Following viral infection, viral antigens bind specifically to receptors on the surface of lymphocytes thereby activating adaptive immunity in the host. An epitope, the smallest structural and functional unit of an antigen, binds specifically to an antibody or antigen receptor, to serve as key sites for the activation of adaptive immunity. The complexity and diverse range of epitopes are essential to study and map for the diagnosis of disease, the design of vaccines and for immunotherapy. Mapping the location of these specific epitopes has become a hot topic in immunology and immune therapy. Recently, epitope mapping techniques have evolved to become multiplexed, with the advent of high-throughput sequencing and techniques such as bacteriophage-display libraries and deep mutational scanning. Here, we briefly introduce the principles, advantages, and disadvantages of the latest epitope mapping techniques with examples for viral antigen discovery.
Adaptive immunity plays a vital role in the elimination of pathogens and the protection of organisms from re-infection. Adaptive immunity relies primarily on two types of lymphocytes, B cells and T cells, which mature in the bone marrow and thymus respectively and enter the peripheral lymphoid organs via the circulatory system. When foreign proteins (e.g. pathogens) are degraded, they are broken down into peptides and presented on the surface of the antigen presenting cell (APC). When these peptides are detected via specific receptors on lymphocytes, the naive lymphocytes are activated, proliferate and differentiate into effector cells and memory cells. The effector cells can specifically recognize ‘non-self’ foreign substances and either directly or indirectly eliminate pathogens or pathogen-infected cells. Different effector cells have their own specific functions. Plasma cells, the predominant effector B cells, secrete antibodies that specifically bind and recognize, thus mediating humoral immunity. Depending on their function, effector T cells are classified as cytotoxic T cells, helper T cells and regulatory T cells. Cytotoxic T cells bind and kill infected cells; helper T cells activate and maintain the function of other immune cells by producing a large number of cytokines; and regulatory T cells negatively regulate immune-mediated hyperinflammation. Memory cells also have the ability to specifically recognize ‘non-self’ foreign peptides, but unlike effector cells which die rapidly after the infection has been eliminated, memory cells can survive for long periods of time, up to 100 years. When re-infected with the same or a highly similar pathogen, memory cells are rapidly activated by the antigen and differentiate into effector cells, triggering a series of immune responses to clear the infection. This is the basis of immunological memory and is the main principle of vaccine immunity.
These foreign poly-peptides that stimulate adaptive immunity are collectively referred to as antigens. There are many different pathogens in nature, which in turn contain a variety of antigens. An effective antigen should have two main functions: immunogenicity, the ability of the antigen to efficiently activate the proliferation and differentiation of naive lymphocytes; antigenicity, the ability of the antigen to bind with high specificity to the immune effector cell. As antigen molecules are difficult to recognize by immune cells, epitopes are recognized by immune cells as an immune active region on the antigen. Epitopes, also known as antigenic determinants, are the smallest structural and functional units of an antigen molecule that bind specifically to an antibody or antigen receptor. Commonly, epitopes consist of 1-6 monosaccharides or 5-8 amino acid residues (B cells) or 8-11 amino acids (for T cells). Thus, an antigenic molecule contains more than one immunologically active region. Even the same pathogen can contain hundreds of different antigens.
B cells and T cells recognize epitopes via different mechanisms. B cells bind directly and specifically to antigens via the membrane surface immunoglobulins, called B cell receptors (BCRs), and differentiate into plasma cells and memory B cells with the same antigenic specificity. Plasma cells secrete antibodies that bind specifically to antigens and are important effector molecules in mediating humoral immunity against pathogens. The structure of an antibody is closely linked to its function. Antibody structures can be divided into variable region (V region) and constant region (C region). The region of an antibody that has a highly variable amino acid composition is the V region. Both recombination and somatic hypermutation in the genes encoding immunoglobulins result in the high diversity of the V region, giving rise to diverse antibody repertoires. Theoretically, the human immune system can produce up to 10 26 antibodies in different sequence combinations. When a person is infected, a large number of antigenic epitopes of the pathogen continuously stimulate specifically recognized T/B cells. During massive proliferation and replication, activated B cells undergo somatic hypermutation, which alters the affinity of the antibodies. This occurs via gradual mutational optimization of complementarity-determining regions (CDR)-antigen interactions. During infections such as SARS-CoV-2, this has been shown to increase overtime to gradually evolve our antibody responses against a pathogen. Plasma cells that produce high-affinity antibodies are retained and proliferate, while plasma cells with poor-affinity are eliminated. Antibodies can control and clear infections by directly neutralizing pathogens or toxins, activating complement, mediating antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. At high viral loads, viral genomes are highly susceptible to mutation, and these ongoing mutations may evade adaptive immunity and reduce its effectiveness.
