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Superantigen

Superantigens (SAgs) are a class of antigens which cause non-specific activation of T-cells resulting in polyclonal T cell activation and massive cytokine release. SAgs can be produced by pathogenic microbes (including viruses, mycoplasma, and bacteria) as a defense mechanism against the immune system. Compared to a normal antigen-induced T-cell response where .0001.001% of the bodys T-cells are activated, these SAgs are capable of activating up to 25% of the bodys T-cellsFurthermore, Anti-CD3 and Anti-CD28 Antibodies (CD28-SuperMAB) have also shown to be highly potent superantigens (and can activate up to 100% of T cells). The large number of activated T-cells generates a massive immune response which is not specific to any particular epitope on the SAg thus undermining one of the fundamental strengths of the adaptive immune system, that is, its ability to target antigens with high specificity. More importantly, the large number of activated T-cells secrete large amounts of cytokines, the most important of which is Interferon gamma. This excess amount of IFN-gamma in turn activates the macrophages. The activated macrophages, in turn, over-produce proinflammatory cytokines such as IL-1, IL-6 and TNF-alpha. TNF-alpha is particularly important as a part of the body's inflammatory response. In normal circumstances it is released locally in low levels and helps the immune system defeat pathogens. However when it is systemically released in the blood and in high levels (due to mass T-cell activation resulting from the SAg binding), it can cause severe and life-threatening symptoms, including shock and multiple organ failure.

Structure
SAgs are produced intracellularly by bacteria and are released upon infection as extracellular mature toxins. The sequences of these toxins are relatively conserved among the different subgroups. More important than sequence homology, the 3D structure is very similar among different SAgs resulting in similar functional effects among different groups. Crystal structures of the enterotoxins reveals that they are compact, ellipsoidal proteins sharing a characteristic two-domain folding pattern comprising an NH2-terminal barrel globular domain known as the oligosaccharide / oligonucleotide fold, a long -helix that diagonally spans the center of the molecule, and a COOH terminal globular domain.[ The domains have binding regions for the Major Histocompatibility Complex Class II (MHC Class II) and the T cell receptor (TCR), respectively.

Binding
Superantigens bind first to the MHC Class II and then coordinate to the variable alpha or beta chain of T-cell Receptors (TCR)

MHC Class II
SAgs show preference for the HLA-DQ form of the molecule. Binding to the -chain puts the SAg in the appropriate position to coordinate to the TCR. Less commonly, SAgs attach to the polymorphic MHC class II -chain in an interaction mediated by a zinc ion coordination complex between three SAg residues and a highly conserved region of the HLA-DR chain. The use of a zinc ion in binding leads to a higher affinity interaction. Several staphylococcal SAgs are capable of cross-linking MHC molecules by binding to both the and chains. This mechanism stimulates cytokine expression and release in antigen presenting cells as well as inducing the production of costimulatory molecules that allow the cell to bind to and activate T cells more effectively.

T-cell receptor
T-cell binding region of the SAg interacts with the Variable region on the Beta chain of the Tcell Receptor. A given SAg can activate a large proportion of the T-cell population because the human T-cell repertoire comprises only about 50 types of V elements and some SAgs are capable of binding to multiple types of V regions. This interaction varies slightly among the different groups of SAgs.[6] Variability among different people in the types of T-cell regions that are prevalent explains why some people respond more strongly to certain SAgs. Group I SAgs contact the V at the CDR2 and framework region of the molecule. SAgs of Group II interact with the V region using mechanisms that are conformation-dependent. These interactions are for the most part independent of specific V amino acid side-chains. Group IV SAgs have been shown to engage all three CDR loops of certain V forms. The interaction takes place in a cleft between the small and large domains of the SAg and allows the SAg to act as a wedge between the TCR and MHC. This displaces the antigenic peptide away from the TCR and circumvents the normal mechanism for T-cell activation. The biological strength of the SAg (its ability to stimulate) is determined by its affinity for the TCR. SAgs with the highest affinity for the TCR elicit the strongest response. SPMEZ-2 is the most potent SAg discovered to date.[

T-cell signaling
The SAg cross-links the MHC and the TCR inducing a signaling pathway that results in the proliferation of the cell and production of cytokines. Low levels of Zap-70 have been found in Tcells activated by SAgs, indicating that the normal signaling pathway of T-cell activation is impaired It is hypothesized that Fyn rather than Lck is activated by a tyrosine kinase, leading to the adaptive induction of anergy. Both the protein kinase C pathway and the protein tyrosine kinase pathways are activated, resulting in upregulating production of proinflammatory cytokines
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This alternative signaling pathway impairs the calcium/calcineurin and Ras/MAPkinase pathways slightly, but allows for a focused inflammatory response.

