Bio 151 lec 2 2012 2013

ANTIGENS & ANTIBODIES

Biology 151 lecture 2AY 2012-2013_1st

Main Reference for this Topic: Immunology by KUBY et al.

Monday, June 25, 2012

ANTIGENS


What are antigens?

• any molecule that can bind specifically to an antibody

• substances that can be recognized by the immunoglobulin receptor of B cells or by the T-cell receptor when complexed with MHC

• name arises from their ability to generate antibodies


• includes sugars, lipids, intermediary metabolites, hormones, complex carbohydrates, phospholipids, nucleic acids and proteins

• found in surface or parts of a microbe or from the environment (e.g. food, pollen, etc)

What are antigens?


Immunologic Properties of Antigens• Allergenicity:

• having the capacity to induce allergy (hypersensitivity)

• Tolerogenicity:

• capable of inducing immunological tolerance (immune system does not attack the antigen)

• Immunogenicity*

• Antigenicity*Monday, June 25, 2012

ARE YOU ALLERGIC TO SOME DRUGS?

Biology 151Introduction to Immunology




Antigens and Immunogens• ANTIGEN

• A molecule which can be specifically recognized and bound by an antibody

• “Antigenic”

• IMMUNOGEN

• A molecule which can elicit the production of specific antibody upon injection into a suitable host

• “Immunogenic”

• ALL molecules that are immunogenic are also antigenic..BUT...not all antigenic molecules are immunogenic! (Example: HAPTENS!)


Antigens: Haptens & Immunogens

• HAPTENS (incomplete antigen)

• antigens that by themselves do not elicit antibody production

• unable to induce an immune response alone but able to react with products (Abs)

• have the property of antigenicity but not immunogenicity (elicit immune response)

• NOTE: could be rendered immunogenic by covalently linking them to a carrier molecule




Antigens: Haptens & Immunogens

• IMMUNOGENS (complete antigen)

• antigens that can elicit antibody production

• stimulate B and/or T cell arms of the immune response and react with products (Abs)

• both induces an immune response and reacts with the products of it (Abs)


Factors that Influence Immunogenicity

• Nature of the Immunogen

• foreignness

• molecular size

• chemical composition and heterogeneity

• lipids as antigens

• susceptibility to antigen processing and presentation


Foreigness• Recall: “in order to elicit an immune response, a

molecule must be recognized as NON-SELF by the biological system”

• tolerance for SELF-antigens

• The greater the phylogenetic distance between two species, the greater the structural disparity between them

• EXAMPLE: bovine serum albumin not immunogenic to cow bt is on chicken (cow > goat > chicken)


Molecular Size

• The most ACTIVE immunogens:

•100,000 Da

• Substances with a molecular mass of 5,000-10,000 Da are poor immunogens

• EXEMPTIONS: few substances with a molecular mass less than 1,000 Da have proven to be immunogenic


Chemical Composition & Heterogeneity

• chemical complexity contributes to immunogenicity


Lipids as Antigens

• appropriately presented lipoidal antigens can induce both B-cell and T-cell responses

• Example: lipid-protein conjugates (lipids are used as haptens)


Lipids as Antigens


Susceptibility to Antigen Presentation & Processing

• the development of both humoral and cell-mediated immune responses requires interaction of T-cells with antigen that has been processed and presented together with MHC molecules

• LARGE, INSOLUBLE macromolecules are generally more immunogenic than SMALL, SOLUBLE ones

• larger molecules are more readily phagocytosed and processed

• degradative enzymes within antigen-presenting cells can degrade only proteins containing L-amino acids, polymers of D-amino acids cannot be processed


Factors that Influence Immunogenicity

•Biological System

• genotype of the recipient animal

• immunogen dosage and and route of administration

• adjuvants


Genotype of Recipient Animal• MHC gene products = determines the degree

to which an animal responds to an immunogen (immune responsiveness)

• response influenced by genes that encode B-cell and T-cell receptors

• response influenced by genes that encode various proteins involved in immune regulatory mechanisms

