2nd seminar: the antigen definition and properties antigenic determinant (epitope) hapten, carrier...

2nd seminar:2nd seminar:THE ANTIGENTHE ANTIGEN

Definition and properties

Antigenic determinant (epitope)

Hapten, carrier

Antigen recognition by B and T cells

Superantigens

ACUTE ACUTE INFLAMMATIONINFLAMMATION

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

DEFINITIONSDEFINITIONS

• ANTIGENICITY– capability of an antigen to bind to bind specificallyspecifically with certain product of the adaptive immunity: TCR or BCR/antibody,

– immunogenicity - capability of an antigen to induceinduce an (adaptive) immune response,

– tolerogenicity - capability to induce immunological tolerance, specific immune non-responsiveness

DEFINITIONSDEFINITIONS

FACTORS INFLUENCING FACTORS INFLUENCING IMMUNOGENICITY I.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 FACTORS INFLUENCING IMMUNOGENICITY II.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 FACTORS INFLUENCING IMMUNOGENICITY IIIMMUNOGENICITY IIII..

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 antigensT 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 (ANTIGENIC DETERMINANT (==EPITOPE)EPITOPE)

B cell epitopeB cell epitope T cell epitopeT 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 epitopesAntigens 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 2Epitope 1

„carrier” (1)

Epitope 2

„carrier” (2)

Antigén

EPITOPE AND „CARRIER”

Ab1Ab2

hidden/revealed determinant

denaturation

new/neoantigen determinant

conformational determinant

cleveage

conformational/linear determinant

TYPES (STRUCTURE) OF ANTIGEN DETERMINANTSTYPES (STRUCTURE) OF ANTIGEN DETERMINANTS

surface/accessible determinants

linear determinantlinear determinant conformational determinantconformational determinant

(TCR, BCR, Ig) (BCR, Ig)

LPS – antigen or PAMP?!

Antigenif recognised by TCR/BCR

PAMPif recognized by PRR (TLR4)

LPS

Fc

specific antibody reactive to the glucoseamin epitope of LPS

Fab

Fab

side view

top view

ANTIGEN RECOGNITION ≠ CELL ACTIVATIONANTIGEN RECOGNITION ≠ CELL ACTIVATION

ANTIGEN RECOGNITION BY NAIVE T CELLS REQUIRES ANTIGEN RECOGNITION BY NAIVE T CELLS REQUIRES PRESENTATION VIA MHC MOLECULESPRESENTATION VIA MHC MOLECULES

Recognition/No activation

Recognition/Activation

T CELLT CELL--DEPENDENT BDEPENDENT B CELL ACTIVATIONCELL ACTIVATION

Polysacharides are not presented!

B cell

cytokines

CD4TCR

MHCII+peptide T cell

2

1

T-INDEPENDENT ANTIGENTI-1

T-INDEPENDENT ANTIGENTI-2

Strong crosslinking of BCRcrosslinking of BCR by repetitive polysaccharide or

protein epitopes

Simultaneous activation of BCR and BCR and other receptorsother 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 CELLSB CELL ACTIVATION WITHOUT THE HELP OF T CELLS

B CELL ACTIVATION WITHOUT THE HELP OF T CELLSB 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 haptens haptens attached to a carrier

0

HAPTEN

receptor cross-linking signalization

carrier specific hapten specific carrier + hapten specific

carrier + haptenantibodies

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

toxinGlu Gly

toxoid

complex antigen of vaccine

toxoid+

B cellspecific to bacterial polysaccharid

polysacharid

T cellspecific to

toxin/toxoid epitope

BCRTCRMHCII

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

SUPERANTIGENSSUPERANTIGENS

SUPERANTIGENSSUPERANTIGENS

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

1:104 - 1:105

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

107 – 108 / 1011 1010 / 1011activated T cells

SUPERANTIGENSSUPERANTIGENS

SUPERANTIGENSSUPERANTIGENS

Classification Sources

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*•Viral superantigens:  (a) Mouse leukemia virus                                         (b) IDDMK1222- Ppol-ENV-U3                                         (c) HIV-Nef                                        (d) Rabies virus-nucleoside protein   

 .

ACUTE INFLAMMATIONACUTE INFLAMMATION

AND AND

ACUTE-PHASE RESPONSEACUTE-PHASE RESPONSE

THE INFLAMMATORY 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 INFLAMMATIONACUTE 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 chemoattractantMMP: matrix metalloproteinase

MIGRATION OF NEUTROPHILS 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 PUS

CONSEQUENCECONSEQUENCESS OF OF MACROPHAGE AMACROPHAGE ACTIVATIONCTIVATIONSYNTHESIS OF SYNTHESIS OF CCYTOKINESYTOKINES

ACUTE-PHASE REACTIONACUTE-PHASE REACTION

proinflammatory cytokines

hypothalamic control of body temperature

increased ‚set-point’ value

fever

Liver

Mannose binding

lectin/proteinMBL/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

ComplementIL-6

ACUTE PHASE REACTIONACUTE PHASE REACTION

ACUTE-PHASE RESPONSEACUTE-PHASE RESPONSEPentraxin family:CRP – opsonization, complement activationSAP – 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, LTB4, chemokines (e.g. IL-8)

Fever• IL-1, IL-6, TNFα, 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 INFLAMMATIONRESOLUTION OF ACUTE INFLAMMATION

SEPTIC SHOCKSEPTIC 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 SHOCKSEPTIC SHOCK

The key molecule of the process: TNF-alpha

TNF-alpha and other inflammatory cytokines

capillar permeability blood pressure

DIChigh fever multiorgan failure

Therapy: anti-TNF-alpha antibody

disseminated intravascular

coagulation

DICDICDDisseminated isseminated IIntravascular ntravascular CCoagulationoagulation

• pathologic activation of thrombotic process

• distress of thrombotic process, bleeding

• other causes: snake bite, septic abortion, acute obstetric complications, malignant tumors, leukemias

DIC: DIC: DDisseminated isseminated IIntravascular ntravascular CCoagulationoagulation