DEPARTMENT OF IMMUNOBIOLOGY Antigens (Ag) Xiaowu Hong Department of Immunology Shanghai Medical College of Fudan University

I Definition and characteristics of antigen II Properties of Ag: Foreignness and specificity III Factors that influence immunogenicity IV Classification of antigen V Nonspecific stimuli for immune response antigen

Antigen (Ag) Chapter 1 Definition of antigen combine specifically induce Substances that combine specifically with a B or T cell’s antigen-binding receptors can then induce an immune response are called antigens.

Antigen (Ag) Antigen binding causes cytotoxic T cells Memory T cell Activated cytotoxic T cell Antigen binding triggers release of antibodies Memory B cell Activated B cell Antibodies

Chapter 2 Characteristics of antigen （ 1 ） immunogenicity （ 2 ） antigenicity The antigen molecule generally pose two natures, that is

(1) Immunogenicity Immunogenicity is the ability to induce a humoral and / or cell mediated immune response, including induce production of antibody and effector lymphocytes. Activated B cell antibody Activated CTL

(2) antigenicity combine specifically Antigenicity is the ability to combine specifically with the final products of the above responses (i.e., antibody and /or cell-surface receptors).

antigenicity immuogenicity T T T Ag

Antigen and Adaptive immunity B cells+ antigen Effector B cells + memory B cells (Plasma cell) T cells+ antigen Effector T cells + memory T cells (CTLs, Th)

1 Specificity Specificity of antigen The characteristic that antigen only binds the antibody and sensitized lymphocyte specifically, is called specificity of antigen （ Specificity ）

Antigenic determinants or epitopes are the immunologically active regions of an immunogen that bind to antigen-specific membrane receptors on lymphocytes (TCR/BCR) or to secreted antibodies. (1) Antigenic determinants or epitopes

Epitope or Antigenic Determinant antigen

1 Conformational epitope surface Nonsequential polypeptides or polysaccharide on the surface of the molecules, Native conformation Native conformation, 2 liner epitope A sequential amino acid fragment, Linear determinant Linear determinant, Inside Inside of the antigen molecule Structure of epitopes

Conformational and Linear Epitope

T cell Epitope and B cell Epitope In immune response, the epitopes that TCR and BCR recognize is different, and are called T cell epitope and B cell epitope, separately. （ 1 ） human pancreatic glucagon was inoculated into the mouse NH COOH （ 2 ） Production of specific antibody and T effector cell.

antibody ： aim directly at 1~17 amino acids in the N terminal T effector cell ： aim directly at 18~29 amino acids in the C terminal No epitope was found to be recognized by T cell and B cell simultaneously. *natural, *on the surface of the molecule * conformational or sequential determinant * tertiary structure of the molecule COOH NH * Antigenic peptide presented by APC * linear determinant

T cell Epitope and B cell Epitope T cell Epitope B cell Epitope Digested

**on surface of molecule antigen antibody CDR3 epitope B cell epitope is recognized by BCR and antibody ** conformational determinants (i) B cell epitope

Determinants recognized by B cells 1. Composition - Antigenic determinants recognized by B cells and the antibodies secreted by B cells are created by the primary sequence of residues in the polymer ( linear or sequence determinants ) and/or by the secondary, tertiary or quaternary structure of the molecule ( conformational determinants ). 2. Size - In general antigenic determinants are small and are limited to approximately 4-8 residues. (amino acids and or sugars). The combining site of an antibody will accommodate

(ii) T cell epitope The peptide that is presented by MHC molecule and recognized by TCR is called T cell epitope. TCR MHC HIV peptide

TCR recognize MHC/antigenic peptide TCR Antigenic peptide MHC-II T cell APC

Ep1Ep2Ep3 T Cell epitope Antigenic peptides recognized by T cell form trimolecular complexes with a TCR and an MHC molecule Depend on the amino acid sequence of the peptide Relevant to the expression of MHC molecule of the host

Determinants recognized by T cells 1.Composition - Antigenic determinants recognized by T cells are created by the primary sequence of amino acids in proteins. 2.T cells do not recognize polysaccharide or nucleic acid antigens. 3. The determinants need not be located on the exposed surface of the antigen since recognition of the determinant by T cells requires that the antigen be proteolytically degraded into smaller peptides. 4.Free peptides are not recognized by T cells, rather the peptides associate with molecules coded for by the major histocompatibility complex (MHC) and it is the complex of MHC molecules + peptide that is recognized by T cells.

