Fundamentals of Immunology - Lecture Course by M. V. Skok - Kyiv 2002

Chapter I. Immunochemistry

Lecture 2. Antigens and Structure of Antigenic Determinants

Antigen is a functional concept. It refers to any substance capable of inducing an Immune Response.

Proteins are potent Antigens. This is logical, as most immune recognition processes are Protein-Structure/156.html">Protein Interactions. Bacterial and snake toxins, viral proteins, and tissue antigens are all protein and peptide antigens. CARBOHYDRATES found in bacterial Cell walls are also readily recognized. Since most Membrane Proteins in higher animals are Glycoproteins, Blood Group Antigens, for instance, are polysaccharide-based. Nucleic Acids are not immunogenic on their own, but they become so when complexed with proteins. For example, the autoimmune response in systemic lupus erythematosus involves a response to Nucleoproteins. Lipids can be effectively recognized when presented via the surface CD1 cellular molecule.

Classification of antigens.

1. Complete and incomplete (haptens).

2. Soluble and particulate.

3. Exogenous and endogenous.

4. T-dependent and T-independent.

5. Hetero-, iso(allo)-, and autoantigens.

1. A distinction must be made between antigenicity and immunogenicity.

Antigenicity (antigenic Specificity) is The ability to bind complementarily to antigen-specific receptors of B AND T Cells. It is characteristic of almost all known substances. Peptides, Amino Acids, Vitamins, and even ATP, dinitrophenol, or Metal Ions can act as antigens. However, simply introducing one of these low-molecular-weight substances into an Organism will not trigger an immune response. Such antigens are termed incomplete antigens, or haptens. Haptens elicit an immune response only after conjugation with high-molecular-weight carriers. Immunogenicity is the ability to provoke an immune response, meaning to stimulate the cascade of events necessary for the activation of immune cells. Immunogenicity depends both on The structure of the antigen (molecular weight, spatial conformation) and on the state of the recipient's immune system (the repertoire of histocompatibility proteins and T-cell receptors).

The strength of an Immune Response to a weak antigen can be enhanced using adjuvants—substances that promote non-specific stimulation of The Immune System, such as mineral oils (Freund's adjuvants) or aluminum oxide.

2. Soluble (foreign proteins, toxins, degradation products of Viruses and Bacteria) and particulate (bacteria, viruses, foreign Eukaryotic cells) antigens are perceived differently by the immune system and, as noted in Lecture 1, elicit different forms of the immune response. This division is based on the mode of antigen presentation to the Cells of the immune system (Lecture 6). Thus, soluble antigens are perceived and presented as exogenous (external), and the result of their recognition is the activation of B lymphocytes and antibody synthesis. Bacterial and viral antigens are often synthesized inside infected cells and are therefore perceived by the immune system as endogenous (internal). The result of their recognition is the activation of cytotoxic T lymphocytes, which destroy the infected cells along with the infectious agent.

3. T-dependent antigens—which comprise the majority—require the participation of T lymphocytes for their recognition. Certain antigens containing repeatedly occurring fragments (bacterial Polysaccharides) are capable of providing a sufficient signal to B lymphocytes without T-cell involvement and are termed T-independent. They induce antibody synthesis exclusively.

4. With respect to the recipient organism, antigens are classified into autoantigens (self), allo- or isoantigens (of the same species), and hetero- or xenoantigens (of a different species). The potency of the immune response increases progressively from autoantigens to heteroantigens.

The term tissue antigens is also used. This refers to a complex of antigens characteristic of a specific organ or tissue, based on the diverse physiological Functions of Organs that determine their distinct biochemical profile. Tumor antigens are specific soluble or cell-associated substances that appear in the body during tumor growth. They are often referred to as oncofetal antigens because they are also characteristic of embryonic development and appear in a woman's body during Pregnancy. Examples of such antigens include alpha-fetoprotein and trophoblast-specific globulin. Their detection is important for diagnosing both pregnancy and tumor growth.

METABOLISM/2.html">THE CONCEPT OF the antigenic determinant, or epitope.

