Hepatic tolerance in chronic equine hepacivirus infection
Final Report Abstract
In this project, we successfully constructed and characterized recombinant Modified Vaccinia Ankara (MVA) vaccine candidates expressing major Equine Hepacivirus (EqHV) antigens using our well-established MVA-vector virus. To activate humoral immune responses, the EqHV- E2 glycoprotein from the wildstorm isolate was targeted, while cellular immune responses were targeted via non-structural proteins NS3/NS4A and NS5B. Full-length cDNAs were synthesized with silent mutations that removed vaccinia virus transcription termination signals and were tagged with influenza HA epitopes to facilitate detection. These sequences were integrated site-specifically into the MVA genome under synthetic early/late promoters through homologous recombination. Fluorescent marker mCherry and GFP allowed for the selection of recombinant MVA-EqHV vaccine candidates, with marker removal from the final construct and genetic stability confirmed by PCR. The recombinant MVA vaccine candidates (MVA-E2, MVA-NS3/NS4A, MVA-NS5) were replication-deficient in human and equine cell lines, supporting their BSL-1 classification. In addition, the MVA vaccine candidates replicated to high titers in permissive chicken embryo fibroblasts, demonstrating suitability for industrial vaccine production. Immunofluorescence and Western blotting confirmed strong antigen expression at expected molecular weights, with comparable intracellular localization patterns. The MVA-EqHV vaccine candidates were prepared in accordance with EMA guidelines and are now ready for in vivo immunogenicity testing. The evolutionary dynamics of EqHV were analyzed and compared with those of human hepatitis C virus (HCV) through deep sequencing of the E1/E2 envelope regions from naturally and experimentally infected horses, as well as from human HCV patients. EqHV showed markedly lower intra-host genetic diversity than HCV, even during prolonged viremia, consistent with its lower chronicity rate. Chronic EqHV infections displayed increased diversity compared to acute cases, mirroring HCV infection patterns. Notably, EqHV lacks the hypervariable region 1 (HVR1) in its E2 glycoprotein, a key feature of HCV that enables immune evasion and persistence. Structural and sequence analyses confirmed HVR1’s uniqueness to HCV among hepaciviruses, suggesting a specialized adaptation for chronic infection. Additionally, minimal viral compartmentalization between serum and liver samples in horses indicated a homogeneous intra-host viral population, primarily restricted to the liver. In vivo studies in ponies confirmed the safety and immunogenicity of MVA-based vaccines expressing the different EqHV antigens. Both MVA-E2 and MVA-NS3/NS4A were well tolerated, causing only mild transient adverse effects without significant laboratory abnormalities. Vaccination with MVA-NS3/NS4A vaccine candidate induced robust antigen-specific cellular responses and higher NS3-specific antibody levels compared to MVA-E2 and controls. Therapeutic vaccination in EqHV-infected ponies resulted in a notable viral load reduction from week 9 post-infection, with viral clearance achieved in some vaccinated animals by week 18, contrasting with persistent viremia in controls. MVA-NS3/NS4A also accelerated NS3 antibody production, highlighting its therapeutic potential. Our work provides crucial insights into hepacivirus biology, advances vaccine development against EqHV, and informs strategies for tackling HCV persistence in humans.
Publications
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Dose-Dependent Hepacivirus Infection Reveals Linkage between Infectious Dose and Immune Response. Microbiology Spectrum, 10(5).
Gömer, André; Delarocque, Julien; Puff, Christina; Nocke, Maximilian K.; Reinecke, Birthe; Baumgärtner, Wolfgang; Cavalleri, Jessika M. V.; Feige, Karsten; Steinmann, Eike & Todt, Daniel
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Experimental cross-species infection of donkeys with equine hepacivirus and analysis of host immune signatures. One Health Outlook, 4(1).
Gömer, André; Puff, Christina; Reinecke, Birthe; Bracht, Stephanie; Conze, Maria; Baumgärtner, Wolfgang; Steinmann, Jörg; Feige, Karsten; Cavalleri, Jessika M. V.; Steinmann, Eike & Todt, Daniel
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Intra-host analysis of hepaciviral glycoprotein evolution reveals signatures associated with viral persistence and clearance. Virus Evolution, 8(1).
Gömer, André; Brown, Richard J. P.; Pfaender, Stephanie; Deterding, Katja; Reuter, Gábor; Orton, Richard; Seitz, Stefan; Bock, C.- Thomas; Cavalleri, Jessika M. V.; Pietschmann, Thomas; Wedemeyer, Heiner; Steinmann, Eike & Todt, Daniel
