Interferons

Discovery of Interferons
1957 Isaacs and Lindenmann Did an experiment using chicken cell cultures Found a substance that interfered with viral replication and was therefore named interferon Nagano and Kojima also independently discovered this soluble antiviral protein

What are Interferons?

Naturally occurring proteins and glycoproteins Secreted by eukaryotic cells in response to viral infections, tumors, and other biological inducers Produce clinical benefits for disease states such as hepatitis, various cancers, multiple sclerosis, and many other diseases Strucurally, they are part of the helical cytokine family which are characterized by an amino acid chain that is 145-166 amino acids long

General Action of Interferons

Interferons are small proteins released by macrophages, lymphocytes, and tissue cells infected with a virus. When a tissue cell is infected by a virus, it releases interferon. Interferon will diffuse to the surrounding cells. When it binds to receptors on the surface of those adjacent cells, they begin the production of a protein that prevents the synthesis of viral proteins. This prevents the spread of the virus throughout the body.

Three types of interferons: alpha, beta and gamma.

Induction of Interferon
dsRNA Most RNA viruses Some DNA viruses Lipopolysaccharide (LPS) Other components of certain bacteria Mycoplasma Protozoa

Type I Interferons
Type I: alpha and beta
Alpha interferons are produced by leukocytes Beta interferons are produced by fibroblasts Both bind to interferon cell receptors type 1 and both encoded on chromosome 9 They have different binding affinities but similar biological effects Viral infection is the stimulus for alpha and beta expression Used to mobilize our 1st line of defense against invading organisms Largest group and are secreted by almost all cell types

 Type II interferon
Interferon g Made by cells of immune system (NK, T cells) Have anti-viral actions Immuno-regulatory functions

Interferon Genes
IFN-a : 20 intronless genes on chromosome 9 166 amino acids Stable at pH 2

IFN-b : One single intronless gene on chromosome 9 30-45% of homology for amino acids and nucleotides with IFN-a

IFN-g : One single gene with three introns on chromosome 12 Acid labile Share little homology with type I interferons

The exact mechanism of type I interferons are not fully understood, but this is an idea of what happens:
Alpha and beta bind to heterodimeric receptor on cell surface. Alpha receptor is made up of at least 2 polypeptide chains: IFNa-R1 and IFNa-R2 IFNa-R1 is involved in signal transduction IFNa-R2 is the ligand-binding chain that also plays a role in signal transduction Ligation induces oligomerisation and initiation of the signal transduction pathway This results in phosphorylation of signal transductors and activators of transcription proteins, which translocate to the nucleous as a trimeric complex, ISGF-3. ISGF-3 activates transcription of interferon stimulated genes, with many biological effects.

Type II Interferon (gamma)

Bind to type 2 receptors and its genes are encoded on chromosome 12

Initially believed that T helper cell type 1 lymphocytes, cytotoxic lymphocytes and natural killer cells only produced IFNg, now evidence that B cells, natural killer T cells and professional antigen-presenting cells secrete IFNg also. Gamma production follows activation with immune and inflammatory stimuli rather than viral infection.
This production is controlled by cytokines interleukin 12 and 18.

Interferon Gamma Receptor

Composed of two ligand binding IFNg-R1 chains associated with two signal transducing IFNg-R2 chains The IFNg-R2 chain is generally the limiting factor in IFNg responsiveness, as the IFNg-R1 chain is usually in excess. The IFNg-R1 intracellular domain contains binding spots for the Jak 1, latent cytosolic factor, signal transducer and activator of transcription (Stat1).

Interferon Gamma Receptor and Signalling Pathway

Receptors are encoded by separate genes (IFNGR1 and IFNGR2, respectively) that are located on different chromosomes.
As the ligand-binding (or a) chains interact with IFN-g they dimerise and become associated with two signal-transducing chains. Receptor assembly leads to activation of the Janus kinases JAK1 and JAK2 and phosphorylation of a tyrosine residue on the intracellular domain of IFN-gR1. This leads to the recruitment and phosphorylation of STAT1, which forms homodimers and translocates to the nucleus to activate a range of IFN-g-responsive genes. After this, the ligand-binding chains are internalised and dissociate. The chains are then recycled to the cell surface.