• THUS: genetic variability affects immunogenicity in different animals


Dosage and Route• experimental immunogen exhibits unique dose-

response curve

• DOSE

• insufficient dose will not stimulate an immune response (fails to activate lymphocytes or tolerance)

• excessively high dose = tolerance

• THUS....repeated adminsitrations or BOOSTERS are done if a single dose will not induce a strong response = increase clonal proliferation of antigen-specific T cells or B-cells = increase the lymphocyte populations SPECIFIC for the immunogen


Dosage and Route• experimental immunogen exhibits unique dose-response curve

• ROUTE

• generally administered parenterally = other than the GIT

• Subcutaneous route > Intramuscular > Intraperitoneal > Intraveous > Oral route

• Can you recall the route of your vaccine shots?

• strongly influence which immune organs and cell populations will be involved in the response

• intravenous = carried first to the spleen

• subcutaneous = moves first to local lymph nodes


Adjuvants

• substances that, when mixed with an antigen and injected with it, ENHANCE the immunogenicity of that antigen

• used to boost the immune response when an antigen has LOW IMMUNOGENICITY or when only SMALL AMOUNTS of an antigen is available


Adjuvants


IN SUMMARY...Parameter Increased

ImmunogenicityDecreased

Immunogenicity

Size Large >10,000; best >100,000

Small MW<2500

Dose Intermediate High or Low

Route Subcutaneous/IM > Intraperitoneal >

Intravenous > Oral or intragastric

Composition Complex Simple

Form ParticulateDenatured

SolubleNative

Similarity to self proteins Multiple differences Few differences

Adjuvants Slow releaseBacteria

Rapid releaseNo bacteria

Interaction with host MHC Effective Ineffective


EPITOPES: Antigenic Determinants


Antigenic Determinants or Epitopes

• DEFINITION:

• immunologically active regions of an immunogen that interacts with the specific antigen binding site in the variable region of the antibody molecule (PARATOPE) or to secreted antibodies

• EXCELLENT FIT between epitope and paratope: based upon their 3-D interaction and non covalent union

• are discrete site on the macromolecule recognized by the lymphocytes (B-lymphocytes/ T-lymphocytes)

• NOTE: B and T cells recognize DIFFERENT epitopes on the SAME antigenic molecule

• THUS: the ability to function as a B-cell epitope is determined by the nature of the ANTIGEN-BINDING site of the antibody molecules DISPLAYED by B-cells


Antigenic Determinants or Epitopes

• An antigen molecule has 2 or more epitopes or antigenic determinants per molecule

• Epitopes consist of approximately 6 amino acids or 6 monosaccharides

• Immunodominant Epitope: stimulate a greater antibody response



APPLICATIONS: Epitope Mapping

• Assignment: Diagram an experiment on how to carry out epitope mapping and determination of epitopes

• QUESTION: How will you know which fraction/epitope will be the most highly immunogenic/antigenic?

• Handwritten: Yellow Paper to be submiited on Tuesday


Antigen Classification• T cell dependent Ags or TD

• much more complex than TI Ags

• usually proteins

• stimulate a full complement of immunoglobulins with all five classes represented

• elicit an anamnestic or memory response & are present in most pathogenic organisms


bacterial protein = flagellin

Fungal proteins = keratinases

Viral proteins = HA and NA


Antigen Classification

•T cell independent Ags or TI

• often polysaccharides or lipopolysaccharides

• elicit an IgM response only and fail to stimulate an amnestic response


capsular polysaccharide

Fungal Mannans & Glucans

Bacterial membrane/OM components


Example: FungiGroup of fungi Fungi Polysaccharide Ag (TI) Protein Ag (TD)