As early as the 1930s, it was demonstrated that a protein molecule could bind several antibody molecules simultaneously. By the 1950s, it became clear that Antibodies interact with discrete regions On the surface of the protein molecule, which were termed antigenic determinants. This raised a fundamental question: what constitutes an antigenic determinant? What properties enable a particular region of a protein to be recognized as foreign and trigger an immune response?

Initially, short synthetic peptides were used as a model. It turned out that linear amino acid homopolymers (such as (Ala-Ala)_n) are non-immunogenic, but after conjugation with a carrier protein, they behave like haptens, meaning they possess antigenic specificity. Branched amino acid heteropolymers are highly immunogenic and induce the synthesis of antibodies directed against surface Regions of the molecule. Peptides in ordered or denatured Conformations exhibited different antigenic specificities. If a synthetic antigen carried charged groups, the antibodies directed against it possessed the opposite charge.

It was concluded that antigenic determinants are located on The surface of the molecule, possess a specific conformation, and feature amino acid residues capable of forming non-covalent bonds with the antibody.

Pioneering work on the antigenic Introduction/12.html">Structure of Globular proteins was conducted in the 1970s and 1980s. As a result, it was established that an antigenic determinant or epitope is a discrete region on the surface of a protein molecule comprising 6-7 amino acid residues. No correlation was found with any specific amino acid residues; the composition of antigenic determinants included those

amino acids typically located on the protein surface. It was found that each antigenic determinant traces a line 23-25 Å in length across the protein surface and possesses defined N- and C-termini.

A distinction is made between sequential (linear) and discontinuous (conformational) antigenic determinants.

Sequential - defined by the order of amino acids. Antibodies against such epitopes readily interact with a linear peptide of the same sequence. In their pure form, they are found in fibrous PROTEINS AND PEPTIDES. In Globular proteins, surface sequential regions possess a specific conformation. Antibodies raised against peptides often recognize native proteins, meaning they can adapt in certain ways to the conformation of surface fragments.

Discontinuous antigenic determinants consist of amino acid residues that are distant from one another in the polypeptide chain, but brought into close proximity by the Tertiary Structure of the protein, primarily Disulfide Bonds. Such antigenic determinants cannot be mimicked by a linear peptide.

Not all amino acids comprising an epitope are equally important for recognition: as a rule, specificity is determined by 1-2 residues (immunodominant), while the others play a role in maintaining the proper conformation of the epitope.

As examples, let us consider the antigenic structure of sperm whale Myoglobin and chicken egg-white Lysozyme—the first protein antigens to be studied in detail.

Myoglobin is a heme-containing Muscle protein with a Molecular Weight of 18 kDa, consisting of 153 amino acid residues and lacking disulfide bonds. Five linear epitopes were identified in the myoglobin molecule: fragments 16-21, 56-62, 94-99, 113-119, and 146-151. Their composition included hydrophilic polar amino acids: Lys, Arg, Glu, His.

Lysozyme is an enzyme found in mammalian secretory Body Fluids and bird egg white, with a molecular weight of 14 kDa and four disulfide bonds. Three discontinuous antigenic determinants corresponding to the following fragments were identified in lysozyme:

22-34 and 113-116, brought together by the 30-115 disulfide bond;

62-68 and 74-96, brought together by the 76-94 and 64-80 bonds;

6-13 and 126-129, brought together by the 6-127 bond.

To study these antigenic determinants, a special experimental approach was proposed—surface-simulation synthesis. Thus, to mimic a discontinuous epitope, residues identified as immunodominant were cross-linked into a single peptide, joining the individual fragments using a Glycine spacer:

116 113 114 34 33

Lys Asn Arg Phe Lys

Lys-Asn-Arg-Gly-Phe-Lys

Such a peptide effectively blocked the binding of specific antibodies to the protein, meaning it resembled a natural discontinuous epitope.

In the 1980s, it became clear that the entire surface of a protein can be antigenic; that is, if synthetic peptides are used for immunization, antibodies can be raised against any surface region. However, upon immunization with the whole protein, antibodies were formed only against specific regions. The Use of Monoclonal Antibodies of strictly defined specificity showed that each antigenic determinant actually consists of several potentially overlapping antigenic regions. These epitopes are now referred to by the more accurate term immunodominant regions.

Naturally, the question arose as to which factors determine immunodominance.

