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Epitope
Epitope
Epitope
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An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that binds to the epitope is called a paratope. Although epitopes are usually non-self proteins, sequences derived from the host that can be recognized (as in the case of autoimmune diseases) are also epitopes.[1]

The epitopes of protein antigens are divided into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the paratope.[2] Conformational and linear epitopes interact with the paratope based on the 3-D conformation adopted by the epitope, which is determined by the surface features of the involved epitope residues and the shape or tertiary structure of other segments of the antigen. A conformational epitope is formed by the 3-D conformation adopted by the interaction of discontiguous amino acid residues. In contrast, a linear epitope is formed by the 3-D conformation adopted by the interaction of contiguous amino acid residues. A linear epitope is not determined solely by the primary structure of the involved amino acids. Residues that flank such amino acid residues, as well as more distant amino acid residues of the antigen affect the ability of the primary structure residues to adopt the epitope's 3-D conformation.[3][4][5][6][7] 90% of epitopes are conformational.[8]

Function

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T cell epitopes

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T cell epitopes[9] are presented on the surface of an antigen-presenting cell, where they are bound to major histocompatibility complex (MHC) molecules. In humans, professional antigen-presenting cells are specialized to present MHC class II peptides, whereas most nucleated somatic cells present MHC class I peptides. T cell epitopes presented by MHC class I molecules are typically peptides between 8 and 11 amino acids in length, whereas MHC class II molecules present longer peptides, 13–17 amino acids in length,[10] and non-classical MHC molecules also present non-peptidic epitopes such as glycolipids.

B cell epitopes

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The part of the antigen that immunoglobulin or antibodies bind to is called a B-cell epitope.[11] B cell epitopes can be divided into two groups: conformational or linear.[11] B cell epitopes are mainly conformational.[12][13] There are additional epitope types when the quaternary structure is considered.[13] Epitopes that are masked when protein subunits aggregate are called cryptotopes.[13] Neotopes are epitopes that are only recognized while in a specific quaternary structure and the residues of the epitope can span multiple protein subunits.[13] Neotopes are not recognized once the subunits dissociate.[13]

Cross-activity

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Epitopes are sometimes cross-reactive. This property is exploited by the immune system in regulation by anti-idiotypic antibodies (originally proposed by Nobel laureate Niels Kaj Jerne). If an antibody binds to an antigen's epitope, the paratope could become the epitope for another antibody that will then bind to it. If this second antibody is of IgM class, its binding can upregulate the immune response; if the second antibody is of IgG class, its binding can downregulate the immune response.[citation needed]

Epitope mapping

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Main article: Epitope mapping

T cell epitopes

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MHC class I and II epitopes can be reliably predicted by computational means alone,[14] although not all in-silico T cell epitope prediction algorithms are equivalent in their accuracy.[15] There are two main methods of predicting peptide-MHC binding: data-driven and structure-based.[11] Structure based methods model the peptide-MHC structure and require great computational power.[11] Data-driven methods have higher predictive performance than structure-based methods.[11] Data-driven methods predict peptide-MHC binding based on peptide sequences that bind MHC molecules.[11] By identifying T-cell epitopes, scientists can track, phenotype, and stimulate T-cells.[16][17][18][19]

B cell epitopes

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There are two main methods of epitope mapping: either structural or functional studies.[20] Methods for structurally mapping epitopes include X-ray crystallography, nuclear magnetic resonance, and electron microscopy.[20] X-ray crystallography of Ag-Ab complexes is considered an accurate way to structurally map epitopes.[20] Nuclear magnetic resonance can be used to map epitopes by using data about the Ag-Ab complex.[20] This method does not require crystal formation but can only work on small peptides and proteins.[20] Electron microscopy is a low-resolution method that can localize epitopes on larger antigens like virus particles.[20]

Methods for functionally mapping epitopes often use binding assays such as western blot, dot blot, and/or ELISA to determine antibody binding.[20] Competition methods look to determine if two monoclonal antibodies (mABs) can bind to an antigen at the same time or compete with each other to bind at the same site.[20] Another technique involves high-throughput mutagenesis, an epitope mapping strategy developed to improve rapid mapping of conformational epitopes on structurally complex proteins.[21] Mutagenesis uses randomly/site-directed mutations at individual residues to map epitopes.[20] B-cell epitope mapping can be used for the development of antibody therapeutics, peptide-based vaccines, and immunodiagnostic tools.[20][22]

