2nd seminar: THE ANTIGEN Definition and properties Antigenic determinant (epitope) Hapten, carrier Antigen recognition by B and T cells Superantigens ACUTE INFLAMMATION

Any chemical structure Soluble or corpuscle Simple or complex Originated from the body or comes from outside Genetically self or non-self Natural or artificial ANTIGEN (Ag) - any substance, which is recognized by the mature immune system of a given organism DEFINITIONS

to bind specificallyANTIGENICITY– capability of an antigen to bind specifically with certain product of the adaptive immunity: TCR or BCR/antibody, induce –immunogenicity - capability of an antigen to induce an (adaptive) immune response, –tolerogenicity - capability to induce immunological tolerance, specific immune non-responsiveness DEFINITIONS

FACTORS INFLUENCING IMMUNOGENICITY I. From the aspect of our body: Genetics (self/non-self) –species (evolutionarily nonconserved molecules) –individual differences (e.g. MHC polymorphism – see later) Age –newborn – less reactive immune system –elderly – no new lymphocytes Physiological condition (pl. immunodeficiencies, starvation)

FACTORS INFLUENCING IMMUNOGENICITY II. From the aspect of the antigen: Physical-chemical properties of the Ag –size/complexity (bigger  more epitopes, role of carrier) –corpuscular (cell, colloid) or soluble –denatured or native (different epitopes!) –degradability (by APCs) Availability (crystalline proteins of the eye are not presented to lymphocytes)

FACTORS INFLUENCING IMMUNOGENICITY III. From the aspect of vaccination: Dose Route –intradermal/subcutan > intravenous > oral > intranasal (oral vaccine against polio virus!) Adjuvant –enhance the response given to the antigen e.g.: alum, Freund-adjuvant, TLR ligands Complex effects: depot effect  long-lasting presence of antigen activation of innate immunity activation of bystander cells

T B BCR (membrane Ig) antibodies (serum Ig) TCR APC MHC B cells recognise native antigens T cells recognise processed antigens Antigen ANTIGEN RECOGNITION BY LYMPHOCYTES

Part of the antigen which directly interacts with the antigen- binding site of a defined immunoglobulin (BCR / antibody) or TCR ANTIGENIC DETERMINANT (=EPITOPE)

B cell epitope T cell epitope recognized by B cells proteins polysaccharides lipids DNA steroids etc. (many artificial molecules) cell or matrix associated or soluble recognized by T cells proteins mainly (8-23 amino acids) requires processing by APC

Antigens may have several different epitopes

Epitopes Theoretical structure of complex antigens „Carrier” no direct interaction with the antigen-binding site These terms can only be used to describe the interaction of particular antigenic determinant and single immunoglobulin or T cell receptor

Antibody 1 Antibody 2 Epitope 1 „carrier” (1) Epitope 2 „carrier” (2) Antigén EPITOPE AND „CARRIER”

Ab1 Ab2 hidden/revealed determinant denaturation new/neoantigen determinant conformational determinant cleveage conformational/linear determinant TYPES (STRUCTURE) OF ANTIGEN DETERMINANTS surface/accessible determinants linear determinant conformational determinant (TCR, BCR, Ig)(BCR, Ig)

LPS – antigen or PAMP?! Antigen if recognised by TCR/BCR PAMP if recognized by PRR (TLR4) LPS Fc specific antibody reactive to the glucoseamin epitope of LPS Fab side view top view

ANTIGEN RECOGNITION ≠ CELL ACTIVATION

ANTIGEN RECOGNITION BY NAIVE T CELLS REQUIRES PRESENTATION VIA MHC MOLECULES Recognition/ No activation Recognition/ Activation

T CELL-DEPENDENT B CELL ACTIVATION Polysacharides are not presented! B cell cytokines CD4 TCR MHCII +peptide T cell 2 1

