Human Interferon Genes

HIG
THE INTERFERONS…..

• Interferons (IFNs) were the first family of cytokines to be

discovered.
• In 1957 researchers observed that if susceptible animal cells were
exposed to a colonizing virus, these cells immediately become
resistant to attack by other viruses.
• This resistance was induced by a substance secreted by virally-
infected cells, which was named ‘interferon’ (IFN).
• Humans produce at least three distinct classes, IFN-a, IFN-b and
IFN-g.
• Biological effects, Induction of cellular resistance to viral attack.
• Regulation of most aspects of immune function.
• Regulation of growth and differentiation of many cell types.
• Sustenance of early phases of pregnancy in some animal species.
 Interferon
• Interferons are an extraordinary group of protein with important
effects on immune system.
• Their actions affect both innate and adaptive immunity.
• It includes induction of an increase in expression of both Class I
MHC (IFN α and IFN β) and Class II MHC (IFN γ) molecules and
augmentation of NK cell activity.
• Interferon was named for its ability to interfere with viral
proliferation.
• The various forms of interferon are the body’s most rapidly
produced and important defence against viruses.
• Interferon can also combat bacterial and parasitic infections,
inhibit cell division, and promote or impede the differentiation of
cells.
• They are produced by all vertebrate animals and possibly by
some invertebrates as well.
• Interferons are categorized as cytokines, small proteins that are
involved in intercellular signalling.
• Interferon is secreted by cells in response to stimulation by a virus or
other foreign substance, but it does not directly inhibit the virus’s
multiplication.
• Rather, it stimulates the infected cells and those nearby to produce
proteins that prevent the virus from replicating within them.
• Further production of the virus is thereby inhibited and the infection
is stemmed.
• Interferons also have immunoregulatory functions—they inhibit B-
lymphocyte (B-cell) activation, enhance T-lymphocyte (T-cell) activity,
and increase the cellular-destruction capability of natural killer cells.
• Interferons are classified into 2 types:
• Type I interferon
• Type II interferon
History

• In 1950’s Yasu-Ichi Nagano and Yashuhiko Kojima, Japanese

virologist discovered the viral inhibitory factors.
• Meanwhile, in London, Alick Issacs and Jean Lindenman were
growing live influenza virus on chick egg chorio allantoic
membranes and noticed that exposure of their membrane to the
heat inactivated form of influenza interfered with the subsequent
growth of a live virus preparation on that surface preparation.
• Hence, name interferon came into action as it interfered with the
growth of the live virus.
Type I interferon:
• It includes interferon α, a family of about 20 related proteins and
interferon β, which are secreted by activated macrophages and
dendritic cells and virus-infected cells.
• Type I IFNs are dimers of 18-20 kDa polypeptides,
predominantly helical in structure and some members of this
family are naturally glycosylated.
• IFN α and IFN β are also secreted by virally infected cells after
recognition of viral components by PRRs located either at the
cell surface or inside the cell.
• IFN α are produced by leukocytes and IFN β are produced by
fibroblasts.
• It is used to mobilize our first line of defense against invading
organisms.
Type II interferon:

• It is produced by activated T cells and NK cells.

• It is a powerful modulator of the adaptive immune response,
biasing T cell help toward TH1 type and inducing the activation
of macrophages with subsequent destruction of any intracellular
pathogens and differentiation of cytotoxic T cell.

• IFN λ:
• Type III IFN contains three members IFNλ1, IFNλ2, IFNλ3.
• It up-regulates the expression of the genes controlling viral
replication and host cell proliferation.
 Interferon therapy
• IFNα:
• for the treatment of hepatitis C and hepatitis B.
• In cancer therapy
• In a type of B cell leukemia known as hairy cell, leukemia responds well to
IFNα.
• In the treatment of kaposis sarcoma.

• IFNβ:
• In the clinical improvement of Multiple sclerosis (MS).