The antigen recognition receptors on the surface of T cells are also membrane surface immunoglobulins and are known as T cell receptors (TCRs). Similar to BCRs, TCRs are also divided into V regions and C regions, and therefore TCRs have the ability to recognize, bind to and deliver activation signals to the cell. TCRs can generally only recognize antigens loaded into MHC molecules and so cannot recognize and bind to antigens directly. The key molecule, major histocompatibility complex (MHC), is therefore essential for processing and presentation of antigens by APCs. MHC-I primarily mediates antigen presentation of endogenous antigens and is expressed by all nucleated cells; MHC-II is expressed only by professional APCs. The antigens are degraded into short antigenic peptide fragments in APCs, usually 8-11 amino acids. These subsequently bind to MHC and present themselves on the cell surface as an antigenic peptide-major histocompatibility complex (p-MHC), ultimately activating the T cells via TCR engagement.
Both B cells and T cells have the capacity to recognize various epitopes that suit a variety of functions. Epitopes may be further classified into the following classifications: linear epitopes and conformational epitopes, based on their structural distinctions. Linear epitopes, also known as continuous epitopes, consist of amino acid residues arranged in a continuous sequence. Conformational epitopes, also known as a discontinuous epitope, consists of amino acid residues that are not continuously arranged but are close to each other in spatial structure. While B cell epitopes can be linear or conformational, T cell epitopes are usually linear due to digestion and processing by APCs, though recently evidence for the importance of structural conformations in T cell epitope affinity has been observed.
During infection, diverse viral epitopes stimulate the host to produce a wide variety of antibodies and cytotoxic T cells. Antigenic variation occurs between different strains and genotypes of the same virus. For example, Influenza A virus, whose major antigenic proteins, hemagglutinin and neuraminidase, vary between strains, generates subtype-specific immune responses. As viruses continue to replicate, mutations in the viral genome accumulate. Under immune selection pressure, viral mutants may acquire immune-evasion to survive, further increasing epitope diversity. In addition, the epitopes targeted by antibodies and cytotoxic T cells vary between individuals, depending on a number of factors, such as age, history of previous infections, affinity maturation and the diversity of MHC genes. Adaptive immunity may not have the capacity to protect the host when Original Antigenic Sin, or failure to mount a response against an evolving pathogen, is present. As such, an optimal epitope is one that induces effective cellular and humoral immunity, no longer requires further adaptations, and plays a key role in the development of efficient vaccines or immunotherapies.
Epitope mapping technology is essential to clarify the natural antigenic landscape and evaluate the most potent and effective epitopes for an appropriate immune response. Common B cell epitope mapping techniques include Deep Mutational Scanning, peptide/protein microarrays, bacteriophage peptide/protein display and other peptide display techniques. Common T cell epitope mapping techniques are divided into three categories, peptide-MHC multimer-based, cell-based and yeast-based epitope mapping techniques. Here, we cover the latest in multiplexed epitope mapping technology and discuss their importance to the advancement of immunological therapeutics.
Epitope mapping technologies.
The table summarizes the characteristics of each epitope mapping technique. “Y” is yes and “N” is no. ^Partial conformation/structure of linear epitopes is tolerated. "+" indicates the degree of time/cost; sequentially from +,++ to +++ as the most expensive/longest time.
Traditional immunology has relied on specific antibodies generated against a single, we
