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antigens

1. Antigens

2. index : INTRODUCTION TO ANTIGENS EPITOPES ANTIGEN CAPTURE AND PRESENTATION TO B CELLS AND T CELLS. HAPTENS HETEROPHILE ANTIGENS SUPERANTIGENS TUMOR ANTIGENS HLA AND HLA TYPING ANTIGEN DETECTION VIRAL ANTIGENS

3. Antigens – “shapes” recognized by the immune system Antigen are defined as any substance that satisfies two distinct immunologic properties: IMMUNOGENICITY AND ANTIGENICITY . OR to any self or non self substance that elicits an immune response. 1. Immunogenicity : It is the ability of an antigen to induce immune response in the body. It depends on many factors. 1. Properties of an immunogen 2. Properties or traits of host 3. Route and dose of immunogen 4. Adjuvants

4. 1. TRAITS OF IMMUNOGEN : Foreignness : Immunogen molecule should be recognized as non self. More distant taxonomically the source of antigen, better the immunogen b.) Molecular size: Immunogen should have a molecular size of at least 10,000 daltons . This is proved by substance such as insulin and glucagon with mol. Wt less that 10,000 are known to be poor immunogens. Commonly used immunogens such as tetanus toxoid, Bovine serum albumin have mol. Wt. of 1,50,000 and 69,000 respectively.

5. c.) Susceptibility to antigen processing and presentation: The antigen, very often, is needed to be degraded and the specific fragment of it containing suitable sequence or epitope is combined with MHC molecules. This Ag-MHC complex is then recognized by T-cells in order to generate an immune response. If the antigen is such that it cannot undergo this processing and presentation, it will be a poor immunogen. This is proved by D-amino acid polymers as compared to L-amino acid polymers. Large insoluble molecules, being easily phagocytosed, are potent immunogens than small and soluble ones. So macromolecular aggregation by heating or complexing with insoluble substances is a commonly used method for increasing the immunogenicity.

6. 2. Traits of host : Age : extremes of age immunogenicity is decreased Nutrition : Malnutrition/undernutrition Genetic make up of the host: The basis of this genetic influence is the genetic make up, including MHC genes, which regulates immune response. Besides MHC, the genes encoding T cells and B cell receptors are also involved. 3. DOSAGE OF IMMUNOGEN : Optimal dose: An antigen is immunologically active only in the optimal dose range. A too little dose fails to elicit immune response and a too large dose leads to development of immunological tolerance.

7. : Repeated immunogen exposure and booster doses: Booster doses increase the clonal proliferation of T and B-cells to augment response. Route of administration: -T he immune response is better induced following parenteral administration, however it also affects the type of antibody produced -IgA are better induced following oral administration of antigens. -Inhalation of pollen antigens induces IgE synthesis; whereas the same antigens given parenterally, elicits IgG antibodies. Site of injection may influence immunogenicity: The hepatitis B vaccine is more immunogenic following deltoid injection than gluteal injection. This may be due to the paucity of antigen presenting cells (APCs) in gluteal fat.

8. 4. ADJUVANTS : - substances used to potentiate the action of an immunogen. - usually mixed and injected with antigen to enhance immune response. - Adjuvants act through the following ways: 1. Delaying the release of antigen 2. By activating phagocytosis 3. By activating TH cells 4. By granuloma formation

9. Examples of Adjuvant Activity Alum (aluminum hydroxide or phosphate) Mineral oil (liquid paraffin) Freund’s incomplete adjuvant: It is a water-in-oil emulsion containing a protein antigen in the aqueous phase. Freund’s complete adjuvant is the mixture of Freund’s incomplete adjuvant and suspension of killed tubercle bacilli in the oil phase Lipopolysaccharide (LPS) fraction of gram-negative bacilli, e.g. LPS of Bordetella pertussis acts as an excellent adjuvant for diphtheria and tetanus toxoids. This explains the reason for using combined immunization for diphtheria, pertussis and tetanus in the form of DPT vaccine. Other bacteria or their products: Mycobacterium bovis Toxoid (diphtheria toxoid and tetanus toxoid act as adjuvant for Haemophilus influenzae (– type b) Nonbacterial products: silica particles, beryllium sulfate , squalene and thiomersal.

