Identification of disease-specific motifs in the antibody specificity repertoire via next-generation sequencing

Pantazes, Robert J.; Reifert, Jack; Bozekowski, Joel; Ibsen, Kelly N.; Murray, Joseph A.; Daugherty, Patrick S.

doi:10.1038/srep30312

Cited by 40 publications

(57 citation statements)

References 41 publications

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“…Then, we identified antibody-binding peptide sequences using NGS. To allow for the manipulation of 20 5 (3.2 million) k-mers rather than full-length peptides, we processed peptides into subsequences and evaluated the enrichments of all k-mers of length 5 [29]. Next, K-TOPE tiled candidate antigen sequences, such as from a proteome, into overlapping k-mers.…”

Section: Resultsmentioning

confidence: 99%

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A general approach for identifying protein epitopes targeted by antibody repertoires using whole proteomes

Paull

Johnston

Ibsen

et al. 2019

Preprint

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28Antibodies are essential to functional immunity, yet the epitopes targeted by antibody 29 repertoires remain largely uncharacterized. To aid in characterization, we developed a 30 generalizable strategy to identify antibody-binding epitopes within individual proteins and entire 31 proteomes. Specifically, we selected antibody-binding peptides for 273 distinct sera out of a 32 random library and identified the peptides using next-generation sequencing. To identify 33 antibody-binding epitopes and the antigens from which these epitopes were derived, we tiled the 34 sequences of candidate antigens into short overlapping subsequences of length k (k-mers). We 35 used the enrichment over background of these k-mers in the antibody-binding peptide dataset to 36 identify antibody-binding epitopes. As a positive control, we used this approach, termed K-mer 37 Tiling of Protein Epitopes (K-TOPE), to identify epitopes targeted by monoclonal and polyclonal 38 antibodies of well-characterized specificity, accurately recovering their known epitopes. K-39 TOPE characterized a commonly targeted antigen from Rhinovirus A, identifying three epitopes 40 recognized by antibodies present in 83% of sera (n = 250). An analysis of 2,908 proteins from 41 400 viral taxa that infect humans revealed seven enterovirus epitopes and five Epstein-Barr virus 42 epitopes recognized by >30% of specimens. Analysis of Staphylococcus and Streptococcus 43 proteomes similarly revealed six epitopes recognized by >40% of specimens. These common 44 viral and bacterial epitopes exhibited excellent agreement with previously mapped epitopes.45 Additionally, we identified 30 HSV2-specific epitopes that were 100% specific against HSV1 in 46 novel and previously reported antigens. The K-TOPE approach thus provides a powerful new 47 tool to elucidate the organisms, antigens, and epitopes targeted by human antibody repertoires. 48 49 50 Introduction 51 52 Immunological memory allows for rapid antibody responses towards diverse antigens 53 long after initial exposure. For example, the adaptive immune response to many vaccinations is 54 often sustained throughout an individual's lifetime [1]. This immunological information is 55 archived within the genes encoding B-cell and T-cell receptors along with the corresponding 56 receptor structures, but has proven difficult to characterize in a comprehensive manner. The 57 ability to more fully interrogate immunological memory could reveal exposures to pathogens, 58 commensal organisms, and allergens. Such information has proven useful for correlating 59 antibody responses with disease outcomes to design more effective vaccines [2]. A detailed 60 record of immune exposures can also facilitate the identification of biomarkers to diagnose 61 infectious [3], autoimmune [4], and allergic conditions [5]. Furthermore, the capability to 62 broadly characterize antibody repertoires at the epitope level could be used to identify conserved 63 pathogen epitopes [6] and tumor specific antigen epitopes [7] to aid in therapeutic disco...

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Section: Resultsmentioning

confidence: 99%

“…The bacterial peptide display screening protocol was carried out as previously described [29,62]. Briefly, an E. coli library displaying approximately 8 billion different 12-mer peptides was combined with 1:100 diluted serum.…”

Section: Methodsmentioning

confidence: 99%

A general approach for identifying protein epitopes targeted by antibody repertoires using whole proteomes

Paull

Johnston

Ibsen

et al. 2019

Preprint

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“…A large, random (8 × 10 9 independent transformants) 12-mer peptide library displayed on the N-terminus of a transmembrane protein scaffold of E. coli (eCPX) [Pantazes et al , 2016; Rice & Daugherty, 2008] was used in this study. The screening protocol described here was based on previously reported methods [Kenrick et al , 2007] and is detailed in Figure S1.…”

