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. 2014 Jul 29;143(1):13–20. doi: 10.1111/imm.12323

A high-throughput shotgun mutagenesis approach to mapping B-cell antibody epitopes

Edgar Davidson 1, Benjamin J Doranz 1,✉
PMCID: PMC4137951  PMID: 24854488

Abstract

Characterizing the binding sites of monoclonal antibodies (mAbs) on protein targets, their ‘epitopes’, can aid in the discovery and development of new therapeutics, diagnostics and vaccines. However, the speed of epitope mapping techniques has not kept pace with the increasingly large numbers of mAbs being isolated. Obtaining detailed epitope maps for functionally relevant antibodies can be challenging, particularly for conformational epitopes on structurally complex proteins. To enable rapid epitope mapping, we developed a high-throughput strategy, shotgun mutagenesis, that enables the identification of both linear and conformational epitopes in a fraction of the time required by conventional approaches. Shotgun mutagenesis epitope mapping is based on large-scale mutagenesis and rapid cellular testing of natively folded proteins. Hundreds of mutant plasmids are individually cloned, arrayed in 384-well microplates, expressed within human cells, and tested for mAb reactivity. Residues are identified as a component of a mAb epitope if their mutation (e.g. to alanine) does not support candidate mAb binding but does support that of other conformational mAbs or allows full protein function. Shotgun mutagenesis is particularly suited for studying structurally complex proteins because targets are expressed in their native form directly within human cells. Shotgun mutagenesis has been used to delineate hundreds of epitopes on a variety of proteins, including G protein-coupled receptor and viral envelope proteins. The epitopes mapped on dengue virus prM/E represent one of the largest collections of epitope information for any viral protein, and results are being used to design better vaccines and drugs.

Keywords: envelope, epitope, G protein-coupled receptor, high-throughput, mapping, shotgun mutagenesis

Introduction

Characterizing monoclonal antibody (mAb) epitopes on protein targets can aid in the discovery and development of new therapeutics, elucidate cancer-specific epitope markers and define the protective (and in some cases pathogenic) effects of vaccines. For example, the identification of mAb epitopes has enhanced our understanding of the therapeutic mechanisms of anti-cancer mAbs that target Her-21,2 and vascular endothelial growth factor,3,4 and is helping to improve the design of vaccines against HIV and influenza virus.5–7 Epitope characterization can also help to elucidate mAb mechanisms of action and strengthen intellectual property claims. In addition, a major increase in the number of characterized B-cell epitopes, correlated with their mechanisms of action, could facilitate the development of more robust algorithms and mathematical models for predicting B-cell epitopes and antibody-mediated immune responses.8

Recent technological improvements have greatly increased the ability to obtain large numbers of mAbs. The rapid isolation of mAbs from infected individuals, by cloning directly from selected B cells and by deep sequencing of human genomes, has enabled the isolation of dozens to hundreds of mAbs at a time from individual patients.9,10 For example, some of the most highly potent and broadly neutralizing HIV-1 mAbs identified to date were isolated directly from HIV-1-infected donors using new, large-scale mAb screening methods.11–15 In addition, phage display libraries, created using cDNA derived from patient B cells, allow the screening of hundreds of millions of different mAbs to isolate both common and rare mAb variants, and to precisely control the screening conditions to facilitate isolation of mAbs that recognize unique epitopes. As a result of these advancements, many laboratories now have hundreds to thousands of relevant mAbs, and the continued evolution of mAb isolation technologies promises to provide even greater numbers.

In contrast to the large increase in mAbs being isolated, high-throughput mAb characterization techniques have not kept pace. Obtaining detailed epitope maps for functionally relevant antibodies can be challenging, particularly for conformational epitopes on structurally complex proteins. G protein-coupled receptor (GPCRs), for example, are embedded in the cell membrane and often have short antigenic regions that fold correctly only within the context of the entire protein in the lipid bilayer. Similarly, most viral envelope proteins contain disulphide bonds that are critical for maintaining their native structure, are modified with O-linked and N-linked sugars that shield conserved regions of the proteins, and form oligomers in the lipid membrane. These types of structures are difficult to accurately recapitulate in bacterial, insect and yeast expression systems that do not fully support human post-translational modifications or native folding. In the absence of a high-throughput methodology for epitope mapping, the epitopes of most mAbs will remain uncharacterized, leaving a major gap between the growing ability to isolate relevant mAbs and the ability to molecularly define the immunogenic structures that gave rise to them.