Epitope tags

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Epitopes are often used in proteomics and the study of other gene products. Using recombinant DNA techniques genetic sequences coding for epitopes that are recognized by common antibodies can be fused to the gene. Following synthesis, the resulting epitope tag allows the antibody to find the protein or other gene product enabling lab techniques for localisation, purification, and further molecular characterization. Common epitopes used for this purpose are Myc-tag, HA-tag, FLAG-tag, GST-tag, 6xHis,[23] V5-tag and OLLAS.[24] Peptides can also be bound by proteins that form covalent bonds to the peptide, allowing irreversible immobilisation.[25] These strategies have also been successfully applied to the development of "epitope-focused" vaccine design.[26][27]

Epitope-based vaccines

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Main article: Peptide vaccine

The first epitope-based vaccine was developed in 1985 by Jacob et al.[28] Epitope-based vaccines stimulate humoral and cellular immune responses using isolated B-cell or T-cell epitopes.[28][22][17] These vaccines can use multiple epitopes to increase their efficacy.[28] To find epitopes to use for the vaccine, in silico mapping is often used.[28] Once candidate epitopes are found, the constructs are engineered and tested for vaccine efficiency.[28] While epitope-based vaccines are generally safe, one possible side effect is cytokine storms.[28]

Neoantigenic determinant

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A neoantigenic determinant is an epitope on a neoantigen, which is a newly formed antigen that has not been previously recognized by the immune system.[29] Neoantigens are often associated with tumor antigens and are found in oncogenic cells.[30] Neoantigens and, by extension, neoantigenic determinants can be formed when a protein undergoes further modification within a biochemical pathway such as glycosylation, phosphorylation or proteolysis. This, by altering the structure of the protein, can produce new epitopes that are called neoantigenic determinants as they give rise to new antigenic determinants. Recognition requires separate, specific antibodies.[citation needed]

See also

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References

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Revisions and contributorsEdit on WikipediaRead on Wikipedia

Epitope

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An epitope, also known as an antigenic determinant, is the specific region on an —a capable of eliciting an —that is recognized and bound by components such as , B-cell receptors, or T-cell receptors, thereby triggering humoral or cellular immunity. These sites are typically small, consisting of 5–8 residues for protein antigens or 1–6 monosaccharides for antigens, and must be accessible on the antigen's surface for effective binding. Epitopes play a central role in adaptive immunity by enabling the to distinguish self from non-self , such as pathogens or foreign proteins, and are essential for processes like production and antigen clearance. Epitopes are broadly classified into two main types based on their structural nature: linear (continuous) epitopes, which consist of a sequential stretch of along the antigen's primary , and conformational (discontinuous) epitopes, which depend on the three-dimensional folding of the antigen and involve brought into proximity by . Linear epitopes are often 9–12 residues long and are more stable to denaturation, while conformational epitopes, typically 15–22 residues, predominate in native proteins and account for about 90% of B-cell epitopes. Additionally, epitopes are categorized by the immune receptor they engage: B-cell epitopes are recognized directly by antibodies or B-cell receptors to stimulate humoral responses, whereas T-cell epitopes are presented by (MHC) molecules to T-cell receptors, divided into MHC class I (for CD8+ T cells, focusing on intracellular pathogens) and MHC class II (for CD4+ T cells, aiding broader coordination). The identification and characterization of epitopes are fundamental to , underpinning design, diagnostic assays, and therapeutic development by targeting immunodominant sites that elicit protective responses. For instance, techniques, including experimental methods like and cryo-electron microscopy alongside predictions, help predict and avoid unwanted responses in biologics. Variations in epitope accessibility, influenced by factors like antigen denaturation or host (e.g., MHC alleles), can modulate immune efficacy, highlighting their dynamic role in disease and immunity.

Definition and Fundamentals

Definition

An epitope, also known as an antigenic determinant, is defined as the specific portion of an molecule that is recognized and bound by the of an or by a . For B-cell recognition, linear epitopes typically comprise 5 to 17 in continuous sequences, while T-cell epitopes are shorter peptides of 8–11 residues for or 13–25 for ; discontinuous epitopes involve spatially proximate residues from non-adjacent parts of the polypeptide chain that form a functional in the native . While often described for protein antigens, epitopes can also occur on carbohydrates (typically 1–6 monosaccharides), , or other molecules. The concept of the epitope emerged from early studies on antigen-antibody interactions, with the term first introduced by immunologist Niels Kaj Jerne in his 1960 paper "Immunological Speculations," where he described it as the surface feature of an capable of eliciting a specific . The of an epitope is influenced by several key physicochemical properties, including its surface to immune molecules, hydrophilicity that promotes solvent exposure, and flexibility allowing conformational adaptation during binding. These characteristics ensure that epitopes are positioned on the antigen's exterior and can form stable, specific interactions with immune receptors, distinguishing them from non-immunogenic regions of the same molecule. For instance, in the model protein hen , monoclonal antibodies such as HyHEL-10 target epitopes involving critical residues like Arg21, Asp101, and Tyr53, which contribute to the binding interface through hydrogen bonding and van der Waals contacts. While an epitope represents a targeted site on an , it is distinct from the full , which encompasses the entire immunogenic entity capable of provoking a broader .