Molds Aspergillus galactomannan glycoproteins

Yeasts Candida albicans Alpha mannan proteinase

Yeasts Cryptococcus neoformans galactoxylomannan

Dermatophytes Trichophyton Galactomannan peptides Keratinases I, II. III

Zygomycetes Rhizopus peptidofucomannan Proteinase

Dimorphic systemic fungi Histoplasma capsulatum galactomannan “h” & ‘m” factors

“ Paracoccodioides galactomannan E2 factor

“ Coccidioides imitis Methyl-mannose polymer Coccidiodin factor

Subcutaneous Sporothrix schenckii Peptido-L-rhamno-D-mannan


Summary of Antigen ClassificationThymus dependent Thymus independent

Structural properties

Complex Simple

Chemistry Proteins; protein-nucleoprotein conjugates;

glycoproteins; lipoproteins

Polysaccharide of pneumococcus; dextran

polyvinyl pyrolidone; bacterial Lipolysaccharide

Antibody class induced

IgG, IgM, IgA, (+ IgD and IgE)

IgM

Immunological memory response

YES NO

Present in most pathogenic microbes

YES NO


• a substance such as a bacterial toxin capable of stimulating MANY CD4+ T lymphocytes leading to the release of relatively large quantities of cytokines that provoke pathophysiologic manifestations

• NOTE: Superantigens are TD antigens = THUS, Do not require phagocyte processing

• stimulate multiple T cells that augment a protective T & B cell response

Superantigens


Superantigens


• Examples:

• Staphylococcal enterotoxins (food poisoning)

• Staphylococcal toxic shock toxin (toxic shock syndrome)

• Staphylococcal exfoliating toxins (scalded skin syndrome)

• Streptococcal pyrogenic exotoxins (shock)

• The diseases associated with exposure to superantigens are, in part, due to hyper activation of the immune system and subsequent release of biologically active cytokines by activated T cells

Superantigens


Mitogens• substance, often derived from plants, that

causes DNA synthesis and induces blast transformation and division by mitosis

• Lectins, representing plant-derived mitogens or phytomitogens are used in experimental and clinical immunology to evaluate T and B lymphocyte function in vitro


MitogensCharacteristic Concanavalin A

(Con A)Phytohemagglutinin (PHA)

Pokeweed mitogen (PWM)

Source Jack beans Kidney beans Pokeweed

Molecular Structure

Tetramer Tetramer Polymeric

Ligand A-D-mannose & a-D-glucose

N-acetylgalactosamine

Di-N-acetylchitobiose

Target cell/s T cells T cells T cells and B cells


SPECIAL ISSUE: Antigenic Variation



How are antigens recognized?

• Pattern Recognition Receptors


DIFFER IN INNATE AND IN ADAPTIVE RESPONSES



ANTIBODIES (Immunoglobulins or Igs)


What are antibodies?

• Antibodies : antigen-binding proteins present on the B-cell membrane and secreted by plasma cells

• when bound confers antigenic specificity on B-cells

• Common to all antibodies:

• structural features

• binds to antigen

• participate in effector function


Antibody: Name Game

Activate complement Complement fixing

antibodies

Neutralize viruses Neutralizing antibodies

! phagocytosis Opsonins

Lyses cells Lysins

Ppt soluble antigens Precipitins

Clumps cells Agglutinins

Neutralize toxins Antitoxin


• Opsonization: promotion of phagocytosis of antigens by macrophages and nuetrophils

• Fc receptors (in surfaces of macrophages and nuetrophils) bind to Ig molecules which induces a signal transduction pathway resulting in phagocytosis of the Ag-Ab complex

• Actions: enzymatic digestion, oxidative damage, membrane-disrupting effects of bacterial peptides

Ab-mediated Effector Functions



• Complement Activation (IgM and IgG): induces a collection of proteins that can perforate cell membranes

• C3b: important by-product; binds non-specifically to cell and Ag-Ab complexes

• many cell types have receptors for C3b (e.g. macrophages leading to phagocytosis)

• significance: removal and killing of pathogens



• Antibody-dependent cell-mediated Cytotoxicity (ADCC): kills cells

• Ab acts as a newly acquired receptor enabling the attacking cell to recognize and kill the target cell

• Pre-requisite: linking of Ab bound to target cells (e.g. virus in host cells) with the Fc receptor of many cell types can direct the cytotoxic activities of the effector cell against the target cell




Basic Structure of Antibodies

• Antibodies found in serum protein fractions

• electrophoretic mobility revealed four peaks: albumin, alpha, beta and gamma

• Gamma globulin factors = immunoglobulins (IgG) which contains serum antibodies


Antibodies are Heterodimers

• consist of two identical side light chains & two identical heavy chains linked by disulfide bonds

