Premed · Premed · Immunology
Lecture 9: Antigens and Immunogenicity
Immunology
Learning Objectives
By the end of this lecture, students will be able to:
- Define antigen, immunogen, hapten, and epitope
- Describe the factors that influence immunogenicity
- Distinguish between T-dependent and T-independent antigens
- Explain the concepts of antigenic determinants (epitopes) and their types
- Describe superantigens and their mechanism of action
Lecture Content
I. Key Definitions
An antigen is any molecule that can be recognized by the adaptive immune system -- that is, it can bind antibodies or TCRs. An immunogen is a substance capable of inducing an immune response; all immunogens are antigens, but not all antigens are immunogens. A hapten is a small molecule that can bind antibodies but cannot induce an immune response on its own. Haptens must be conjugated to a carrier protein to become immunogenic, as exemplified by penicillin, which acts as a hapten when it binds to serum proteins and can trigger allergic reactions. The antibody response may be directed against the hapten, the carrier, or both.
An epitope (antigenic determinant) is the specific portion of an antigen recognized by an antibody or TCR. A tolerogen is a substance that induces immunological tolerance rather than an immune response, and an allergen is an antigen that triggers allergic (hypersensitivity) responses.
II. Types of Epitopes
B cell epitopes, recognized by antibodies and BCRs, can be conformational (discontinuous), formed by amino acids that are distant in the primary sequence but brought together by protein folding, or linear (continuous/sequential), formed by a contiguous stretch of amino acids. Most B cell epitopes on native proteins are conformational, typically encompassing 5-7 amino acids or equivalent sugar residues, and they must be located on the surface of the antigen to be accessible to antibodies. Antibodies can recognize proteins, carbohydrates, lipids, and nucleic acids.
T cell epitopes, recognized by the TCR, are always linear peptide fragments because proteins must first be processed and degraded before presentation. These peptides are presented by MHC molecules on antigen-presenting cells: MHC class I presents peptides of 8-10 amino acids to CD8+ T cells, while MHC class II presents peptides of 13-25 amino acids to CD4+ T cells. Consequently, T cells can only recognize protein antigens that have been processed into peptides.
<image>A comparison diagram of B cell and T cell epitopes. Panel A (B cell epitopes): A native protein is shown in its 3D folded structure. A conformational epitope is highlighted where amino acids from different parts of the primary sequence come together on the protein surface (labeled as "discontinuous -- requires native folding"). A linear epitope is shown as a continuous stretch of amino acids accessible on the surface. An antibody (Y-shaped) is shown binding each type. Panel B (T cell epitopes): The same protein is shown being processed (unfolded and cleaved) into small linear peptide fragments. A peptide fragment is loaded into the groove of an MHC class II molecule on the surface of an APC. A CD4+ T cell's TCR is shown recognizing the peptide-MHC complex. A note emphasizes: "T cells only see linear peptides presented by MHC; B cells can see native 3D structures."</image>
III. Factors That Influence Immunogenicity
Several factors determine how immunogenic a substance will be. Foreignness is critical: the more phylogenetically distant an antigen is from the host, the more immunogenic it will be, because self-molecules are generally non-immunogenic due to tolerance. For example, bovine serum albumin (BSA) is highly immunogenic in rabbits but not in cattle. Molecular size matters because larger molecules are generally more immunogenic -- molecules exceeding 100 kDa are excellent immunogens, while those below 5-10 kDa are poor immunogens (haptens). Larger molecules present more epitopes and are processed more efficiently by APCs. Chemical complexity also plays a role: proteins are the best immunogens due to their diverse amino acid composition and multiple epitopes, polysaccharides can be immunogenic (often T-independently), and lipids and nucleic acids are generally poor immunogens alone. Homopolymers composed of a single repeating unit are poor immunogens compared to heteropolymers.
Degradability is important because antigens must be processed by APCs and presented on MHC molecules; D-amino acid polymers, which resist protease degradation, are poor immunogens. Dose and route of administration have significant effects: very low doses may induce tolerance or no response, very high doses can also cause tolerance (high-zone tolerance), and an optimal intermediate dose produces a strong immune response. Subcutaneous and intradermal routes are the most immunogenic because they provide access to skin-draining lymph nodes, the intravenous route can be tolerogenic, and the oral route may induce oral tolerance through regulatory T cells.
Adjuvants are substances that enhance the immune response to an antigen through mechanisms including depot effect (slow antigen release), APC activation, and cytokine induction. Examples include alum (aluminum salts), MF59, AS04 (MPL plus alum), and Freund's adjuvant (experimental only). TLR agonists such as CpG (TLR9) and MPL (TLR4) are also used as adjuvants. Finally, genetic factors of the host play a role, since MHC genotype determines which peptides can be presented, and MHC polymorphism means that different individuals respond differently to the same antigen -- what were historically called "immune response genes" (Ir genes) are in fact MHC genes.
