title: Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG.
abstract: A recombinant strain HCV1 (hepatitis C virus [HCV] genotype 1a) gpE1/gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high-affinity and scalable method for isolating E1E2. We generated an Fc tag-derived (Fc-d) E1E2 that was selectively captured by protein G Sepharose, with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to those formed by GNA-purified wild-type (WT) E1E2 and exhibited nearly identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26, and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited anti-E2 antibody titers and neutralization of HCV pseudotype viruses similar to those with WT E1E2. Competition enzyme-linked immunosorbent assays (ELISAs) showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d E1E2-immunized mice exhibited stronger competition for AR3B and AR5A than the WT, whereas the levels of competition for HC84.26 and AR4A were similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using protein A/G-based chromatography.
  
  
  OBJECTIVE
  A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine (genotype 1a) elicited cross-neutralizing antibodies from human volunteers. A challenge for isolating the E1E2 antigen is the reliance on GNA, which is unsuitable for large scale-up and global vaccine delivery. We have generated a novel Fc domain-tagged E1E2 antigen that forms functional heterodimers similar to those with native E1E2. Affinity purification and removal of the Fc tag from E1E2 resulted in an antigen with a nearly identical profile of cross-neutralizing epitopes. This antigen elicited anti-HCV antibodies that targeted conserved neutralizing epitopes of E1E2. Owing to the high selectivity and cost-effective binding capacity of affinity resins for capture of the Fc-tagged rE1E2, we anticipate that our method will provide a means for large-scale production of this HCV vaccine candidate.
authors: Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada logan@ualberta.ca michael.houghton@ualberta.ca.
authors: Michael
authors: M
authors: Logan
authors: Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada.
authors: John
authors: J
authors: Law
authors: Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada.
authors: Jason Alexander Ji-Xhin
authors: JAJ
authors: Wong
authors: Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada.
authors: Darren
authors: D
authors: Hockman
authors: Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada.
authors: Amir
authors: A
authors: Landi
authors: Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada.
authors: Chao
authors: C
authors: Chen
authors: Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada.
authors: Kevin
authors: K
authors: Crawford
authors: Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada.
authors: Juthika
authors: J
authors: Kundu
authors: Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada.
authors: Lesley
authors: L
authors: Baldwin
authors: Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada.
authors: Janelle
authors: J
authors: Johnson
authors: Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada.
authors: Anita
authors: A
authors: Dahiya
authors: Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada.
authors: Gerald
authors: G
authors: LaChance
authors: Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey, USA.
authors: Joseph
authors: J
authors: Marcotrigiano
authors: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA.
authors: Mansun
authors: M
authors: Law
authors: Department of Pathology, Stanford University School of Medicine, Stanford, California, USA.
authors: Steven
authors: S
authors: Foung
authors: Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada.
authors: Lorne
authors: L
authors: Tyrrell
authors: Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada logan@ualberta.ca michael.houghton@ualberta.ca.
authors: Michael
authors: M
authors: Houghton
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry; D057134:Antibodies, Neutralizing / Q000096:biosynthesis / Q000737:chemistry; D000956:Antigens, Viral / Q000737:chemistry / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D002847:Chromatography, Agarose / Q000379:methods; D003429:Cross Reactions; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D016174:Hepacivirus / Q000737:chemistry / Q000276:immunology*; D006526:Hepatitis C / Q000276:immunology / Q000517:prevention & control* / Q000821:virology; D018937:Hepatitis C Antibodies / Q000096:biosynthesis* / Q000737:chemistry; D006801:Humans; D007106:Immune Sera / Q000737:chemistry; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D009500:Neutralization Tests; D017510:Protein Folding; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014611:Vaccination; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014761:Viral Hepatitis Vaccines / Q000008:administration & dosage / Q000096:biosynthesis / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000956:Antigens, Viral; D001426:Bacterial Proteins; C087035:E1 protein, Hepatitis C virus; D000939:Epitopes; D018937:Hepatitis C Antibodies; C034284:IgG Fc-binding protein, Streptococcus; D007106:Immune Sera; D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins; D014761:Viral Hepatitis Vaccines; C091015:glycoprotein E2, Hepatitis C virus
keywords: envelope glycoproteins; epitopes; hepatitis C virus; neutralizing antibodies; vaccines