• heavy chain: has an amino-terminal variable region followed by a constant region


Antibodies are Heterodimers


Immunoglobulin Classes

• in any given antibody molecule, the constant region contains one of five basic heavy chain sequences called isotypes

• the heavy chain isotype determines the class of an antibody


Immunoglobulin Classes

• IgG

• IgD

• IgE

• IgA

• IgM


Antibody Classes


Antibody Classes : IgG


• PROPERTIES:

• major serum immunoglobulins (systemic immunity)

• major immunoglobulin in extravascular spaces

• does not require antigen binding during placental transfer (IgG2)

• fixes complement (IgG4)

• binds to Fc receptors (iGg2 and IgG4)

• phagocytes - opsonization

• Killer cells - ADCC

Antibody Classes : IgG


• PROPERTIES

• pentamer

• 3rd highest serum immunoglobulin

• first immunoglobulin made by fetus and B cells

• fixes complement

• Figure: fixation of C1 by IgG and IgM

• agglutinating immunoglobulin

• binds to Fc receptors

• B-cell surface immunoglobulins

C1r C1s

C1q C1r C1s

C1q

No activation Activation

Antibody Classes : IgM


Antibody Classes : IgM & IgG


PROPERTIES OF IgA• serum monomer

• secretions (sIgA)

• 2nd highest serum immunoglobulins

• major secretory Ig (tears, saliva, gastric and pulmonary secretions) = mucous and local immunity

• DO NOT fix complement (unless aggregated

• binds to Fc receptors on some cells

J Chain Secretory Piece

Antibody Classes : IgA


FORMATION OF SECRETORY IgA





PROPERTIES OF IgD

• monomer

• tail piece

• 4th highest serum Ig

• B-cell surface Ig

• DOES NOT BIND complement

Tail Piece

Antibody Classes : IgD


PROPERTIES OF IgE• monomer

• with extra domain

• least common serum Ig

• binds to basophils and mast cells (DO NOT require antigen binding)

• allergic reaction

• parasitic infections (helminths)

• binds to Fc receptors on eosinophils

• DOES NOT fix complement

C!4

Antibody Classes : IgE


IMMUNOGLOBULINS & ALLERGIES

Allergies



MONOCLONAL ANTIBODIES

Polyclonal antibodies : arise from MANY B-cell clones and have a

HETEROGENOUS collection of binding

sites

Monoclonal antibodies : derived from a SINGLE B-cell

clone and is a HOMOGENOUS

collection of binding sites

Georges Kohler and Cesar Milstein in 1975



CLINICAL UTILITY OF MONOCLONAL ANTIBODIES


Parungao-Balolong 2010


CLINICAL UTILITY OF MONOCLONAL ANTIBODIES • DIAGNOSTICS:

detect small amounts of drugs, toxins or hormones, e.g. monoclonal antibodies to human chorionic gonadotropin (HCG) are used in pregnancy test kits (Biotech, 1989); diagnosis of AIDS by the ELISA test.

• THERAPEUTICS: radioimmunodetection and radioimmunotherapy of cancer, and some new methods can even target only the cell

membranes of cancerous cells (Chaudhari et al, 1994);

cancer drug based on monoclonal antibody technology = Ritoxin, approved by the FDA in November 1997 (Orrs, 1997)

radioimmunodetection and radioimmunotherapy of cancer, and some new methods can even target only the cell membranes of cancerous cells (Chaudhari et al, 1994); cancer drug based on monoclonal antibody technology = Ritoxin, approved by the FDA in November 1997 (Orrs, 1997)

viral diseases, traditionally considered "untreatable" = AIDS (P/S/L, 1997).

• TRANSPLANTATIONOKT3, an antibody to the T3 antigen of T cells, is used to alleviate the problem of organ rejection in patients

who have had organ transplants (Transweb, 1996).


NEXT MEETINGInnate and Adaptive

Response



Technology

Bio 151 lec 2 2012 2013