Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326)
IntroductionSyphilis, a sexually transmitted infection caused by the spirochete Treponema pallidum (Tp), is resurging globally. Tp’s repertoire of outer membrane proteins (OMPs) includes BamA (β-barrel assembly machinery subunit A/TP0326), a bipartite protein consisting of a 16-stranded β-barrel wit...
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| Format: | Article |
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Frontiers Media S.A.
2023-08-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2023.1222267/full |
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| author | Mary R. Ferguson Kristina N. Delgado Shannon McBride Isabel C. Orbe Carson J. La Vake Melissa J. Caimano Melissa J. Caimano Melissa J. Caimano Qiana Mendez Trevor F. Moraes Anthony B. Schryvers M. Anthony Moody M. Anthony Moody M. Anthony Moody Justin D. Radolf Justin D. Radolf Justin D. Radolf Justin D. Radolf Justin D. Radolf Michael P. Weiner Kelly L. Hawley Kelly L. Hawley Kelly L. Hawley Kelly L. Hawley |
| author_facet | Mary R. Ferguson Kristina N. Delgado Shannon McBride Isabel C. Orbe Carson J. La Vake Melissa J. Caimano Melissa J. Caimano Melissa J. Caimano Qiana Mendez Trevor F. Moraes Anthony B. Schryvers M. Anthony Moody M. Anthony Moody M. Anthony Moody Justin D. Radolf Justin D. Radolf Justin D. Radolf Justin D. Radolf Justin D. Radolf Michael P. Weiner Kelly L. Hawley Kelly L. Hawley Kelly L. Hawley Kelly L. Hawley |
| author_sort | Mary R. Ferguson |
| collection | DOAJ |
| description | IntroductionSyphilis, a sexually transmitted infection caused by the spirochete Treponema pallidum (Tp), is resurging globally. Tp’s repertoire of outer membrane proteins (OMPs) includes BamA (β-barrel assembly machinery subunit A/TP0326), a bipartite protein consisting of a 16-stranded β-barrel with nine extracellular loops (ECLs) and five periplasmic POTRA (polypeptide transport-associated) domains. BamA ECL4 antisera promotes internalization of Tp by rabbit peritoneal macrophages.MethodsThree overlapping BamA ECL4 peptides and a two-stage, phage display strategy, termed “Epivolve” (for epitope evolution) were employed to generate single-chain variable fragments (scFvs). Additionally, antisera generated by immunizing mice and rabbits with BamA ECL4 displayed by a Pyrococcus furiosus thioredoxin scaffold (PfTrxBamA/ECL4). MAbs and antisera reactivities were evaluated by immunoblotting and ELISA. A comparison of murine and rabbit opsonophagocytosis assays was conducted to evaluate the functional ability of the Abs (e.g., opsonization) and validate the mouse assay. Sera from Tp-infected mice (MSS) and rabbits (IRS) were evaluated for ECL4-specific Abs using PfTrxBamA/ECL4 and overlapping ECL4 peptides in immunoblotting and ELISA assays.ResultsEach of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of PfTrxBamA/ECL4. One mAb, containing a unique amino acid sequence in both the light and heavy chains, showed activity in the murine opsonophagocytosis assay. Mice and rabbits hyperimmunized with PfTrxBamA/ECL4 produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides, including S2. In contrast, Abs generated during Tp infection of mice and rabbits poorly recognized the peptides, indicating that S2 contains a subdominant epitope.DiscussionEpivolve produced mAbs target subdominant opsonic epitopes in BamA ECL4, a top syphilis vaccine candidate. The murine opsonophagocytosis assay can serve as an alternative model to investigate the opsonic potential of vaccinogens. Detailed characterization of BamA ECL4-specific Abs provided a means to dissect Ab responses elicited by Tp infection. |
| first_indexed | 2024-03-12T13:56:36Z |
| format | Article |
| id | doaj.art-269f6fb6d31f482c8031d99450697c02 |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-03-12T13:56:36Z |
| publishDate | 2023-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-269f6fb6d31f482c8031d99450697c022023-08-22T13:55:51ZengFrontiers Media S.A.Frontiers in Immunology1664-32242023-08-011410.3389/fimmu.2023.12222671222267Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326)Mary R. Ferguson0Kristina N. Delgado1Shannon McBride2Isabel C. Orbe3Carson J. La Vake4Melissa J. Caimano5Melissa J. Caimano6Melissa J. Caimano7Qiana Mendez8Trevor F. Moraes9Anthony B. Schryvers10M. Anthony Moody11M. Anthony Moody12M. Anthony Moody13Justin D. Radolf14Justin D. Radolf15Justin D. Radolf16Justin D. Radolf17Justin D. Radolf18Michael P. Weiner19Kelly L. Hawley20Kelly L. Hawley21Kelly L. Hawley22Kelly L. Hawley23Department of Molecular Sciences, Abbratech, Branford, CT, United StatesDepartment of Medicine, UConn Health, Farmington, CT, United StatesResearch and Development Abcam, Branford, CT, United StatesDepartment of Pediatrics, UConn Health, Farmington, CT, United StatesDepartment of Pediatrics, UConn Health, Farmington, CT, United StatesDepartment of Medicine, UConn Health, Farmington, CT, United