Section: Methodsmentioning

confidence: 99%

“…Libraries from all rounds of MACS were prepared for NGS as previously described [Pantazes et al, 2016]. Briefly, plasmids were extracted from cells grown overnight using a plasmid miniprep kit (Qiagen).…”

Section: Methodsmentioning

confidence: 99%

“…Using the IMUNE processor developed in the Daugherty lab [Pantazes et al ., 2016], the generated DNA sequences were translated into a set of unique peptide sequences for each antibody. First, the processor searches for the constant upstream and downstream annealing regions of the DNA and translates the intervening sequence to generate a peptide sequence.…”

Section: Methodsmentioning

confidence: 99%

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Prediction of antibody structural epitopes via random peptide library screening and next generation sequencing

Ibsen

Daugherty

2017

Journal of Immunological Methods

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Next generation sequencing (NGS) is widely applied in immunological research, but has yet to become common in antibody epitope mapping. A method utilizing a 12-mer random peptide library expressed in bacteria coupled with magnetic-based cell sorting and NGS correctly identified more than 75% of epitope residues on the antigens of two monoclonal antibodies (trastuzumab and bevacizumab). PepSurf, a web-based computational method designed for structural epitope mapping was utilized to compare peptides in libraries enriched for monoclonal antibody (mAb) binders to antigen surfaces (HER2 and VEGF-A). Compared to mimotopes recovered from Sanger sequencing of plated colonies from the same sorting protocol, motifs derived from sets of the NGS data improved epitope prediction as defined by sensitivity and precision, from 18% to 82% and 0.27 to 0.51 for trastuzumab and 47% to 76% and 0.19 to 0.27 for bevacizumab. Specificity was similar for Sanger and NGS, 99% and 97% for trastuzumab and 66% and 67% for bevacizumab. These results indicate that combining peptide library screening with NGS yields epitope motifs that can improve prediction of structural epitopes.

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Alzheimer's disease‐associated CD83(+) microglia are linked with increased immunoglobulin G4 and human cytomegalovirus in the gut, vagal nerve, and brain

Readhead,

Mastroeni,

Wang

et al. 2024

Alzheimer's & Dementia

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INTRODUCTIONWhile there may be microbial contributions to Alzheimer's disease (AD), findings have been inconclusive. We recently reported an AD‐associated CD83(+) microglia subtype associated with increased immunoglobulin G4 (IgG4) in the transverse colon (TC).METHODSWe used immunohistochemistry (IHC), IgG4 repertoire profiling, and brain organoid experiments to explore this association.RESULTSCD83(+) microglia in the superior frontal gyrus (SFG) are associated with elevated IgG4 and human cytomegalovirus (HCMV) in the TC, anti‐HCMV IgG4 in cerebrospinal fluid, and both HCMV and IgG4 in the SFG and vagal nerve. This association was replicated in an independent AD cohort. HCMV‐infected cerebral organoids showed accelerated AD pathophysiological features (Aβ42 and pTau‐212) and neuronal death.DISCUSSIONFindings indicate complex, cross‐tissue interactions between HCMV and the adaptive immune response associated with CD83(+) microglia in persons with AD. This may indicate an opportunity for antiviral therapy in persons with AD and biomarker evidence of HCMV, IgG4, or CD83(+) microglia.Highlights Cross‐tissue interaction between HCMV and the adaptive immune response in a subset of persons with AD. Presence of CD83(+) microglial associated with IgG4 and HCMV in the gut. CD83(+) microglia are also associated presence of HCMV and IgG4 in the cortex and vagal nerve. Replication of key association in an independent cohort of AD subjects. HCMV infection of cerebral organoids accelerates the production of AD neuropathological features.

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Identification of disease-specific motifs in the antibody specificity repertoire via next-generation sequencing

Cited by 40 publications

References 41 publications

A general approach for identifying protein epitopes targeted by antibody repertoires using whole proteomes

A general approach for identifying protein epitopes targeted by antibody repertoires using whole proteomes

Prediction of antibody structural epitopes via random peptide library screening and next generation sequencing

Alzheimer's disease‐associated CD83(+) microglia are linked with increased immunoglobulin G4 and human cytomegalovirus in the gut, vagal nerve, and brain

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