Comparison of epitope of T and B cell T cell epitope B cell epitope Receptor TCR BCR MHC molecule required to display none required processed antigen character liner peptides natural polypeptide,LPS, polysaccharide, organic compound 5~15 amino acids, 5~7 monosaccharide or 5~7 nucleotide Epitope type liner conformational, liner side any on the surface size 8 ~12 amino acids (CD8 + ),12 ~17 amino acids (CD4 + )

(2) Common antigen and cross-reaction Each B cell determinant induces production of a specific antibody. Thereby a complicated antigen can induce production of multiply antibodies. complicated antigen

A same or similar antigenic determinant that exists between the two different antigens is called common antigen.

The reaction that antibody reacts with same or similar antigenic determinant between the different antigens is called cross-reaction.

Cowpox vaccine VACCINE Smallpox virus Bm cell

Immunological memory A B cowpox D E Anti-Cowpox antibody cowpox Bm cell Anti- Cowpox antibody against Smallpox Smallpox Bm cell cowpox Bm cell

days First response to antigen Cowpox Antigen Cowpox immunity Antigen Smallpox Antigen B immunity Antibody concentration First response to antigen B Secondary response to antigen Smallpox

Foreignness is the centre of immunogen. The greater of the phylogenitic distance between two species, the greater the structural disparity between them. **Various pathogens,animal protein **homologuous ** Self-molecule that has not been exposed to immature lymphocytes during lymphocyte development may be later recognized as nonself, or foreign,by the immune system. 2 Foreignness

1.3. Hapten and Carrier 1 Hapten ： some small molecules, called haptens, are antigenic but incapable,by themselves, of inducing a spedific immune response. In other words, they lack immunogenicity 2 carrier ： Chemical coupling of hapten to a large protein yields an immunoginic hapten-carrier conjugate. The substance that can make the hapten be immunogenic ( is capable of inducing immune response) are called carrier.

Hapten ： Haptens have the property of antigenicity but not immunogenicity. A substance that is non- immunogenic but which can react with the products of a specific immune response. small molecules immune response + carrier hapten+carrier=complete antigen

Chapter 3 Factors influencing immunogenicity

1 Physicochemical nature of antigens (1) chemical nature glycoprotein, lipoprotein 、 polysaccharide 、 LPS DNA ， chromosome 、 histone in the activated lymphocyte Auto-antibody for self-DNA, histone Antinuclear antibody (ANA) Autoimmune diseases

(2) molecule size >10kD >100kD <10kD immunogens Strong immunogens Poor immunogens The higher is the molecule weight The more are antigenic determinants The more complex is the antigenic structure Not be degradated easilier Stimulating immune cell continuously High immune response

(3) complicated chemical structure Glutin MW>100kD ） Straight chain amino acid Add Aromatic amino acid （ 2% tyrosine ） （ poor immunogenicity ） （ better immunogenicity ）

(4) conformation and accessibility of the molecule +++ ± +++ Tyrosine poly-alanine Glutan poly-lysine immunogenicity

(1) Genetic characteristics of the host Genetic control of immune responsiveness, largely confined to genes within the MHC. (2) Age, sex, and state of health Infection, use of immune inhibitor 2 Effect of host

Chapter 4 Classification of antigens 1. Depend on whether need the Th for producing the antibody ** thymus dependent antigen, TD-Ag **thymus independent antigen, TI-Ag

