Description of Module . Paper : 16 Immunology Module : 7 Epitopes, Antigenicity and Haptens Principal Investigator Dr. Sunil Kumar Khare,Professor Dept. of Chemistry, I.I.T. Delhi Paper Coordinator Dr. M.N.Gupta, Emeritus Professor and Dept. of Biochemical Engg. and Content Writer Biotechnology, I.I.T. Delhi Content Reviewer: Dr. Prashant Mishra, Professor Dept. of Biochemical Engg. and Biotechnology, I.I.T. Delhi 1 Immunology Biochemistry Epitopes, Antigenicity and Haptens Subject Name Biochemstry Paper Name 16 Immunology Module Name/Title Epitopes, Antigenicity and Haptens Dr. Vijaya Khader Dr. MC Varadaraj 2 Immunology Biochemistry Epitopes, Antigenicity and Haptens 1. Objectives How antigens/infection agents invade our bodies There are three ways epithelia protects us What are the features of a good antigen? What are epitopes and Haptens? 2. Concept Map Figure 1 3 Immunology Biochemistry Epitopes, Antigenicity and Haptens 3. Description Antigen is a substance which is recognized by the immune system as a foreign one. The result is the immune response of the body. All non-self substances do not provoke this immune response and hence do not constitute antigens. For example, a low molecular weight poison if consumed, may not elicit immune response. For dealing with such substances, the body has detoxification mechanisms which largely consist of converting the foreign substance into soluble bioconjugates which can be excreted. Please recall, the well-known experiment by Knoop in 1904 in which phenyl derivatives of the even numbered fatty acids when fed to the dogs, the animals excreted the glycine conjugate of phenylacetic acid called phenyl aceturic acid. This experiment, which later on led to the development of the β-oxidation pathway of fatty acids involved the detoxification mechanism. Figure 2 4 Immunology Biochemistry Epitopes, Antigenicity and Haptens So what qualifies as a foreign substance to be recognized as an antigen? The features which improve “antigenicity” of the foreign substance have been established after many decades of experience with immunization. In early days of immunology, the knowledge about cell mediated immunity was not available. IN that period, the term antigen implied a foreign substance which can result in the production of a specific antibody. Thus, antigen meant sort of a antibody generating foreign molecule. Figure 3 5 Immunology Biochemistry Epitopes, Antigenicity and Haptens We know now that: The specific antibody generation in fact consisted of multiple antibody molecules. We will learn in this module why it happens that way. However, it is for this reason that the resulting antibody preparation is referred to as polyclonal antibodies. Each specific antibody is generated by a single kind of B-cell. The antigen can trigger immune response either by B-cells or T-cells or both. Before we discuss many features characteristic of a good antigen, let us consider how antigens enter the body and what do these non-self substances encounter immediately. Physical Barriers to the Entry Infectious diseases occur when an invading microorganism enters through mucosal surfaces or external epithelia. Mucosal surfaces are present in airways, gastrointestinal tract and reproductive tract. Figure 4 6 Immunology Biochemistry Epitopes, Antigenicity and Haptens The external epithelia consists of external surface, wounds/abrasions (which disturb the integrity of the external surface) and insect bites. Figure 5: Healing of a wound In many cases the infection remains local. For example, Athlete‟s foot In other cases, infectious agents spread through blood or lymphatics or by releasing toxins 7 Immunology Biochemistry Epitopes, Antigenicity and Haptens Let us look at the routes of entry in a little more detail. The respiratory tract (airway) mucosa obviously is the entry point of airborn organisms. The organism can be inhaled as droplets. Examples are influenza virus and Neisserice meningitis which cause influenza and Meningococcal memningitis. Contaminated food or drinks (including water!) involve the invasion of the gastrointestinal tract. The well known examples are Salmonella typhii (causing typhoid fever) or Rotavirus which causes Diarrhoea. Physical contacts are necessary for the invasion of the reproductive tract by pathogens. Traponema palladium causing syphilis is a well known example of this. These three tracts (the respiratory, gastrointestinal and the reproductive) have internal epithelial surfaces. The infection occurs either by just colonization or by crossing the internal epithelia. For colonizing pathogens, the binding to the internal epithelia has to be firm enough so that the pathogens are not dislodged by flow of air of fluids. The internal epithelia are called mucosal epithelia as these secret mucus containing mucin glycoproteins. The function of this mucus is to prevent adherence of the pathogen. In the respiratory tract, mucus flows through beating of the epithelial cilia. Thus, some lung infections can be caused by defects in the mucus secretions or ciliary movements in some people. 