Lecture Notes Classification of Immunity by Spontaneity Innate Immunity: Inborn Complement Classical Pathway Alternative Pathway Adaptive Immunity:

Stimulated by Exposure to Antigen Presenting Cells Dendritic Cells Macrophages B Lymphocytes Classification of Immunity by Specificity Non-Specific Immunity: Direct recognition by complement proteins (C-reactive) Specific Immunity: Antigen signal amplification (using Ig) for recognition by forming major histocompatibility complex (MHC) Classification by Immunity Focus Cell Mediated: Under T lymphocyte control, tends to be local Humoral: requires B lymphocytes, tends to be systemic nnate Immune System First-line defense: physical and chemical barrier The first-line defense includes barriers to infection, such as skin and mucus coating of the gut and airways. Second-line defense: Phagocytic cells phagocytic cells (Myeloid cells) (macrophages and neutrophil granulocytes) can destroy (phagocytose) foreign substances. Phagocytosis involves digestion of the bacterium by using enzymes. Anti-microbial proteins Anti-microbial proteins are activated if a pathogen passes through the barrier offered by skin. There are several classes of antimicrobial proteins, such as acute phase proteins (for example, proteins that enhance phagocytosis), lysozyme, and the complement system. The complement system is a very complex group of serum proteins, which is activated in a cascade fashion. Three different pathways are involved in complement activation:

A cascade of protein activity follows complement activation -> destruction of the pathogen and inflammation Interferons are also anti-microbial proteins. -> secreted by virus-infected cells -> diffuse rapidly to neighboring cells -> inhibit the spread of the viral infection. The adaptive immune system, also called the "acquired immune system", ensures that most mammals that survive an initial infection by a pathogen are generally immune to further illness caused by that same pathogen. The adaptive immune system is based on dedicated immune cells termed leukocytes (white blood cells) that are produced by stem cells in the bone marrow, and mature in the thymus and/or lymph nodes. It is in the lymph nodes where antigen is usually presented to the immune system. What Happens during an infection? Innate Immunity activated macrophages slip between cells [extravasation] to arrive cytokine chemicals attract other troops [chemotaxis] histamine chemicals dilate blood vessels for easier access to injury [vasodilatation]

Phagocytic cells able to ingest small foreign invaders neutrophils monocyte macrophages they release cytokines that enhance the immune response Mast cells /basophils release histamine that dilates blood vessels causes redness [erythrema], swelling [edema], and heat [fever] Call for help from the ADAPTIVE IMMUNE System -> results in a coordinated successful defense

Adapative response 1. Humoral immune system: -> acts against bacteria and viruses using immunoglobulins (also known as antibodies) -> produced by B cells.

2. Cellular immune system: -> destroys intracellular pathogens (such as virus-infected cells and mycobacteria causing tuberculosis) using T cells (also called "T lymphocytes"; "T" means they develop in the thymus). There are two major types of T cells: Cytotoxic T cells (TC cells): -> recognize infected cells by using T cell receptors to probe cell surfaces (-> Major Histocompatability Complex [MHC]) . If they recognize an infected cell, they release granzymes (proteases) to trigger that cell to become apoptotic ("commit suicide") Helper T cells (TH cells): -> activate macrophages and also produce cytokines (interleukins) that induce the proliferation of B and T cells. -> recognize infected cells of the immune system by using HelperT cell receptors to probe cell surfaces (-> Major Histocompatability Complex [MHC]) Passive and Active Immunization Passive Immunization -> Natural maternal serum/milk -> Artificial immune serum Active Immunization -> Natural infection -> Artificial infection*: Attenuated organisms (live) inactivated organisms (dead) Cloned genes of microbiological antigens Purified microbial macromolecules Synthetic peptides DNA *Artificial refers to steps involving human intervention Vaccination Goal is to establish resistance to virus/pathological organism by evoking an immune response 1. Give host a foreign organism/protein in non-infectious form (active immunization) 2. Antibodies are generated Ab binds to surface proteins of organism (passive immunization) Other vaccine components: Adjuvant: chemicals in the vaccine solution that enhance the immune response

Alum Ag in the vaccine clumps with the alum such that the Ag is released slowly, gives more time for memory cells to form

Traditional Vaccines: Grow in animals (vaccinia in calves for smallpox; rabbit brains for rabies) Simple bacterial culture (Cholera vibrio) then inactivation Grow in eggs (influenza, vaccinia) then inactivate Limitations -cant grow all organisms in culture -safety to lab personnel -Expense -insufficient attenuation (living agent altered to become harmless or less virulent) -reversion to infectious state -need refrigeration -do not work for all infectious agents -infants/children receive them immature immunity New Generation of Vaccines ! ! ! Recombinant DNA technology is being used to produce a new generation of vaccines. Virulence genes are deleted and organism is still able to stimulate an immune response. Live nonpathogenic strains can carry antigenic determinants from pathogenic strains. If the agent cannot be maintained in culture, genes of proteins for antigenic determinants can be cloned and expressed in an alternative host e.g. E. coli.