StatesDepartment of Pediatrics, UConn Health, Farmington, CT, United StatesDepartment of Molecular Biology and Biophysics, UConn Health, Farmington, CT, United StatesDepartment of Molecular Sciences, Abbratech, Branford, CT, United StatesDepartment of Biochemistry, University of Toronto, Toronto, ON, CanadaDepartment of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, CanadaDuke Human Vaccine Institute, Durham, NC, United StatesDepartment of Pediatrics, Duke University Medical Center, Durham, NC, United States0Department of Integrative Immunobiology, Duke University Medical Center, Durham, NC, United StatesDepartment of Medicine, UConn Health, Farmington, CT, United StatesDepartment of Pediatrics, UConn Health, Farmington, CT, United StatesDepartment of Molecular Biology and Biophysics, UConn Health, Farmington, CT, United States1Department of Immunology, UConn Health, Farmington, CT, United States2Department of Genetics and Genome Sciences, UConn Health, Farmington, CT, United StatesDepartment of Molecular Sciences, Abbratech, Branford, CT, United StatesDepartment of Medicine, UConn Health, Farmington, CT, United StatesDepartment of Pediatrics, UConn Health, Farmington, CT, United States1Department of Immunology, UConn Health, Farmington, CT, United States3Division of Infectious Diseases and Immunology, Connecticut Children’s, Hartford, CT, United StatesIntroductionSyphilis, a sexually transmitted infection caused by the spirochete Treponema pallidum (Tp), is resurging globally. Tp’s repertoire of outer membrane proteins (OMPs) includes BamA (β-barrel assembly machinery subunit A/TP0326), a bipartite protein consisting of a 16-stranded β-barrel with nine extracellular loops (ECLs) and five periplasmic POTRA (polypeptide transport-associated) domains. BamA ECL4 antisera promotes internalization of Tp by rabbit peritoneal macrophages.MethodsThree overlapping BamA ECL4 peptides and a two-stage, phage display strategy, termed “Epivolve” (for epitope evolution) were employed to generate single-chain variable fragments (scFvs). Additionally, antisera generated by immunizing mice and rabbits with BamA ECL4 displayed by a Pyrococcus furiosus thioredoxin scaffold (PfTrxBamA/ECL4). MAbs and antisera reactivities were evaluated by immunoblotting and ELISA. A comparison of murine and rabbit opsonophagocytosis assays was conducted to evaluate the functional ability of the Abs (e.g., opsonization) and validate the mouse assay. Sera from Tp-infected mice (MSS) and rabbits (IRS) were evaluated for ECL4-specific Abs using PfTrxBamA/ECL4 and overlapping ECL4 peptides in immunoblotting and ELISA assays.ResultsEach of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of PfTrxBamA/ECL4. One mAb, containing a unique amino acid sequence in both the light and heavy chains, showed activity in the murine opsonophagocytosis assay. Mice and rabbits hyperimmunized with PfTrxBamA/ECL4 produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides, including S2. In contrast, Abs generated during Tp infection of mice and rabbits poorly recognized the peptides, indicating that S2 contains a subdominant epitope.DiscussionEpivolve produced mAbs target subdominant opsonic epitopes in BamA ECL4, a top syphilis vaccine candidate. The murine opsonophagocytosis assay can serve as an alternative model to investigate the opsonic potential of vaccinogens. Detailed characterization of BamA ECL4-specific Abs provided a means to dissect Ab responses elicited by Tp infection.https://www.frontiersin.org/articles/10.3389/fimmu.2023.1222267/fullsyphilisTreponema pallidumouter membrane proteinBamA ECL4opsonic antibodymonoclonal antibody |
| spellingShingle | Mary R. Ferguson Kristina N. Delgado Shannon McBride Isabel C. Orbe Carson J. La Vake Melissa J. Caimano Melissa J. Caimano Melissa J. Caimano Qiana Mendez Trevor F. Moraes Anthony B. Schryvers M. Anthony Moody M. Anthony Moody M. Anthony Moody Justin D. Radolf Justin D. Radolf Justin D. Radolf Justin D. Radolf Justin D. Radolf Michael P. Weiner Kelly L. Hawley Kelly L. Hawley Kelly L. Hawley Kelly L. Hawley Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326) Frontiers in Immunology syphilis Treponema pallidum outer membrane protein BamA ECL4 opsonic antibody monoclonal antibody |
| title | Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326) |
| title_full | Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326) |
| title_fullStr | Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326) |
| title_full_unstemmed | Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326) |
| title_short | Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326) |
| title_sort | use of epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of treponema pallidum bama tp0326 |
| topic | syphilis Treponema pallidum outer membrane protein BamA ECL4 opsonic antibody monoclonal antibody |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2023.1222267/full |
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