Relation to Antigens and Antibodies

An epitope represents a discrete subset of an , serving as the specific molecular region recognized by components of the . Antigens are typically large macromolecules, such as proteins or , that contain one or more such epitopes capable of eliciting an . In contrast, haptens are small molecules, like certain chemicals or peptides, that lack inherent on their own but acquire antigenic properties when covalently attached to a larger carrier protein, which provides the necessary structural context and T-cell epitopes for immune activation. This conjugation transforms the hapten into an epitope within the composite , enabling B-cell recognition and production. The functional relationship between epitopes and antibodies centers on the precise interaction between the epitope and the —the antigen-binding site located in the variable regions of the antibody's Fab fragment or on T-cell receptors. This binding occurs through non-covalent forces, including bonds formed by polar residues like serine and threonine, van der Waals interactions dominated by aromatic side chains such as and , and electrostatic attractions that facilitate initial orientation over distances up to several nanometers. These interactions collectively bury a significant surface area (often ~1600 Ų) at the interface, expelling molecules and stabilizing the complex without covalent linkages. The strength of epitope-paratope binding is quantified by affinity, defined as the (K_d), which measures the equilibrium between associated and dissociated states; lower K_d values indicate higher affinity. For monoclonal antibodies, typical K_d values range from 20 pM to 300 pM against epitopes, reflecting strong monovalent interactions driven by cumulative weak forces totaling ~12 kcal/mol in . , the cumulative binding strength from multiple epitope-paratope engagements (e.g., in IgM or multimeric antigens), amplifies this affinity by orders of magnitude through cooperative effects, enhancing overall immune efficacy. This relational dynamic underpins immune specificity, analogous to the lock-and-key model, wherein the epitope's three-dimensional structure complements the 's and chemical properties for a rigid, highly selective fit with minimal conformational adjustment upon binding. Aromatic residues in the often form the core "lock," interacting with backbone and side-chain atoms on the epitope to discriminate against non-cognate structures, while surrounding hydrophilic contacts fine-tune discrimination. Such precision ensures targeted immune responses while avoiding off-target effects.

Types of Epitopes

Linear Epitopes

Linear epitopes, also known as sequential epitopes, consist of continuous stretches of , typically 5 to 15 residues long, derived from the primary structure of a protein . These epitopes are recognized by antibodies based solely on their linear sequence, without reliance on the protein's folded conformation. A key property of linear epitopes is their resistance to denaturation; unlike conformational epitopes, they remain intact and accessible even when the protein is unfolded by heat, changes, or chemical agents, making them detectable in denatured samples. Linear epitopes constitute approximately 10% of all B-cell epitopes, though they are more prevalent in certain antigens such as bacterial toxins and viral proteins. For instance, in bacterial toxins, a linear epitope spanning residues 40-62 of the epsilon toxin from Clostridium perfringens has been identified as immunogenic when fused with cholera toxin B subunit for vaccine development. In viral proteins, a well-characterized example is the linear epitope within amino acids 93-104 of the VP1 capsid protein of poliovirus type 1, which is recognized by neutralizing monoclonal antibodies and contributes to antiviral immunity. These examples highlight how linear epitopes in pathogens can elicit protective antibody responses, particularly in exposed or linear regions of the antigen. One advantage of linear epitopes is their ease of synthesis as short peptides, which facilitates their use in immunological assays and design without the need for complex . However, a disadvantage is that synthetic linear peptides may not fully mimic the native antigenic context, potentially leading to reduced or specificity compared to epitopes in the intact . In contrast, conformational epitopes require the protein's three-dimensional fold for recognition and are disrupted under denaturing conditions. Experimental evidence for linear epitopes often comes from techniques involving synthetic peptides, such as where overlapping peptides are screened for binding, confirming sequential recognition. Similarly, peptide scanning methods, which use arrays of systematically overlapping peptides covering the sequence, have identified linear epitopes in viral capsid proteins by detecting specific interactions. These approaches provide direct validation of the continuous nature of such epitopes in various pathogens.

