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).
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).
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DOI：
10.3389/fimmu.2023.1222267
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发表时间：
2023
影响因子：
7.3
通讯作者：
中科院分区：
文献类型：
--
作者：
Syphilis, 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. Three 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. Each 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. Epivolve 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.
影响因子：
2.2
作者：
Holland, Erika G.;Acca, Felicity E.;Belanger, Kristina M.;Bylo, Mary E.;Kay, Brian K.;Weiner, Michael P.;Kiss, Margaret M.
通讯作者：
Kiss, Margaret M.
影响因子：
2.7
作者：
Dan Z;Tan Z;Xia H;Wu G
通讯作者：
Wu G
影响因子：
6.7
作者：
Cai F;Chen WH;Wu W;Jones JA;Choe M;Gohain N;Shen X;LaBranche C;Eaton A;Sutherland L;Lee EM;Hernandez GE;Wu NR;Scearce R;Seaman MS;Moody MA;Santra S;Wiehe K;Tomaras GD;Wagh K;Korber B;Bonsignori M;Montefiori DC;Haynes BF;de Val N;Joyce MG;Saunders KO
通讯作者：
Saunders KO