Chapter 6 Experimental Systems and Methods in Immunology PDF

CHAPTER 6: EXPERIMENTAL SYSTEMS AND METHODS IN IMMUNOLOGY IMMUNIZATION AND VACCINES Epidemiology  Epidemiology is the study (scientific, systematic, and data-driven) of the distribution (frequency, pattern) and determinants (causes, risk factors) of health-related states and events...

CHAPTER 6: EXPERIMENTAL SYSTEMS AND METHODS IN IMMUNOLOGY IMMUNIZATION AND VACCINES Epidemiology  Epidemiology is the study (scientific, systematic, and data-driven) of the distribution (frequency, pattern) and determinants (causes, risk factors) of health-related states and events (not just diseases) in specified populations (neighborhood, school, city, state, country, global). It is also the application of this study to the control of health problems (Source: Principles of Epidemiology, 3rd Edition)  Part of the control of infectious diseases is immunization and the administration of vaccines to the population  Immunization is the process whereby a person is made immune or resistant to an infectious disease, typically by the administration of a vaccine Why immunization is important? https://coronavirus.jhu.edu/map.html   A vaccine is a biological preparation 7 that improves immunity to a particular disease. Edward Jenner  vaccinologist (1749-1823) What is the right thing to do? Herd immunity: is a form of indirect protection from infectious disease that occurs when a large percentage of a population has become immune to an infection, thereby providing a measure of protection for individuals who are not immune. Immunological memory Characteristics of vaccines  Effective protection against the intended pathogen without significant danger of causing the disease or sever side effects  Long lasting protection  Induces an immune response against the intended pathogen  Neutralizing Ab must be stimulated in order to minimize the re-infection  Economically feasible to produce  Suitably stable for storage, transport, and use Route of administration  Intramuscular (IM) injection administers the vaccine into the muscle mass. Vaccines containing adjuvants should be injected IM to reduce adverse local effects.  Subcutaneous (SC) injection administers the vaccine into the subcutaneous layer above the muscle and below the skin.  Intradermal (ID) injection administers the vaccine in the topmost layer of the skin. BCG is the only vaccine with this route of administration.  Oral administration of vaccine makes immunization easier by eliminating the need for a needle and syringe.  Intranasal spray application of a vaccine offers a needle free approach through the nasal mucosa of the vaccine. Factors affecting the immune response Types of vaccines  Live  Attenuated  Killed  Extract  Recombinant  DNA/RNA  ….  Live vaccines  Attenuated vaccines  Are vaccines based on  Their virulence and living organism capable ability to replicate are of normal infection and reduced by treatment replication with heat, chemicals or  Not appropriate for other methods pathogens that are  Can cause subclinical or capable of causing mild symptoms sever or life-  Can re-mutate and threatening diseases revert to the wild type.  Killed/inactivated  Extract vaccines vaccines  Do not contain whole  Include organisms that are dead because of organisms but composed treatments with physical or of materials isolated chemical agents. from disrupted or lysed  Should be incapable of infection, replication and organism. function (Toxoids)  This type is most suitable  It is difficult to guarantee for providing protection that all organisms in the treatment are dead against diseases  Recombinant vaccines  DNA/RNA vaccines  Made based on  Naked pathogen DNA/RNA extract injected molecular biology to the host techniques by modifying  The DNA/RNA is also the pathogen’s virulence modified, and some genes or reproduction at the removed gene level  Host cells take up pathogen’s DNA and  Such a pathogen can express proteins to evoke infect host cells or even the immune system replicate but cannot  Last longer than other induce disease vaccines Microbial evasion of the immune system  Ability to hide from the immune sys.  Latency  infect immunopriviledged tissues  Kill immune cells/phagocytes  Secreted modulators or toxins (eg. Virokines occupy binding sites on host receptors)  Block acquired immunity  Block antigen presentation  Down-regulation of MHC I or II  Rapid polymorphism of antigenic makeup  Inhibit cytokines/interferon/chemokines Components of a vaccine Antigens The components derived from the structure of disease-causing 20 organisms, which are recognized as 'foreign' by the immune system and trigger a protective immune response to the vaccine. Stabilizers Used to help the vaccine maintain its effectiveness during storage. Factors affecting stability are temperature and acidity or alkalinity of the vaccine (pH). Stabilizing agents include MgCl2 (for OPV), MgSO4 (for measles), lactose-sorbitol and sorbitol-gelatin. Adjuvants An ingredient added to a vaccine to improve the immune response it produces. 21 They can bind to the antigens in the vaccine, help keep antigens at the site of injection, and help deliver antigens to the lymph nodes, where immune responses to the antigens are initiated. Antibiotics Antibiotics (in trace amounts) are used during the manufacturing phase to prevent bacterial contamination of the tissue culture cells in which the viruses are grown. Preservatives Preservatives are added to multidose vaccines to prevent bacterial and fungal growth. They include a variety of substances, for example thiomersal, formaldehyde, or phenol derivatives Example of vaccine mechanism of action. IMMUNOASSAYS Immunoassay  Immunoassay is a technique which incorporates the binding reaction of a target substance (antigen) with an antibody.  This interaction in vitro is used for diagnostic purposes in order to identify specific antigen or antibody.  Factors affecting immunoassays in vitro are as follows:  Affinity: Strength of reaction between a single antigenic determinant and a single antibody combining site;  Avidity: The overall strength of binding of antigen with multivalent antibodies;  Antigen – Antibody ratio  Physical form of antigen Agglutination  The interaction between soluble antibody and insoluble particulate antigen results in agglutination (clumping).  The highest dilution of serum that still causes agglutination but beyond which no agglutination occurs is termed “Titer” Precipitation reactions  The interaction between an antibody and a soluble antigen in aqueous solution forms a lattice that eventually develops a visible precipitation.  