Conformational Epitopes

Conformational epitopes consist of discontinuous residues that are spatially adjacent only in the three-dimensional structure of the native , formed by rather than contiguous sequences in the primary chain. These epitopes typically involve clusters of 10 to 20 or more brought together in close proximity, often exceeding 15 residues due to their reliance on tertiary or folding. Unlike linear epitopes, which depend solely on primary and can persist under denaturing conditions, conformational epitopes are highly sensitive to denaturation processes such as or chemical disruption, which abolish their structure and prevent antibody recognition. The structural basis of conformational epitopes lies in their location on the exposed surfaces of folded proteins, where disparate residues from secondary structural elements like beta-sheets, loops, and helices converge to form accessible binding sites for antibodies. These epitopes often span regions involving beta-sheet strands connected by loops, creating concave or convex patches that facilitate specific interactions. A prominent example is found in the HIV-1 glycoprotein gp120, where conformational epitopes at the CD4-binding site and variable loops (such as V1/V2) are critical for recognition by broadly neutralizing antibodies, enabling potent viral inhibition through precise targeting of the trimer's native conformation. Conformational epitopes represent approximately 90% of B-cell epitopes on native proteins, underscoring their dominance in humoral immune responses to folded . Their recognition necessitates an intact, non-denatured structure, as disruption of folding eliminates the spatial arrangement required for binding, highlighting their role in mimicking physiological . Biophysically, these epitopes are maintained by stabilizing features of the protein's tertiary structure, including disulfide bonds that covalently link distant cysteines to lock conformations and hydrophobic cores that bury nonpolar residues internally, shielding the epitope's solvent-exposed face. bridges, in particular, enhance rigidity in loop regions common to epitopes, while hydrophobic interactions contribute to the overall stability of surface patches, ensuring persistence under physiological conditions.

Immune Recognition Mechanisms

B Cell Epitopes

B cell epitopes are regions on the surface of that are directly recognized and bound by receptors (BCRs), which are membrane-bound immunoglobulins, or by secreted antibodies produced by plasma cells in the humoral immune response. This recognition occurs without the need for , focusing on native, extracellular epitopes that are accessible in their three-dimensional structure. BCR-antigen interactions typically take place in a two-dimensional environment, where the antigen may be anchored, facilitating initial binding and subsequent immune signaling. These epitopes are predominantly conformational, comprising approximately 90% of known cases, and are characterized by their solvent-exposed nature on the antigen surface, allowing interaction with the paratope of the BCR or antibody. In terms of physical dimensions, B cell epitopes typically encompass about 15 amino acid residues on average, with an elliptical surface area of roughly 400 Ų and a thickness of around 8 Å, corresponding to a diameter of 15-22 Å that fits within the antigen-binding site of an immunoglobulin. This preference for exposed, discontinuous structures arises because B cells target protruding or flexible regions rich in coils and charged residues, rather than buried or linear sequences. Upon binding, effective B cell activation requires the cross-linking of multiple BCRs by multivalent antigens, which oligomerizes the receptors and initiates intracellular signaling cascades leading to B cell proliferation, differentiation into antibody-secreting plasma cells, and affinity maturation. This cross-linking threshold ensures that only high-avidity interactions trigger a robust response, amplifying the humoral immune defense against pathogens or allergens. Representative examples include epitopes on the capsules of such as or , which are recognized by antibodies to promote opsonization and , eliciting protective . In allergic contexts, protein epitopes from allergens like Amb a 1 serve as targets for IgE antibodies, driving reactions in sensitized individuals.