Jak-STAT Pathway
Interferon a/b and g Jak (Janus Kinase) family of protein tyrosine kinases ( Jak1, Jak2 and Tyk2) STAT (Signal Transducers and Activators of Transcription) family (STAT1 and 2) Interferon Stimulated Response Elements (ISRE) Cis-acting DNA sequence Regulate IFN inducible gene expression

Action of Interferons

2,5 oligoadenylate synthetase (2-5 OAS)

Minimal basal expression Increase 10-1000 fold in response to IFN In the presence of ds RNA (formed during virus infection), synthesize 2-5-linked oligoadenylates [2-5 (A)] 2-5 (A) binds and dimerizes inactivated RNase L (latent cellular endoribonuclease) to become activated Activated RNase L degrades mRNA Block viral protein synthesis Inhibits virus replication

Double-stranded RNA-dependent Protein Kinase (PKR)

All cells contain basal level IFN induces PKR (inactivated) In the presence of ds RNA, PKR becomes autophosphorylated and activated Activated PKR then phosphorylate eIF-2a (initiation factor of protein synthesis) and render it inactivate Inhibit protein synthesis ( cellular or viral proteins) Inhibit virus replication

Mx protein
Highly conserved in mammals, birds and fish Produced by mouse cells after treatment with IFN Specifically protect mouse from influenza virus infection, but not other viruses IFN turn on specific gene that express specific protein and block specific virus replication

Applications of IFN
Anti-viral agent Negative cell growth regulator Immunomodulator Inhibitors of non-viral pathogens

Immunomodulation effects
Activate NK cells Increase MHC class I expression IFN-g activate macrophages kill cells Increase MHC II expression, antigen presentation to helper T cell

Treatment of non-viral diseases

Activation of macrophages
Toxoplasmosis Leishmaniasis

Anti-proliferative effects
Melanoma, Kaposis sarcoma

Cloned and expressed IFN

IFN genes cloned into plasmid and produced in large quantity IFN action is very cell species specific

Treatment of viral diseases

Hairy cell leukemia ( caused by HTLV-2) Hepatitis B virus Hepatitis C virus Human papillomavirus (warts) Human Rhinovirus Herpesvirus

Side effects of IFN treatment

Fever, malaise, fatigue, muscle pains High level toxicity Kidney, liver, bone marrow and heart

Different Interferon Drugs

Interferons are broken down into recombinant versions of a specific interferon
subtype and purified blends of natural human interferon. Many of these are in clinical use and are given intramuscularly or subcutaneously Recombinant forms of alpha interferon include: Alpha-2a drug name Roferon Alpha-2b drug name Intron A

Alpha-n1 drug name Wellferon

Alpha-n3 drug name AlferonN Alpha-con1 drug name Infergen Recombinant forms of beta interferon include:

Beta-1a drug name Avonex

Beta-1b drug name Betaseron Recombinant forms of gamma interferon include: Gamma-1b drug name Acimmune

Alpha Interferon-2a (Roferon A)

Protein chain that is 165 amino acids long Produced using recombinant DNA technology Non-glycosylated protein Short half life, short terminal elimination of half life, a large volume of distribution, and a larger reduction in renal clearance. These problems were resolved by pegylating alpha-2a resulting in peginterferon alpha-2a that is named Pegasys.

Pegylated Interferon-2a (Pegasys)

Pegasys is recombinant interferon alpha-2a that is covalently conjugated with bismonomethoxy polyethylene glycol (PEG)

Background:
First developed by Davis, Abuchowski and colleagues in the 1970s
In early 1990s PEG attached to alpha-2a, but it lacked the required profile of improving pharmacokinetics Pegylation of interferon alpha-2b was achieved with the addition of a linear PEG, designed to degrade to allow the full potency of the interferon, while achieving a longer half-life.

Structure:
PEG moieties are inert, longchain amphiphilic molecules that are produced by linking repeating units of ethylene oxide. Can be linear or branched in their structure

Increasing the size with PEG, the absorption and life are prolongued and the clearance of the IFN is decreased.
Goal of pegylation is to decrease clearence, retention of biological activity, get a stable linkage and enhance water solubility

CH3(OCH2CH2)n--OH
O

Pegylation is achieved by
the covalent attachment of PEG derivatives that utilize amino groups of lysines and the N-terminus of polypeptide molecules as the modification site

mPEGOO2CCNH mPEGOO2CNH(CH2)4

Interferon Beta-2a (Avonex)

FDA approval on May 17 1996 for Relapsing
Remitting MS Clinical trials showed that it slowed MS progression and had an extra benefit of slowing or preventing the development of MS-related brain atropy. The exact mechanism of IFN beta activity in treating MS is unknown, but studies have shown that interlukin 10 levels in the cerebrospinal fluid were increased in patients Structurally IFNb-2a is a 166 amino acid glycoprotein. Produced by recombinant DNA technology. Amino acid sequence is the same as human beta interferon. They are both glycosylated at the asparagines residue at position 80

Some side effects include:

Flu-like symptoms Muscle aches

Chills

Conclusion
Interferons have overlapping but different biological activities Their mechanisms of action are not fully understood, therefore there is a lot of room for future growth within this field Interferon based strategies can possibly be further tailored to each individual patient according to early response dynamics

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