PHARMACEUTICAL PRODUCTS OF RECOMBINANT DNA TECHNOLOGY: AN

OVERVIEW

INTRODUCTION:
A few decades ago, it was realized that certain proteins could be used as pharmaceutical
agents for the treatment of human diseases. e.g. insulin for diabetes mellitus, interferon for
viral diseases. However, the availability of such therapeutic/ pharmaceutical products was
limited due to costly and cumbersome procedures involved in their isolation. Further, their
use in humans was associated with several complications. For instance, administration of pig
insulin to diabetic patients results in the development of antibodies.

The advent of recombinant DNA technology heralded a new chapter for the production of a
wide range of therapeutic agents in sufficient quantities for human use. The commercial
exploitation of recombinant DNA (rDNA) technology began in late 1970s by
biotechnological companies to produce proteins.There are around 400 different proteins
being produced by rDNAtechnologyand as of now around 30 have been approved for human
use.

Recombinant DNA technology involves using microorganisms, macroscopic organisms, or

hybrids of tumor cells and leukocytes:

 to create new pharmaceuticals;

 to create safer and/or more effective versions of conventionally produced
pharmaceuticals; and
 to produce substances identical to conventionally made pharmaceuticals more cost-
effectively than the latter pharmaceuticals are produced.

Recombinant DNA technology enables modifying microorganisms, animals, and plants so

that they yield medically useful substances, particularly scarce human proteins (by giving
animals human genes, for example). This review, however, focuses not on pharmaceutical
biotechnology’s methods but on its products, notably recombinant pharmaceuticals.

TYPES:
The pharmaceutical products of rDNA technology are broadly divided into following three
types
1. Human protein replacements
2. Therapeutic agents for human diseases
3. Vaccines

1) HUMAN PROTEIN REPLACEMENTS

The synthesis of the cellular proteins is ultimately under the control of genes. Any defect in a
gene produces an incorrect protein or no protein at all. Thus, gene defects will result in
inherited or genetically linked diseases.

Identification of defective or deficient proteins in the causation of inherited diseases is very

important. The rDNA technology can be fruitfully employed to produce human proteins that
can be used for the treatment of genetically linked diseases. This is referred to as human
protein replacement strategy in biotechnology.

EXAMPLES:

INSULIN:
The hormone insulin is produced by the β-cells of islets of Langerhans of pancreas. Human
insulin contains 51 amino acids, arranged in two polypeptide chains. The chain A has 21
amino acids while b has 30 amino acids. Both are held together by disulfide bonds.

Insulin is central to regulating carbohydrateand fat metabolism in the body. Insulin causes
cells in the liver, skeletal muscles, and fat tissue to absorb glucose from the blood. In the liver
and skeletal muscles, glucose is stored as glycogen, and in fat cells (adipocytes) it is stored as
triglycerides.

Insulin stops the use of fat as an energy source by inhibiting the release of glucagon. With the
exception of the metabolic disorder diabetes mellitus and metabolic syndrome, insulin is
provided within the body in a constant proportion to remove excess glucose from the blood,
which otherwise would be toxic. When blood glucose levels fall below a certain level, the
body begins to use stored sugar as an energy source through glycogenolysis, which breaks
down the glycogen stored in the liver and muscles into glucose, which can then be utilized as
an energy source. As a central metabolic control mechanism, its status is also used as a
control signal to other body systems (such as amino acid uptake by body cells). In addition, it
has several other anabolic effects throughout the body.

When control of insulin levels fails, diabetes mellitus can result. As a consequence, insulin is
used medically to treat some forms of diabetes mellitus. Patients with type 1 diabetes depend
on external insulin (most commonly injected subcutaneously) for their survival because the
hormone is no longer produced internally. Patients with type 2 diabetes are often insulin
resistant and, because of such resistance, may suffer from a "relative" insulin deficiency.
Some patients with type 2 diabetes may eventually require insulin if other medications fail to
control blood glucose levels adequately. Over 40% of those with Type 2 diabetes require
insulin as part of their diabetes management plan.