T Cell Epitopes

T cell epitopes are short peptide fragments derived from intracellular or extracellular antigens that are processed and presented on the surface of antigen-presenting cells (APCs) or infected cells in complex with (MHC) molecules, enabling recognition by T lymphocytes. Unlike B cell epitopes, which are recognized directly on the native structure of antigens by B cell receptors, T cell epitopes require proteolytic processing into peptides typically ranging from 8 to 25 in length before presentation. This processing ensures that T cells monitor intracellular events, such as viral infections or aberrant protein production, by surveying the peptide-MHC (pMHC) complexes displayed on cell surfaces. The recognition process involves two primary pathways corresponding to MHC class I and class II molecules. For MHC class I presentation, endogenous antigens in the cytosol—such as viral or self-proteins—are degraded by the proteasome into peptides of 8-11 amino acids, which are then transported into the endoplasmic reticulum (ER) via the transporter associated with antigen processing (TAP), trimmed, and loaded onto MHC class I molecules for display on the cell surface to CD8+ cytotoxic T cells. In contrast, exogenous antigens taken up by endocytosis are processed in lysosomes or endosomes by proteases into longer peptides (12-25 amino acids), which bind to MHC class II molecules after removal of the invariant chain (CLIP) peptide, and are presented to CD4+ helper T cells primarily on professional APCs like dendritic cells. These pathways allow T cells to distinguish between intracellular threats (via MHC I) and extracellular pathogens (via MHC II), with peptide lengths accommodating the closed groove of MHC I and the open-ended groove of MHC II. MHC restriction is a fundamental principle dictating that T cell recognition is allele-specific, as epitopes must bind with sufficient affinity to particular MHC variants to form stable pMHC complexes; for instance, the HLA-A*02:01 , prevalent in about 50% of certain populations, frequently presents viral epitopes due to its binding motif favoring hydrophobic residues at anchor positions. This polymorphism in (HLA) genes—encoding MHC molecules—means that epitope immunogenicity varies across individuals, influencing immune responses to pathogens. Only peptides that fit the specific binding pockets of an individual's MHC s can elicit a T cell response, underscoring the of MHC diversity in population-level immunity.00701-7) Upon encounter, the (TCR) on a naïve T cell binds to the cognate pMHC complex, initiating through the CD3 complex and zeta chain, but full activation requires a second co-stimulatory signal, such as interacting with B7 molecules on APCs, to prevent anergy or . For + T cells, this leads to differentiation into cytotoxic effectors that release perforin and granzymes to induce target cell , thereby eliminating infected or malignant cells. CD4+ T cells, conversely, become helper cells that secrete cytokines to orchestrate broader immune responses, including activation and recruitment. A well-characterized example is the matrix protein 1 (M1) epitope GILGFVFTL (residues 58-66), presented by HLA-A*02:01 in the pathway, which elicits a potent + cytotoxic response to clear infected respiratory epithelial cells.

Cross-Reactivity

Cross-reactivity in epitopes occurs when antibodies or T cell receptors (TCRs) bind to similar but non-identical epitopes on different antigens, primarily through the mechanism of molecular mimicry. In this process, epitopes share key structural residues or motifs that allow cross-binding, such as conserved sequences in viral proteins that mimic host or other structures. For instance, TCRs can recognize epitopes with partial homology if the core binding residues align sufficiently, enabling an against related pathogens. This phenomenon has significant implications for both protective and detrimental immune responses. Beneficially, it underpins heterosubtypic immunity in vaccines, where antibodies elicited against one strain provide partial protection against others due to shared epitopes, as demonstrated in studies showing cross-neutralization across subtypes. Conversely, it can drive pathological , such as in , where streptococcal M protein epitopes mimic cardiac , leading to cross-reactive antibodies that attack heart tissue. Factors influencing include exceeding 70% or structural similarity in epitope conformation, often quantified by cross-reactivity indices from assays like or , which measure binding affinity to heterologous antigens. High homology in anchor residues for MHC presentation further enhances T cell cross-reactivity. A notable example is in infections, where cross-reactive antibodies from a primary bind to epitopes on a secondary , facilitating (ADE) that worsens disease severity by promoting viral entry into immune cells. This highlights the double-edged nature of epitope cross-reactivity in flavivirus immunity.

Epitope Mapping Techniques

B Cell Epitope Mapping

B cell epitope mapping involves a suite of experimental techniques designed to identify the specific regions on recognized by -derived antibodies, focusing on both linear and conformational epitopes central to . libraries represent a cornerstone method, where random or antigen fragment libraries are expressed on surfaces to screen for binding interactions, enabling the isolation of epitope mimics or direct antigen sequences that elicit specific antibody responses. This approach has been instrumental in design by revealing immunodominant B cell epitopes from pathogens. Structural methods provide atomic-level insights into epitope-paratope interfaces. determines the three-dimensional structure of -antigen complexes, often achieving resolutions below 2.5 Å to delineate contacting residues and conformational details. For instance, crystallographic studies have mapped epitopes on viral proteins, highlighting buried and solvent-exposed interactions critical for binding. Complementing this, cryo-electron microscopy (cryo-EM) excels for larger complexes, resolving structures at near-atomic resolution without crystallization; notable examples include mapping neutralizing epitopes on the , where cryo-EM revealed conformational epitopes targeted by memory B cells during . Functional assays quantify binding and validate epitope contributions. Enzyme-linked immunosorbent assay () and () measure antibody- affinity, with competition formats binning antibodies by overlapping epitopes and assessing binding kinetics in real-time. further refines mapping by systematically altering residues and monitoring loss of binding, pinpointing critical within epitopes; high-throughput variants, such as , accelerate identification of both linear and discontinuous sites. A key challenge in epitope mapping is ensuring native-like to capture conformational epitopes, which predominate in structured proteins and are often lost in linear peptide-based assays or denatured forms. Techniques like and cryo-EM mitigate this by preserving tertiary structures, but variability in antigen folding and affinity can complicate comprehensive mapping, necessitating integrated approaches for reliable results.