Based on the recognized function of the immune system to distinguish "self" from "non-self", the first principle underlying immunodominance was the principle of antigen foreignness relative to the recipient's proteins. To test the validity of this principle, series of homologous proteins were studied—that is, proteins found in many organisms and differing by individual Amino Acid Substitutions. Cytochromes c proved ideal for such experiments.

Cytochromes c are heme-containing Proteins of the mitochondrial Respiratory Chain with a molecular weight of 13 kDa, consisting of about 100 amino acid residues. They appeared very early in the Evolution of the living world, with the first cytochromes c found in bacteria. The Protein Structure proved so successful that it has essentially been preserved up to higher animals. Mammalian cytochromes c differ among themselves by individual amino acid residues, meaning they can be viewed as point mutants. A direct correlation was found between the immunogenicity of cytochrome c and the number of residues distinguishing the antigen from the recipient's homologous cytochrome c. However, regarding the Specificity of the antibodies produced, this correlation did not prove absolute. For instance, rabbits immunized with their own cytochrome modified with glutaraldehyde produced antibodies against epitopes of their own cytochrome. When animals of different species were immunized with a single type of cytochrome, antibodies were produced against the exact same regions. Consequently, another principle of immunodominance was considered—the relationship with the Structural Features of the antigen, accessibility, charge, and specific Location at the bends of the polypeptide chain. Algorithms for predicting immunodominant regions based on hydrophilicity and atomic mobility principles were proposed. Subsequent experiments revealed a link between hydrophilicity and mobility with evolutionary Variability; amino acid substitutions fixed during evolution must not disrupt the BIOLOGICAL FUNCTIONS OF cytochrome c and are therefore localized precisely in surface, highly flexible regions where the Introduction of a different amino acid is safest and can be compensated for by molecular flexibility.

As a result of these studies, it was concluded that although the entire surface of a protein can, in principle, be antigenic, natural immunization with the native protein induces antibodies only against specific epitopes whose immunodominance is determined by their structural features, primarily hydrophilicity and atomic mobility (flexibility).

Antibodies (and B lymphocytes) bind the native antigen and recognize so-called B epitopes on its surface. Yet, during the immune response, the antigen is also recognized by T lymphocytes. Moreover, the specificity of T lymphocytes determines which immunodominant regions will be recognized as B epitopes. The regions of the antigen recognized by T lymphocytes are called T epitopes. Their position and structure are not as easily determined as those of B epitopes because T cells recognize antigens in a completely different manner.

1. For recognition by T lymphocytes, the antigen must be processed (cleaved). Processing takes place inside specialized Cells under the action of Proteolytic Enzymes. THE SPECTRUM OF peptides generated depends on the type of proteases, which vary among different cell types.

2. The processed peptide must be presented in a complex with Major Histocompatibility Complex proteins; the Selection of the antigenic peptide depends on the structure of these proteins, which are highly polymorphic and differ even among different individuals of the same species.

3. Recognition of the presented peptide depends on the repertoire of T-cell receptors, which results from positive and negative selection in a given individual.

As a result, a T epitope is not necessarily a surface structure; it is a linear peptide rather than a conformation-dependent one. Its position is not related to the hydrophilicity or mobility of the polypeptide chain. It depends both on the STRUCTURE OF THE native protein (potential proteolysis sites, peptide motifs corresponding to histocompatibility protein-binding sites) and on the immune system state of the individual recipient (the repertoire of histocompatibility proteins and T-cell receptors). T epitopes are more closely associated with sites of antigen foreignness relative to the recipient's proteins than B epitopes, since the T-cell receptor repertoire undergoes more stringent negative selection.

Determining the structure and localization of B and T epitopes is of more than just fundamental interest. It is essential for The Development of effective Vaccines and immunodiagnostics.

Abstract.

The immune system is capable of recognizing almost any substance from the macroorganism's surrounding environment. For this to occur, the antigen must be properly presented to immune cells. B lymphocytes and antibodies recognize conformation-dependent surface epitopes located in regions of the polypeptide chain with the highest hydrophilicity and flexibility. T lymphocytes recognize internal linear peptide fragments generated as a result of proteolysis (processing) of the native antigen.