Diabetes mellitus affects about 2-3% of the general population.it is a genetically linked
disease characterized by the increased blood glucose concentration (hyperglycemia). Insulin
facilitates the cellular uptake and utilization of glucose for release of energy. In the absence
of insulin, glucose accumulates in the blood stream at higher concentration, usually when the
blood glucose concentration exceeds about 180mg/dl, glucose is excreted into urine. The
patients of diabetes ate weak and tired since the production of energy (i,e ATP) is very much
depressed.The more serious complications of uncontrolled diabetis include kidney damage
(neuropathy), nerve diseases (neuropathy), and circulatory diseases (atheroscelerosis,stroke).

Production of recombinant insulin:

Attempts to produce insulin by rDNA technology started in late 1970s.the basic technique
consisted of inserting human insulin gene and the promoter gene of lac operon on to the
plasmids of E.coli. Recently the procedure employed for synthesis involves insertion of
genes for insulin A chain and B chain separately to the plasmids of differentE.colicultures.the
lac operon system( consisting of inducer gene, promoter gene, operator gene and structural
gene Z for –galactosidase)is used for expression of both genes.the presence of llactose in the
culture mediuminduces the synthesis of insulin A and B chains in separate cultures. The so
formed insulin chains can be isolated, purified and joined together to give a full-fledged
human insulin.
Fig: Production of recombinant insulin in E.coli

Forms of human insulin:

Human insulin is available in two forms, a short acting (regular) form and an intermediate
acting (NPH) form. NPH (Neutral Protamine Hagedorn) insulin, also known as isophane
insulin, is a suspension meaning that the insulin vial should be rolled or repeatedly turned
upside down to ensure the solution is uniformly cloudy.

Some examples of human insulin:

 Regular (short acting): Humulin S, Actrapid, Insuman Rapid

 NPH (intermediate acting):Humulin I, Insuman basal, Insulatard

 Premixed human insulins:Humulin M2, M3 and M5, Insuman Comb 15, 25 and 50

As a medication
Insulin vial
Biosynthetic "human" insulin is now manufactured for widespread clinical use using
recombinant DNA technology. More recently, researchers have succeeded in introducing the
gene for human insulin into plants and in producing insulin in them, to be specific safflower.
This technique is anticipated to reduce production costs.

Several of these slightly modified versions of human insulin, while having a clinical effect on
blood glucose levels as though they were exact copies, have been designed to have somewhat
different absorption or duration of action characteristics. They are usually referred to as
"insulin analogues". For instance, the first one available, Humalog(insulin lispro), does not
exhibit a delayed absorption effect found in regular insulin, and begins to have an effect in as
little as 15 minutes. Other rapid-acting analogues are NovoRapid and Apidra, with similar
profiles. All are rapidly absorbed due to a mutation in the sequence that prevents the insulin
analogue from forming dimers and hexamers. Instead, the insulin molecule is a monomer,
which is more rapidly absorbed. Using it, therefore, does not require the planning required for
other insulins that begin to take effect much later (up to many hours) after administration.
Another type is extended-release insulin; the first of these was Lantus(insulin glargine).
These have a steady effect for the entire time they are active, without the peak and drop off
effect in other insulins; typically, they continue to have an insulin effect for an extended
period from 18 to 24 hours. Likewise, another protracted insulin analogue (Levemir) is based
on a fatty acid acylation approach. A myristyric acid molecule is attached to this analogue,
which in turn associates the insulin molecule to the abundant serum albumin, which in turn
extends the effect and reduces the risk of hypoglycemia. Both protracted analogues need to
be taken only once-daily, and are very much used in the type 1 diabetes market as the basal
insulin. A combination of a rapid acting and a protracted insulin is also available for the
patients, making it more likely for them to achieve an insulin profile that mimics that of the
body´s own insulin release.Insulin is usually taken as subcutaneous injections by single-use
syringes with needles, via an insulin pump, or by repeated-use insulin pens with needles.