T-INDEPENDENT ANTIGEN TI-1 T-INDEPENDENT ANTIGEN TI-2 crosslinking of BCR Strong crosslinking of BCR by repetitive polysaccharide or protein epitopes BCR and other receptors Simultaneous activation of BCR and other receptors on B cells (i.e. LPS binding protein /CD14) induces the B cells to proliferate and differentiate B cell B CELL ACTIVATION (extensive receptor-aggregation)(extra activation signal) B CELL ACTIVATION WITHOUT THE HELP OF T CELLS

carrier + haptens hapten (e.g. DNP: dinitrofenil) - + HAPTEN molecules that are too small to provoke an immune response unless they are attached to carriers

free haptenshaptens attached to a carrier 0 HAPTEN receptor cross-linking  signalization

carrier specifichapten specificcarrier + hapten specific carrier + hapten antibodies Antibody response generated against a hapten- carrier conjugate

Purified bacterial polysacharides used for vaccination do not lead to long-lasting immunity because the activation of T cells is required for memory B cell formation Hence the polysaccharide chains are conjugated to protein carriers which can activate T cells Carrier: CRM197  modified diphteria toxin (toxoid) (a single aminoacid change (Glu  Gly) in the toxin can abolish toxicity) The toxoid acts the same way the toxin does; it activates specific T cells and may lead to the production of antitoxins by plasmacells EXAMPLE (Prevenar - pneumococus vaccine) polysaccharides of different Streptococcus pneumoniae strains toxin Glu  Gly toxoid complex antigen of vaccine toxoid +

B cell specific to bacterial polysaccharid polysacharid T cell specific to toxin/toxoid epitope BCR TCRMHCII cytokines, CD40-CD40L peptide antigen derived from toxoid formation of pneumococcus- specific memory B cells toxoid

Microbial proteins that bind to and activate all the T cells that express a particular set or family of TCR molecules resulting in a polyclonal activation SUPERANTIGENS

SUPERANTIGENS Microbial proteins that bind to and activate all the T cells that express a particular set or family of TCR molecules resulting in a polyclonal activation. Interaction is not via the peptide binding cleft of MHC molecule. Hypotension Rash Desquamation Fever

conventional antigen monoclonal/oligoclonal T cell response 1: :10 5 superantigen polyclonal T cell response 1:4 - 1:10 Microbial proteins that bind to and activate all the T cells in an individual that express a particular set or family of TCR molecules 10 7 – 10 8 / / activated T cells SUPERANTIGENS

SUPERANTIGENS ClassificationSources Endogenous Exogenous 1.Mouse mammary tomor virus (MMTV) 2.Epstein-Barr virus (EBV) 1.Staphylococcal enterotoxins (SEs): A, B, C1 to C3, D, E, G to Q 2.Staphylococcal toxic shock syndrome toxin-1 (TSST-1) 3.Staphylococcal exfoliative toxins: exoliatin A, exfoliatin B 4.Staphylococcal enterotoxin-like toxins formed due to recombination within enterotoxin gene cluster: U2, V 5.Streptococcal pyrogenic exotoxins (SPEs): A1 to A4, C, G to M 6.Streptococcal mitogenic exotoxins: SMEZ 7.Streptococcal superantigen :SSA 8.Yersinia pseudotuberculosis: Yersinia pseudotuberculosis-derived mitogen (YAM) 9.Mycoplasma species: Mycoplasma arthritidis-derived mitogen (MAM) 10.Cholera toxin: subunit A of cholera toxin 11.Prevotella intermedia* 12.Mycobacterium tuberculosis* 13.Viral superantigens: (a) Mouse leukemia virus (b) IDDMK1222- Ppol-ENV-U3 (c) HIV-Nef (d) Rabies virus-nucleoside protein.

