Applied Immunology Lecture notes Aftab Jasir: European Centre for Disease Prevention and Control (ECDC) European Public Health Microbiology training program (EUPHEM) Introductory course, Menorca,2012

Objectives Define basic components of immunology Describe important terms in immunology Explain major applications of immunology

What is immunology? Immunology is a broad branch of biomedical science that covers the study of all aspects of the immune system in all living organisms. It deals with the physiological functioning of the immune system in states of both health and disease The specificity of the bond between antibody and antigen has made it an excellent tool in the detection of substances in a variety of diagnostic techniques. Antibodies specific for a desired antigen can be conjugated with a radiolabel, fluorescent label, or color-forming enzyme and are used as a "probe" to detect it. However, the similarity between some antigens can lead to false positives and other errors in such tests by antibodies cross-reacting with antigens that aren't exact matches

The immune system is the ministry of defence of the human/animal body What is the immune system? The immune system is the ministry of defence of the human/animal body

Immunologically Naive No previous experience No memory Must be educated

Immunodeficiency and Immunosuppression Immunodeficiencyis a state in which the immune system's ability to fight infectious disease is low or entirely absent. Immunosuppression involves an act that reduces the activation or efficacy of the immune system. immunosuppression may occur as an adverse reaction to treatment of other conditions.

Major defence components of the human immune system Cells Immunoglobulins

Overview of the immune system Innate (nonspecific) 1st line of defense Cellular components Humoral components Adaptive (specific) 2nd line of defense

Definitions/terminology Antigens (Ag) Large molecules, is anything that obtain the formation of a specific immune response (Anomy) Ag determinants (epitopes) are the particular chemical groups on a molecule that are antigenic Antibody(Ab)/immunoglobulin (Ig). A special group of soluble proteins that are produced in response to foreign antigens (substances) Anomy and forgion

Antigen and antibody

IgM (first exposure, large, not passing placenta, huge amont) 5 classes of IGs IgM (first exposure, large, not passing placenta, huge amont) IgG (secondary exposure, small, passing placenta) IgD (proteins in the plasma membranes of mature B-lymphocytes, same time as IgM) IgA (mucosal immunity, respiratory tract) IgE (Allergy and parasites) IgG is the most abundant immunoglobulin and is approximately equally distributed in blood and in tissue liquids, comprising 75% of serum immunoglobulins in humans, IgG molecules are synthesized and secreted by plasma B cells. Immunoglobulin A (IgA) is an antibody that plays a critical role in mucosal immunity. More IgA is produced in mucosal linings than all other types of antibody combined In biology, Immunoglobulin E (IgE) is a class of antibody (or immunoglobulin "isotype") that has only been found in mammals. IgE is a monomeric antibody with 4 Ig-like domains (CH1->CH4). [1] It plays an important role in allergy, and is especially associated with type 1 hypersensitivity.[2] IgE has also been implicated in immune system responses to most parasitic worms Immunoglobulin D (IgD) is an antibody isotype that makes up about 1% of proteins in the plasma membranes of mature B-lymphocytes where it is usually coexpressed with another cell surface antibody called IgM. IgD is also produced in a secreted form that is found in very small amounts in blood serum.

Haptens Vaccine conjugate

Ministry of defence of the human body

Antibody Protection of the Host

Primary and secondary antibodies P: antibodies raised against an antigenic target of interest and are typically unconjugated. S: antibody that binds to primary antibodies or antibody fragments. They are typically labeled with probes that make them useful for detection, Primary antibodies are antibodies raised against an antigenic target of interest (a protein, peptide, carbohydrate, or other small molecule) and are typically unconjugated (unlabelled). secondary antibody is an antibody that binds to primary antibodies or antibody fragments. They are typically labeled with probes that make them useful for detection

Immune Precipitation Antigen Antibody Study Guide Why do soluble antigens and antibodies precipitate? Antigen Antibody

Agglutination RBC RBC RBC IgM Antibody RBC RBC RBC RBC RBC Study Guide What is the difference between precipitation and agglutination? IgG Antibody RBC RBC RBC

Factors influencing immunogenicity Contribution of immunogen Contribution of biological system Method of administration

Class Switching Biological mechanism that changes a B cell's production of antibody from one class to another, for example, from IgM to IgG. antibody titer Study Guide What is the significance of class switching? IgM IgG time

Four phases of the primary response lag phase where no antibody is detected log phase in which the antibody titer rises logarithmically plateau phase during which the antibody titer stabilizes phase (decline) during which the antibody is cleared or catabilized

Kinetics of the Ab Response T-dependent Ag; 1o Response D a y s A f t e r I m m u n i z a t i o n A b T i t e r LAG LOG DECLINE PLATEAU Lag Log Plateau Decline

Kinetics of the Ab Response T-dependent Ag; 2o Response 1o Ag 2o Ag D a y s A f t e r I m m u n i z a t i o n A b T i t e r Lag Log Plateau Decline

Major practical applications of immunology Use of antiserum and vaccination to provide protection against disease. Diagnostic tool to detect disease. Epidemiological investigation of vaccine preventable diseases

My face is my fortune Where are you going, my pretty maid? I’m going a-milking, sir, she said May I go with you, my pretty maid? You’re kindly welcome, sir, she said What is your father, my pretty maid? My father is a farmer, sir, she said What is your fortune, my pretty maid? My face is my fortune, sir, she said Illustration of the famous first vaccination experiment. In 1796 Edward Jenner infected eight-year-old James Phipps with fluid from a lesion on the hand of Sarah Nelmes, who had caught cowpox. The word ‘vaccination’ comes from the Latin word ‘vaccinia’ for cowpox. This was a successful experiment, because the boy was subsequently protected against a human smallpox infection. Years later Louis Pasteur proposed that all inoculations intended to protect against infectious diseases should be called vaccinations, in honour of Jenner.