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Unlike many medicines, insulin currently cannot be taken orally because, like nearly all other
proteins introduced into the gastrointestinal tract, it is reduced to fragments (even single
amino acid components), whereupon all activity is lost. There has been some research into
ways to protect insulin from the digestive tract, so that it can be administered orally or
sublingually. While experimental, several companies now have various formulations in
human clinical trials, and one, the India-based Biocon, has formed an agreement with BMS to
produce an oral-insulin alternative.

HUMAN GROWTH HORMONE:

Growth hormone is produced by the pituitary gland. It regulates the growth and development.
Growth hormone stimulates overall body growth by increasing the cellular uptake of amino
acids , and protein synthesis, and promoting the use of fat as body fuel.insufficient human
growth hormone (HGH) in young children results in retarded growth, clinically referred to
as pituitary dwarfism.

Growth hormone is used as a prescription drug in medicine to treat children's growth

disorders and adult growth hormone deficiency. In the United States, it is only available
legally from pharmacies, by prescription from a doctor. In recent years in the United States,
some doctors have started to prescribe growth hormone in GH-deficient older patients (but
not on healthy people) to increase vitality. While legal, the efficacy and safety of this use for
HGH has not been tested in a clinical trial.

Function
Effects of growth hormone on the tissues of the body can generally be described as anabolic
(building up). Like most other protein hormones, GH acts by interacting with a specific
receptor on the surface of cells.

Increased height during childhood is the most widely known effect of GH. Height appears to
be stimulated by at least two mechanisms:
1. Because polypeptide hormones are not fat-soluble, they cannot penetrate cell membranes.
Thus, GH exerts some of its effects by binding to receptors on target cells, where it activates
the MAPK/ERK pathway. Through this mechanism GH directly stimulates division and
multiplication of chondrocytes of cartilage.

2. GH also stimulates, through the JAK-STAT signaling pathway,[30] the production of

insulin-like growth factor 1(IGF-1, formerly known as somatomedin C), a hormone
homologous to proinsulin The liver is a major target organ of GH for this process and is the
principal site of IGF-1 production. IGF-1 has growth-stimulating effects on a wide variety of
tissues. Additional IGF-1 is generated within target tissues, making it what appears to be both
an endocrine and an autocrine/paracrine hormone. IGF-1 also has stimulatory effects on
osteoblast and chondrocyte activity to promote bone growth.

Main pathways in endocrine regulation of growth.

In addition to increasing height in children and adolescents, growth hormone has many other
effects on the body:
* Increases calciumretention, and strengthens and increases the mineralization of bone
* Increases muscle mass through sarcomerehypertrophy
* Promotes lipolysis
* Increases protein synthesis
* Stimulates the growth of all internal organs excluding the brain
* Plays a role in homeostasis
* Reduces liveruptake of glucose
* Promotes gluconeogenesisin the liver
* Contributes to the maintenance and function of pancreatic islets
* Stimulates the immune system

Production of recombinant HGH

Biotechnologists can now produce HGH by genetic engineering. The technique adopted is
quite comparable with that of insulin production. The procedure essentially consists of
inserting HGH gene into E.coli plasmid, culturing the cells and isolation of the HGH from the
extracellular medium.
Limitation in HGHproduction : The HGH is a protein comprised of 191 amino acids. During
the course of its natural synthesis in the body.,HGH is tagged with a single peptide (with 26
amino acids) The signal peptide is removed during secretion to release the active HGH for
biological functions. The entire process of HGH synthesis goes on in an orderly fashion in the
body. However, signal peptide interrupts HGH production by recombinant technology. The
complementary DNA (cDNA) synthesized from the mRNA encoding HGH is inserted into
the plasmid. The plasmid containing E.coli when cultured, produces full length HGH along
with signal peptide.ButE.coli cannot remove the signal peptide. Further, it is also quite
difficultto get rid of signal peptide by various other means. Theoretically, cDNA encoding
signal peptide can be cut to solve these problems. Unfortunately, there is no restriction
endonuclease to do this job, hence this is not possible.