10. 2. Antigenicity (immunological reactivity ): It is the ability of an antigen to combine specifically with the final products.(i.e. antibodies and/ or T cell-surface receptors). An antigen can induce formation of antibody and also produce a specific and observable reaction with the antibody so produced. Antigens can be proteins, carbohydrates, lipids, nucleic acids, small chemical groupings referred to as haptens, in fact virtually anything. The antigens may be a component of microorganisms, of larger infectious agents such as parasitic worms, of ingested substances such as foods, of inhaled substances such as pollens, of transplanted organs or tissues, or even our own body components (“self” antigens).

11. Types of antigens Two types: exogenous and endogenous antigens EXOGENOUS ANTIGENS: - They enter into the body from outside and are not produced by host cell. - Microorganisms, drugs, pollens, pollutants,sometimes food items. ENDOGENOUS ANTIGENS: - Produced by host cells - may belong to host’s own body or maybe foreign origin. - genetically mutated cells or cancerous cells

12. ANTIGENIC DETERMINANTS OR EPITOPES : The immune system does not recognize an infectious agent or foreign molecule as a whole, but reacts to structurally distinct areas: antigenic determinants or epitopes. It is defined as a small area present on the antigen comprising of few (four to five) amino acids or monosaccharide residues, that is capable of sensitizing T and B cells and reacting with specific site of T cell receptor or an antibody. The interaction between cells of the immune system and antigens takes place at many levels and complexity of antigen is mirrored by epitope.

13. Epitopes may be grouped into two types: 1. Sequential or linear epitope : It presents as a single linear sequence of few amino acid residues. 2. Conformational or non-sequential epitopes are found on the flexible region of complex antigens having tertiary structures. In general, T cells recognize sequential epitopes, while B cells bind to the conformational epitopes.

14. B cell epitopes: Antigenic determinants recognized by B cells. B cell epitope can combine with its receptor only if the antigen molecule is in its native state. Smaller molecules usually fit within a particular depression or groove in the antigen binding site of antibody molecule. When an antigen enters the body, it is confronted by a dazzling array of B‐lymphocytes all bearing different antibodies each with its own individual recognition site. The antigen will only bind to those receptors with which it makes a good fit. B‐lymphocytes whose receptors have bound antigen receive a triggering signal and can then develop into either plasma cells or memory B‐cells.

15. Antigen Capture and Delivery to B Cells Antigen may be delivered to naive B cells in lymphoid organs by multiple routes . Small antigens are delivered to B cells in follicles through afferent lymphatics and via conduits. larger antigens by subcapsular sinus macrophages or by dendritic cells in the medulla.

16. This process is referred to as clonal selection and ensures that only the relevant, antigen‐specific, lymphocytes are triggered to produce the appropriate effector cells and memory cells. In the case of the antibody‐producing B‐lymphocytes they use a cell surface version of the antibody, which directly binds native antigen, as the B‐cell receptor (BCR).

17. ANTIGEN PRESENTATION TO T LYMPHOCYTES: It is impossible for the few T cells specific for any antigen to constantly patrol all possible tissues where antigens may enter or be produced. The cells that capture antigens and display them to T lymphocytes are called as Antigen-presenting cells (APC). The task of displaying host cell-associated antigens for recognition by CD4 and CD8 T cells is performed by specialized proteins called MHC – Major histocompatibility complex molecules, which are expressed on host cells.

18. MHC CLASS I MHC CLASS II Expressed on all nucleated cells (except sperms) and platelets. Expressed on antigen presenting vells Consists of a polymorphic α , heavy chain, linked non-covalently to   β 2- macroglobulin Heterodimer consisting of non-covalently associated α and β chains, both polymorphic . α chains are encoded by three genes- HLA-A, HLA-B, AND HLA-C Encoded by HLA-D, with sub-regions: HLA-DP, HLA-DQ, HLA-DR. Display antigenic peptides that are derived from viral and tumor proteins. Present antigens that are internalized into vesicles, and derived from extracellular microbes. Class I complexes are recognized by CD8+ Tcells . CD4+ Tcells Antigen binding site is present between α 1/ α 2 groove Present between α 1/ β 1 groove.