Antibody that forms precipitation is known as precipitin. This process is called as precipitation reaction. Formation of an Ag-Ab lattice depends on the valency (relative capability to act upon, react, bind) of both antigen and antibody. The antibody should be bivalent. The antigen must be either bivalent or multivalent. Zone of equivalence is a point at which the maximum precipitation occurs. Ouchterlony Double Immuno-diffusion  Used in the detection, identification and quantification of antibodies and antigens.  Immuno-diffusion procedures are precipitation reactions carried out in an agar-gel medium.  Antibody and antigen are loaded in different wells and diffuse through the medium.  At optimal antigen-antibody ratio, a visible band appears in the gel.  The antiserum is placed in the central well and different antigens in the surrounding well.  If two adjacent antigens are identical, the lines of precipitate formed by them will fuse and prove “V” shaped curve.  If they are unrelated, the lines will cross each other and provide “X” shaped curve.  Cross reaction or partial identify is indicated by spur formation and provide “Y” shaped curve. Radial Immuno-diffusion  antiserum is incorporated in agar gel poured on a flat surface of slide or Petri dish.  The antigen is added to the wells cut on the surface of the gel.  The diameter of the rings can be compared to that of a known concentration of antigen to gives an estimate of the concentration of the unknown antigen.  The antigen diffuses radially from the well and forms ring shaped bands of precipitation concentrically around the well. Western Blotting  Protein mixture is electrophoretically separated on Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS- PAGE).  The protein bands are transferred to a nitrocellulose membrane. The antigen-antibody complexes that form on the band containing the protein recognized by the antibody.  After binding of the enzyme antibody conjugate, addition of a chromogenic substrate that produces a highly colored and insoluble product causes the appearance of a colored band at the site of the target antigen. Enzyme Linked Immunosorbent Assay (ELISA)  It was first developed for the measurement of antibodies it has since been modified for measurements of other molecules including hormones.  This assay is sensitive, cheap and easy to perform and there is no danger of radioactive hazard. Most widely used procedure in serology.  An enzyme conjugated with an antibody reacts with a colorless substrate to generate a colored reaction product. Such a substrate is called a chromogenic substrate.  Indirect ELISA  This technique is used to detect or quantify antibody levels. Serum or other sample containing primary antibody (Ab1) is added to an antigen-coated microtiter well. The presence of antibody bound to the antigen is detected by adding an enzyme-conjugated secondary antibody (Ab2), which binds to the primary antibody. Substrate for the enzyme is added. The amount of colored reaction product that forms is measured by specialized spectrophotometer.  Sandwich ELISA  This method can detect or measure antigens. The antibody is immobilized on a microtiter well. A sample containing antigen is added and allowed to react with the immobilized antibody. Second enzyme-linked antibody specific for a different epitope on the antigen is added and allowed to react with the bound antigen. After any free second antibody is removed by washing, substrate is added, and the colored reaction product is measured.  Competitive ELISA  Antibody is first incubated in solution with a sample containing antigen. The antigen-antibody mixture is then added to an antigen coated microtiter well. The more antigens present in the sample, the less free antibody will be available to bind to the antigen-coated well. Addition of an enzyme-conjugated secondary antibody (Ab2) specific for the primary antibody can be used to determine the amount of primary antibody bound to the well as in an indirect ELISA. Immuno-fluorescence  Fluorescence is the property of absorbing light rays of one particular wavelength and emitting rays with a different wavelength.  Fluorescent dyes show up brightly under ultraviolet light as they convert ultraviolet into visible light.  Fluorescent dyes can be conjugated to antibodies and that such labeled antibodies can be used to locate and identify antigens in tissue.  Can be direct or indirect  The most commonly used fluorescent dyes are fluorescein isothiocyanate (FITC) phycoerythrin (PE). Flow cytometry  The flow cytometer uses a laser beam and light detector to count single intact cells in suspension. Every time a cell passes the laser beam, light is deflected from the detector, and this interruption of the laser signal is recorded. Those cells having a fluorescently tagged antibody bound to their cell surface antigens are excited by the laser and emit light that is recorded by a second detector system located at a right angle to the laser beam. Polyclonal antibodies production  injecting a lab animal such as a rabbit or a goat with a specific antigen. Within a few weeks, the animal’s immune system will produce high levels of antibodies specific for the antigen. These antibodies can be harvested in an antiserum, which is whole serum collected from an animal following exposure to an antigen.  Because most antigens are complex structures with multiple epitopes, they result in the production of multiple antibodies in the lab animal. This so-called polyclonal antibody response is also typical of the response to infection by the human immune system. Antiserum drawn from an animal will thus contain antibodies from multiple clones of B cells, with each B cell responding to a specific epitope on the antigen Monoclonal antibodies production  monoclonal antibodies are produced in vitro using tissue-culture techniques. mAbs are produced by immunizing an animal, often a mouse, with a specific antigen. B cells from the spleen of the immunized animal are then removed. B-cells fused myeloma cells, to yield hybridoma cells. The hybridomas, which are capable of growing continuously in culture while producing antibodies, are then screened for the desired mAb. Those producing the desired mAb are grown in tissue culture; the culture medium is harvested periodically and mAbs are purified from the medium. This is a very expensive and time- consuming process. It may take weeks of culturing and many liters of media to provide enough mAbs for an experiment or to treat a single patient. mAbs are expensive