HGH Vial

A novel approach for HGH production:

Biotechnologists have resolved the problem of signal peptide interruption by a novel
approach. The base sequence in cDNA encoding signal peptide ( 26 amino acids ) plus the
neighbouring 24 amino acids is cut by restriction endonuclease ECoRI. Now a gene (cDNA )
for 24 amino acid sequence of HGH is freshly synthesized and ligated to the remaining
HGHcDNA. The so constituted cDNA , attached to a vector, is inserted into a bacterium such
as E. coli for culture and production of HGH. In this manner, the biologically functional
HGH can be produced by DNA technology.

2) THERAPEUTIC AGENTS FOR HUMAN DISEASES

Biotechnology is very useful for the production of several therapeutic products for treating
human diseases. A selected list of rDNAderived therapeutic agents along with trade names
and their uses in human are given below…..

rDNA Product Trade name Application / Uses

Insulin Humulin Diabetes

Growth hormone Protropin/Humatrope Pituitary dwarfism

Interferon Intron A Hairy cell leukemia

Hepatitis B vaccine Recombinax HB/ Hepatitis B

Engerix

TissuEplasminogen Activase Myocardial infarction

activator

Factor vIII Kogenate/Recombinate Hemophilia

Dnase Pulmozyme Cystic fibrosis

Erythropoietin Epogen/rocrit Severe anemia with kidney

damage

INTERFERONS:

Interferons(IFNs) are proteins made an/d released by host cells in response to the presence
of pathogenssuch as viruses, bacteria, parasites or tumor cells. They allow for communication
between cells to trigger the protective defenses of the immune system that eradicate
pathogens or tumors.

IFNs belong to the large class of glycoproteins known as cytokines. Interferons are named
after their ability to "interfere" with viral replication within host cells. IFNs have other
functions: they activate immune cells, such as natural killer cells and macrophages; they
increase recognition of infection or tumor cells by up-regulating antigen presentation to T
lymphocytes; and they increase the ability of uninfected host cells to resist new infection by
virus. Certain symptoms, such as aching muscles and fever, are related to the production of
IFNs during infection.

Functions
All interferons share several common effects; they are antiviral agents and can fight tumors.
As an infected cell dies from a cytolytic virus, viral particles are released that can infect
nearby cells. However, the infected cell can warn neighboring cells of a viral presence by
releasing interferon. The neighboring cells, in response to interferon, produce large amounts
of an enzymeknown as protein kinase R(PKR). This enzyme phosphorylates a protein known
as eIF-2 in response to new viral infections; the phosphorylated eIF-2 forms an inactive
complex with another protein, called eIF2B, to reduce protein synthesis within the cell.
Another cellular enzyme, RNAse L— also induced following PKR activation—destroys
RNA within the cells to further reduce protein synthesis of both viral and host genes.
Inhibited protein synthesis destroys both the virus and infected host cells. In addition,
interferons induce production of hundreds of other proteins—known collectively as
interferon-stimulated genes (ISGs)—that have roles in combating viruses. They also limit
viral spread by increasing p53 activity, which kills virus-infected cells by promoting
apoptosis. The effect of IFN on p53 is also linked to its protective role against certain
cancers.

Another function of interferons is to upregulate major histocompatibility complex molecules,

MHC Iand MHC II, and increase immunoproteasomeactivity. Higher MHC I expression
increases presentation of viral peptides to cytotoxic T cells, while the immunoproteasome
processes viral peptides for loading onto the MHC I molecule, thereby increasing the
recognition and killing of infected cells. Higher MHC II expression increases presentation of
viral peptides to helper T cells; these cells release cytokines (such as more interferons and
interleukins, among others) that signal to and co-ordinate the activity of other immune cells.