19. Functions of different antigen-presenting cells.

20. Antigen is delivered from tissues to the lymph node via the afferent lymphatics.

21. Haptens: Haptens are low molecular weight molecules that lack immunogenicity but retain antigenicity or immunological reactivity (i.e. can bind to their specific antibody or T cell receptor). Haptens may be classified as complex or simple: Complex haptens contain two or more epitopes; they can react with specific antibodies and the hapten-antibody complex can be visualized by various methods such as precipitation reaction. Simple haptens usually contain only one epitope (univalent). Such haptens can bind to the antibodies but the hapten antibody complex cannot be visualized, as it is believed that precipitation reaction to occur, it requires the antigen to have at least two or more epitopes.

22. Haptens can activate B cells when covalently bound to a carrier protein. When bound with a carrier molecule, they form an immunogenic hapten-carrier conjugate. When lymphocytes are stimulated by combined hapten carrier molecules, they can react to either the hapten or the larger carrier molecule. One example of a hapten is penicillin. By itself, penicillin is a small molecule that is not antigenic. However, when it is combined with certain serum proteins in sensitive individuals, it becomes immunogenic, activates lymphocytes, and initiates a severe and sometimes fatal allergic immune reaction. In these instances, the hapten is acting as an antigenic determinant or epitope.

23. ANTIGEN-HOST RELATIONSHIP Based on the antigen-host relationship, antigens can be grouped into two groups as follows: 1 . Self or autoantigens : They belong to the host itself; hence they are not immunogenic. I mmunological tolerance . However, sometimes, the self antigens are biologically altered (e.g. as in cancer cells) and can become immunogenic.

24. 2. Non-self or foreign antigens : They are immunogenic and are of three types based on their phylogenetic distance to the host: Alloantigens are species specific. Tissues of all individual in a species contain species-specific antigens. Isoantigens are type of antigens which are present only in subsets of a species, e.g. blood group antigens and histocompatibility antigens. The histocompatibility antigens are highly specific as they are unique to every individual of a species. Heteroantigens : Antigens belonging to two different species are called heteroantigens , e.g. antigens of plant or animal or microorganisms, etc.

25. Heterophile Antigens A type of heteroantigens that are present in two different species; but they share epitopes with each other. Antibody produced against antigen of one species can react with the other and vice versa. Diagnostic Application Heterophile antigens can be used in various serological tests. Antibody against one antigen can be detected in patient’s serum by employing a different antigen which is heterophile (cross reactive) to the first antigen. For example: 1.Weil-Felix reaction is done for typhus fever. Antibodies against rickettsial antigens are detected by using cross reacting Proteus antigens (OXK, OX2, and OX19)

26. 2. Paul-Bunnell test is done for infectious mononucleosis (caused by Epstein-Barr virus). Here, sheep red blood cell (RBC) antigens are used to detect cross-reacting antibodies in patient’s sera. 3. Cold agglutination test and Streptococcus MG test are done for primary atypical pneumonia. Here, antibodies against Mycoplasma pneumoniae are detected by using human O blood group RBCs and Streptococcus MG antigens respectively.

27. Superantigens: Superantigens Are a Special Class of T-Cell Activators Superantigens are the third variety of biological class of antigens. The unique feature of superantigens is, they can activate T cells directly without being processed by antigen-presenting cells (APCs).  In a normal adaptive immune response, only around 0.0001% of T-cells are activated. In contrast, superantigen exposure can activate up to 30% of the T-cell pool, leading to severe pathologies following infection. The variable β region of T cell receptor (vβ of TCR) appears to be the receptor for superantigens. They directly bridge non-specifically between major histocompatibility complex (MHC)-II of APCs and T cells. Non-specific activation of T cells leads to massive release of cytokines known as “cytokine storm,” which include inflammatory mediators such as interferon γ, IL-1, IL-6, TNF- α, and TNF- β

28. Superantigen-mediated cross-linkage of T cell receptor and class II MHC molecules. A superantigen binds to all TCRs bearing a particular V sequence regardless of their antigen specificity. Exogenous superantigens are soluble secreted bacterial proteins, including various exotoxins. Endogenous superantigens are membrane-embedded proteins produced by certain viruses; they include minor lymphocyte-stimulating ( Mls ) antigens encoded by mouse mammary tumor virus.

