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).

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 (ß-barrel assembly machinery subunit A/TP0326), a bipartite protein consisting of a 16-stranded ß-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 (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. Results: 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. 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.

Ligand-Directed GPCR Antibody Discovery.

Developing affinity reagents recognizing and modulating G-protein coupled receptors (GPCR) function by traditional animal immunization or in vitro screening methods is challenging. Some anti-GPCR antibodies exist on the market, but the success rate of development is still poor compared with antibodies targeting soluble or peripherally anchored proteins. More importantly, most of these antibodies do not modulate GPCR function. The current pipeline for antibody development primarily screens for overall affinity rather than functional epitope recognition. We developed a new strategy utilizing natural ligand affinity to generate a library of antibody variants with an inherent bias toward the active site of the GPCR. Instead of using phage libraries displaying antibodies with random CDR sequences at polymorphism sites observed in natural immune repertoire sequences, we generated focused antibody libraries with a natural ligand encoded within or conjugated to one of the CDRs or the N-terminus. To tailor antibody binding to the active site, we limited the sequence randomization of the antibody in regions holstering the ligand while leaving the ligand-carrying part unaltered in the first round of randomization. With hits from the successful first round, the second round of randomization of the ligand-carrying part was then performed to eliminate the bias of the ligand. Based on our results on three different GPCR targets, the proposed pipeline will enable the rapid generation of functional antibodies (both agonists and antagonists) against high-value targets with poor function epitope exposures including GPCR, channels, transporters as well as cell surface targets whose binding site is heavily masked by glycosylation.

MILKSHAKE: novel validation method for antibodies to post-translationally modified targets by surrogate Western blot.

Antibody (Ab) validation is the procedure in which an Ab is thoroughly assayed for sensitivity and specificity in a given application. Validation of Abs against post-translationally modified (PTM) targets is particularly challenging because it requires specifically prepared antigen. Here we describe a novel validation method using surrogate proteins in a Western blot. The surrogate protein, which we termed 'MILKSHAKE,' is a modified maltose binding protein enzymatically conjugated to a peptide from the chosen target that is either modified or nonmodified at the residue of interest. The certainty of the residue's modification status can be used to confirm Ab specificity. This method also allows for Ab validation even in the absence or limited availability of treated cell lysates.

Mechanical performance of hydrogel contact lenses with a range of power under parallel plate compression and central load.

When a contact lens is compressed between two parallel plates (PPC) or under a central load (CLC), the constitutive relation depends not only on the mechanical properties such as elastic modulus, E, of the hydrogel materials, but also the lens power, d, or thickness variation, h(ϕ0), along the meridional direction ϕ0. Hyperopic lenses (d>0) are thicker at the apex along the optical axis and thin out gradually along the meridian, while myopic lenses (d<0) are thinnest at the apex. Mechanical deformation is quantified by the inter-relationship between applied force, F, vertical displacement of the external load, w0, contact or dimple radius, a, and the deformed profile, w(r). Force responses show that lenses with positive d are apparently stiffer in the initial loading but become more compliant as load increases. Conversely, lenses with negative d are more deformable initially and becomes gradually more resistant to loading. This is consistent with the theoretical shell model using the same E. The mechanical behavior has significant impacts in defining the degree of comfort of contact lenses as well as the lens adhesion to the corneal epithelium.