Zhijian Cai, Ph.D Institute of immunology, ZJU Lecture 2 Antigen Zhijian Cai, Ph.D Institute of immunology, ZJU

Introduction Antigenicity and specificity Requirements for immunogenicity Types of antigens Superantigen ,immunological adjuvants and mitogen

Introduction 1.Antigen (Ag) – A substance that induces a specific immune response and can specifically bind to antibodies and effect T and B cells. Alternative name - Immunogen 2.Property of Ag - immunogenicity and antigenicity Complete Ag has immunogenicity and antigenicity.

Introduction 3.Hapten - A substance that is non-immunogenic but which can react with the products of a specific immune response. Haptens are small molecules which could never induce an immune response when administered by themselves but which can when coupled to a carrier molecule. Haptens have the property of antigenicity but not immunogenicity. 4. Tolerogen,allergen

Antigenicity and specificity The ability of a compound to bind with antibodies or with cells of the immune system. This binding is highly specific.

Antigenicity and specificity 1. Antigenic determinant (epitope) - the portion of the antigen that binds specifically with the binding site of an antibody or a receptor on a lymphocyte. - the size of an epitope is approximately equivalent to 5-7 amino acids.

Antigenicity and specificity description example One epitope haptens Many epitopes of the same specificity polysaccharides Many epitopes of different specificity proteins Reprensentation of some possible antigenic structures

Antigenicity and specificity 2.structure of epitopes conformational determinant sequential or linear determinant

B cell epitope and T cell epitope 3. Epitopes recognized by B cells Epitopes recognized by T cells B cell epitope Antigenic peptide Degradation T cell epitope T cell epitope Natural protein B cell epitope and T cell epitope

T and B cell epitopes in a protein antigen

Antigenicity and specificity T cell epitopes B cell epitopes Receptor TCR BCR MHC necessary non-necessary Character linear short natural polypeptide peptide polysaccharides Size 8～12 aa(CD8+T) 5 ～15 aa 12～17aa(CD4+T) 5 ～7monosaccharides Type linear determinant conformational determinant ; linear determinant Location everywhere of Ag surface of Ag

Antigenicity and specificity 4. Hapten-carrier effect

Antigenicity and specificity *common antigen and cross-reaction

Factors influencing immunogenicity A. Contribution of the Immunogen 1. Foreignness - The immune system normally discriminates between self and non-self such that only foreign molecules are immunogenic.

Factors influencing immunogenicity A. Contribution of the Immunogen 2.Chemical Composition - In general, the more complex the substance is chemically the more immunogenic it will be. Carbohydrates（polysaccharides）：haptens Lipids：rarely immunogenic Nucleic acids：poor immunogens Proteins：virtually all proteins are immunogenic

Factors influencing immunogenicity Contribution of the Immunogen 3. Size - There is not absolute size above which a substance will be immunogenic. In general, the larger the molecule the more immunogenic it is likely to be. high molecular weight：>6000 Da

Factors influencing immunogenicity 4.Chemical Complexity- an increase in the chemical complexity of a compound is accompanied by an increase in its immunogenicity. Thus copolymers of several amino acids, such as polyglutamic, alanine, tend to be highly immunogenic. primary structure: amino acid sequence secondary structure :backbone of the polypeptide chain tertiary structure: three-dimensional configuration quaternary structure: more than one protein subunit

Four structures of protein

Factors influencing immunogenicity 5.Degradability and accessibility For antigen that activate T cells to stimulate immune responses, interactions with MHC molecules expressed on APCs must occur. 6. Physical form - In general particulate antigens are more immunogenic than soluble ones and denatured antigens more immunogenic than the native form.

Factors influencing immunogenicity B. Contribution of the Biological System 1. Genetic Factors - Some substances are immunogenic in one species but not in another. Similarly, some substances are immunogenic in one individual but not in others (i.e.responders and non-responders). 2. Age - Age can also influence immunogenicity. Usually the very young and the very old have a diminished ability to mount and immune response in response to an immunogen.

Factors influencing immunogenicity C. Method of Administration Dose - The dose of administration of an immunogen can influence its immunogenicity. 2. Route - Generally the subcutaneous route is better than the intravenous or intragastric routes. The route of antigen administration can also alter the nature of the response 3. Adjuvants

Types of antigens Th: Thymus-dependent antigen(TD-Ag) Thymus-independent antigen(TI-Ag) Relative:Xenogenic antigen Allogenic antigen Autoantigen Heterophilic antigen

Types of antigens TD-Ag T-dependent antigens are those that do not directly stimulate the production of antibody without the help of T cells. Proteins are T-dependent antigens.

Types of antigens TI-Ag T-independent antigens are antigens which can directly stimulate the B cells to produce antibody without the requirement for T cell help In general,polysaccharides are T-independent antigens. The responses to these antigens differ from the responses to other antigens.

Types of antigens TI-Ag T-independent antigens can be subdivided into Type 1 and Type 2 based on their ability to polyclonally activate B cells. Type 1 T independent antigens are polyclonal activators while Type 2 are not.

(A) TI-1 Ag (B) TI-2 Ag

Types of antigens Xenogenic Ag -An antigen found in more than one species.

