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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 mechanismagainst the immune system. Compared to a normal antigen-induced T-cell response where .0001-

.001% of the body’s T-cells are activated, these SAgs are capable of activating up to 25% of the

 body’s T-cellsFurthermore, Anti-CD3 and Anti-CD28 Antibodies (CD28-SuperMAB) have alsoshown 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 notspecific 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 suchas 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 theimmune 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. Moreimportant 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 β barrel globular domain

known as the oligosaccharide / oligonucleotide fold, a long α-helix that diagonally spans thecenter 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)

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MHC Class II

SAgs show preference for the HLA-DQ form of the molecule. Binding to the α-chain puts the

SAg in the appropriate position to coordinate to the TCR.

Less commonly, SAgs attach to the  polymorphic MHC class II β-chain in an interactionmediated by a zinc ion coordination complex between three SAg residues and a highly conserved

region of the HLA-DR  β 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 α and β chains. This mechanism stimulates cytokine expression and release inantigen 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 T-

cell Receptor. A given SAg can activate a large proportion of the T-cell population because thehuman T-cell repertoire comprises only about 50 types of Vβ elements and some SAgs are

capable of binding to multiple types of Vβ regions. This interaction varies slightly among the

different groups of SAgs.[6]

  Variability among different people in the types of T-cell regions thatare prevalent explains why some people respond more strongly to certain SAgs. Group I SAgs

contact the Vβ at the CDR2 and framework region of the molecule. SAgs of Group II interact

with the Vβ region using mechanisms that are conformation-dependent. These interactions are

for the most part independent of specific Vβ amino acid side-chains. Group IV SAgs have beenshown to engage all three CDR loops of certain Vβ 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 theTCR. 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 T-cells 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 mainlyinflammatory, focused on the action of Th1 T-helper cells. Some of the major products are IL-1, 

IL-2,  IL-6, TNF-α, gamma interferon (IFN-γ), macrophage inflammatory protein 1α (MIP-1α),

MIP-1β, and monocyte chemoattractant protein 1 (MCP-1).

This excessive uncoordinated release of cytokines, (especially TNF-α), 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-α is another product of prolonged SAg exposure. This

cytokine is closely linked with induction of autoimmunity, and the autoimmune diseaseKawasaki Disease is known to be caused by SAg infection.

SAg activation in T-cells leads to production of CD40 ligand which activates isotype switchingin B cells to IgG and IgM and IgE. 

To summarize, the T-cells are stimulated and produce excess amounts of cytokine resulting incytokine-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.

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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-α, leading to increased expression of adhesionmolecules 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 thresholdfor 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 SAgresult 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 toan 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. Atetanus,  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'stoxins, not to the aluminium, but would not respond enough without the help of the aluminiumadjuvant.

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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 inorder 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 phosphatehydroxide, 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.