Comparison of motif-based and whole-unique-sequence-based analyses of phage display library datasets generated by biopanning of anti-Borrelia burgdorferi immune sera

Abstract

Detection of protection-associated epitopes via reverse vaccinology is the first step for development of subunit vaccines against microbial pathogens. Mapping subunit vaccine targets requires high throughput methods, which would allow delineation of epitopes recognized by protective antibodies on a large scale. Phage displayed random peptide library coupled to Next Generation Sequencing (PDRPL/NGS) is the universal platform that enables high-yield identification of peptides that mimic epitopes (mimotopes). Despite being unsurpassed as a tool for discovery of polyclonal serum mimotopes, the PDRPL/NGS is far inferior as a quantitative method of immune response. Difficult-to-control fluctuations in amounts of antibody-bound phages after rounds of selection and amplification diminish the quantitative capacity of the PDRPL/NGS. In an attempt to improve the accuracy of the PDRPL/NGS method, we compared the discriminating capacity of two approaches for PDRPL/NGS data analysis. The whole-unique-sequence-based analysis (WUSA) involved generation of 7-mer peptide profiles and comparison of the numbers of sequencing reads for unique peptide sequences between serum samples. The motif-based analysis (MA) included identification of 4-mer consensus motifs unifying unique 7-mer sequences and comparison of motifs between serum samples. The motif comparison was based not on the numbers of sequencing reads, but on the numbers of distinct 7-mers constituting the motifs. Our PDRPL/NGS datasets generated from biopanning of protective and non-protective anti-Borrelia burgdorferi sera of New Zealand rabbits were used to contrast the two approaches. As a result, the principle component analyses (PCA) showed that the discriminating powers of the WUSA and MA were similar. In contrast, the unsupervised hierarchical clustering obtained via the MA classified the preimmune, non-protective, and protective sera better than the WUSA-based clustering. Also, a total number of discriminating motifs was higher than that of discriminating 7-mers. In sum, our results indicate that MA approach improves the accuracy and quantitative capacity of the PDRPL/NGS method.

A White Plaque, Associated with Genomic Deletion, Derived from M13KE-Based Peptide Library Is Enriched in a Target-Unrelated Manner during Phage Display Biopanning Due to Propagation Advantage

“…In the current work, we report the isolation of a fast-propagating phage clone displaying the peptide WSLGYTG, which was enriched through screening of the commercial Ph.D. TM -7 phage display library against recombinant mouse CD4 protein. A literature search revealed that this peptide sequence has already been isolated in biopanning on two other targets, including human recombinant CD3 Ɛ protein (the epsilon chain of T-cell surface glycoprotein CD3) [ 15 ] and protective and non-protective anti- Borrelia burgdorferi sera of New Zealand White (NZW) rabbits [ 16 ]. However, the chance of obtaining a true binder on different targets in biopanning of a library containing billions of peptides is very unlikely, and the peptide might be a nonspecific ligand.…”

Modeling the sequence dependence of differential antibody binding in the immune response to infectious disease

“…A number of groups have looked at overlapping peptides presented on microarrays or in phage display libraries generated by tiling antigens or entire proteomes [35][36][37][38]. Bio-panning of phage or bacterial peptide display libraries coupled with next generation sequencing have provided broader binding profiles [39,40]. The advantage of tiling and panning approaches is that one is starting with known or suspected binding sequences, and thus the dataset is naturally rich in strong binding information.…”

Modeling the Sequence Dependence of Differential Antibody Binding in the Immune Response to Infectious Disease

“…A number of groups have looked at overlapping peptides presented on microarrays or in phage display libraries generated by tiling antigens or entire proteomes (22)(23)(24)(25)(26)(27). Panning of phage or bacterial peptide display libraries coupled with next generation sequencing have provided broader binding profiles (28,29). The advantage of tiling and panning approaches is that one is starting with known or suspected binding sequences, and thus the dataset is naturally rich in strong binding information.…”