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Antigens/Microbial antigens

Dr. Klára Megyeri

University of Szeged, Faculty of Medicine, Department of Medical Microbiology and

Immunobiology

2014/2015

An ANTIGEN is any structure/molecule/cell that is

recognized by the mature immune system.•The immune system is tolerant (fails to respond) to self antigens.

Tolerance to self antigens is an essential feature of the immune

system; when tolerance is lost, the immune system can destroy self tissues,

as happens in autoimmune disease.

•Non-self antigens elicit an immune response.

ANTIGENICITY is the ability of the antigen to interact with the

antibodies or T lymphocytes in a specific manner.

IMMUNOGENICITY is the ability of an antigen to elicit an immune

response.

TOLEROGENICITY is the ability of an antigen to induce specific

immunologic nonresponsiveness.

1. Basic concepts

EPITOPES

CARRIER

WHOLE ANTIGEN

SPECIFIC REACTIONSPECIFIC REACTIONSPECIFIC REACTIONSPECIFIC REACTION IMMUNOGENICITYIMMUNOGENICITYIMMUNOGENICITYIMMUNOGENICITY

Conventional antigens

Theoretically every antigen is composed of two parts. One part is a carrier, while

the other part is the epitope or antigenic determinant.

The epitope is the smallest identifiable part of an antigen molecule that can be

recognized by a given TCR or BCR/antibody.

Antigenic determinants recognized by B cells and the antibodies 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).

Antigenic determinants recognized by T cells are created by the primary

sequence of amino acids in proteins. Free peptides are not recognized by T cells,

rather the peptides associate with molecules encoded by the major

histocompatibility complex (MHC), and it is the complex of MHC molecules +

peptide that is recognized by T cells. Some T cells can recognize lipids in

conjugation with a MHC-like molecule called CD1.

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HaptensSmall molecules (Mw < 1000 Da) behave as incomplete antigens, ie they can not elicit an

antibody response alone but can do so if they are coupled to larger molecular weigth carriers.

Such small molecules were defined haptens by K. Landsteiner in order to distinguish them

from bona fide antigens.

Eg:

•Poison ivy

•Penicillin

2. Factors affecting immunogenicity

Chemical nature of immunogen

A. Proteins – The vast majority of immunogens are proteins. These may be

pure proteins or they may be glycoproteins or lipoproteins. In general,

proteins are usually very good immunogens.

B. Polysaccharides – Pure polysaccharides and lipopolysaccharides are

good immunogens.

C. Nucleic acids – Nucleic acids are usually poorly immunogenic. However

they may become immunogenic when they are single stranded or

complexed with proteins.

D. Lipids – In general lipids are non-immunogenic, although they may be

haptens. Some glycolipids and phospholipids can stimulate T cells and

produce a cell-mediated immune response.

E. Metal ions, synthetic chemical compound (drugs) – In general small

chemicals are non-immunogenic, but can be haptens.

Based on their chemical structure, antigens

eliciting antibody production can be classified

to two groups:

1. Thymus-dependent (TD), protein antigens

2. Thymus-independent (TI), non-protein antigens

TD antigens are those that do not directly stimulate the production of

antibody without the help of the T cells. Proteins are TD antigens.

Structurally these antigens are characterized by a few copies of

many different antigenic determinants. Following binding of

BCR to the TD antigen, B-cells present it for T-cells as a peptide-

MHCII complex. T-B interaction leads to mutual activation of

both cell types; and T-cells provide the second activation signal

for B-lymphocyte activation. Thus, antibody production occurs

under the influence of T-cells, and involves isotype switching,

affinity maturation and memory B cell development.

TI antigens are antigens, which can directly stimulate the B-cells to

produce antibody without the requirement for T cell help. TI

antigens are polyclonal B-cell activators. Structurally these

antigens are characterized by repetitive polymer structure. TI

antigens are not presented by the B-cells, and antibody

production occurs in the absence of T-cell signals, therefore

isotype switching, affinity maturation and memory B-cell

production can not take place.

Complexity of immunogenMore complex immunogens elicit more intense the immune response

Increase in complexity increases the heterogenicity of immune response

Molecular weight of immunogenLarge Mw antigens are strong immunogens, while small Mw antigens are weak

immunogens or haptens.

≥100 kDa strong immunogen; ≤5-10 kDa hapten

Polyclonal antibody production in response to large, complex immunogen

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Foreigness•Autoantigen: self antigen recognized by the immune system•Alloantigen: an antigen present in some but not all individuals of a species

•Xenoantigen: an antigen found in more than one species•Heterophile/Forssman antigen: an antigen common to more than one species

and whose species distribution is unrelated to its phylogenetic distributionEg: Rickettsia prowazekii and Proteus vulgaris OX19 (Weil-Felix test)

Epstein-Barr virus and sheep red blood cells (Paul-Bunnel test)

Dose and route of antigenToo low dose / too high dose; toleranceImmunogenicity: subcutan > intraperitoneal > intravenous / per os

An antigen administered per os can be an antigen (not only parenterally administered

immunogens elicit immunity)

Genotype of hostImmune response to a specific antigen is genetically determined.