• IFNγ:
• For treating malignancies that include lymphoma and myeloma.
• In treating chronic granulomatous disease(CGD).
• In the treatment of osteopetrosis, a life threatening congenital disease that is
characterized by overgrowth of bone.
Interferons

• Interferons (IFNs) are a group of signalling proteins made and

released by host cells in response to the presence of
pathogens, such as viruses, bacteria, parasites, or tumor cells.
• In a typical scenario, a virus-infected cell will release interferons
causing nearby cells to heighten their anti-viral defenses.
Human Interferon Genes (HIG)

• For the first time, Isaacs and Lindenmann isolated the interferon
in 1957.
• Definition and nomenclature of interferon have been
recommended by a committee of experts (Anonymous, 1980).
• Interferon is defined as "a protein which exerts virus non-
specific antiviral activity, at least in homologous cells through
cellular metabolic procedure involving the synthesis of both
RNA and protein.
• " Thus, interferon is secreted by human cells just to resist the
immediate invasion by virus and multiplication of abnormal
cells.
• Interferon is used to cure many viral diseases such as common
cold and hepatitis. It is species specific. In man there are 3
classes of interferon:
• Alpha interferon (IFN-a) or leukocyte interferon (leukocytes of
blood)
• Beta interferon (IFN-b) or fibroblast interferon (fibroblast of
connective tissue).
• Gamma interferon (IFN-γ) or immune interferon (by
lymphocytes of blood) and lymphoblastoid interferon by
transformed leukocytes.
• According to origin of cells they are classified into two major
groups : leukocyte interferon and fibroblast interferon.
• Leukocyte interferon is produced on large scale but major
difficulty is that mass production of IFN-a cannot be done.
Production of Recombinant Interferons
• Recombinant DNA technology has proved the most satisfactory
route to the large scale production of human interferons.
• The genes of all three types of HuIFN have been cloned in
micro-organisms and expression obtained.
• HuIFNβ and γ produced in this manner lack the glycosylation
present in the naturally occurring substances but this does not
affect their specific activity.
• Greatly improved methods of purification, including immuno-
adsorption chromatography on monoclonal antibody columns,
are now available so there should be no difficulty in supplying
adequate amounts of very pure interferon of all three types
although, up till now, only HuIFNα has been readily available.
• A DNA sequence coding for the product was synthesized and
inserted into E. coli.
• The recombinant product accumulates intracellularly as
inclusion bodies.
• Large-scale manufacture entails an initial fermentation step.
• After harvest, the E. coli cells are homogenized and the
inclusion bodies recovered via centrifugation.
• After solubilization and refolding, the interferon is purified to
homogeneity by a combination of chromatographic steps.
• The final product is formulated in the presence of a phosphate
buffer and sodium chloride.
• It is resented as a 30 mg/ml solution in glass vials and displays
a shelf life of 24 months when stored at 2–8°C`.
• In 1980, IFN-a and IFN-b were successfully produced from
genetically engineered E. coli cells (by isolation of mRNA from
leukocytes and fibroblasts, production of cDNA, its integration
into pBR322 and incorporation and cloning into E. coli cells).
• Production was estimated to be about 1,000 to 100,000
molecules of IFN-b per cell.
• The Swedish firm, Biogene, produced IFN-a and IFN-b through
recombinant DNA techniques which are now under clinical
trials.
• It was found that genes responsible for the production of IFN- a
and IFN-b had 865 and 836 nucleotides, respectively.
• Later on hybrid plasmid containing cDNA of IFN-b genes was
built up which needed a promoter site on plasmid to express
in E. coli cells (Derynck et al, 1980).
• Similarly, hybrid plasmids were also prepared that contained
IFN- genes with trap promoter between the leader and
ribosome binding sites, so that expression of interferon could be
done.
• Expression of both the interferon could be optimized by varying
the spacing sequence between trap Shine-Dalgarno sequence
and the initiator codon (Glover, 1984).
• IFN-b produced by genetically engineered microorganism
showed lower specific activity and decreased stability than
natural one.
• Enhanced specific activity and stability was obtained when the
cysteine at position 17 was replaced by a series of site specific
mutagenesis resulting in ‘IFN-b-Ser’ molecule.
• It was stable for two years and well tolerant in cancer patients.
Moreover, genetically engineered E. coli is reported to yield 5-
10 million units/ml of IFN-b-Ser in a 200 litre batch reactor
within 2-3 days of fermentation.