Interferons, such as interferon gamma, directly activate other immune cells, such as
macrophages and natural killer cells. Interferons can inflame the tongue and cause
dysfunction in taste bud cells, restructuring or killing taste buds entirely.

Interferon therapy
Three vials filled with human leukocyte interferon

The immune effects of interferons have been exploited to treat several diseases. Agents that
activate the immune system, such as small imidazoquinoline molecules that activate TLR7,
can induce IFN-α. Imidazoquinoline is the main ingredient of Aldara (Imiquimod) cream, a
treatment approved in the United States by the Food and Drug Administration (FDA) for
actinic keratosis, superficial basal cell carcinoma, papilloma and external genital warts.
Synthetic IFNs are also made, and administered as antiviral, antiseptic and anticarcinogenic
drugs, and to treat some autoimmune diseases.

New research has shown that imiquimod's anti-proliferative effect is totally independent of
immune system activation or function. Imiquimod exerts its effect by increasing levels of the
opioid growth factor receptor (OGFr). Blocking OGFr function with siRNA technology
resulted in loss of any antiproliferative effect of imiquimod.

Interferon beta-1a and interferon beta-1b are used to treat and control multiple sclerosis, an
autoimmune disorder. This treatment is effective for slowing disease progression and activity
in relapsing-remitting multiple sclerosis and reducing attacks in secondary progressive
multiple sclerosis.

Interferon therapy is used (in combination with chemotherapy and radiation) as a treatment
for many cancers. This treatment is most effective for treating hematological malignancy;
leukemia and lymphomas including hairy cell leukemia, chronic myeloid leukemia, nodular
lymphoma, cutaneous T-cell lymphoma.[21]Patients with recurrent melanomas receive
recombinant IFN-α2b. Type I IFNs have a therapeutic potential for the treatment of a wide
variety of leukemias and solid tumors due to their antiproliferative and apoptotic effects, their
anti-angiogenic effects and their ability to modulate an immune response specifically
activating dendritic cells, cytolytic T cells and NK cells. Research in this area is receiving
intensive investigation. Interferon a 2b is also being used for treatment of ocular surface
squamous neoplasia (OSSN) in the form of perilesional injection followed by topical
interferon a 2b drops at Lahore General Hospital Eye unit II.[

Both hepatitis B and hepatitis C are treated with IFN-α, often in combination with other
antiviral drugs. Some of those treated with interferon have a sustained virological response
and can eliminate hepatitis virus. The most harmful strain—hepatitis C genotype I virus—can
be treated with a 60-80% success rate with the current standard-of-care treatment of
interferon-α, ribavirin and recently approved protease inhibitors such as Telaprevir (Incivek)
or Boceprevir (Victrelis). Biopsies of patients given the treatment show reductions in liver
damage and cirrhosis. Some evidence shows giving interferon immediately following
infection can prevent chronic hepatitis C, although diagnosis early in infection is difficult
since physical symptoms are sparse in early hepatitis C infection. Control of chronic hepatitis
C by IFN is associated with reduced hepatocellular carcinoma.

Administered intranasally in very low doses, interferon is extensively used in Eastern Europe
and Russia as a method to prevent and treat viral respiratory diseases such as cold and flu.
However, mechanisms of such action of interferon are not well understood; it is thought that
doses must be larger by several orders of magnitude to have any effect on the virus. Although
most scientists are skeptical of any claims of good efficacy recent findings suggest that
interferon applied to mucosa may act as an adjuvant against influenza virus, boosting the
specific immune system response against the virus. A flu vaccine that uses interferon as
adjuvant is currently under clinical trials in the US.