Technical approaches for obtaining mAb epitopes face several challenges. First, an epitope mapping technique should enable mapping of both linear and conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in the target protein, while conformational epitopes are composed of amino acids that are discontinuous in the primary sequence but are brought together upon three-dimensional protein folding. Since many therapeutically important mAbs target conformational epitopes formed only in the native structure of a protein, the ability to map conformational epitopes is crucial. Second, an epitope mapping technique should be able to delineate epitopes at high resolution so that individual amino acid contact points can be accurately identified. Third, epitope mapping should ideally be able to distinguish the functionally relevant residues that account for most of the energetic contributions of mAb binding.16–18 For example, the average epitope contains 20 contact residues, but only two to five of these are typically energetically critical for the interaction (the so-called ‘hotspot’ residues of the epitope).16,19,20 Finally, epitope mapping must be applicable to conformationally complex targets, including membrane proteins, proteins that are post-translationally modified and oligomers.

Numerous methods have been used to obtain information about the location and structural components of epitopes, and such techniques have been well reviewed elsewhere19,21–23 (Table 1). Crystallography, nuclear magnetic resonance, and cryo-electron microscopy can provide high-resolution maps of antibody–antigen interactions, and a high-resolution structure of a mAb in complex with its target is the gold standard for determining the atomic-level contact points on the target protein that define an epitope. However, these techniques are not always feasible because of the difficulty of obtaining sufficient quantities of correctly folded, natively processed, and antibody-complexed protein. Additionally, these strategies are often time-consuming and expensive, do not distinguish the energetically critical residues that define the functional epitope, and cannot usually be used to epitope map dozens or hundreds of mAbs. For viral antigens, escape mutations are often used to localize epitope residues, but require a significant amount of work, growing and sequencing live viruses over time and then re-introducing single mutations into infectious clones to confirm mutations of interest. Identifying escape mutations is also less feasible when the mutation of epitope residues disrupts viral functions. Simple epitope mapping can be performed using peptide libraries, but this approach is limited to mapping linear, not conformational, epitopes.

Table 1.

Epitope mapping technologies

Technique Time requirement Conformational epitopes High resolution Complex proteins
X-ray crystallography +++ ✓ ✓ ✓/–
Peptide scanning + – – ✓
H/D exchange ++ ✓ – –
Yeast display ++ ✓ ✓ –
Conventional site-directed mutagenesis +++ ✓ ✓ ✓
Viral escape +++ ✓ ✓ ✓
Shotgun mutagenesis epitope mapping + ✓ ✓ ✓
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Each epitope mapping technique offers relative advantages and disadvantages, summarized here. Shotgun mutagenesis epitope mapping enables mapping of both linear and conformational epitopes at amino-acid resolution directly within cells, even for the most complex eukaryotic proteins.

High-throughput shotgun mutagenesis epitope mapping

To enable rapid epitope mapping of conformationally complex proteins, we have developed a high-throughput strategy, shotgun mutagenesis, that enables the identification of both linear and conformational epitopes in a fraction of the time required by other approaches. Shotgun mutagenesis epitope mapping technology is based on large-scale mutagenesis and rapid cellular testing of natively folded proteins. Shotgun mutagenesis begins with the creation of a plasmid mutation library for a target gene, each clone in the library bearing a defined amino acid mutation, such as an alanine substitution (Fig. 1). At the time of library creation, each clone is sequenced and tested for expression. Hundreds of plasmid clones from the mutation library are individually arrayed in 384-well microplates (one mutation per well, a ‘mutation array’), expressed within human cells, and tested for mAb reactivity, allowing amino acids critical for a mAb of interest to be readily identified by a loss of immunoreactivity. These critical residues are then mapped onto the protein structure to visualize the epitope. The value of this approach is that it provides a fast and efficient method for mapping new epitopes. Shotgun mutagenesis is particularly suited for studying structurally complex proteins, since proteins are expressed in their native form directly within human cells, maintaining proper oligomerization, disulphide bonding, glycosylation and other post-translational modifications.

