Norway rat hepacivirus resembles hepatitis C virus in terms of intra-host evolution and escape from neutralizing antibodies

Abstract

Background & aims: Norway rat hepacivirus 1 (NrHV) is an attractive surrogate model for evaluating vaccine strategies against hepatitis C virus (HCV). Yet the immune response in NrHV infections remains poorly understood, particularly the role of neutralizing antibodies (nAbs). Here, we explore nAb development and viral evolution during chronic NrHV infection of inbred rats to understand neutralization and viral escape dynamics.

Methods: Lewis rats inoculated with the NrHV RHV-rn1 strain were monitored for >52 weeks. Viremia was quantified by reverse-transcription quantitative PCR, and NrHV nAbs were characterized by infectious cell culture-based neutralization assays and challenge experiments. Viral evolution was followed over time by whole open reading frame deep sequencing.

Results: In most animals, high levels of nAbs appeared after 20 to 45 weeks of infection, coinciding with the emergence of numerous mutations in the envelope proteins. Incorporation of these E1/E2 mutations into cell culture-adapted RHV-rn1 reduced sensitivity to neutralization by autologous contemporary serum. Five key recurrent E1/E2 substitutions (E209K, R224Q, V275I, T500K, and L569P) were identified, collectively impairing neutralization by serum, with E209K in E1 alone proving sufficient for escape from neutralization. In contrast, NrHV-infected rats devoid of nAbs displayed fewer envelope mutations. Finally, pretreatment of cells with rat serum with high-titer nAbs led to partial control of NrHV infection, and passive immunization with such sera protected SCID mice from subsequent challenge.

Conclusions: This study demonstrates the correlation between nAbs and viral evolution during long-term NrHV infection. The observed humoral immunity for NrHV infection closely resembles that of chronic HCV infection, where late-emerging high-level nAbs fail to clear evolving viral populations, thereby contributing to evasion from adaptive immune responses. Preexisting antibodies do, however, protect from infection.

Impact and implications: The findings from this study provide compelling scientific justification for using Norway rat hepacivirus (NrHV) as a model to investigate immune responses and associated vaccine strategies against hepatitis C virus (HCV). By demonstrating that delayed neutralizing antibody development and viral escape through envelope mutations mirror patterns observed in HCV infections, this research offers valuable insights into the adaptive immune dynamics underlying chronic hepacivirus infections. These results are particularly important for researchers and vaccine developers aiming to better understand immune evasion mechanisms and refine HCV vaccine candidates. Practical applications include utilizing NrHV as a preclinical platform to test and optimize vaccine formulations and evaluate passive immunization strategies aimed at controlling HCV-related disease burdens while considering limitations related to host and viral variability.

Keywords: Animal hepacivirus; HCV; NrHV; RHV-rn1; escape mutations; passive immunization; rodent hepacivirus; surrogate model; vaccine development; viral evolution.