title: Epitope mapping by random peptide phage display reveals essential residues for vaccinia extracellular enveloped virion spread.
abstract: BACKGROUND
  A33 is a type II integral membrane protein expressed on the extracellular enveloped form of vaccinia virus (VACV). Passive transfer of A33-directed monoclonal antibodies or vaccination with an A33 subunit vaccine confers protection against lethal poxvirus challenge in animal models. Homologs of A33 are highly conserved among members of the Orthopoxvirus genus and are potential candidates for inclusion in vaccines or assays targeting extracellular enveloped virus activity. One monoclonal antibody directed against VACV A33, MAb-1G10, has been shown to target a conformation-dependent epitope. Interestingly, while it recognizes VACV A33 as well as the corresponding variola homolog, it does not bind to the monkeypox homolog. In this study, we utilized a random phage display library to investigate the epitope recognized by MAb-1G10 that is critical for facilitating cell-to-cell spread of the vaccinia virus.
  
  
  RESULTS
  By screening with linear or conformational random phage libraries, we found that phages binding to MAb-1G10 display the consensus motif CEPLC, with a disulfide bond formed between two cysteine residues required for MAb-1G10 binding. Although the phage motif contained no linear sequences homologous to VACV A33, structure modeling and analysis suggested that residue D115 is important to form the minimal epitope core. A panel of point mutants expressing the ectodomain of A33 protein was generated and analyzed by either binding assays such as ELISA and immunoprecipitation or a functional assessment by blocking MAb-1G10 mediated comet inhibition in cell culture.
  
  
  CONCLUSIONS
  These results confirm L118 as a component of the MAb-1G10 binding epitope, and further identify D115 as an essential residue. By defining the minimum conformational structure, as well as the conformational arrangement of a short peptide sequence recognized by MAb-1G10, these results introduce the possibility of designing small molecule mimetics that may interfere with the function of A33 in vivo. This information will also be useful for designing improved assays to evaluate the potency of monoclonal and polyclonal products that target A33 or A33-modulated EV dissemination.
authors.affiliation: Laboratory of Plasma Derivatives, Division of Hematology, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, FDA/CBER/OBRR/DH/LPD, HFM-345, 1401 Rockville Pike, Rockville, MD 20852, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: He
authors.forename: Yonggang
authors.initials: Y
authors.lastname: Wang
authors.forename: Evi B
authors.initials: EB
authors.lastname: Struble
authors.forename: Pei
authors.initials: P
authors.lastname: Zhang
authors.forename: Soma
authors.initials: S
authors.lastname: Chowdhury
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Reed
authors.forename: Michael
authors.initials: M
authors.lastname: Kennedy
authors.forename: Dorothy E
authors.initials: DE
authors.lastname: Scott
authors.forename: Robert W
authors.initials: RW
authors.lastname: Fisher
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D047468:Immunoprecipitation; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D050505:Mutant Proteins / Q000235:genetics / Q000276:immunology; D019151:Peptide Library; D017354:Point Mutation; D011485:Protein Binding; D014616:Vaccinia virus / Q000276:immunology* / Q000472:pathogenicity*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*; D014771:Virion / Q000276:immunology*
chemical_list: C446940:A33R protein, Vaccinia virus; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000939:Epitopes; D008562:Membrane Glycoproteins; D050505:Mutant Proteins; D019151:Peptide Library; D014759:Viral Envelope Proteins