•The MHC molecules shape the TCR repertoire.•MHC genotype determines the host’s efficiency in presenting particular epitopes to T-cells.

•Host genotype can also affect the structure of the cellular receptors to which parasites

attach.

Duration of antigen persistenceExtension of antigen persistence increases the intensity of immune response and alters

several features of immunity.

AdjuvantsSubstances that increase the immune response to a particular antigen by:

•causing slow release of antigen in tissues

•increasing immunogenicity of antigen in a non-specific manner:

-they increase migration of macrophages and other immune cells thereby recruit

them to the site of injection

-they stimulate PRR on macrophages and other immune cells, thereby induce

cytokine production, which in turn enhances antigen-specific B- and T-cell

responses

++--LPS

?+--Bordetella pertussis

?++-Mycobacterium

tuberculosis

+++++Freund’s complete

++++Freund’s incomplete

?+++Aluminium salts

Increased co-stimulationNon-specific stimulation of

lymphocytes

Induction of granuloma

formation

Increase in the

duration of antigen

persistance

Mode of actionAdjuvants

Microbial antigens can be present on pathogenic and commensalmicroorganisms in the human body.

Pathogen colonization is transient; and antigen recognition on pathogenic

microorganisms facilitates the removal of the harmful microorganisms.

Humans harbor a complex population of commensal microorganisms in the GI

tract, on the skin and urogenital tract.

•This complex population of commensal microorganisms is termed the

microbiota.Human Microbiome Project (HMP) and the European Metagenomics of the Human

Intestinal Tract (MetaHIT) Project have analyzed these human-associated microbial

communities and their genes termed the microbiome.

�The continuous presence of commensal microorganisms shapes host immunity.

�Alterations in microbiota composition may lead to increased susceptibility to

various diseases, including:

-inflammatory (inflammatory bowel disease, Crohn’s disease),

-metabolic (diabetes, obesity, metabolic syndrome),

-allergic (asthma, atopy),

-autoimmune (arthritis, multiple sclerosis), and

-psychological/neurologic (autism)

3. Microbial antigens – Friends or foes?

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Host – microbiota dialog

Microbiota shape host immunityImmune system control of microbiota

Bacterium• Component

-cell wall - (O) antigen

-flagellar - (H) -

-pili -

-capsular (polysaccharide, D-glutamic acid polymer, hyaluronic acid)

• Product

-exotoxin (diphtheria-, tetanus-)

-enzyme (lecitinase, fibrinolyzin, streptolyzin O, coagulase, hemolyzin)

Virus• Capsid protein

• Envelope protein

• Non-structural proteins

Fungus/Protozoa/Helminth• Component

-cell wall/cell membrane components

-capsid

• Products (enzymes, metabolites)

3. Microbial antigens present on commensal and

pathogenic microorganisms

4. Superantigens

Features:

•They are NOT processed and presented

•They interact with T-cells in a non-

MHC-restricted fashion

•They bind directly to the outer part of

MHC II and TCR-Vβ chain

•The interaction of superantigens with T-

cells leads to a „cytokin storm” and

intensive lymphocyte proliferation

Superantigens are polyclonal T-cell activators having the ability to

activate a large proportion (5-20%) of T-cells

Microbial superantigens

Bacteria

Staphylococcus aureus: toxic shock syndrome toxin

Staphylococcus aureus: enterotoxin A, B, C, D, E, F and H

Staphylococcus aureus: exfoliative toxin

Streptococcus pyogenes: pyrogenic exotoxin A, B and C

Streptococcus pyogenes: erythrogenic toxin

Mycoplasma arthritidis: mitogen I

Yersinia enterocolitica: not identified

Yersinia pseudotuberculosis: YPM (Y. pseudotuberculosis-derived mitogen)

Viruses

Epstein-Barr virus superantigen

Cytomegalovirus superantigen

Rabiesvirus nucleocapsid protein

HIV superantigen

Human endogenous retrovirus (HERV): HERV-K18env

Fungi

Malassezia furfur superantigen

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INFECTION

TSST-1

T-cell activation

Monocyte activationIFN-γγγγ

TNF-ααααIL-1

Capillary permeability ⇑⇑⇑⇑

Hypotension

Brain edemaRenal

failure

Respiratory

failure Heart failure

DEATH

Fever Endotoxin sensitivity ⇑⇑⇑⇑

5. Microbial antigens as triggers of autoimmune

processes

Immunological mechanisms implicated in PIFAS:

1. Molecular mimicry

2. Epitope spreading

3. Adjuvant effect

4. Bystander activation

5. Superantigenic effect

6. Immortalization of autoreactive T-cells by certain microbes

PICIS=Post-infectious clinical and immunological syndrome

•PIFSIS=Post-infectious secondary immunodeficiency syndome

•PIFAS=Post-infectious autoimmune syndrome

•PIFSIS+PIFAS

Mimicry

Based on structural similarity between microbial antigens and human

molecules.

