Immunology

IMMUNOGENS AND ANTIGENS - DELFIN, RMT

 Immunogen: Any agent capable of inducing an immune response

 Antigen: Any agent capable of interacting specifically to components of the immune response such as lymphocytes and antibodies.

Properties of Immunogen

 Immunogenecity: Inherent ability of a substance (immunogen) to induce as specific response resulting in the formation of antibodies or immune lymphocytes:

 Antigenecity: Property of a substance (antigen) to react specifically with the antibody.  Immunogenic substances are always antigenic whereas antigens are not necessary

immunogenic.

 Antigens/ Immunogens are found on  RBC surface (Group A and B)

 WBC surface(Human Leukocyte antigens)

 Platelets

 Tissue cells

 Semen

 Saliva

 Antigens/Immunogens can be introduced by:  Blood transfusion

 Tissue grafting and organ transplantation

 Transplacental leakage

 Ingestion

Antigenic Determinant (Epitope)

 The portion that binds specifically with the binding site of an antibody (paratope) or to a lymphocyte receptor.

Antigenic Determinant (Epitopes)

 Consists of 4-6 amino acids or 5-7 monosaccharides in length.

 Aromatic amino acids (as tyrosine) provide more immunogenecity that non-aromatic amines

 Polysaccharides are better immunogens than oligosaccahrides because of their complexity.

 Hydrophobic molecules are more immunogenic than hydrophobic molecules.  Configurations of epitopes

 Linear: found within the amino acid sequence of the molecule.

 Conformational: formed by amino acids that appear on the same area of the protein but not adjacent to the peptide chain.

Classes of Immunogens:

1. Thymic dependent antigens:

 Require the help of T cells for the formation of antibody.  They activate B cells with the help of T cells

 Most immunogens are thymic dependent 2. Thymic independent antigens:

 Stimulate antibody without interacting with T cells

 Response to these antigens is of the IgM class with little or no immunologic memory generated.

 T independent antigens are usually carbohydrates which are composed of monotonously repeating epitopes.

Epitopes, T-Independent Antigen

According to epitopes:

 Unideterminant, univalent antigen:

 There is only 1 epitope on the molecule like hapten  Unideterminant, multivalent antigen:

 There is only one kind of epitope but many such epitopes on each molecule such as many polysaccharides and homopolymers

 Multideterminant, univalent antigen:

 There are many different kinds of epitopes but only one of each kind such as protein  Multideterminant, multivalent antigen:

 There are many kinds of determinants and many of each kind such as polymerized proteins

Hapten and Carrier Haptens:

Low molecular weight compounds which by itself cannot elicit the formation of antibodies but can combine with an antibody

Also called incomplete or partial antigens

Has the chemical configurations which gives the antigen its antibody provoking activity Carriers

 Usually proteins which give the hapten the required size

 Combines with a hapten so that it can elicit formation of an antibody

 The hapten temporarily loses it immunogenecity when detached from a carrier and restored if it combines with the carrier.

Requirements of Immunogenecity

 The degree of immunogenecity of a molecule is influenced by several factors. The relationship is expressed as:

1. Foreigness

 An antigen must be foreign or non self to the host to which it is administered. o Autologous Antigens: found within the same individual

 Autograft

o Syngeneic antigens: found in genetically identical individuals  Syngeneic graft or isograft

o Allogenic or Homologous antigens: Found between different individuals with the same specie

 Homograft/allogragft

o Xenogeneic or heterologous antigens: Found between different individuals of different species

 Xenograft

o Sequestered Antigens: Antigens not exposed to antibody producing cells and become immunogenic when exposed to antibody forming tissues.

o Heterogenetic/Heterophilic antigens: Occur in unrelated animal and plant species 2. Chemical Complexity

 Proteins:

o Majority of immunogens

o Strongest immunogens because of the diversity of amino acids which imparts epitopes of different specificities to the molecules

o The total immune response will be the sum of all the antibodies produced by the epitopes  Glycoproteins

o Immunogens

o Best illustrates by RBC blood group antigens

o HLA antigens found on the surface of nucleated body cells comprising both solid tissue and most circulating blood cells.