TI and TD antigen 1 2 CD40/CD40L B cell Th cell TI- antigenTD antigen CD4 1 2

(1) Thymus dependent antigen, TD-Ag T-dependent Antigens - do not directly stimulate the production of antibody without the help of T cells. Proteins. Structurally characterized by a few copies of many different antigenic determinants

(2) Thymus independent antigen, TI-Ag T-independent Antigens - directly stimulate the B cells to produce antibody without the requirement for T cell help Polysaccharides Characterized by the same antigenic determinant repeated many times

2 Depend on the relationship with host (1) Xenogenic Ag ： The antigen from different species is called xenogenic antigen. Microorganism Animal immune serum for therapy ： horse serum (antitoxin) Horse serum antitoxins posses dual natures ： (i). Specific antibody ---- neutralization of toxin (ii). Xenogenic antigen---stimulate production of antibody against horse serum

toxoidtoxin Horse serum antitoxin hypersensitivity antibody antigen

(2) Allogenic Ag ： The antigens from different individuals of the same species are called allogenic antigen. Human allogenic Ag ： blood type （ RBC ） antigen Histocompatibility (transplantation) antigens Blood type ： ABO and Rh A B O AB antigen antibody A B － A,B Anti-B anti-A anti-A,anti-B － Blood type

RBC + complement RBC Type A A antigen Type B Anti-A antibody transfusion reaction

RBC Autoimmune hemolytic anemia drug RBC Auto-antibody RBC Ag-Ab complex Activate complement Membrane of RBC C9 C8C8 C 5b 67 (3) Autoantigen cover Infection, wound and medicine use make the cover antigen release or auto-antigen changed or modified, which induce response for auto-antigen.

(4) Hetorophilic antigen, or Forssam antigen Hemolytic streptococcus B C antigen M antigen Streptococcus infection Acute Glomerulonephritis Glycoprotein of Cardiac valve heart muscle Rheumatic fever Streptococcus A Glomerular basement membrane The hetorophilic antigens are common antigen, which are irrelevant to species and exist in human, animal, plant and microorganism.

Ⅵ Superantigen, SAg 1 conception:Exceed low dose(1 ～ 10ng/ml) of superantigen is needed to active 2 ％～ 20 ％ of T cell clones to initiate very strong immune response. 2 category:Exogenous superantigen eg.Staphylococcus aureus enterotoxin A-E Endogenous superantigen eg. mouse mammary tumor virus protein 3 difference with common antigen ： 1 ） low dose, but strong response 2 ） not constraint by MHC 3 ） recognition is not specific ， and procession and presentation is not required 4 ） induce a lot of cytokines releasing, resulting in severe pathologic disease

Ⅶ Mitogens Substances which can active a cluster of lymphocytes including a lot of clones. Such as ConA, PHA,PWM, LPS and so on. Nonspecific stimuli for immune response

Ⅷ adjuvant 1 conception Adjuvants (from Latin adjuvare, to help) are substances that, when mixed with an antigen and injected with it, enhance the immunogenicity of that antigen. 2 category Biological agents: BCG Inorganic compound: Al(OH)3 Synthesis: cytidine monophosphate(CMP ）， liposome immunostimulating complex （ ISCOM ） CpG et al which is neotype of adjuvant Freund‘s complete adjuvant ( CFA) and Freund’s non-complete adjuvant ( IFA) are commonly used 3 mechanism of action n Prolong antigen persistence n Enhance co-stimulatory signals n Induce granuloma formation n Stimulate lymphocyte proliferation nonspecifically

1.Master the concept, characteristic of the antigen, and the factors influencing immunogenicity ( chemical nature, molecule size, foreignness, and genetic characteristics of the host ). 2. Master antigenic determinant, common antigen. 3.Understand antigen category. 4.know superantigen 5.Understand the important antigen in medicine.

School of Medicine Fudan University