Epitope mapping by random peptide phage display reveals essential residues for vaccinia extracellular enveloped virion spread
<p>Abstract</p> <p>Background</p> <p>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 a...
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doaj-b4b78e5fdc7a4ace89ecb6af5acc2d0f2020-11-25T00:25:27ZengBMCVirology Journal1743-422X2012-09-019121710.1186/1743-422X-9-217Epitope mapping by random peptide phage display reveals essential residues for vaccinia extracellular enveloped virion spreadHe YongWang YonggangStruble Evi BZhang PeiChowdhury SomaReed Jennifer LKennedy MichaelScott Dorothy EFisher Robert W<p>Abstract</p> <p>Background</p> <p>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 <it>Orthopoxvirus</it> 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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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 <it>in vivo</it>. 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.</p> http://www.virologyj.com/content/9/1/217OrthopoxvirusesMonoclonal antibodyB-cell epitopeImmunogenVacciniaPhage display library |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
He Yong Wang Yonggang Struble Evi B Zhang Pei Chowdhury Soma Reed Jennifer L Kennedy Michael Scott Dorothy E Fisher Robert W |
spellingShingle |
He Yong Wang Yonggang Struble Evi B Zhang Pei Chowdhury Soma Reed Jennifer L Kennedy Michael Scott Dorothy E Fisher Robert W Epitope mapping by random peptide phage display reveals essential residues for vaccinia extracellular enveloped virion spread Virology Journal Orthopoxviruses Monoclonal antibody B-cell epitope Immunogen Vaccinia Phage display library |
author_facet |
He Yong Wang Yonggang Struble Evi B Zhang Pei Chowdhury Soma Reed Jennifer L Kennedy Michael Scott Dorothy E Fisher Robert W |
author_sort |
He Yong |
title |
Epitope mapping by random peptide phage display reveals essential residues for vaccinia extracellular enveloped virion spread |
title_short |
Epitope mapping by random peptide phage display reveals essential residues for vaccinia extracellular enveloped virion spread |
title_full |
Epitope mapping by random peptide phage display reveals essential residues for vaccinia extracellular enveloped virion spread |
title_fullStr |
Epitope mapping by random peptide phage display reveals essential residues for vaccinia extracellular enveloped virion spread |
title_full_unstemmed |
Epitope mapping by random peptide phage display reveals essential residues for vaccinia extracellular enveloped virion spread |
title_sort |
epitope mapping by random peptide phage display reveals essential residues for vaccinia extracellular enveloped virion spread |
publisher |
BMC |
series |
Virology Journal |
issn |
1743-422X |
publishDate |
2012-09-01 |
description |
<p>Abstract</p> <p>Background</p> <p>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 <it>Orthopoxvirus</it> 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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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 <it>in vivo</it>. 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.</p> |
topic |
Orthopoxviruses Monoclonal antibody B-cell epitope Immunogen Vaccinia Phage display library |
url |
http://www.virologyj.com/content/9/1/217 |
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