 Polysaccharides: o Most are haptens

o Easily degraded when injected

 Pure polysaccharides substance

 Pneumococcal capsule which are responsible for the protective immune response to pneumococcus

 Lipopolysaccharide substances (endtotoxins)  Nucleic Acids:

o Non immunogenic because of relative simplicity, molecular flexibility and rapid degradation

o Nucleoproteins are stronger immunogens because the nucleic acids are coupled to proteins.

 Lipids

o Non immunogenic

 Relatively simple, lack structural stability 3. Molecular Size

 As a general rule, molecules below 5,000 daltons are non-immunogenic.  Reasonable immune responses will be induced by molecules like

o serum albumin (40,000 daltons) o Gamma globulin (160,000 dlatons) o Hemocyanin (1,000,000)

 Size is important since the number of epitopes increases proportionately with the size or protein.

 Larger molecules from antibodies move rapidly since they are processes and phagocytized by a macrophage.

 Soluble antigen is difficult or impossible to phagocytized and therefore is not immunogenic at times.

 Major Histocompatibility Complex

o Exerts genetic influence of an individual’s ability to respond to an immunogen.

o It has been found that if a particular MHC antigen is not present for an immunogen, there is no specific response.

Route Dosage and Timing

 Intravenous and intraperitoneal routes are effective

 Intradermal route offers a stronger stimulus than the subcutaneous or intramuscular route,  Generally, the smaller the dose, the less likely an immune response

Cross Reactivity

 Denotes a situation in which two or more compounds that may have various degree of dissimilarity, share antigenic determinants/epitope and would therefore react within immune components induced against any one of the compounds.

 A toxoid is a modified form of toxin may have one more antigenic determinants in common with the native toxin

 Immunization with a toxoid leads to an immune response capable of reacting only with the toxoid but also with the native toxin

 Cross reactivity is illustrated by:

o Treponema pallidum and cardiolipin

o Epstein-Barr Virus and sheep red blood cells o Ricketssia and Proteus

Immunologic Adjuvant

 Adjuvant: Substance which when mixed with an Immunogen enhances the immune response against the Immunogen

Types of Adjuvant

1. CFA (Complete Freund’s Adjuvant)

 Consist of water-in-oil emulsion and killed Mycobacterium tuberculosis or butyricum  The antigen in in the water phase.

o Other examples: o BCG

o Corynebacterium parvum o Bordetella pertusis

 These types of adjuvants release the antigen slowly but continously and stimulate certain subpopulation of T lymphocytes and macrophages.

2. Toxins and polysaccharide

a. LPS (lipopolysaccharide or endotoxin) b. Synthetic MDP (muramyldipeptide)

LPS enhances antibody response by stimujlating subpopulation of B cells while MDP stimulates T-cells

3. Alum precipitate

 Consist of alumunum hydroxide on which antigen is absorb

 Has a slight irritant effect which enhances ingestion and processing of antigen by macrophages. Hapten is different from adjuvant

 Hapten becomes immunogenic when conjugated covalently to a carrier but will not become immunogenic when conjugated with an adjuvant.

 An adjuvant enhances immune response to Immunogen but does not confer immunogenecity to Haptens

Second Line of Defense 1. Phagocytosis

2. Inflammation

3. Complement System 4. Acute phase reactants

1. Phagocytosis

 (engulfment and destruction) of microorganism and damaged cells  literally means “eating cell process”

 Phagocytosis –MEMORIZE (ICED)

o Initiation is caused by damage to the tissues, either by trauma or as a result of microbial multiplication.

o Chemotaxis, attraction of leukocytes or other cells by chemicals.

 Opsonization - Opsonization coating a pathogen by substances so as to enhance phagocytosis.