When used in the systemic therapy, IFNs are mostly administered by an intramuscular
injection. The injection of IFNs in the muscle, in the vein, or under skin is generally well
tolerated. The most frequent adverse effects are flu-like symptoms: increased body
temperature, feeling ill, fatigue, headache, muscle pain, convulsion, dizziness, hair thinning,
and depression. Erythema, pain and hardness on the spot of injection are also frequently
observed. IFN therapy causes immunosuppression, in particular through neutropenia and can
result in some infections manifesting in unusual ways.

Drug formulations:

Generic name Trade name

 Interferon alpha 2a Roferon A

Interferon alpha 2b Intron A/Reliferon/Uniferon

Human leukocyte Interferon-alpha (HuIFN-alpha-Le) Multiferon

Interferon beta 1a, liquid form Rebif

Interferon beta 1a, lyophilized Avonex

Interferon beta 1a, biogeneric (Iran) Cinnovex

Interferon beta 1b Betaseron/ Betaferon

Interferon gamma 1b Actimmune

PEGylated interferon alpha 2a Pegasys

PEGylated interferon alpha 2a(Egypt) Reiferon Retard

PEGylated interferon alpha 2b PegIntron

PEGylated interferon alpha 2bplus ribavirin(Canada) Pegetron

Several different types of interferon are now approved for use in humans. By March 10,
2009, Multiferon — a brand name known generically as human leukocyte interferon-alpha
(HuIFN-alpha-Le) — was being used in 14 European countries. This drug was approved for
treatment of patients with high risk (stage IIb–III) cutaneous melanoma, after two treatment
cycles with dacarbazine, following a clinical trial performed in Germany.

In January 2001, the Food and Drug Administration(FDA) approved the use of PEGylated
interferon-alpha in the USA; in this formulation, polyethylene glycolis added to make the
interferon last longer in the body. Initially used for production of PEGylated interferon-alpha-
2b(Pegintron), approval for PEGylated interferon-alpha-2a(Pegasys) followed in October
2002. These PEGylated drugs are injected once weekly, rather than administering three times
per week, as is necessary for conventional interferon-alpha. When used with the antiviral
drugribavirin, PEGylated interferon is effective in treatment of hepatitis C; at least 75%
people with hepatitis C genotypes 2 or 3 benefit from interferon treatment, although this is
effective in less than 50% of people infected with genotype 1 (the more common form of
hepatitis C virus in both the U.S. and Western Europe).[36][37][38]Recently, 2 new Protease
Inhibitors have been approved which improves the outcomes for Genotype 1 hepatitis C -
boceprevirand telaprevir. These drugs are used in addition to PEG-IFN / Ribavirin.

Erythropoietin:
It is also known as erythropoetin or erthropoyetin and or EPO, is a glycoprotein hormone
that controls erythropoiesis, or red blood cell production. It is a cytokine(protein signaling
molecule) for erythrocyte (red blood cell) precursors in the bone marrow. Human EPO has a
molecular weight of 34 kDa.

Also called hematopoietin or hemopoietin, it is produced by interstitial fibroblasts in the

kidney in close association with peritubular capillary and tubular epithelial cells. It is also
produced in perisinusoidal cells in the liver. While liver production predominates in the fetal
and perinatal period, renal production is predominant during adulthood. In addition to
erythropoiesis, erythropoietin also has other known biological functions. For example, it
plays an important role in the brain's response to neuronal injury. EPO is also involved in the
wound healing process.

When exogenous EPO is used as a performance-enhancing drug, it is classified as an

erythropoiesis-stimulating agent (ESA). Exogenous EPO can often be detected in blood, due
to slight differences from the endogenous protein, for example, in features of
posttranslational modification.

Mechanism of action:
Erythropoietin has been shown to exert its effects by binding to the erythropoietin
receptor(EpoR).