Figure 1.

Figure 1

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Shotgun mutagenesis epitope mapping overview: 384-well microplates are prepared so that each well contains a plasmid clone with a single, defined amino acid change from the wild-type sequence, usually an alanine substitution. Human cells are then added, which are transfected with the plasmid to express a defined mutant protein in each well. The use of human cells facilitates native protein expression and folding. The mutation array is tested in a monoclonal antibody (mAb) binding assay to identify the amino acids required for mAb reactivity. Mutations that eliminate the ability of the mAb to bind (but that are otherwise expressed and folded correctly) comprise the mAb's epitope. The mAb epitopes (green residues) can then be visualized on the structure of the protein.

To construct a shotgun mutagenesis library, a parental protein expression vector is mutagenized to create a library of clones, each representing an individual point mutant and cumulatively covering all residues in the protein. Initial libraries were produced using random mutagenesis and a selection of two or three mutants per position (typically including a conserved and non-conserved substitution), whereas subsequent libraries have been constructed using alanine scanning mutagenesis, which provides a more controlled method of defining the side-chain contributions of each residue. Using semi-automated robotic protocols, each mutated plasmid is individually cloned, sequenced, mini-prepped and arrayed in 384-well microplate format for repeated transfection, expression and antibody binding assays in human cells. We have constructed mutation libraries totalling over 10 000 individual point mutations, representing viral envelope proteins [dengue virus-3 (DENV-3) prM/E, DENV-4 prM/E, chikungunya virus E2/E1, hepatitis C E1/E2, hepatitis B virus surface antigen, respiratory syncytial virus F protein and HIV gp160], GPCR proteins (CCR5, CXCR2, CXCR4 and TAS2R16), 4TM proteins (claudin-1 and claudin-4) and other membrane proteins (MCAM-1 and Her-2) (Table 2).

Table 2.

Mutation libraries for high-throughput epitope mapping

Class Target protein Library size (no. of clones) Library type
Virus DENV-3 prM/E 1400 2 mutations/AA
Virus DENV-4 prM/E 660 Ala scan
Virus CHIKV E2/E1 920 Ala scan
Virus HCV E1/E2 553 Ala scan
Virus HIV gp160 679 Ala scan
Virus RSV F protein 1029 2 mutations/AA
Virus HBV sAg 441 2 mutations/AA
GPCR CXCR2 714 Ala scan
GPCR CXCR4 731 2 mutations/AA
GPCR CCR5 734 2 mutations/AA
GPCR TAS2R16 573 2 mutations/AA
4TM Claudin-1 413 2 mutations/AA
4TM Claudin-4 423 2 mutations/AA
Membrane MCAM 545 Ala scan
Membrane Her-2 625 Ala scan
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DENV, dengue virus; CHIKV, chikungunya virus; GPCR, G protein-coupled receptor; HCV, hepatitis C virus; HBV, hepatitis B virus; HIV, human immunodeficiency virus; RSV, respiratory syncytial virus.

Comprehensive mutation libraries constructed at Integral Molecular cover nearly every residue of the indicated proteins, over 10 000 individual mutations in total. Initial libraries were derived using random mutagenesis and selection of two or three mutants per position, while subsequent libraries have been constructed using alanine scanning mutagenesis.

Before mAbs are screened against a shotgun mutagenesis library, the appropriate screening concentration for each individual mAb is determined using an immunofluorescence titration curve against a wild-type target protein. This ensures the ability to measure mAb binding within the linear range of detection and that mAb binding displays a sufficiently high signal-to-background ratio to generate robust reactivity data. Initial screens also determine the optimal reaction conditions for mAb binding, since such differences can sometimes affect the exposure of specific epitopes. Monoclonal antibodies are also often tested at this time for cross-reactivity with related proteins or family members.