29. MICROBES CAN ALSO PROVIDE bcell superantigens: - B-cell super antigens can hyper stimulate a large population of B-cells without necessarily having the ability to crosslink TCRs with MHC Class II receptors; they therefore have a different mechanism and specificity compared to T-cell superantigens. - SpA -IgG is thought to form a ‘lattice’ structure around the B-cells by crosslinking BCR Fab with IgG Fc and other BCR Fab regions promoting a sustained stimulation. - Expression of B-cell superantigens ultimately leads to B-cell depletion and evasion of the immune system: in this sense, they can be considered as virulence factors

30. SUPERANTIGEN APPLICATIONS: 1 . Diagnostics Potential : Superantigens are used to detect IgG in serum, making use of their immunoglobulin binding specificity. Engineering of superantigens to be specific to regions of TCRs or antibody V-region families or isotypes for the development of diagnostic kits could be applied to the quantification of disease associated proteins e.g. IgE in allergy. The ability to specifically bind antibodies can also allow its development in easy-to-use, non technical point-of-care testing home-use devices, as recently applied during the COVID-19 pandemic. Such superantigen-based diagnostics can be coupled with colorimetric, home-made devices [e.g. mobile spectrophotometers]. Given the increasing association of antibody VH families with certain diseases e.g. [VH5 in nickel allergy], superantigens that can differentiate antibody VH families have clear potential in diagnostic kit development.

31. 2. Therapeutic Potential : -Superantigens have also shown promise in the treatment of cancer through a synergistic effect with antibodies in the recruitment of T-cells. - - The SAg Staphylococcal enterotoxin A (SEA) when co-cultured with human peripheral blood mononuclear cells (PBMCs) inhibited the proliferation and induced the death of human lung carcinoma A549 cells24. - The Staphylococcal enterotoxin B (SEB) is an efficient activator of the antitumor immune response that leads to the eradication of tumor growth and inhibition of metastasis25. SEB-Superantigen-activated PBMC (peripheral blood mononuclear cells) significantly induced apoptosis in transitional cell carcinoma cells (TCC).

32. Superantigens. Bacterial superantigen SUPERANTIGENS BACTERIAL SUPERANTIGEN Staphylococcal toxin : tsst-1, exfoliative toxin, enterotoxins Streptococcal pyrogenic exotoxin (SPE)-a and c Mycoplasma arthritidis mitogen-I (MAM) Yersinia enterocolitica Yersinia pseudotuberculosis mitogen ( 2. VIRAL SUPERANTIGENS Epstein- barr virus associated superantigen CMV associated superantugen Rabies nucleocapsid HIV Encoded superantigen 3. FUNGAL SUPERANTIGEN Malassezia furfur

33. Tumor antigens: Tumor antigens stimulate specific adaptive immune responses that can prevent or limit the growth and spread of the tumors. The first experimental demonstration that tumors can induce protective immune responses came from studies of transplanted tumors performed in the 1950s. A sarcoma may be induced in an inbred mouse by painting its skin with the chemical carcinogen methylcholanthrene (MCA). If the MCA induced tumor is excised and transplanted into other syngeneic mice, the tumor grows. In contrast, if cells from the original tumor are transplanted back into the original host, the mouse rejects this transplant and no tumor grows. The same mouse that has become immune to its own tumor does not reject MCA-induced tumors produced in other mice, which have different MCA-induced mutations and express different tumor antigens. Furthermore, transfer of T cells from the tumor-bearing animal to a tumor free animal can impart protective immunity against that tumor. Thus, immune responses to these tumors exhibit the defining characteristics of adaptive immunity—that is, specificity, memory, and a key role of lymphocytes .