8 Immunology Biochemistry Epitopes, Antigenicity and Haptens Figure 7 Peristalsis in the gut ensures not only movement of food but of the pathogens as well. Bacterial overgrowth in the intestinal lumen may result from defective peristalsis. 9 Immunology Biochemistry Epitopes, Antigenicity and Haptens Figure 8 Hairs in the nose and cough as a reflex action also prevent the respiratory tract infections. Epithelial Barriers There are three distinct kinds of barriers as a result of epithelia. The mechanical ones involve the epithelial cells joined by tight junctions. The flow of air, fluid and mucus has already been mentioned as an intrinsic epithelial defence mechanism. The nature of chemical barriers is different at different places in the body. The sweat contains fatty acids which lower its pH. Lysozyme also aids the antimicrobial action of sweat. Lysozyme is also present in saliva and tears. Pepsin in the gut is also a hydrolase. Low pH of the stomach also prevents microbial growth. 10 Immunology Biochemistry Epitopes, Antigenicity and Haptens Figure 9 The small intestine has Paneth cells in the base of the crypts beneath the epithelial stem cells. These cells produce antimicrobial peptides called α-defensins or cryptidines which are both antimicrobial and antifungal. Related peptides β-defensins are produced by the skin and respiratory tracts. Figure 10 11 Immunology Biochemistry Epitopes, Antigenicity and Haptens Two surfactant proteins A and D bath the lung epithelial surface and opsonize the pathogens. This paves the way for their phagocytosis. So, antibodies are not the only protein opsonines. Surfactant property enables these proteins to bind to microbes with different cell surface properties. The microbiological barrier consists of normal microbial flora. Apart from competing with the pathogen for the nutrient, such flora also produce antimicrobial substances like collicins, the peptides produced by E.coli. The use of broad spectrum antibiotics destroys the intestinal flora. Hence, its recolonization is important. Hence, the importance of prebiotics and probiotics in maintaining or re-establishing the intestinal flora. Interestingly, gut flora is being recognized as having a very important role in the health of an individual. Features of a good Antigen After an antigen has succeeded in evading the above barriers, innate immunity and acquired immunity come into play. For the latter, the antigen should have the following features: 1.) Large Size: Larger is better as far as size/molecular weight of the non-self substance is concerned. High molecular weight proteins are good antigens as compared to the peptide hormones. Serum albumins have molecular weight around 60,000 kDa and are good antigens. However, these are known to produce tolerance (see later discussion on tolerance). We will shortly discuss how one can still get specific antibodies against small molecular weight substances. 2.) Complexity in structure: Large molecules with repetitive structures like polysaccharides, homo- α-amino acid polymers and even nucleic acids are poor antigens. Proteins, with diverse side chains are good antigens. Similarly bacterial lipopolysaccharides are good antigens. 3.) Flexibility: Good antigens have optimum flexibility. Enough to bind to the cell receptors but highly flexible structures are unstable. Unstable molecules are not good antigens. A related parameter is degradability. If the antigen is hydrolyzed fast, it does not last long enough to produce immune response. Again, there is a range of optimum vulnerability as antigens, as we will learn later, have to be „processed‟ which requires a protein, for example, to be broken down to the peptides. Inert organic polymers, homo- or co-polymers of D-amino acids thus are poor antigens. 12 Immunology Biochemistry Epitopes, Antigenicity and Haptens 4.) Non-self nature: Last but not the least, it is understood that a substance has to be a non-self one to provoke immune response. The concept of “forgiveness” is just not a qualitative one. A protein far removed from the same host protein on the evolutionary tree is likely to be a better antigen. Also, the concept of self and non-self can be applied in a local context. Damages to testes, sperm antigens enter blood and elicit immune response. This also is common after vasectomy. Epitopes An antigen, thus, is a large complex molecule. As pointed out earlier, it provokes many antibodies. The areas on its surface on which