EPO is highly glycosylated (40% of total molecular weight), with half-life in blood around
five hours. EPO's half-life may vary between endogenous and various recombinant versions.
Additional glycosylation or other alterations of EPO via recombinant technology have led to
the increase of EPO's stability in blood (thus requiring less frequent injections). EPO binds to
the erythropoietin receptor on the red cell progenitor surface and activates a JAK2 signaling
cascade. Erythropoietin receptor expression is found in a number of tissues, such as bone
marrow and peripheral/central nervous tissue. In the bloodstream, red cells themselves do not
express erythropoietin receptor, so cannot respond to EPO. However, indirect dependence of
red cell longevity in the blood on plasma erythropoietin levels has been reported, a process
termed neocytolysis.

Pharmaceutical companies make human recombinanterythropoietin with recombinant

technology, in which genes are inserted to create a custom organism. In this particular case,
bacteria are modified with recombination so that they will produce human erythropoietin
which can be administered to patients. The same technology is used to produce a variety of
other human hormones. These hormones are as effective in the body as hormones of human
or animal origin, but they are easier and safer to produce.

There are side effects associated with human recombinanterythropoietin, especially in

patients who use it for a long time. It can increase the risk of heavy clotting and adverse
cardiovascular events, and it can also lead to iron deficiency and high blood pressure. In
some young athletes, unexpected death has been linked to EPO usage, which is one of the
reasons sports authorities are concerned about blood doping. Recombinant EPO is chemically
slightly different from the made in the body version, and this can be used on blood tests to
determine whether or not an athlete is doping.

3) VACCINES:
Vaccination is the phenomenon of preventive immunization. In the modern concept,
vaccination involves the administration (injection or oral) of an antigen to elicit an antibody
response that will protect the organism against the future infections.

Vaccine is a biological preparation that improves immunity to a particular disease. A vaccine

typically contains an agent that resembles a disease-causing microorganism, and is often
made from weakened or killed forms of the microbe, its toxins or one of its surface proteins.
The agent stimulates the body's immune system to recognize the agent as foreign, destroy it,
and "remember" it, so that the immune system can more easily recognize and destroy any of
these microorganisms that it later encounters.

Vaccines may be prophylactic (example: to prevent or ameliorate the effects of a future

infection by any natural or "wild" pathogen), or therapeutic (e.g. vaccines against cancer are
also being investigated; see cancer vaccine).
The term vaccine derives from Edward Jenner's 1796 use of cow pox (Latin variolavaccinia,
adapted from the Latin vacc?n-us, from vacca, cow), to inoculate humans, providing them
protection against smallpox.

Effectiveness
Vaccines do not guarantee complete protection from a disease.Sometimes, this is because the
host's immune system simply does not respond adequately or at all. This may be due to a
lowered immunity in general (diabetes, steroid use, HIV infection, age) or because the host's
immune system does not have a B cell capable of generating antibodies to that antigen.

Even if the host develops antibodies, the human immune system is not perfect and in any case
the immune system might still not be able to defeat the infection immediately. In this case,
the infection will be less severe and heal faster.

Adjuvants are typically used to boost immune response. Most often aluminium adjuvants are
used, but adjuvants like squalene are also used in some vaccines and more vaccines with
squalene and phosphate adjuvants are being tested. Larger doses are used in some cases for
older people (50–75 years and up), whose immune response to a given vaccine is not as
strong.

Maurice Hilleman's measles vaccine is estimated to prevent 1 million deaths every year.

The efficacy or performance of the vaccine is dependent on a number of factors:

 the disease itself (for some diseases vaccination performs better than for other
diseases)

 the strain of vaccine (some vaccinations are for different strains of the disease)

 whether one kept to the timetable for the vaccinations (see Vaccination schedule)

 some individuals are "non-responders" to certain vaccines, meaning that they do not
generate antibodies even after being vaccinated correctly other factors such as
ethnicity, age, or genetic predisposition.

Recombinant vaccines:
Biotechnology sector has also played its part in developing vaccines against certain diseases.
Such vaccine which makes use of recombinant DNA technology is known as recombinant
vaccines. It is also known as subunit vaccines.