After establishing appropriate screening conditions, each mAb is tested for binding to all of the mutated clones in the appropriate library to identify residues whose mutation results in decreased mAb reactivity. Briefly, the immunoreactivity of a mAb to the target protein is detected in each individual well of the library plates using a fluorescent secondary antibody (Fig. 2a). Fluorescence intensity in each well is quantified by high-throughput flow cytometry (IntelliCyt HTFC; IntelliCyt Corporation, Albuquerque, NM) for sensitive well-by-well quantification of mAb binding with each mutant clone. Cells are generally fixed before mAb binding, but mapping of virus mAbs under different conditions has revealed that some epitopes are exposed only under unfixed conditions. Extracellular staining conditions are generally used for surface-expressed proteins, but intracellular staining can also be used to analyse cytoplasmic and secreted proteins.

Figure 2.

Figure 2

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Shotgun mutagenesis epitope mapping process. (a) Hundreds of plasmid clones (one mutation per well) are expressed within human cells (to maintain proper folding) in 384-well microplates and tested for monoclonal antibody (mAb) reactivity. (b) Binding data obtained for mAb CKV064 is compared with that of a control mAb (CKV063) to identify ‘dropout’ residues (highlighted as red) whose mutation eliminates specific binding of the mAb under analysis. (c) Residues are identified as a component of a mAb epitope if their mutation does not allow candidate mAb binding but does support that of other conformational mAbs or functions. (d) Residues identified as critical for mAb binding (green spheres) are mapped onto the chikungunya virus E2/E1 heterodimer (PDB 3N41) to visualize the epitope (E2 protein is red, E1 protein is yellow, fusion loop is cyan). (e) Over the course of 2 years, 250 epitope maps were generated at an accelerating rate as mutation array design, experimental assays and data analysis were optimized.

To ensure robust data analysis, all array plates contain positive wild-type and negative mock-transfected control wells. The mAb reactivities against each mutant clone are calculated relative to wild-type protein reactivity by subtracting the signal from mock-transfected controls and normalizing to the signal from wild-type controls. Binding data for each clone are expressed as a percentage relative to mAb binding to cells expressing wild-type protein, and represent the average results of replicate experiments.

In the course of our analyses, epitope mapping protocols have been optimized so that epitopes can be derived even under circumstances where mAb reagents are limiting or where mAb binding is far from ideal (e.g. low signal from poorly reactive mAbs or high background from ‘sticky’ mAbs). In several cases, complete epitope maps were obtained with only 1 ml of unpurified hybridoma supernatant. Other mAbs have bound with such high affinity that single mutations under standard screening conditions do not diminish mAb binding, but this can almost always be overcome by screening the mAb under more stringent binding conditions.

Epitope assignment

With 95–100% of a protein's residues mutated in a typical shotgun mutagenesis library, a number of mutations will eliminate mAb binding by causing non-specific disruptions of protein structure. To exclude such mutations from analysis, data obtained for the binding of the candidate mAb to the entire library are compared with that of control mAbs to identify ‘dropout’ residues whose mutation eliminates specific binding (Fig. 2b). The mAb reactivity thresholds for control and experimental antibodies are initially chosen with the aim of identifying a biophysically appropriate number of energetically critical residues (∼3–1224). The effect of each mutation on protein expression, folding and function are also examined to confirm native protein folding. For example, protein functions such as receptor signalling and viral infectivity are used to validate that overall protein folding is not disrupted by the mutation. Protein folding in the vicinity of a candidate critical residue is also evaluated using conformational mAbs that bind in the same protein motif as the mAb being analysed (e.g. a fusion peptide in a viral envelope protein) or in a larger domain that includes the protein motif.

Residues are identified as a component of the epitope if their mutation does not enable candidate mAb binding but does support that of other conformational mAbs or functions (Fig. 2c). Confidence in a critical residue is also based on the reproducibility of its binding, surface accessibility, distance from other critical residues, and the nature of the substituted residue (e.g. Cys substitutions). After final validation, critical residues are mapped onto available protein structures to visualize the epitope (Fig. 2d).