Variolation The word ‘variolation’ comes from the Latin word ‘variola’ for human smallpox. Figure 1. Ancient Oriental print illustrating the technique of variolation. Dried material from scabs of human smallpox is blown into the nose of an unaffected person with a small blowpipe. The word ‘variolation’ comes from the Latin word ‘variola’ for human smallpox.

Discovery of small pox vaccine In 1798, Jenner introduced 1st vaccination (vacca: cow) following his experimentation with isolates of cow pox virus from ‘Blossom’. Cartoon on smallpox vaccination from 1802 entitled “The Cow Pock or the Wonderful Effects of the New Inoculation”. It shows Edward Jenner and persons who are developing cow-like projections as a result of allowing themselves to be inoculated with an ‘animal disease Edward Jenner 1780AD Blossom 28

Types of acquired immunity

Passive – receive Abs made by another 1. natural 2. artificial - γ globulin, hyperimmune serum Natural Artificial

Mode of delivery

Advantages and Disadvantages of Active Immunization Not immediate Immune suppressed/deficiency Long term immunity Herd immunity Risk of infection Risk of contamination Animal ??? Attenuated can revert to their pathogenic form

Advantages and Disadvantages of Passive Immunization no long term protection serum sickness immediate protection risk of hepatitis and HIV graft vs. host disease

Serological tests based on Abs specifically binding to Ag Serology A science that attempts to detect signs of infection in a patient’s serum such as Ab for a specific microbe Serological tests based on Abs specifically binding to Ag Ag of known identity will react with Ab in an unknown serum sample. Known Ab can be used to detect Ag in serum Ag-Ab reactions are visible by clumps, precipitates, color changes or release of radioactivity. The most effective tests have high specificity and sensitivity.

Enzyme (ELISA) Immunoassay Enzyme conjugated to anti-Ig antibody (“second antibody”) Add substrate for enzyme anti-antigen antibody Study Guide Radioimmunoassays (RIA) and Immunfluorescence Assays (IFA) depend on the detection of a radionuclide or a fluorescent probe attached to the second antibody, instead of the enzyme. In those cases, no substrate is needed. How would you use this assay to measure the concentration or detect the presence of an antigen? Antigen

a) The presence of a specific Ab b) Identification of microbes

Specificity, sensitivity, and cross reactivity a) Specificity Ab attaches with great exact-ness to only one type of Ag. b) Sensitivity Ab can locate Ag, even when it is greatly diluted. c) Cross reactivity the ability of an individual antibody combining site to react with more than one antigenic determinant or the ability of a population of antibody molecules to react with more than one antigen.

Examples of serological tests Agglutination tests Precipitation tests Immunoelectrophoresis Western blot tests Complement fixation tests Immunofluorescence testing Immunoassays

agglutination is the clumping of particles agglutination is the clumping of particles. The word agglutination comes from the Latin agglutinare, meaning "to glue to." This occurs in biology in three main examples: The clumping of cells such as bacteria or red blood cells in the presence of an antibody. The antibody or other molecule binds multiple particles and joins them, creating a large complex. The coalescing of small particles that are suspended in a solution; these larger masses are then (usually) precipitated. An allergic reaction type occurrence where cells become more compacted together to prevent foreign materials entering them. This is usually the result of an antigen in the vicinity of the cells.

Acute infection Onset of symptoms Incubation Primary infection Days Modified from Hedman et al. 2000 Primary infection Days Weeks Months Years

Acute infection Onset of symptoms IgM Incubation Primary infection Days Weeks Months Years

Acute virus infection Onset of symptoms IgG IgM Incubation Primary infection Days Weeks Months Years

Avidity = the sum of affinities between: antibody and antigen Acute virus infection Onset of symptoms IgG avidity IgM IgG Incubation Primary infection Days Weeks Months Years

Secondary infection or reactivation Onset of symptoms IgG avidity IgM IgG Incubation Persistence Primary infection Secondary infection or reactivation Days Weeks Months Years

Secondary infection or reactivation Onset of symptoms IgG avidity IgG IgM Viremia Primary infection Secondary infection or reactivation Days Weeks Months Years

Q fever IgM2 IgM1 IgG2 1/800 IgG1 IG titter 1/64 1/40 1 1 2 1 2 Time

What should you have in mind!!! Tack home massage Some times Ag x Ab based tests can results in wrong alarm of outbreak ( Salmonella) Antigen variation is always a problem (Chlamydia, grouping of streptococci) Cross-reactivity can give wrong information of an outbreak Any unusual or unexpected results should be confirmed by genetic test If possible use other methods than serology in an outbreak situation or combine with other methods