Characterisation of an Anti-Vaccinia Virus F13 Single Chain Fragment Variable from a Human Anti-Vaccinia Virus-Specific Recombinant Immunoglobulin Library
Vaccinia virus (VACV) belongs to the genus Orthopoxvirus of the family Poxviridae. There are four different forms of infectious virus particles: intracellular mature virus (IMV), intracellular en-veloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). The...
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MDPI AG
2022-01-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/14/2/197 |
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| author | Henrike P. Ahsendorf Ulrike S. Diesterbeck Sven-Kevin Hotop Michael Winkler Mark Brönstrup Claus-Peter Czerny |
| author_facet | Henrike P. Ahsendorf Ulrike S. Diesterbeck Sven-Kevin Hotop Michael Winkler Mark Brönstrup Claus-Peter Czerny |
| author_sort | Henrike P. Ahsendorf |
| collection | DOAJ |
| description | Vaccinia virus (VACV) belongs to the genus Orthopoxvirus of the family Poxviridae. There are four different forms of infectious virus particles: intracellular mature virus (IMV), intracellular en-veloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). The F13 protein occupies the inner side of the CEV- and EEV-membranes and the outer side of the IEV-membranes. It plays an important role in wrapping progress and EEV production. We constructed a human single-chain fragment variable (scFv) library with a diversity of ≥4 × 108 independent colonies using peripheral blood from four vaccinated donors. One anti-F13 scFv was isolated and characterised after three rounds of panning. In Western blotting assays, the scFv 3E2 reacted with the recombinant F13VACV protein with a reduction of binding under denatured and reduced conditions. Two antigenic binding sites (139-GSIHTIKTLGVYSDY-153 and 169-AFNSAKNSWLNL-188) of scFv 3E2 were mapped using a cellulose membrane encompassing 372 15-mere peptides with 12 overlaps covering the whole F13 protein. No neutralisation capa-bilities were observed either in the presence or absence of complement. In conclusion, the con-struction of recombinant immunoglobulin libraries is a promising strategy to isolate specific scFvs to enable the study of the host-pathogen interaction. |
| first_indexed | 2024-03-09T20:53:04Z |
| format | Article |
| id | doaj.art-eccded8ec0ce49ce89d4445481a02864 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-09T20:53:04Z |
| publishDate | 2022-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-eccded8ec0ce49ce89d4445481a028642023-11-23T22:29:06ZengMDPI AGViruses1999-49152022-01-0114219710.3390/v14020197Characterisation of an Anti-Vaccinia Virus F13 Single Chain Fragment Variable from a Human Anti-Vaccinia Virus-Specific Recombinant Immunoglobulin LibraryHenrike P. Ahsendorf0Ulrike S. Diesterbeck1Sven-Kevin Hotop2Michael Winkler3Mark Brönstrup4Claus-Peter Czerny5Division of Microbiology and Animal Hygiene, Department of Animal Sciences, University of Göttingen, Burckhardtweg 2, 37077 Göttingen, GermanyDivision of Microbiology and Animal Hygiene, Department of Animal Sciences, University of Göttingen, Burckhardtweg 2, 37077 Göttingen, GermanyHelmholtz Centre for Infection Research, Inhoffenstraβe 7, 38124 Braunschweig, GermanyInfection Biology Unit, German Primate Center, Leibniz Institute for Primate Research, Kellnerweg 4, 37077 Göttingen, GermanyHelmholtz Centre for Infection Research, Inhoffenstraβe 7, 38124 Braunschweig, GermanyDivision of Microbiology and Animal Hygiene, Department of Animal Sciences, University of Göttingen, Burckhardtweg 2, 37077 Göttingen, GermanyVaccinia virus (VACV) belongs to the genus Orthopoxvirus of the family Poxviridae. There are four different forms of infectious virus particles: intracellular mature virus (IMV), intracellular en-veloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). The F13 protein occupies the inner side of the CEV- and EEV-membranes and the outer side of the IEV-membranes. It plays an important role in wrapping progress and EEV production. We constructed a human single-chain fragment variable (scFv) library with a diversity of ≥4 × 108 independent colonies using peripheral blood from four vaccinated donors. One anti-F13 scFv was isolated and characterised after three rounds of panning. In Western blotting assays, the scFv 3E2 reacted with the recombinant F13VACV protein with a reduction of binding under denatured and reduced conditions. Two antigenic binding sites (139-GSIHTIKTLGVYSDY-153 and 169-AFNSAKNSWLNL-188) of scFv 3E2 were mapped using a cellulose membrane encompassing 372 15-mere peptides with 12 overlaps covering the whole F13 protein. No neutralisation capa-bilities were observed either in the presence or absence of complement. In conclusion, the con-struction of recombinant immunoglobulin libraries is a promising strategy to isolate specific scFvs to enable the study of the host-pathogen interaction.https://www.mdpi.com/1999-4915/14/2/197scFvvaccinia virusrecombinant antibodyF13epitope mapping |
| spellingShingle | Henrike P. Ahsendorf Ulrike S. Diesterbeck Sven-Kevin Hotop Michael Winkler Mark Brönstrup Claus-Peter Czerny Characterisation of an Anti-Vaccinia Virus F13 Single Chain Fragment Variable from a Human Anti-Vaccinia Virus-Specific Recombinant Immunoglobulin Library Viruses scFv vaccinia virus recombinant antibody F13 epitope mapping |
| title | Characterisation of an Anti-Vaccinia Virus F13 Single Chain Fragment Variable from a Human Anti-Vaccinia Virus-Specific Recombinant Immunoglobulin Library |
| title_full | Characterisation of an Anti-Vaccinia Virus F13 Single Chain Fragment Variable from a Human Anti-Vaccinia Virus-Specific Recombinant Immunoglobulin Library |
| title_fullStr | Characterisation of an Anti-Vaccinia Virus F13 Single Chain Fragment Variable from a Human Anti-Vaccinia Virus-Specific Recombinant Immunoglobulin Library |
| title_full_unstemmed | Characterisation of an Anti-Vaccinia Virus F13 Single Chain Fragment Variable from a Human Anti-Vaccinia Virus-Specific Recombinant Immunoglobulin Library |
| title_short | Characterisation of an Anti-Vaccinia Virus F13 Single Chain Fragment Variable from a Human Anti-Vaccinia Virus-Specific Recombinant Immunoglobulin Library |
| title_sort | characterisation of an anti vaccinia virus f13 single chain fragment variable from a human anti vaccinia virus specific recombinant immunoglobulin library |
| topic | scFv vaccinia virus recombinant antibody F13 epitope mapping |
| url | https://www.mdpi.com/1999-4915/14/2/197 |
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