Recombinant vaccines can be broadly grouped into two kinds:

(i) Recombinant protein vaccines: This is based on production of recombinant DNA which
is expressed to release the specific protein used in vaccine preparation

(ii) DNA vaccines: Here the gene encoding for immunogenic protein is isolated and used to
produce recombinant DNA which acts as vaccine to be injected into the individual.

Steps involved:
Production of recombinant vaccines involves the following steps:
(i) First and foremost, it is important that the protein which is crucial to the growth and
development of the causative organism be identified.
(ii) The corresponding gene is then isolated applying various techniques. Further to this, an
extensive study of the gene explains the gene expression pattern involved in the production of
corresponding protein.
(iii) This gene is then integrated into a suitable expression vector to produce a recombinant
DNA.
(iv) ThisrDNA is used as vaccines or is introduce into another host organism to produce
immunogenic proteins which acts as vaccines.

Recombinant protein vaccines:

A pathogen upon infection produces proteins, vital for its functions, which elicit an immune
response from the infected body. The gene encoding such a protein is isolated from the
causative organism and used to develop a recombinant DNA. This DNA is expressed in
another host organism, like genetically engineered microbes; animal cells; plant cells; insect
larvae etc, resulting in the release of the appropriate proteins which are then isolated and
purified. These when injected into the body, causes immunogenic response to be active
against the corresponding disease providing immunity against future attack of the pathogen.

Based on the proteins involved in evoking immune response recombinant protein vaccines
are of two types:
Whole protein vaccines: The whole immunogenic protein is produced in another host
organism which is isolated and purified to act as vaccines.
Polypeptide vaccines: It is known that in the immunogenic protein produced, the actual
immunogenic property is limited to one or two polypeptides forming the protein. The other
parts of the protein may be successful in evoking an immune response but do not actually
cause the disease. For eg: in the case of cholera caused by Vibrio cholerae, consists of three
polypeptide chains like A1, A2, and B. The A polypeptides are toxic while B is non-toxic.
Thus while producing vaccines, the polypeptide B is produced by rDNA technology and used
for vaccination.

DNA vaccines:
It refers to the recombinant vaccines in which the DNA is used as a vaccine. The gene
responsible for the immunogenic protein is identified, isolated and cloned with corresponding
expression vector. Upon introduction into the individuals to be immunized, it produces a
recombinant DNA. This DNA when expressed triggers an immune response and the person
becomes successfully vaccinated. The mode of delivery of DNA vaccines include: direct
injection into muscle; use of vectors like adenovirus, retrovirus etc; invitro transfer of the
gene into autologous cells and reimplantation of the same and particle gun delivery of the
DNA.

In certain cases, the responsible gene is integrated into live vectors which are introduced into
individuals as vaccines. This is known as live recombinant vaccines. Eg: vaccinia virus. Live
vaccinia virus vaccine (VV vaccine) with genes corresponding to several diseases, when
introduced into the body elicit an immune response but does not actually cause the diseases.

Advantages:
(i) Since it does not involve actual pathogen, recombinant vaccines is considered to be safe
than the conventional vaccines.
(ii) It induces both humoral and cellular immune response resulting in effective vaccination.

Risks involved:
(i) High cost of production.
(ii) Have to be stored at low temperature since heat destabilizes protein. Hence storage and
transportation is tedious.
(iii) Individuals with immunodeficiency may elicit poor immune response.

CONCLUSION:
Recombinant DNA Technology is playing very important role in revolutionizing
medicinei.e., enabling mass production of safe, pure, more effective versions of biochemicals
that human body produces naturallyexamples includes a variety of products such as
hormones, Therapeutic proteins, Vaccines and various Enzymes and also to create new
pharmaceuticals..With sensible regulatory requirements and expeditious product review by
regulatory agencies, biotech pharmaceuticals can within decades become unprecedented
preventers and relievers of human suffering.