Factors that influence molecular mimicry:

the primary structure of antigen

the conformation of antigen

the charge distribution of antigen

Epitope spreading

Each parasite presents a large number of epitopes to the host's immune system. The immune response focuses on only a few of the many potential epitopes, a process called immunodominance.If the immunodominant response fails to clear the targets at first, the immune system will mount a more diversified and possibly long-lasting inflammatory response locally or systemically. This process ofbroadening the initially restricted immune response is called epitope/determinant spreading.Spreading can occur within a single molecule (intramolecular) or among different nearby molecules(intermolecular).Unlike the immunodominant response, where regulation of the few dominant “driver” T cell cloneswould be very efficient, a spreaded T cell response would be more difficult to regulate due to theincreased TCR diversity among the effector T cells. If coupled with malfunctioning regulatory component(s), which are crucial for the downregulationof the dominant T cells, a chronic autoimmune response would lead to irreversible pathogenesis.

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Adjuvant effect

Microbial PAMPs :

activate immune cells

activate co-stimulatory signals

increase the production of pro-inflammatory cytokines

increase antigen-presentation

Bystander activation

Pathogen-activated antigen-presenting cells can display self-antigens from dying

cells to autoreactive T lymphocytes in a process known as bystander activation.

Superantigenic effect

Microbial superantigens trigger the simultaneous activation of a large

proportion of T lymphocytes (5-20%); among them autoreactive T cells

may also become activated.

Increase in the viability of autoreactive T cells in

response to infections

Certain viruses may immortalize lymphocytes, including autoreactive cells.

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Potential role of microbial antigens in induction of

autoimmune processes

Post-Streptococcal diseases

Epitopes present in the cell wall, cell membrane, and the A, B, and C repeat regions of

the streptococcal M protein are immunologically similar to molecules in human myosin,

tropomyosin, keratin, actin, laminin, vimentin, and N-acetylglucosamine. This molecular

mimicry is the basis for the autoimmune response that leads to certain post-streptococcal

diseases, including:

•Acute rheumatic fever

•PANDA (Pediatric Autoimmune Neuropsychiatric

Disorders Associated with Streptococcal infections):

-Obsessive-compulsive disorder (OCD)

-Tourette syndrome

•Other CNS diseases:

-Sydenham’s chorea

-Dystonia

-Myoclonus

-Paroxysmal Dystonic Choreoathetosis

-Motor Stereotypes

-Encephalitis lethargica

-Parkinsonism

The body mounts an antibody responseagainst a pathogen (Campylobacter) protein that resembles neural glycolipids(gangliosides), resulting in an attack on theperipheral nervous system. In the case of Campylobacter jejuniinfection, antibodies will be produced, leading to activation of the complementsystem and phagocytosis of the bacteria. However, in rare cases the antibodiesproduced against certain C. jejuni antigenswill also bind to gangliosides of the neurvoustissue causing complement activation anddamage by phagocytes. This results in damage to peripheral nervoustissues, which leads to demyelination andaxonal damage.

Guillain-Barré szindrómaSimilarity between human ganglioside GM1 and GM1-like epitope

of C. jejuni LOS

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Relationship between infections, anti-ganglioside antibodies

and clinical course of GBS

GBS subtypes

Guillain-Barre Syndrome (GBS) is a rare, post-infectious, inflammatory autoimmune disease that is characterized by an ascending limb weakness and numbness in the extremities that can progress, in somecases, to paralysis. GBS can be divided into three subtypes that can be differentiated through electrodiagnostic techniques: acute inflammatory demyelinating polyradiculoneuropathy (AIDP), acute motor axonal neuropathy(AMAN), acute motor and sensory axonal neuropathy (AMSAN), and Miller Fisher syndrome (MFS).

Insulin-dependent diabetes mellitus

Basis: sequence homology between glutamate decarboxylase (GAD65), an enzyme

expressed in pancreatic beta cells, and coxsackievirus P2-C, an enzyme involved in the

replication of coxsackievirus B leads to mimicry and immunopathology.

Multiple sclerosis

Epitopes present in viral and bacterial peptides are immunologically similar to myelin

basic protein (MBP).

T cells specific to viral or bacterial antigens may also recognize and attack components

of the axonal myelin sheath destroying myelin and the underlying axon.