34. The tumor antigens that elicit T cell immune responses can be classified into : 1. Neoantigens: Antigens Encoded by Mutated Genes Tumor neoantigens are proteins encoded by mutated genes, which appear foreign to the immune system because they do not exist in normal cells and newly arise as a cancer develops. 2. Antigens of Oncogenic Viruses The products of oncogenic viruses function as tumor antigens and elicit specific T cell responses that may serve to eradicate virus-induced tumors.

35. 3.Overexpressed Cellular Proteins Some tumor antigens are the products of genes that are silenced in normal cells and derepressed in tumor cells or are proteins made by normal cells but produced in excessive amounts by tumors. 4. Oncofetal Antigens The two most studied oncofetal antigens are carcinoembryonic antigen (CEA) and α-fetoprotein (AFP).

36. Transplantation antigens The antigens that determine the histocompatibility are of two types: 1. ABO blood group antigens Present on RBCs, epithelial cells, endothelial cells Hence if donor carries antibodies to any of these antigens, the graft will induce a rapid antibody mediated response leading to its necrosis. for this, transplants done with ABO compatible individuals. 2. MHC antigens: Inherited as a complete set called halotype from each parent. In a given population, there is a great degree of MHC polymorphism In a outbred population, it is must that screening for MHC-antigens matching be done before transplantation. This is called tissue typing.

37. Hla: The MHC in humans is known as human leukocyte antigens (HLA) complex . In humans, these alloantigens are present on the surface of leukocytes and are called HLA and the set of genes encoding for them is named the HLA complex. HLA complex of genes is located on short arm of chromosome 6 containing several genes critical to immune function. They are classified into three classes as follows: 1. Class I: HLA-A, HLA-B, and HLA-C. 2. Class II: HLA-DR, HLA-DQ, and HLA-DP. All of these a re present within HLA-D region of HLA complex. 3. Class III: Complement loci that encode for C2, C4, and factor B of complement system and TNFs alpha and beta.

38. HLA loci are usually multiallelic. There are 24 alleles at HLA-A locus and 50 at HLA-B locus. Each allele expresses for a distinct antigen. HLA system is pleomorphic and every person inherits one set of HLA genes from each parent.

39. Hla typing: HLA typing or tissue typing are usually performed to determine the closest MCH match between the donors and recipients before performing transplantation surgery. The methods commonly used in the laboratory include ( a ) molecular methods using DNA sequence, ( b ) serological assays, and ( c ) mixed lymphocyte culture (MLC) techniques. All these methods are used to determine the haplotype, i.e., the class I and class II alleles on both chromosomes of both the donor and recipient. DNA probe and PCR are highly specific and sensitive methods used to detect the different alleles. Serological assays using a battery of antibodies specific for a different class I and class II proteins are also used to demonstrate the alleles. If these two methods fail to provide sufficient data, then additional information can be obtained by performing the MLC technique,also known as mixed lymphocyte reaction.

40. microcytotoxicity method .

41. HLA typing is carried out: ■ usually before tissue transplantation, ■ for determination of paternity in case of dispute, and ■ for finding association of HLA with diseases, such as association of HLA-B27 with ankylosing spondylitis and HLA-DR4 with rheumatoid arthritis, HLA-DR5 with Hashimoto’s thyroiditis etc.

42. Detection methods: 1. EIAs for Antigen Detection: Antigen-capture EIAs are available for the detection of diverse microbial pathogens or virulence factors in several specimen types such as: Diarrheal stool (e.g., EIAs for Shiga-like toxins of enterohemorrhagic E. coli, organism-specific antigens of Giardia , Cryptosporidium , and Helicobacter pylori, rotavirus antigen, and Clostridium difficile antigens and toxins), Urine (EIAs for L. pneumophila serogroup 1 antigen), and Serum ( Aspergillus galactomannan [GM] and HIV-1 p24 antigen).

43. EIA antigen-capture technique for H. pylori . In this technique, antibody directed against the antigen to be detected is bound to the solid phase. S tep 1: A stool specimen is added to the well Antigens present in the sample are “captured” by the solid-phase antibody. S tep 2: After a wash step, anti -H. pylori antibody that is conjugated to an enzyme is added and reacts with the solid-phase antibody-bound antigen. S tep 3: After another wash step, enzyme substrate is added and a visible color is detected.