Shotgun mutagenesis epitope mapping is now sufficiently robust to enable mapping rates of over 20 epitopes/month (Fig. 2e), and is particularly well-suited for analysing large numbers of mAbs, as the confidence of epitope assignment during validation strengthens with the number of mAbs used for cross-comparison. For the DENV envelope protein mutation libraries, we estimate that having binding data for approximately 30 diverse mAbs allowed the prediction of additional epitopes with high confidence levels.

To enable high-throughput epitope mapping, we have developed over 40 standard operating protocols covering each step of mutation library production, validation and testing. All information regarding mutation libraries, microplates, individual clones and assay results are tracked using a custom-designed relational database (Fig. 3). This database enables all shotgun mutagenesis information (involving over 500 000 individual wells for the DENV, chikungunya virus and hepatitis C virus libraries described here) to be managed in a systematic, accurate and efficient manner. The database also performs the statistical calculations used throughout data analysis and epitope refinement, including patented algorithms to derive the final epitopes.

Figure 3.

Figure 3

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Production and management of shotgun mutagenesis monoclonal antibody (mAb) binding data using lims software. Screenshots from our laboratory-information management system (lims) software demonstrating various analyses steps in mapping mAb epitopes. The Import Window imports raw data files from the 384-well plate readers and flow cytometry equipment used to detect and quantify mAb reactivities. The Plate QC Window validates that each plate of imported data displays low variability, high signal-to-noise, and no outlier wells so the plate can be included in analysis. The Data QC Window compares replicate plates run using the same mAb to ensure that experiments are consistent. The Control Selection Window compares each data set to plates run using other mAbs so that epitope residues specific to a given mAb can be identified. Finally, the Analysis Window assembles all epitope data and visualizes the epitope residues on a three-dimensional structure of the target protein in real-time. The database is also programmed to track all details regarding library creation (e.g. parental sequence ID, epitope tags, expression vector, date of creation), individual microplates (e.g. microplate ID, contents, freezer location, date of printing), and individual clones [e.g. clone ID, sequence, sequence quality value (QV), nucleotide mutation, amino acid mutation, and location on a specific microplate].

Analysing epitope structures

To date we have used shotgun mutagenesis to epitope map over 250 mAbs targeting DENV, chikungunya virus and hepatitis C virus, with additional mAb epitopes mapped on hepatitis B virus, respiratory syncytial virus and HIV. In addition, we have used shotgun mutagenesis to map epitopes, drug-binding sites and the functional regions of other complex membrane proteins, such as GPCRs. Our studies on these latter targets include descriptions of atomic-level mAb epitopes on the GPCRs CCR525 and CXCR4,26 the identification of cancer biomarker epitopes on the 4TM proteins claudin-1 and claudin-4, an atomic-level model describing the intramolecular signal transduction pathway of CXCR4, a proposed mechanism for the ligand specificity and sensitivity of the GPCR TAS2R16, mapping of inhibitor-binding sites on TAS2R16,27 and mapping of paratope residues on a clinical antibody against respiratory syncytial virus.28

The majority of epitope contacts in mAb co-crystal structures occur through amino acid side-chains,20 and alanine scanning mutagenesis provides a controlled method to define the contributions of each residue's side-chain to mAb binding. Although this mapping strategy may not identify every epitope residue in contact with an antibody, the critical residues identified using this approach represent amino acids whose side chains make the highest energetic contributions to the mAb–epitope interaction.16,17 Most epitopes that we have characterized are conformational in nature and range in size from 1 to 10 residues.