T Cell Epitope Mapping

T cell epitope mapping involves identifying peptides that, when presented by (MHC) molecules, elicit specific T cell responses, crucial for understanding cellular immunity. This process typically integrates functional assays to detect T cell activation with biochemical methods to assess peptide-MHC interactions, focusing on CD4+ and + T cells that recognize epitopes restricted by and class I molecules, respectively. One key technique is MHC tetramer staining, which uses fluorescently labeled MHC-peptide multimers to directly visualize and quantify epitope-specific T cells via . These tetramers, formed by biotinylated MHC-peptide complexes bound to streptavidin-fluorochrome conjugates, bind to T cell receptors with high specificity, enabling detection of antigen-specific + T cells at frequencies as low as 1:50,000 peripheral blood mononuclear cells (PBMCs) without prior . In -1 studies, MHC tetramers have mapped epitope-specific responses, revealing frequencies of 1-4% of + T cells in chronically infected individuals targeting conserved epitopes like those in protein restricted by *02:01. The enzyme-linked immunospot () assay measures release, such as interferon-gamma (IFN-γ), from T cells activated by -MHC complexes, providing a sensitive readout of epitope-specific responses. In this method, PBMCs are incubated with candidate s in multi-well plates coated with -capture antibodies, where activated T cells form visible spots proportional to their frequency; it can detect responses from as few as 1 in 100,000 cells. is often combined with pools for initial screening, followed by to pinpoint immunogenic epitopes, and has been pivotal in validating T cell responses to HIV-1 antigens. Peptide libraries, particularly overlapping synthetic spanning target proteins, are scanned to identify epitopes capable of MHC binding and T cell . Typically, libraries consist of 15-18 overlapping by 11 residues to ensure coverage of potential 8-11 mer class I or longer class II epitopes; these are tested in pools or matrices to minimize cell usage, with positive hits deconvoluted via iterative testing. For instance, such libraries have identified tumor-specific epitopes like those from melanoma-associated antigens by assessing T cell and production. In vitro binding assays quantify affinity to purified MHC molecules, often integrating predictions to prioritize candidates. Competitive binding assays, such as fluorescence polarization, measure the inhibitory concentration () required for a test to displace 50% of a labeled reference from MHC; lower values (e.g., <500 nM) indicate high-affinity binders likely to form stable complexes for T cell recognition. These assays, performed under physiological conditions (37°C, 5.5 for class II), have refined by validating predicted binders for alleles like HLA-DR1. In HIV-1 , overlapping peptide libraries and have identified immunodominant epitopes restricted by specific HLA alleles, such as the Pol-IY11 (ILKEPVHGVYY) peptide presented by HLA-*12:02, confirmed via and T cell response assays in infected individuals. Similarly, the Nef-MY9 (MARELHPEY) epitope, also HLA-*12:02-restricted, elicits strong + T cell responses, highlighting allele-specific targeting in protective immunity. These approaches underscore the MHC dependency of T cell recognition, where epitopes must bind stably to elicit .

