Synthetic Peptides as Antigens

S. Muller, M.H.V. van Regenmortel

• Publisher: Elsevier Science
• Year: 1999
• Print ISBN: 9780444821751
• eBook ISBN: 9780080858975
• Topic: Medicine
• Subtopic: Immunology

This newest edition to the Laboratory Techniques Series gives current state of the art use of synthetic peptides in molecular biology and practical protocols on how to conjugate peptides, immunize animals with peptides and monitor immune responses to peptides in vitro.It gives background information on antigenic specificity, prediction of antigenic sites in proteins and applications of peptides in immunology and virology, as probes in diagnosis and as vaccines. The book also describes antigenicity of proteins and methods to localize antigenic sites as well as methods for predicting epitoxes, and gives detailed protocols for peptide-carrier conjugation, immunization with peptides, and peptide immunoassays.The volume also describes typical use of antipeptide antibodies in molecular and cellular biology as well as the use of peptides in the diagnosis of viral infections and autoimmune diseases, and the use of peptides as potential synthetic vaccines. An excellent edition to an excellent series, available in hardbound and paperback.

Chapter 1

Molecular dissection of protein antigens and the prediction of epitopes

S. Muller

1.1 Introduction

Interest in the immunological properties of synthetic peptides stems mainly from the fact that peptides can be used to mimic the antigenic sites of proteins. Since most antigens of biological interest are proteins, one of the major goals of molecular immunology has been to elucidate the antigenic structure of proteins. The development by Merrifield (1963) of the solid-phase method of peptide synthesis made it much easier to obtain short fragments of a protein by synthesis rather than by enzymatic or chemical cleavage of the protein. Natural fragments obtained by cleavage of a protein must be separated from each other, and since the level of purity required for immunological studies is extremely high, this is often no mean task. Indeed, if a fragment of a protein devoid of antigenicity is contaminated by a small amount of a highly reactive antigenic fragment derived from the same molecule, this may lead to the erroneous conclusion that the major, inactive peptide component is antigenic. Such misinterpretations are eliminated when synthetic fragments are used to locate antigenicity in proteins.

In an earlier version of this book (Van Regenmortel et al., 1988), a chapter was included which reviewed for the non-specialist the laboratory methods used in solid-phase peptide synthesis. In the intervening years, numerous texts have appeared which can be consulted for guidance on the procedures of peptide synthesis (Fields, 1997; Grant. 1992: Lloyd-Williams et al., 1997; Pennington and Dunn, 1994).

The molecular dissection of protein antigens has been undertaken, not only to increase our understanding of immunological specificity, but also because such knowledge makes it possible to manipulate the immune system and leads to many useful practical applications in molecular biology, biochemistry and microbiology. For instance, increasing knowledge of the location of antigenic sites in toxins, viruses and parasites has led to many attempts to develop new synthetic peptide vaccines (Arnon, 1987; Arnon and Van Regenmortel 1992; Francis, 1994; Nicholson, 1994b; Van Regenmortel and Neurath, 1990). It was found, for instance, that protective immunity could be elicited against foot-and-mouth disease. influenza, hepatitis B and cholera by immunizing animals with synthetic peptides (see Chapter 8, this volume). Synthetic peptides are also increasingly replacing intact proteins as reagents for the diagnosis of viral and autoimmune diseases (see Chapters 6 and 7).

Another major application of synthetic peptides relies on their ability to elicit antipeptide antibodies that cross-react with the corresponding complete protein. Such antibodies have been found to be extremely useful reagents for isolating and characterizing gene products (Boersma et al., 1993; Lerner, 1984; Walter, 1986). Because of advances in gene cloning and sequencing, the information on protein sequences is nowadays nearly always derived from nucleotide sequence analysis. In many cases, the protein is not available in sufficient quantity for conventional chemical studies, or its presence in the cell may even be in doubt. By synthesizing a peptide fragment of the putative protein inferred from nucleic acid sequencing and raising antibodies against it, it is possible to isolate and characterize the protein using appropriate immunoassays (see Chapter 5).

For many years the antigenic properties of proteins were defined only in terms of B-cell epitopes recognized by antibodies and B-cell receptors. Once it was established that antigens were also specifically recognized by T cells, a second type of epitope known as a T-cell epitope was defined which corresponds to linear fragments of the antigen capable of interacting with T-cell receptors. In the absence of further qualification, the term ‘epitope’ is used to denote a B-cell epitope, and this convention will be used in the present text. Sections 1.2–1.6 of this chapter discuss B-cell epitopes, while Section 1.7 is devoted to T-cell epitopes.

1.2 Definition of antigenicity and the concept of epitope

The antigenic reactivity of a protein refers to its capacity to bind specifically to the functional binding sites or paratopes of certain immunoglobulin (Ig) molecules. When such a binding is observed experimentally, the particular Ig becomes known as an antibody specific for the protein.

Immunoglobulin molecules are heterodimers consisting of four polypeptide chains linked by disulphide bridges (Nezlin, 1994). There are two identical heavy (H) chains of 450–600 amino acid residues and two light (L) chains of about 220 residues. The sequence of the N-terminal domain of both H and L chains differs in antibodies of different specificity and are called variable (V) regions, while the remaining domains in each chain are invariant and are called constant (C) regions. Within each V region, three segments exhibit sequence hypervariability and form the complementarity determining region (CDR) of the Ig. The most common type of Ig is known as IgG and contains a H chain called γ. Immunoglobulins can be cleaved at the middle of their H chains by various proteases (Nezlin, 1994). Papain cleaves γ-chains at the N-terminal side of the disulphide bridges that keep the H chains together, thereby generating two fragment antigen binding (Fab) fragments and one fragment crystallizable (Fc) fragment. Each Fab fragment contains at its tip one of the two identical combining sites (or paratopes) of the Ig constituted by the three CDRs of the H chain and the three CDRs of the L chain (Fig 1.1). The CDR loops vary not only in sequence but also in the length from one antibody to...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright page
• Acknowledgements
• List of abbreviations
• Chapter 1: Molecular dissection of protein antigens and the prediction of epitopes
• Chapter 2: Peptide-carrier conjugation
• Chapter 3: Immunization with peptides
• Chapter 4: Peptide immunoassays
• Chapter 5: Use of antipeptide antibodies in molecular and cellular biology
• Chapter 6: The use of peptides for diagnosing viral infections
• Chapter 7: Peptides in diagnosis of autoimmune diseases
• Chapter 8: Synthetic peptides as vaccines
• References
• Subject Index