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)
Bibliographic record
Abstract
Introduction 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. Methods 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 ( Pf Trx BamA/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 Pf Trx BamA/ECL4 and overlapping ECL4 peptides in immunoblotting and ELISA assays. Results Each of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of Pf Trx BamA/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 Pf Trx BamA/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. Discussion 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.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".