Practical Applications

Epitope Tags in

Epitope tags are short sequences derived from known antigens that are genetically fused to recombinant proteins to enable their detection, purification, and analysis using specific antibodies, without the need for custom antibodies against the target protein itself. These tags are particularly valuable in research settings where studying or low-abundance proteins requires reliable tools for visualization and isolation. Common epitope tags, such as HA, , and c-Myc, were selected for their compact size, minimal in expression hosts like mammalian or cells, and the existence of high-affinity monoclonal antibodies that recognize them with high specificity. The , derived from the protein, consists of the 9-amino-acid sequence YPYDVPDYA and was first introduced in 1988 for purifying RAS-responsive complexes in yeast. The is an 8-amino-acid synthetic sequence, DYKDDDDK, developed in 1988 as a marker for hybrid from mammalian cells, incorporating an enterokinase cleavage site for tag removal. The c-Myc tag, a 10-amino-acid EQKLISEEDL from the human c-Myc proto-oncogene product, originated from studies in 1985 and is recognized by the widely used 9E10 . In laboratory applications, epitope tags facilitate techniques such as Western blotting for protein expression confirmation and for studying protein-protein interactions. For instance, the is routinely employed to visualize recombinant proteins expressed in mammalian cells, allowing subcellular localization via without disrupting native function. These tags also support affinity purification using anti-tag resins, enabling isolation of tagged proteins from complex lysates with high yield and purity in systems ranging from to eukaryotic cells.
TagSequenceLength (aa)Origin
HAYPYDVPDYA9 hemagglutinin
FLAGDYKDDDDK8Synthetic (enterokinase site)
c-MycEQKLISEEDL10Human c-Myc proto-oncogene
The primary advantages of epitope tags include their small size, which generally avoids significant alterations to the target protein's structure, folding, or activity, and the option for protease-mediated removal, as with the using enterokinase. This modularity makes them versatile for functional studies, where tags can be positioned at the N- or or internally if tolerated. However, limitations exist, such as potential interference with native or localization if the tag is placed in a critical region, and challenges in immunological where the tag itself may mask genuine epitopes or provoke unintended immune responses. Additionally, reliance on commercial antibodies can introduce variability in detection efficiency across different experimental contexts.

Epitope-Based Vaccines

Epitope-based vaccines are designed to elicit targeted s by incorporating specific antigenic epitopes from , rather than using whole organisms or inactivated pathogens. These vaccines typically employ subunit approaches, such as synthetic peptides representing linear T-cell or B-cell epitopes, or virus-like particles (VLPs) that display conformational epitopes to mimic native structures. For instance, the human papillomavirus () vaccine, such as , utilizes recombinant L1 capsid proteins assembled into VLPs that present conformational epitopes, inducing neutralizing antibodies against the without the risks associated with live-attenuated formulations. This design allows precise selection of immunodominant epitopes to focus the on critical pathogen components. A key advantage of epitope-based vaccines is their enhanced safety profile, as they avoid the inclusion of extraneous pathogen material that could cause adverse reactions or immune interference. By excluding non-essential antigens, these vaccines minimize side effects while enabling the creation of multi-epitope constructs that provide broad protection against variant strains, such as in polyvalent formulations targeting multiple serotypes. Additionally, their modular nature facilitates rapid adaptation to emerging threats through computational epitope prediction and synthesis. Despite these benefits, epitope-based vaccines face challenges, particularly their inherently low , which often necessitates the use of adjuvants to provide T-cell help and enhance responses. For example, the RTS,S incorporates both T-cell and B-cell epitopes from the circumsporozoite protein (CSP) of , combined with the AS01 adjuvant to boost production and cellular immunity, addressing the poor standalone of epitopes. Other hurdles include ensuring proper epitope processing and presentation , as well as overcoming potential in chronic infections. Clinical progress in epitope-based vaccines has demonstrated their potential, with peptide vaccines for inducing robust, epitope-specific CD8+ T-cell responses that correlate with tumor regression in phase I/II trials. These vaccines, often combining helper and cytotoxic T-lymphocyte epitopes, have shown durable immune memory and epitope spreading, where initial responses expand to additional tumor antigens, supporting their role in personalized . Ongoing advancements, including delivery systems, continue to improve efficacy in infectious disease settings beyond cancer.

Neoepitopes in Cancer

Neoepitopes are novel epitopes arising from somatic mutations in tumor cells, which generate unique antigenic peptides not present in normal tissues and capable of eliciting an antitumor immune response. These patient-specific neoepitopes typically result from point mutations, insertions, deletions, or gene fusions that alter protein sequences, leading to altered peptides that can bind to major histocompatibility complex (MHC) molecules for presentation to T cells. Unlike shared tumor antigens, neoepitopes are highly individualized, making them ideal targets for personalized cancer immunotherapies that minimize off-target effects on healthy cells. The generation of neoepitopes begins with tumor genomic sequencing to identify somatic variants, followed by bioinformatics prediction of peptides that can bind to the patient's specific HLA alleles and provoke T cell recognition. For instance, the G12D , prevalent in pancreatic ductal , produces a neoepitope that has been targeted in T cell receptor (TCR) , demonstrating tumor regression in a clinical case of metastatic . Whole-exome sequencing of tumor biopsies, combined with sequencing to confirm expression, enables the selection of high-affinity neoepitopes for therapeutic development. In therapeutic applications, neoepitope-targeted deliver patient-specific peptides or nucleic acids encoding them to stimulate T cell responses against tumors, while chimeric receptor () T cells or TCR-engineered T cells can be designed to recognize neoepitope-MHC complexes. Clinical trials of personalized neoantigen in advanced have reported objective response rates of approximately 30%, with durable responses in a subset of patients when combined with checkpoint inhibitors. As of 2025, expansion cohorts in trials like autogene cevumeran combined with have shown promising ORRs of 33.3% in CPI-naive advanced patients (n=9). Similarly, CAR-T therapies targeting neoepitope-derived peptides, such as those from mutant , have shown preclinical efficacy in solid tumors by enhancing tumor-specific . Key challenges in neoepitope-based include variability in MHC binding affinity, which depends on individual HLA types and can limit neoepitope , as well as intratumoral heterogeneity that allows antigen escape through subclonal mutations. These factors contribute to variable clinical responses, with only a fraction of predicted neoepitopes eliciting robust T cell activation . Addressing tumor and optimizing neoepitope selection remain critical for broader .