44. 2. LATERAL FLOW IMMUNOASSAY . The specimen (e.g., serum, urine) containing the analyte to be detected is placed on the sample pad, which soaks up the specimen fluid. The fluid then migrates to the conjugate pad, which contains antibodies directed against the analyte. These antibodies are conjugated with gold, colored latex, or a chromophore. The analyte is captured by the antibodies on the conjugate pad and then migrates across a nitrocellulose membrane toward the “test” line. As more and more analyte–antibody complexes are captured at the “test” line, the line becomes visible on the membrane. Antibodies that are not analyte-specific are not captured and continue to migrate toward the “control line.” The control line is composed of immobilized antibodies directed against immunoglobulin As more and more uncomplexed antibody passes over the “control” line, the uncomplexed antibodies are captured and become visible at the “control” line. The presence of a “control” line only indicates that the test was performed properly and that the analyte is absent from the specimen. Lines at both the test and control areas indicate a positive test. A single line in the test area without a corresponding control line is an invalid test.

45. 3.Immunofluorescence Techniques for Antigen Detection  : In the DFA method, the antigen (e.g., respiratory specimens for RSV) is placed in the well of an FA slide and reacted directly with a fluorescein-conjugated monoclonal antibody directed against the antigen. After incubation and washing, the slide is examined for characteristic fluorescence. Reagents for either DFA or IFA detection of antigens are commercially available for a variety of agents, including HSVs, VZV, respiratory viruses (i.e., influenza, RSV, parainfluenza viruses), Pneumocystis jirovecii , G. lamblia , and C. parvum.

46. indirect immunofluorescence assay (IFA) for antigen detection. In this method, the specimen (e.g., sputum for Legionella ) is reacted with an excess of unlabeled antibodies directed against the antigen. After a wash step, fluorescein-conjugated antibodies directed against the species of antibody used in the initial reaction (e.g., fluorescein-labeled antirabbit IgG raised in goats) are overlaid on the FA slide. After washing, the slide is examined for specific fluorescence. With Legionella , for example, unlabeled rabbit antibodies against a large number of serotypes can be used for the first step in the procedure, but only a single fluorescein-conjugated goat antirabbit Ig is required for the second step. If Legionella organisms were to be detected by a DFA method, separate fluorescein-labeled conjugates for each serotype would be required.

47. Viral Antigens *Hepatitis B surface antigen (HBsAg): HBsAg was previously called Australia antigen as it was first observed in the serum of an Australian Aboriginal person (1965). HBsAg is antigenically complex and contains two components— common group reactive antigen ‘a’ epitope. two pairs of type specific antigens d/y and w/r; only one of each pair being present at a time. Thus, there are four subtypes of HBsAg— adw , ayw , adr , and ayr . *Hepatitis B core antigen ( HBcAg ): HBcAg forms the intracellular core protein. It is not secreted and does not circulate in blood, but can be demonstrated in hepatocytes by immunofluorescence. *Hepatitis B precore antigen ( HBeAg ): HBeAg is a nonparticulate soluble antigen possessing a signal protein which enables it to be secreted. It is therefore present in circulation. -The major determinants for infectivity of HBV are located in the N terminal 55 amino acids of L (envelope protein).

48. NS1 ANtigen - NS1, a protein that is absent from the virion, is secreted at high levels into the extracellular environment during flavivirus infection, predominantly as a hexamer, with significant accumulation (up to 50 mg/ml) in the sera of DENV-infected patients. - In addition, soluble NS1 can bind back to the plasma membrane of cells through an interaction with speciic sulfated glycosaminoglycans. Furthermore, NS1 is expressed directly on the surface of infected cells, possibly via glycosyl phosphatidyl inositol (GPI) linkage, lipid raft association, or through an as-yet undeined mechanism. - ELISA and ICT formats are available for detecting NS1 antigen in serum. They gained recent popularity because of the early detection of the infection. -NS1 antigen becomes detectable from day 1 of fever and remains positive up to 18 days -Highly specific: It differentiates between flaviviruses. It can also be specific to different dengue serotypes.

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