 Adherence - firm contact between phagocyte and microorganism. o Engulfment into cytoplasm and enclosed in a vacuole.

o Digestion enzymatic contents in vacuole destroy the microorganism.  INITIATION is caused by damage to the tissues, either by trauma or as a result of

microbial multiplication.

 activated phagocyte has increased surface receptors that allow for adherence (adhesive factor)

o CR3

o Lamin receptor

o Leucyformyl-methionyl phenylalanine

 CHEMOTAXIS attraction of leukocytes or other cells by chemicals. ( chemotaxin)  Chemotaxin

o Interleukin 1 - released by macrophage

o Histamine - released by basophils, tissue mast cells o Complement (C5a)

 2 kinds

o Positive – movement towards the stimulate o Negative – movement away from the stimulate

 Opsonization - a process of coating a pathogen by substances so as to enhance phagocytosis.

o “to prepare for eating.”

o Opsonins are serum proteins that attach to a foreign substance and help prepare it for phagocytosis

o Opsonin:  antibody

 Complement component  C- reactive protein

 Adherence - firm contact between phagocyte and microorganism.

 ENGULFMENT

 Phagosome (vacuole) - contained the engulfed materials  Phagolysosome - fusion of phagosome and lysosome  DIGESTION

o enzymatic contents in vacuole destroy the microorganism.

 Number of killing mechanisms operating in the vacuoles of phagocytic cells.

 One of the major mechanisms involves hydrogen peroxide which, acting along with an intracellular enzyme, is rapidly lethal to many bacteria.

Destruction of antigen

1. Formation of the phagolysosome leading to the release of lysosomal contents including  defensins,

 lactoferrin  lysozyme.

2. Nitric oxide produce by macrophage which is toxic to microorganism

3. Activation of the NADPH oxidase, which is present in the phagosome membrane, leading to the production of reactive oxygen intermediates, superoxide anion ,H2O2 and hyrogen radicals which are cytotoxic for microorganism.

Second Line of Defense Phagocytosis

 Inflammation  Complement System  Acute phase reactant

INFLAMMATION

A nonspecific exaggerated physiological response by phagocytic cells to infection/injury.

 Rubor (redness) is caused by increased circulation and vasodilation in the injured tissues;  Calor (warmth) is the heat given off by the increased flow of blood;

 Tumor (swelling) is caused by increased extracelluar fluid accumulating in the tissues  Dolor (pain) is caused by the stimulation of nerve endings from the pressure of swelling or

by chemical mediators

 Functio laesa (loss of function)

STAGES

 Vascular response  Cellular response Vascular response

 dilation of blood vessels to increase blood flow in the area of injury  hyperemia

 redness/ heat

 increase capillary permeability  plasma leakage to tissue Cellular response

 first to migrate  short lived  acute inflammation Monocyte/ Macrophage  2nd _{to migrate}  long lived  chronic inflammation

 act as APC (antigen presenting cell)  APC (antigen presenting cell)

o release interleukin 1 (IL1) o Effects of IL1

 Act in hypothalamus (FEVER)  Increase acute phase reactants ACUTE PHASE REACTANTS

REPAIR AND RESOLUTION Initiated by fibroblast proliferation

 affected area may be totally repaired  injury may lead to formation of abscess  granuloma may be formed

Antimicrobial substances  Complement  Properdin  Interferon

 Tumor necrosis factor  Betalysin

Type 1 – non- immune, produced primarily in the initial innate response to viral infection o alpha IFN

 aka leukocyte interferon

 produce by virus induced leukocyte culture (NULL lymphocyte) o beta IFN

 aka fibroblast & epithelial/ fibroepithelial IFN  produced by fibroblast cells

Type 2 – immune, primarily produced as a component of the specific immune response to viral and other pathogens

o Gamma IFN

 aka immune IFN

 produced by immunologically stimulated lymphocyte 2. Tumor Necrotic factor

 cytotoxic against tumor cells and virally infected cells  TNF alpha

o aka cachetin

o produced by macrophage  TNF beta

o aka lymphotoxin

o produced by CD4 and CD8 cells

3. Complement - complex series of more than 30 soluble and cell-bound proteins that interact in a very specific way to enhance host defense mechanisms against foreign cells.

 Function

o mediate inflammation o activate immune system o opsonization

o cell lysis

4. Properdin - serum protein that exerts bactericidal and viricidal effect in the presence of C3 and Mg

5. Betalysin

 released by platelets during coagulation

 substance with bactericidal activity found in serum (not in plasma).

Interleukin - cytokines (chemical messengers) produced by leukocytes that affect the inflammatory process through an increase in soluble factors or cells

Cellular versus Humoral Immunity

 Cellular - foreign substances were removed by specialized cells in a process known as phagocytosis.

