino acids was described in patients infected with genotype 1b. We aimed at determining whether HVR1 insertions may be underreported due to extreme HVR1 variability. Approach and Results: We observed a 0.7% HVR1 insertion prevalence in routine NGS patient contigs. Thus, we performed direct sequence analysis of E1E2 sequences from 131 HCV infected patients. Interestingly, we observed that 3% of patients harbored viruses (genotype 1a, 2b, 3a) with dominant HVR1 insertions. Insertion of longer non-canonical HVR1s into HCV cell culture recombinants frequently caused loss of fitness. However, culture-viable viruses with HVR1 insertions were fully viable in vivo. Interestingly, in adapted genotype 1b recombinants with HVR1 insertions, we found internal HVR1 deletions, that increased antibody sensitivity, which surprisingly correlated more with reduced LDLr than reduced SR-BI dependency, indicating a role of LDLr in NAb evasion. Conversely, HVR1 insertions had no effect on receptor dependency, however, they modulated epitope-specific NAb sensitivity. Conclusions: HVR1 insertion prevalence and NAb sensitivity modulation indicate they represent a mechanism by which HCV evades emerging NAbs during infection.