Types of antigens Allogenic Ag -individuals of a species differ in alleles (are allogeneic) and the antigenic diffences will cause an immune response to allografts. The antigens concerned are often of the MHC and are referred to as alloantigens.

Types of antigens Autoantigen -The antigens of an organisms own cells and cell products are self antigens to the immune system of that organism. - Clones of immune cells reactive with self antigens are normally eliminated.

Types of antigens Heterophilic Ag(Forssman antigen) - A cross-reacting antigen that appears in widely ranging species such as humans and bacteria. - Postinfectious glomerulonephritis: group A Streptococcus glomerular basement membrane

Nonspecific immune activators Superantigen Adjuvant Mitogen

Superantigen (SAg) When the immune system encounters a conventional TD-Ag, only a small fraction (1 in 106 -104) of the T cell population is able to recognize the antigen and become activated (monoclonal/oligoclonal response). However, there are some Ags which polyclonally activate some subtypes of the T cells (up to 25%). These Ags are called superantigens. Such as SEA~SEE, Mls (minor lymphocyte stimulating Ag)

Types of SAgs T cell SAgs: TCR SAgs: HSP TCR SAgs Endogenous: Mls B cell SAgs: SPA, gp120 (HIV) bind BCR Endogenous: Mls Exogenous: SE, etc.

staphylococcal enterotoxin (SE) Superantigens bind directly to T-cell receptors and to MHC molecules.

Conventional Antigen Superantigen T cell reactivity 1/105-------1/106 1/20-------1/5 TCR binding site CDR3 of TCR α,β chain CD1、CD2 of TCR Vβ MHC binding site peptide-binding cleft (α-helix)outside MHC restriction + _ APC

Types of SAgs B cell SAgs: SPA, gp120 (HIV) BCR cross-linking SPA (Staphylococcus aureus): 25%-46% B cell conventional Ag: 0.1% B cell

SAgs in human diseases Food poisoning: SEA-SEE are potent gastrointestinal toxins responsible for staphylococcal food poisoning. Toxic shock syndrome (TSS): TSS toxin (TSST) is regarded as the primary causative agent for TSS which is mediated through TNF-.

Streptococcal toxic shock syndrome (STSS): STSS, caused by S Streptococcal toxic shock syndrome (STSS): STSS, caused by S. pyogenes, is the most severe form of invasive streptococcal disease, with mortality rates of up to 50%. Streptococcal SAgs have implicated in STSS. Acute rheumatic fever (ARF): ARF is a cross-reactive immune response to the host’s cardiac tissue and it has been proposed that the reactive T cells might be driven by SAgs. Several novel streptococcal SAgs have been identified from ARF-associated serotypes.

Autoimmune diseases: It has been proposed that SAgs might contribute to the pathogenesis of autoimmune disease by activating T cells that are specific for self antigens. SAgs could, under the right conditions, break the tolerance and suppression of autoreactive T cell clones and induce a state of autoimmunity. For example, administration of SEB to mice recovering from experimental autoimmune encephalomyelitis (EAE) triggered direct stimulation of the V3+ autoreactive MBP peptide specific T cells resulting in a rapid relapse of the disease. 实验性自身免疫性脑脊髓炎 髓鞘碱性蛋白

Adjuvants It is a substance which, when mixed with an immunogen, enhances the immune response against the immunogen. The use of adjuvants, however, is often hampered by undesirable side effects such as fever and inflammation. CFA, IFA, BCG, LPS, CpG, etc.

Types of adjuvants Conventional: Freund adjuvants, including complete and incomplete Freund adjuvants. Novel: 1) Cytokine- Protein: IL-2 - Gene: IL-12, IFN- 2) Liposome;nanoparticle

3) CpG C: Cytosine (Cyt), G: Guanine (Gua) Immunological characteristic of CpG DNA Activate a variety of immune cells, including M, NK cell and B cell Induce cytokine production: IL-6, IL-12, IFN- Inhibit cell apoptosis and promote cell proliferation

4) HSP (heat shock protein) HSP100、HSP90、HSP70、HSP60 and small molecular HSP Function: serve as adjuvant or carrier for Ag, participate in synthesis, folding, assembly, transportation and degradation of proteins.

Mitogen PHA (phytohemagglutinin, T, human) ConA (concanavalin A, T, mice) LPS (lipopolysaccharide, B,mice) SPA ( staphylococcus protein, B,human) PWM (pokeweed mitogen, T/B)

summary Immunogenicity is the capacity of a compound to induce an immune response. Antigenicity refers to the ability of a compound to bind with antibodies or with the immune system. The smallest unit of antigen that is capable of binding with antibodies is called epitope. Adjuvants are substances that can accelerate,prolong and enhance the quality of specific immune responses. Superantigen are a class of antigens which cause non-specific activation of T-cells resulting in polyclonal T cell activation and massive cytokine release.

Important items antigen; immunogenicity; immunoreactivity; cete antigen; hapten; epitope; squential epitope; conformational epitope; cross antigen; thymus dependent antigen, TD-Ag; thymus independent antigen, TI-Ag; heterophilic antigen; superantigen, SAg; adjuvant