Journal of Molecular Biology
Volume 334, Issue 1, 14 November 2003, Pages 87-101
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The Mapping and Reconstitution of a Conformational Discontinuous B-cell Epitope of HIV-1

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Abstract

A method for the discovery of the structure of conformational discontinuous epitopes of monoclonal antibodies (mAbs) is described. The mAb is used to select specific phages from combinatorial phage-display peptide libraries that in turn are used as an epitope-defining database that is applied via a novel computer algorithm to analyze the crystalline structure of the original antigen. The algorithm is based on the following: (1) Most contacts between a mAb and an antigen are through side-chain atoms of the residues. (2) In the three-dimensional structure of a protein, amino acid residues remote in linear sequence can juxtapose to one another through folding. (3) Tandem amino acid residues of the selected phage-displayed peptides can represent pairs of juxtaposed amino acid residues of the antigen. (4) Contact residues of the epitope are accessible to the antigen surface. (5) The most frequent tandem pairs of amino acid residues in the selected phage-displayed peptides can reflect pairs of juxtaposed amino acid residues of the epitope. Application of the algorithm enabled prediction of epitopes. On the basis of these predictions, segments of an antigen were used to reconstitute an antigenic epitope mimetic that was recognized by its original mAb.

Introduction

An effective humoral response towards an infectious agent is the ability of antibodies to bind and inactivate the pathogen. Vaccines designed to induce the production of such antibodies are typically derivatives of the pathogen; i.e. killed whole cells, attenuated live pathogens, fragments of antigens or DNA corresponding to the latter.1., 2. Whatever the modality, the purpose of the vaccine is to stimulate neutralizing immunity in the naive individual in preparation for future encounters with fully virulent field-isolates of the pathogen. Correspondence between the vaccine and the field-isolate of the pathogen must be substantial, therefore, to ensure its efficacy. In cases where the pathogen undergoes extensive genetic variation, the ability to formulate an effective vaccine may present what appears to be an insurmountable obstacle. Such seems to be the case, for example, for human immunodeficiency virus type 1 (HIV-1), the etiological agent of the AIDS epidemic, that is continuously selected for its ability to evade immune surveillance.3., 4., 5.

HIV-1, as a result of each infectious cycle, accumulates numerous random mutations providing it with an endless source of variants.6., 7. Nonetheless, over the years a few examples of highly cross-reactive and neutralizing anti-HIV monoclonal antibodies (mAbs) have been described, illustrating that protective immunity is possible.8., 9., 10., 11., 12., 13., 14. This has been substantiated by experiments in which cocktails of mixtures of these mAbs, administered as passive immunotherapy, have proven effective in preventing the infection of CD4+ lymphocytes both in vitro and in vivo.8., 9. Thus a rational approach to the design of a cross-reactive antibody response against AIDS can be proposed as follows: (1) First, one must accumulate a collection of genuine broadly cross-reactive and neutralizing mAbs (to date, at least four exist10., 11., 12., 13., 14.). (2) These in turn are used to discover their corresponding epitopes within the antigens of HIV-1. (3) Once mapped, the epitopes are to be reconstituted as synthetic versions that must be both antigenic (i.e. recognized by the original mAbs) and immunogenic (i.e. able to elicit in the naive individual the production of antibodies that are as effective as the original mAbs).

Unfortunately, such a protocol turns out to be a very difficult task as the “interesting” mAbs against HIV-1 (and in fact against most pathogens) typically correspond to highly conformational epitopes that are comprised of discontinuous segments of the viral antigen.15 Very often, even linear epitopes show conformational preferences and dependence on the context of a protein antigen.16 Thus, we are faced with a fundamental problem; namely, how can one discover the precise molecular design of conformational discontinuous epitopes of highly desirable mAbs of clinical importance?

Here, we describe the initial steps of a systematic approach designed to solve precisely this problem. It is based on using HIV-specific mAbs to screen combinatorial phage display peptide libraries. The mAb-specific phages were then used as an epitope-defining database that was applied via a novel computer algorithm to analyze the crystalline structures of the viral antigens. In this manner, candidate epitopes were mapped to the surface of the antigens. On the basis of this mapping, bona fide segments of the viral antigen were used to reconstitute an epitope mimetic that was recognized by the original mAb.

Section snippets

Experimental rationale

Our experimental rationale was based on the assumption that affinity-selected peptides derived from a vast collection of random peptides, due to their specific binding to the mAb of interest, must reflect structural elements of the original epitope. Initially, this might appear to be a trivial assumption, as one could expect to obtain peptides that show linear homologies with the immunogenic antigen. However, in the case of highly conformation-dependent mAbs, the peptides obtained are often

Discussion

“The elucidation of protein antigenic structures is presently a difficult, uncertain and time-consuming task”. Thus, over 20 years ago, Hopp & Woods open their article on the prediction of antigenic-determinants.29 This statement is still very true today.

Antibody:antigen interfaces have been assumed, generally, to be hydrophilic and transiently accessible to the surrounding milieu bathing the antigen and, as such, distinct from the subunit:subunit interface of multimeric protein complexes.

DNA preparations

Single-stranded DNA of filamentous phages was isolated using the QIAprep Spin M13 Kit® (QIAGEN GmbH, Germany).

Monoclonal antibodies

CG10, CG1 and CG9 mAbs used in this study were produced at Tel Aviv University.27 Human mAb 17b was produced at Tulane University Medical Center.47 The mAb 13b5 is a product of bioMérieux, France.25

Library construction and biopanning

Three 12mer cysteine-constrained peptide libraries were constructed: one in ftac88 and the other two in fth1.46 Protein G (Sigma Chemical Co., St Louis, MO) was used to coat the bottom of 35

Acknowledgements

The authors thank Professor James Robinson for providing us with 17b mAb and Dr François Mallet for providing 13b5 mAb. This research was supported by The Israel Science Foundation. The authors are grateful to Dr Edward Spigelman for useful discussions and consultation.

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    Present address: D. Enshell-Seijffers, Cutaneous Biology Research Center, Massachusetts General Hospital 149 Bld, 13th Street, Charlestown, MA 02129, USA.

    D.E.-S. and D.D. contributed equally to this work.

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