Computational Tools

Epitope Prediction Methods

Epitope prediction methods employ computational algorithms to identify potential antigenic sites from protein sequences, primarily focusing on T cell and B cell epitopes. For T cell epitopes, models such as NetMHC predict peptide binding affinity to ( molecules by estimating the half-maximal inhibitory concentration (), classifying peptides with IC50 values below 500 nM as strong binders likely to elicit + T cell responses. These artificial neural network-based approaches, initially developed using quantitative binding data, have evolved to incorporate pan-specific predictions for diverse MHC alleles, enhancing their utility in personalized immunotherapies. B cell epitope prediction tools, such as BepiPred, target linear epitopes by integrating sequence-based features like hydrophilicity, surface accessibility, and flexibility, which are derived from propensity scales and hidden Markov models trained on experimentally validated epitopes. This method scores segments for their likelihood of being exposed and immunogenic, prioritizing regions with high solvent exposure to facilitate recognition, though it performs best for sequential rather than discontinuous epitopes. Recent advances in epitope prediction leverage , particularly models trained on large datasets from the Immune Epitope Database (IEDB), to improve accuracy by capturing complex motifs and structural nuances. For instance, convolutional neural networks and transformers have been integrated into tools like MHCflurry and subsequent iterations, achieving predictive accuracies of approximately 70-80% for T cell epitopes when evaluated on independent benchmarks for binding and . These AI-driven methods outperform traditional models by reducing false positives through multi-layer feature extraction from peptide-MHC interactions. To validate predictions, especially for conformational epitopes, computational pipelines often combine sequence-based scoring with in silico molecular docking simulations that model antigen-antibody or peptide-MHC complexes. Tools employing rigid or flexible docking, such as those based on energy minimization algorithms, assess binding stability and epitope exposure, providing structural rationale for experimental prioritization and bridging the gap between linear predictions and three-dimensional antigen presentation.

Epitope Databases

The Immune Epitope Database (IEDB) serves as a central repository for experimentally validated immune epitopes, encompassing data on recognition, T cell responses, and (MHC) interactions derived from over 25,000 publications. Established in 2004 and funded by the National Institute of Allergy and Infectious Diseases (NIAID), the IEDB currently holds more than 2.2 million entries as of 2025, covering epitopes associated with infectious diseases, allergies, , and transplantation. Epitopes in the IEDB are richly annotated with detailed attributes, including sequences, associated MHC alleles (such as HLA class I and II specificities), and experimental outcomes like binding affinities, immunogenicity measurements, and functional responses in cellular s. For instance, researchers can query the database for viral epitopes by specifying the , such as retrieving T cell epitopes from restricted to specific HLA alleles, facilitating targeted immunological studies. Specialized databases complement the IEDB for allergen epitopes, such as the Structural Database of Allergenic Proteins (SDAP 2.0), which curates over 1,600 sequences with associated epitope mappings, structural models, and data derived from experimental validations. Similarly, the COMPARE database, as of its 2025 release, focuses on clinically relevant protein sequences with descriptions and citation support, enabling comparative analysis for allergenicity assessment in food and environmental contexts to support safety evaluations and . These databases are instrumental in immunological research, particularly for training machine learning-based epitope prediction tools by providing large-scale, validated datasets that improve model accuracy in forecasting MHC binding and immunogenicity. They also enable benchmarking of experimental epitope mapping techniques, such as comparing high-throughput sequencing results against curated assay data to validate novel discoveries. The IEDB undergoes regular curation updates, with significant expansions post-2020 incorporating thousands of COVID-19-related epitopes from and T cell studies, reflecting the surge in research and enhancing resources for design and variant surveillance. Allergen databases like SDAP are similarly maintained with periodic releases to include emerging data from clinical trials and structural analyses.

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