The > 150 epitopes we have mapped against DENV represent one of the largest collections of epitope information against a viral protein (prM/E). Over half of the DENV epitopes are unique (containing non-identical epitope residues) and the epitopes can be grouped into 14 non-overlapping immunodominant regions. Many of the epitope residues localize on or immediately adjacent to residues known to be involved in envelope protein functionality, helping to explain how mAbs neutralize this virus and providing specific envelope protein amino acid targets for structurally based drug and vaccine development. Our analyses of the mechanisms of action of these epitopes (i) demonstrate that mAbs against conserved fusion loop epitopes can broadly neutralize all four DENV serotypes as well as related flaviviruses such as West Nile virus and yellow fever virus,29 (ii) explain how a unique epitope adjacent to the fusion loop mediates highly potent and broadly cross-reactive neutralization,30 (iii) explain how a mAb with micromolar binding affinity is still one of the most potently neutralizing DENV antibodies identified to date,31 (iv) demonstrate that epitopes on the DENV-4 serotype are shielded from neutralization,32 and (v) explain how neutralizing epitopes block functional regions of envelope protein.33 In addition, by incorporating each of the DENV-3 envelope protein mutants into infectious dengue reporter virus particles34 and testing them in independent assays for budding and infectivity, we were able to identify critical residues throughout DENV prM/E that are required for infectivity and propose atomic-level models of how envelope protein mediates fusion.35

Throughout our studies, we have mapped a number of mAbs that have been well characterized previously by other groups, and found that epitopes obtained using shotgun mutagenesis compare extremely well. For example, we identified almost the same DENV epitope for mAb 5J733 as was independently published by viral escape mutation;36 we identified nearly the same hepatitis C virus epitopes for mAbs HCV003 and HCV004 as were independently published using site-directed mutations;37,38 we identified the same epitope for mAb HCV1 as was independently derived using co-crystallography with a hepatitis C virus peptide, viral escape,39 and peptide mutagenesis40 (Fig. 4a); and we identified the same epitope for the very well characterized DENV mAb 1A1D-2 as was derived using co-crystallography,41 yeast display42 and amino acid mutations on virus particles43 and recombinant protein44 (Fig. 4b). Comparison of the 1A1D-2 epitope in energetic studies44 also confirmed that shotgun mutagenesis identified the ‘hotspot’ residues that are the energetically critical residues for binding.16,17 Many cross-reactive DENV mAbs have been mapped using both our random mutagenesis DENV-3 library and our alanine scan DENV-4 library, with essentially identical results. These results validate the utility of shotgun mutagenesis and provide confidence that this technology can rapidly provide accurate epitope maps.

Figure 4.

Figure 4

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Epitope validation. (a) Critical residues (red) for hepatitis C virus (HCV) monoclonal antibody (mAb) HCV1 identified using various techniques39,40 match epitope residues identified using shotgun mutagenesis, including three of the four residues identified using co-crystallography with an HCV E2 peptide. (b) Critical residues for dengue virus (DENV) mAb 1A1D-2 identified using various techniques41–43 match results derived using shotgun mutagenesis. Several of the differences between the exact residues identified are due to serotype differences between the studies.

Conclusion

We have developed a high-throughput strategy, shotgun mutagenesis epitope mapping, that enables the identification of both linear and conformational epitopes in a fraction of the time required by other approaches. The value of this strategy is that it provides a fast and efficient method for mapping new epitopes. Shotgun mutagenesis is particularly suited for studying structurally complex proteins, as proteins are expressed in their native form directly within human cells. We have used the methods outlined here to epitope map mAb binding sites on a wide variety of proteins, including over 250 mAbs targeting envelope proteins from DENV, HIV, and chikungunya, hepatitis C, hepatitis B and respiratory syncytial viruses, many of which have been deposited in the Immune Epitope Database.45 These epitopes have increased our understanding of the envelope epitopes that provoke an immune response, and provide insights into the mechanism by which mAbs neutralize viruses. In addition, we have also used shotgun mutagenesis to map epitopes, drug-binding sites and the functional regions of other complex membrane proteins, such as GPCRs. The ability to map mAb epitopes efficiently, accurately, and on structurally complex targets is being used to support the discovery and development of antibody therapeutics and recombinant vaccine strategies.

Acknowledgments

This work was supported by the National Institute of Allergy and Infectious Diseases contract HHSN272200900055C.

Glossary

DENV

dengue virus

GPCR

G protein-coupled receptor

mAb

monoclonal antibody

Disclosures

ED and BJD are current employees of Integral Molecular. BJD is a shareholder of Integral Molecular.

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