IV. T-Dependent vs. T-Independent Antigens
T-dependent (TD) antigens are primarily protein antigens that require T helper cell assistance for full B cell activation and antibody production. They are processed and presented on MHC class II to CD4+ T helper cells, and T cell help drives isotype switching (IgM to IgG, IgA, or IgE), affinity maturation through somatic hypermutation in germinal centers, and memory B cell generation. TD antigens generate robust, long-lasting immune responses with immunological memory.
T-independent (TI) antigens activate B cells without CD4+ T cell involvement and come in two types. TI-1 antigens are polyclonal B cell activators (mitogens) at high concentrations but activate only antigen-specific B cells at lower concentrations; LPS is a classic example, activating B cells through both the BCR and TLR4. TI-2 antigens are highly repetitive structures that extensively cross-link BCRs, such as bacterial capsular polysaccharides (pneumococcal polysaccharide) and polymeric flagellin. TI-2 antigens are primarily handled by marginal zone B cells and B-1 cells. TI responses mainly produce IgM with limited isotype switching, little or no affinity maturation, and poor immunological memory. They are poorly immunogenic in children under 2 years, whose marginal zone B cells are not yet fully developed. Conjugate vaccines address this limitation by linking TI-2 polysaccharide antigens to protein carriers, converting them to TD antigens and eliciting robust responses with memory -- as exemplified by the Hib vaccine and pneumococcal conjugate vaccine.
<image>A side-by-side comparison of T-dependent and T-independent B cell activation. Left panel (T-dependent): A protein antigen is internalized by a B cell via BCR, processed, and presented on MHC II. A CD4+ T helper cell recognizes the peptide-MHC II complex via its TCR, with CD40L-CD40 co-stimulation and cytokine release (IL-4, IL-21). This leads to germinal center reactions, isotype switching (IgM to IgG), affinity maturation, and memory B cell and long-lived plasma cell generation. Right panel (T-independent type 2): A bacterial polysaccharide capsule with repeating epitopes extensively cross-links multiple BCRs on a B cell surface, providing a strong signal 1 without T cell help. This leads to rapid IgM production by marginal zone B cells and B-1 cells, but minimal isotype switching, no affinity maturation, and poor memory. An inset shows the conjugate vaccine strategy: polysaccharide linked to a carrier protein, converting the response to T-dependent.</image>
V. Superantigens
Superantigens are microbial proteins that activate a vastly larger fraction of T cells than conventional antigens -- up to 20% compared to less than 0.01% for a typical antigen. They achieve this by binding simultaneously to MHC class II on APCs (outside the peptide-binding groove) and to the Vβ domain of the TCR on T cells, activating all T cells that express a particular Vβ family regardless of their antigen specificity. No processing is required, as superantigens bind as intact proteins. The consequences are devastating: massive polyclonal T cell activation triggers a cytokine storm (TNF-α, IL-1, IL-2, IFN-γ), producing fever, hypotension, shock, and multi-organ failure, followed by T cell anergy or deletion through exhaustion. Important examples include staphylococcal enterotoxins (SEA, SEB, SEC), which cause food poisoning and toxic shock syndrome; toxic shock syndrome toxin-1 (TSST-1) from Staphylococcus aureus; streptococcal pyrogenic exotoxins (SPE-A, SPE-C) from Group A Streptococcus, which cause scarlet fever and streptococcal toxic shock syndrome; and Mycoplasma arthritidis mitogen (MAM).
VI. Adjuvants in Detail
Alum (aluminum hydroxide/phosphate) is the most widely used adjuvant in human vaccines. It works through a depot effect, activation of the NLRP3 inflammasome, and enhanced antigen uptake by APCs, and it primarily enhances Th2/antibody responses. MF59, an oil-in-water emulsion used in influenza vaccines, enhances APC recruitment and activation. AS01 (liposome plus MPL plus QS-21) is used in the shingles vaccine (Shingrix) and the malaria vaccine and produces strong Th1 and CD8+ responses. AS04 (MPL plus alum) is used in the HPV vaccine Cervarix and some hepatitis B vaccines; MPL (monophosphoryl lipid A) is a detoxified LPS derivative that acts as a TLR4 agonist. CpG oligodeoxynucleotides, which are TLR9 agonists, are used in the hepatitis B vaccine Heplisav-B. Freund's adjuvant is used only in research: complete Freund's adjuvant (CFA) contains mineral oil plus killed mycobacteria and is potent but too reactogenic for humans, while incomplete Freund's adjuvant (IFA) contains mineral oil alone.
