Fine-tuning of hepatitis C virus immune evasion through hypervariable region 1 insertions

Hepatitis C virus (HCV) remains a major cause of chronic liver disease and hepatocellular carcinoma worldwide. Although direct-acting antivirals can eliminate HCV, a significant hepatocellular carcinoma risk persists in patients with cirrhosis, even after a viral cure. Another challenge is the limited of access of direct-acting antivirals in priority populations, such as people who inject drugs, and in low-income and middle-income countries, hampering the elimination of the virus on a population level.1 Therefore, an effective vaccine for HCV is urgently needed to prevent chronic HCV infection and support global HCV elimination efforts. However, evasion of neutralizing antibodies (NAbs) by HCV poses a significant challenge for vaccine development efforts. In particular, HVR1, a stretch of 27 amino acids at the N-terminal of HCV envelope glycoprotein E2, exhibits high heterogeneity and is linked to the evasion of broadly reactive NAbs (bNAbs). The presence of HVR1 also modulates interactions with HCV entry receptors, particularly CD81, scavenger receptor class B type I (SR-BI), and low-density lipoprotein receptor (LDLr). Interestingly, insertions in HVR1 have been documented in the literature,2 although the prevalence and functional implications of these insertions have remained unclear. To address these open questions important for vaccine development, Olesen et al3 examined the prevalence of HVR1 insertions in HCV-infected individuals across different HCV genotypes. HVR1 insertions of 1, 3, or 4 amino acids were found in 3% of patients from a Danish cohort, and in 1.2% of E2 sequences from the Los Alamos HCV database.4 Insertions were found across genotypes. Through in-depth next-generation sequencing, the authors found that the viral lineage with the HVR1 insertion was dominant in most patients where insertions were detected, suggesting a functional role for the HVR1 insertions in HCV infection and/or immune evasion. Given the known roles of HVR1, it is possible that these insertions could modulate HCV entry and receptor dependency or evasion of neutralizing antibodies.5,6 To assess the functional role of the HVR1 insertions, the authors introduced patient-derived noncanonical HVR1 sequences or specific amino acid insertions at the appropriate sites into HCVcc core-NS2 recombinants of genotype 1a (H77), 1b (DH5), 2b (DH8) or 3a (DBN).2 Although the presence of the noncanonical HVR1 region decreased infectivity to varying degrees for all recombinants, this was likely due to the chimeric nature of the HVR1 swap,7 as the specific amino acid insertions themselves did not affect infectivity. The authors next attempted to adapt the HVR1 chimeric viruses through long-term cell culture adaptation and identified potential substitutions, as well as HVR1 internal deletions in the adapted recombinants of the DH5 (genotype 1b) strain, that rescued infectivity. Importantly, the HVR1 insertions, as well as the HVR1 internal deletions, were genetically stable and maintained over time in cell culture and in human liver chimeric mice. The impact of HVR1 insertions on receptor dependency was assessed using blocking antibodies against CD81, SR-BI, and LDLr. The introduction of the specific amino acid insertions in HVR1 in the context of DBN (genotype 3a) or H77 (genotype 1a) did not affect receptor dependency, as entry of these viruses was still inhibited by the blockade or absence of the indicated receptors. However, insertion of the entire noncanonical HVR1 sequences reduced dependency on SR-BI, as these chimeric viruses retained their ability to enter cells even in the presence of SR-BI-blocking antibodies. Interestingly, the cell culture adaptive internal HVR1 deletions in DH5 (genotype 1b) also reduced dependency on SR-BI or LDLr, further supporting a functional relationship between HVR1 and these lipoprotein receptors.8 Olesen and colleagues went on to explore the effect of the HVR1 insertions and deletions on sensitivity to well-established bNAbs AR3A, AR4A, and AR5A, which bind non-overlapping neutralization epitopes in E2 or E1/E2. The presence of the adaptive internal HVR1 deletions significantly increased sensitivity to bNAbs, which appeared to correlate with reduced dependency on LDLr. These findings hint at a potential relationship between receptor dependency and antibody evasion. Since HVR1 partially shields the E2 CD81-binding region5 and has been suggested to mediate interactions with LDLr,8 it is possible that binding to LDLr minimizes exposure of vulnerable epitopes during subsequent CD81 engagement and downstream entry steps. A reduced dependency on LDLr may support enhanced CD81 binding while resulting in increased exposure of neutralization epitopes to antibodies. However, further mechanistic studies are needed to specifically test these possibilities and to assess whether these particular HVR1 insertions and deletions affect binding to LDLr. Intriguingly, HVR1 insertions differentially modulated bNAb sensitivity in an epitope-specific manner, which was illustrated by the DBN (genotype 3a) chimeric viruses. While the presence of the entire noncanonical HVR1 region increased the sensitivity of DBN to all bNAbs irrespective of the insertions, introducing specific amino acid insertions (GSG or TTNS) into the corresponding sites in DBN had a more variable effect on antibody sensitivity. For instance, the presence of the GSG insertion in the context of the DBN backbone substantially increased resistance to AR3A, without affecting sensitivity to AR4A or AR5A. In contrast, the introduction of the GSG insertion in the context of another genotype 3a strain (S52) increased sensitivity to all 3 bNAbs. These findings highlight the complex role of HVR1 in mediating bNAb evasion. Moreover, they underscore the importance of the overall E1/E2 context, rather than intrinsic HVR1 sequences, in modulating sensitivity to NAbs. Overall, this study provides the first functional characterization of HVR1 insertions, which are more prevalent in HCV patients than previously thought (Figure 1). Olesen and colleagues perform a thorough assessment of the identified HVR1 insertions, which paves the way for further mechanistic studies to fully understand how these insertions impact antibody binding and neutralization. Although it remains to be determined what selection pressures drive HVR1 insertions, as well as how generalizable the findings are to other genotypes, this study provides new insight into HVR1 that will have important implications for vaccine development. While a successful HCV vaccine will likely need to elicit both antibody and T-cell responses, effective bNAbs are crucial for protecting against infection in a pan-genotypic manner. Dissecting the enigmatic functions of HVR1 is crucial for an effective HCV vaccine since the presence of HVR1 in E2 is required to elicit effective NAbs following immunization.9 HVR1 also protects neutralizing epitopes at least in part by shielding those epitopes from antibodies,10 which could also involve properly orchestrated interactions with entry receptors. Ultimately, the success of an HCV vaccine is predicated on a better understanding of the role of HVR1 in HCV entry and antibody evasion. As such, these findings on the role of HVR1 insertions and deletions contribute valuable new insights into HVR1 biology to inform the development of an effective broadly neutralizing HCV vaccine to ultimately allow the global elimination of HCV.FIGURE 1: Schematic highlighting the prevalence and functional role of HVR1 insertions in HCV entry and antibody evasion investigated by Olesen et al.3 These findings will contribute to HCV vaccine development efforts through a better understanding of antibody evasion mechanisms mediated by HVR1 of the HCV E2 protein. Created with Biorender.com. Abbreviations: E1, envelope glycoprotein 1; E2, envelope glycoprotein 2.

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