 Humoral - substances in the blood provided protection from microorganisms, humoral immunity

ANTIBODIES - specific proteins referred to as immunoglobulins

 Many are isolated in the gamma globulin fraction of protein (electrophoresis separation)  Found in the plasma and many body fluids (eg. Tears, saliva, colostrums)

 Immunoglobulin (Ig)  Function

o 1° - body defense: combine w/ antigen, enough to neutralize bacterial toxins or some viruses

Immunoglobulins

 Classes differ from characteristics such as MW and sedimentation coefficients 1. IgM (SC: Σ, of 19)

 10 % of Ig pool

 Largely confined to intravascular pool and blood due to its large size  Produced early in immune response

 Effective in Agglutination & Cytolytic reactions

 5 individual HEAVY chains (65,000 Da, whole: 900,000 Da)

 Decreased in 1° (genetically determined) Ig disorders as well as 2° Ig deficiencies  Increased in

o Infectious diseases, such as subacute bacterial endocarditis, infectious mononucleosis, leprosy, trypanosomiasis, malaria, and actinomycosis o Collagen disorders, such as scleroderma

o Hematologic disorders, such as polyclonal gammopathies, monocytic leukemia, and monoclonal gammopathies(e.g., Waldenström’s macroglobulinemia)

 PENTAMER 2. IgG (SC: 7S)

 Major Ig in normal serum

 Diffuses more readily than other Igs into extravascular spaces and neutralizes toxins or binds to microorganisms in extravascular spaces

 Only Ig that cross placenta except IgG2  MW: 150,000 Da

 Cord blood and CSF

 Decrease in 1° (genetically) or 2° (acquired) Ig deficiencies  Significant Increase in

o Infectious diseases, such as hepatitis, rubella, and infectious mononucleosis o Collagen disorders, such as rheumatoid arthritis and systemic lupus

erythematosus

o Hematologic disorders, such as polyclonal gammopathies, monoclonal gammopathies, monocytic leukemia, and Hodgkin’s disease

 MONOMER 3. IgA

 15&-20% total circulatory Ig pool

 Predominant Ig in secretion of tears, saliva, colostrums, milk and intestinal fluids

 Synthesized largely by plasma cells on BODY SURFACES

 May pass directly into intestinal lumen or diffuse into blood circulation

 Transported through intestinal epithelial cells (hepatocytes) = binds to glycoprotein called SECRETORY COMPONENT

o Protects Ig from digestion by GI proteolytic enzymes

o Comple IgA - critical in protecting body surfaces against invading miciroorganisms o Presence of seromucous secretion (tears, saliva, nasal fluids, colostrums)

 Monomer and Dimer

 Decrease in 1° (genetically) or 2° Ig deficiencies

 Significant increase in serum

o Infectious diseases, such as tuberculosis and actinomycosis o Collagen disorders, such as rheumatoid arthritis

o Hematologic disorders, such as polyclonal gammopathies, monocytic leukemia, and monoclonal gammopathy (e.g., IgA myeloma)

o Liver disease, such as Laennec’s cirrhosis and chronic active hepatitis

4. IgD

 Low concentration in plasma, <1% total Ig pool  Extremely susceptible to proteolysis

 Primary a cell membrane found on the SURFACE of B lymphocytes in association w/ IgM  MONOMER

5. IgE

 Trace plasma protein found in blood plasma of unparasitized individuals  MW: 188,000 Da

 Mediates some types of hypersensitivity (allergic reactions, allergies, and anaphylaxis  Binds strongly to receptor on mast cells and basophils

 w/ antigen, mediates release of histamines and heparin  MONOMER

Antibody Structure

 Homology unit or Domain o 12 Domains

o 2 Heavy Chains and 2 Light Chains linked through cysteine residues by disulfide bonds o Variable Region – N-terminal end (heterogeneity); antigen-binding portion

o Constant Region – constant amino acid sequences  Globular Regions

o 2 Fab and Fc region linked by Hinge Region o 2 Fab fragments – retain ability to bind antigen o Fc portion – homogenous, crystallizable