ACUTE INFLAMMATION AND ACUTE-PHASE RESPONSE

THE INFLAMMATORY RESPONSE

A rapid response to an injurious agent that serves to deliver leukocytes and plasma proteins to the site of injury ACUTE INFLAMMATION

 Infections  Trauma  Physical and Chemical agents (thermal injury, irradiation, chemicals)  Tissue Necrosis  Foreign bodies (splinters, dirt, sutures)  Hypersensitivity or autoimmune reactions 1.Vascular response:  Increased vascular diameter  Increased flood flow.  Endothelial cell activation  increased permeability that permits plasma proteins and leukocytes to leave the circulation and enter the tissue  edema  increased expression of cell adhesion molecules e.g. E-selectin, ICAM 2.Cellular response:  Migration of leukocytes (diapedesis/extravasation), accumulation, effector functions MAJOR COMPONENTS OF INFLAMMATION: TRIGGERS OF ACUTE INFLAMMATION:

Redness (rubor) Swelling (tumor) Heat (calor) Pain (dolor) Loss of function (functio laesa) CLASSICAL SYMPTOMES OF ACUTE INFLAMMATION:

NEUTROPHIL GRANULOCYTES 68% of circulating leukocytes, 99% of circulating granulocytes Phagocytic cells Are not present in healthy tissues Migration  elimination of pathogens (enzymes, reactive oxygen intermediates) Main participants of acute inflammatory processes

NEUTROPHIL CHEMOTAXIS acPGP: N-acetyl Proline-Glycine-Proline – neutrophil chemoattractant MMP: matrix metalloproteinase

MIGRATION OF NEUTROPHILS

Neutrophil Transendothelial Migration (Diapedesis)

Pus is a whitish-yellow, yellow, or yellow-brown exudate produced by vertebrates during inflammatory pyogenic bacterial infections. Pus consists of a thin, protein-rich fluid, known as liquor puris, and dead cells. PUS

CONSEQUENCES OF MACROPHAGE ACTIVATION SYNTHESIS OF CYTOKINES

ACUTE-PHASE REACTION proinflammatory cytokines hypothalamic control of body temperature increased ‚set-point’ value fever

Liver Mannose binding lectin/protein MBL/MBP Fibrinogen Serum amyloid protein (SAP) C-reactive protein (CRP) UNDER THE INFLUENCE OF IL-6 THE LIVER PRODUCES A BUNCH OF ACUTE-PHASE PROTEINS Complement IL-6 ACUTE PHASE REACTION

ACUTE-PHASE RESPONSE Pentraxin family: CRP – opsonization, complement activation SAP – opsonization, complement activation, binding of mannose/galactose Collectin family: MBL – part of the complement system (SP-A/D – collectins of lungs) Complement proteins (C1-C9) Fibrinogen  blood clotting

 Vasodilation –Prostaglandins (PG), nitric oxide (NO)  Increased vascular permeability –vasoactive amines (histamine, serotonin), C3a and C5a (complement system), bradykinin, leukotrienes (LT), PAF  Chemotactic leukocyte activation –C3a, C5a, LTB 4, chemokines (e.g. IL-8)  Fever IL-1, IL-6, TNF α, PGE2  Pain Prostaglandins, bradykinin  Tissue damage Neutrophil and Macrophage products –lysosomal enzymes –Reactive oxygen species (ROS) –NO CHEMICAL MEDIATORS NSAIDs and Paracetamol: inhibiting COX-1 and COX-2  preventing the synthesis of prostaglandins NSAIDs and Paracetamol: inhibiting COX-1 and COX-2  preventing the synthesis of prostaglandins

RESOLUTION OF ACUTE INFLAMMATION

SEPTIC SHOCK Triggering factors : systemic infection (bacteraemia) microbial cell wall products and/or toxins released from the pathogens Result: Systemic activation of neutrophils and macrophages  High level of cytokine (TNF-alpha) production : „cytokine storm”  Excessive inflammatory response

SEPTIC SHOCK The key molecule of the process: TNF-alpha TNF-alpha and other inflammatory cytokines capillar permeability blood pressure DIC high fever multiorgan failure Therapy: anti-TNF-alpha antibody disseminated intravascular coagulation

DIC Disseminated Intravascular Coagulation pathologic activation of thrombotic process distress of thrombotic process, bleeding other causes: snake bite, septic abortion, acute obstetric complications, malignant tumors, leukemias

DIC: Disseminated Intravascular Coagulation