Direct effects
SAg stimulation of antigen presenting cells and T-cells elicits a response that is mainly inflammatory, focused on the action of Th1 T-helper cells. Some of the major products are IL-1, IL-2, IL-6, TNF-, gamma interferon (IFN-), macrophage inflammatory protein 1 (MIP-1), MIP-1, and monocyte chemoattractant protein 1 (MCP-1). This excessive uncoordinated release of cytokines, (especially TNF-), overloads the body and results in rashes, fever, and can lead to multi-organ failure, coma and death. Deletion or anergy of activated T-cells follows infection. This results from production of IL-4 and IL-10 from prolonged exposure to the toxin. The IL-4 and IL-10 downregulate production of IFN-gamma,MHC Class II, and costimulatory molecules on the surface of APCs. These effects produce memory cells that are unresponsive to antigen stimulation. One mechanism by which this is possible involves cytokine-mediated suppression of T-cells. MHC crosslinking also activates a signaling pathway that suppresses hematopoiesis and upregulates Fas-mediated apoptosis. IFN- is another product of prolonged SAg exposure. This cytokine is closely linked with induction of autoimmunity, and the autoimmune disease Kawasaki Disease is known to be caused by SAg infection. SAg activation in T-cells leads to production of CD40 ligand which activates isotype switching in B cells to IgG and IgM and IgE. To summarize, the T-cells are stimulated and produce excess amounts of cytokine resulting in cytokine-mediated suppression of T-cells and deletion of the activated cells as the body returns to homeostasis. The toxic effects of the microbe and SAg also damage tissue and organ systems, a condition known as Toxic Shock Syndrome. If the initial inflammation is survived, the host cells become anergic or are deleted, resulting in a severely compromised immune system.

Superantigenicity independent (indirect) effects


Apart from their mitogenic activity, SAgs are able to cause symptoms that are characteristic of infection. One such effect is emesis. This effect is felt in cases of food poisoning, when SAg-producing bacteria release the toxin, which is highly resistant to heat. There is a distinct region of the molecule that is active in inducing gastrointestinal toxicity.[ This activity is also highly potent, and quantities as small as 20-35ug of SAg are able to induce vomiting.

SAgs are able to stimulate recruitment of neutrophils to the site of infection in a way that is independent of T-cell stimulation. This effect is due to the ability of SAgs to activate monocytic cells, stimulating the release of the cytokine TNF-, leading to increased expression of adhesion molecules that recruit leukocytes to infected regions. This causes inflammation in the lungs, intestinal tissue, and any place that the bacteria have colonized. While small amounts of inflammation are natural and helpful, excessive inflammation can lead to tissue destruction. One of the more dangerous indirect effects of SAg infection concerns the ability of SAgs to augment the effects of endotoxins in the body. This is accomplished by reducing the threshold for endotoxicity. Schlievert demonstrated that, when administered conjunctively, the effects of SAg and endotoxin are magnified as much as 50,000 times. This could be due to the reduced immune system efficiency induced by SAg infection. Aside from the synergistic relationship between endotoxin and SAg, the double hit effect of the activity of the endotoxin and the SAg result in effects more deleterious that those seen in a typical bacterial infection. This also implicates SAgs in the progression of sepsis in patients with bacterial infections.[

Adjuvant.
An adjuvant (from Latin, adiuvare: to aid) is a pharmacological and/or immunological agent that modifies the effect of other agents. Adjuvants are inorganic or organic chemicals, macromolecules or entire cells of certain killed bacteria, which enhance the immune response to an antigen. They may be included in a vaccine to enhance the recipient's immune response to the supplied antigen, thus minimizing the amount of injected foreign material. Adjuvants are also used in the production of antibodies from immunized animals. The most commonly used adjuvants include aluminum hydroxide and paraffin oil.

Immunologic adjuvants
Immunologic adjuvants are added to vaccines to stimulate the immune system's response to the target antigen, but do not in themselves confer immunity. Adjuvants can act in various ways in presenting an antigen to the immune system. Adjuvants can act as a depot for the antigen, presenting the antigen over a long period of time, thus maximizing the immune response before the body clears the antigen. Examples of depot type adjuvants are oil emulsions. Adjuvants can also act as an irritant which causes the body to recruit and amplify its immune response. A tetanus, diphtheria, and pertussis vaccine, for example, contains minute quantities of toxins produced by each of the target bacteria, but also contains some aluminium hydroxide.Such aluminium salts are common adjuvants in vaccines sold in the United States and have been used in vaccines for over 70 years. The body's immune system develops an antitoxin to the bacteria's toxins, not to the aluminium, but would not respond enough without the help of the aluminium adjuvant.

Mechanisms of adjuvants
Adjuvants are needed to improve the routing and adaptive immune responses to antigens. This reaction is mediated by two main types of lymphocytes, B and T cells. Adjuvants can apply their effects through different mechanisms. Some adjuvants, such as alum, function as delivery systems by generating depots that trap antigens at the injection site, providing slow release in order to continue the stimulation of the immune system.

Adjuvants as stabilizing agents


Although immunological adjuvants have traditionally been viewed as substances that aid the immune response to antigen, adjuvants have also evolved as substances that can aid in stabilizing formulations of antigens, especially for vaccines administered for animal health.

Types of adjuvants

Inorganic compounds: aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, beryllium Mineral oil: paraffin oil Bacterial products: killed bacteria Bordetella pertussis, Mycobacterium bovis, toxoids Nonbacterial organics: squalene, thimerosal Delivery systems: detergents (Quil A) Cytokines: IL-1, IL-2, IL-12 Combination: Freund's complete adjuvant, Freund's incomplete adjuvant

The mechanism of immune stimulation by adjuvants


Adjuvants can enhance the immune response to the antigen in different ways:

extend the presence of antigen in the blood help absorb the antigen presenting cells antigen activate macrophages and lymphocytes support the production of cytokines

Alum as an adjuvant
Alum is the most commonly used adjuvant in human vaccination. It is found in numerous vaccines, including diphtheria-tetanus-pertussis, human papillomavirus, and hepatitis vaccines.