Recombinant vaccines 1. Subunit Vaccines peptide vaccines Genetic immunization 2. Attenuated Vaccines 3. Vector Vaccines 4. Bacterial Antigen Delivery Systems -> ghosts -> delivery systems Subunit/peptide vaccines It has been showed that the capsid or envelope proteins are enough to cause an immune response: - Herpes simplex virus envelop glycoprotein O.

- Foot and mouth disease virus capsid protein (VP1) - Extracellular proteins produced by Mycobacterium tuberculosis. Antibodies usually bind to surface proteins of the pathogen or proteins generated after the disruption of the pathogen. Binding of antibodies to these proteins will stimulate an immune response. Therefore proteins can be use to stimulate an immune response. Design: Do not use entire virus or bacteria (pathogenic agent) Use components of pathogenic organism instead of whole organism Advantage: no extraneous pathogenic particles i.e. DNA Is protein the same as in situ? Cost? Disadvantage: Example: Tuberculosis is caused by Mycobacterium tuberculosis. The bacterium forms lesions in the tissues and organs, causing cell death. Often the lung is affected. About 2 billion people are infected and there are 3 million deaths/year. Currently tuberculosis is controlled by a vaccine called BCG (Bacillus CalmetteGuerin) which is a strain of M. bovis. M. bovis often responds to diagnostic test for M. tuberculosis. Six extracellular proteins of M. tuberculosis were purified. Separately and in combinations these proteins were used to immunized guinea pigs. These animals were then challenged with M. tuberculosis. After 9-10 weeks examination showed that some combinations of the purified proteins provided the same level of protection as the BCG vaccine. Delivery: CARRIER PROTEINS Problem - Small Peptides are often degraded - no strong immune response - Carrier Proteins Make more Stable + stronger immune response Make fusion protein of carrier + vaccine peptide - inert carrier or highly immunogenic carrier (hepatitis B core protein) - Other delivery vehicles? Attenuated vaccines Attenuated vaccines often consists of a pathogenic strains in which the virulent genes are deleted or modified. Live vaccines are more effective than a killed or subunit (protein) vaccines.

Example - Vaccine against Cholera

cholera is caused by infection withVibrio cholerae and is transmitted through contaminated water. V. cholerae produces a enterotoxin with an A1 subunit and 5 B subunits -> causes disease Presently the cholera vaccine consist of a phenol-killed V. cholerae and it only last 3-6 months. A live vaccine would be more effective.

Vector vaccines - use a Virus as Antigen Gene Delivery System - Vaccinia good candidate for a live recombinant viral vaccine benign virus replicate in cytoplasm (viral replication genes) easy to store The vaccinia virus is generally nonpathogenic. The procedure involves: The DNA sequence for the specific antigen is inserted into a plasmid beside the vaccinia virus promoter in the middle of a non-essential gene e.g. thymidine kinase. A number of antigen genes have been inserted into the vaccinia virus genome e.g. - Rabies virus G protein - Hepatitis B surface antigen - Influenza virus NP and HA proteins. A recombinant vaccinia virus vaccine for rabies is able to elicit neutralizing antibodies in foxes which are a major carrier of the disease.

Bacterial Antigen Delivery Systems Use live nonpathogenic bacterium which contains antigen (Salmonella or epitope from cholera) Insert antigen gene into flagellin gene Epitope is expressed on the flagellum surface - Flagellin-engineered bacteria is VACCINE Advantage - Oral Where to target vaccines for cancer and other diseases? Dendritic cells are the educators of the immune system - found in lymphoid or immune organs, and at the interfaces between our bodies and the environment; epidermal layer of the skin has a rich network of dendritic cells; also line the surfaces of the airway and intestine, where they function as sentinels that sample proteins and particulates from the environment.

- When the body is challenged by injury and infection, dendritic cells travel from body surfaces to immune or lymphoid tissues, where they home to regions rich in T cells. There, dendritic cells deliver two types of information: they display antigens, the substances that are recognized by T cells, and they alert these lymphocytes to the presence of injury or infection. This directs the T cells to make an immune response that is matched to the challenge at hand. - dendritic cells patrol the body seeking out foreign invaders, whether these are bacteria, viruses, or dangerous toxins. After capturing the invaders (antigens) dendritic cells convert them into smaller pieces and display the antigenic fragments on their cell surfaces (APCs). - dendritic cells then travel to lymph nodes or the spleen where they stimulate other cells of the immune system to make vigorous responses, in particular, the B cells that make antibodies to neutralize the invaders and killer T cells that launch specific attacks to destroy them. How to target? One possibility is to target the toll-like receptors (TLRs) - part of innate immunity, recognize structurally conserved molecules derived from microbes (cannot be found on self) ligands: bacterial cell-surface lipopolysaccharides, lipoproteins, lipopeptides, lipoarabinomannan; proteins such as flagellin from bacterial flagella; double-stranded RNA of viruses; unmethylated CpG islands found in promoters of eukaryotic DNA; other RNA and DNA molecules;