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Dept of Pharmacognosy
Shri D.D. Vispute College of Pharmacy, Mumbai.
Immunization is the process whereby a person is
made immune or resistant to an infectious
disease, typically by the administration of a
Vaccines : vaccines are preparations of antigenic
materials, which are administered with the
objective of inducing in the recipient specific
and active immunity against infectious
microorganisms or toxins produced by them
They contain living or killed microorganisms,
bacterial toxoids or antigenic material from the
particular parts of bacterum, rickettsia, or virus
Types of Vaccines
Live attenuated vaccines
Inactivated or killed vaccines
Classification of vaccines
I. Live attenuated
Eg. Measles, Mumps, Rubella, Varicella, Rotavirus,
II. Inactivated or whole killed
Eg. Hepatitis A, Some influenza vaccines
III. Subunit: sub classified as
a) Toxoid: Diphtheria, tetanus
b) Polysaccharide: Pneumococcal (23-
valent), meningococcal ACYW-135
c) Conjugate: Pneumococcal (10- and 13-valent),
Haemophilus nfluenzae type b,meningococcal C
d) Recombinant: Hepatitis B, human
e) Other subunit: Pertussis, acellular influenza
I. Live attenuated vaccines
To produce a live vaccine, the wild or disease-
causing virus is attenuated (weakened),
traditionally by repeated culture in the
The virulence properties of the virus are reduced
so that it does not cause disease in healthy
individuals. The attenuated vaccine virus
multiplies to a limited extent in host tissue and
induces an immune response similar to wild virus
infection in the majority of subjects. Live
vaccines are generally very effective and induce
Eg. Measles, Mumps, Rubella, Varicella,
Rotavirus, Tuberculosis (BCG)
II. Killed and inactivated vaccines
The term ‘killed’ is generally used for bacterial
vaccines and the term ‘inactivated’ for viral
vaccines. These vaccines are prepared by treating the
whole cell or virus with chemicals that cause
inactivation. Generally these organisms remain intact
and whole. They generate an immune response (to a
broad range of antigens) but cannot cause an
infection because they are dead and so cannot
Eg. Hepatitis A, Some influenza vaccines
III. Subunit vaccines
Subunit vaccines are developed using only the
antigens known to elicit protective immunity. They
can be further categorised as follows.
In some bacterial infections (eg, diphtheria,
tetanus), the clinical manifestations of
disease are caused not by the bacteria
themselves but by the toxins they secrete.
Toxoid vaccines are produced by harvesting a
toxin and altering it chemically (usually with
formaldehyde) to convert the toxin to a
toxoid. The toxoid is then purified. Toxoid
vaccines induce antibodies that neutralise
the harmful exotoxins released from these
Recombinant vaccines, such as those used against hepatitis B and HPV,
are made using a gene from the (disease-causing) pathogen as an
antigen, which generates a protective immune response. The gene is
inserted into a cell system capable of producing large amounts of the
protein of interest.
There are four genetically-engineered vaccines are currently available:
the gene for the hepatitis B surface antigen is inserted into yeast cells,
which replicate and produce large amounts of the hepatitis B surface
antigen. This is purified and used to make vaccine. The advantage of
this approach is that it results in a very pure vaccine that is efficient to
Human papillomavirus vaccines are produced by inserting genes for a
viral coat protein into either yeast (as the hepatitis B vaccines) or into
insect cell lines. Viral-like particles are produced and these indice a
protective immune response.
Live typhoid vaccine (Ty21a) is Salmonella typhi bacteria that has been
genetically modified to not cause illness.
Live attenuated influenza vaccine (LAIV) has been engineered to
replicate effectively in the mucosa of the nasopharynx but not in the
Vaccine is an antigenic substance
prepared from the causative agent of a
disease or a synthetic substitute, used
to provide immunity against one or
Contains dead bacteria or weak
bacteria or toxins.
stimulates the body to make
Gaining immunity after a period.
Remain immune for long.
Use a series of complex methods to
separate and purify the
virus and extract the required part,
reduce the low virulent or inactivated
and produced vaccine.
A vaccine consists of normally a single
type of antibodies( usually monoclonal)
or may be for a particular disease
The clear, pale
yellow liquid that separates from
the clot in the coagulation of blood
does not contain bacteria or toxins.
contains anti formed in another animal.
acquired immune immediately.
remain a short time.
specific animal by immunization, whole
blood collected in, but the serum is a
nonspecific mixture obtained after
Polio vaccines are vaccines used to prevent poliomyelitis (polio), 2
1. Inactivated poliovirus and is given by injection (IPV) The
inactivated polio vaccines are very safe. Mild redness or pain
may occur at the site of injection.
2. Weakened poliovirus and is given by mouth (OPV). Oral polio
vaccines result in vaccine-associated paralytic poliomyelitis in
about three per million doses.[
The World Health Organization recommends all children be
vaccinated against polio. The two vaccines have eliminated
polio from most of the world, and reduced the number of cases
each year from an estimated 350,000 in 1988 to 74 in 2015.
The first polio vaccine was the inactivated polio vaccine. It was
developed by Jonas Salk and came into use in 1955. The oral
polio vaccine was developed by Albert Sabin and came into
commercial use in 1961
Salk vaccine Sabin vaccine
Jonas Salk developed
An inoculation of
dead polio virus
It is administered as
includes a satisfactory
Albert Sabin developed
An inoculation of
attenuated live polio
It is administered oraly
1 or 2 doses of oral
vaccine gives 90%-100%
Local immunity in gut
• The original Salk polio vaccine is an example of an inactivated
(killed) vaccine. The Salk vaccine, or inactivated poliovirus
vaccine (IPV), is based on three wild, virulent reference strains,
Mahoney (type 1 poliovirus), MEF-1 (type 2 poliovirus), and Saukett
(type 3 poliovirus),
• It is made by growing virulent polio virus in tissue culture
[monkey kidney tissue culture (Vero cell line)] , by the microcarrier
• Then the viruses are concentrated, purified and inactivated by
treating the virus with formaldehyde so that it cannot reproduce in
the person who receives the vaccine.
• Each 0.5 ml dose of trivalent vaccine is formulated to contain 40 D
antigen units of Type 1. 8 D antigen units of Type 2. and 32 D antigen
units of Type 3 poliovirus. 0.5% phenoxyethanol and a maximum of
O.02X formaldehyde. Neomycin. streptomycin·and polymyxin B are
used in vaccine production
• Neutralizing antibody produced to polio virus is very efficient at
blocking the ability of the virus to infect host cells and offers good
protection from infection.
• The Sabin oral polio vaccine is attenuated
• To make an attenuated vaccine, the pathogen is
grown in animals or tissue culture under
conditions that make it less virulent. Steps
The pathogenic virus is isolated from a patient
and grown in human cultured cells
The cultured virus is used to infect monkey cells
The virus acquires many mutations that allow it
to grow well in monkey cells
The virus no longer grows well in human cells(it
is attenuated) and can be used as vaccine
The attenuated Sabin poliovirus vaccine
replicates very efficiently in the gut, the
primary site of infection and replication, but
is unable to replicate efficiently
within nervous system tissue.
In 1961, type 1 and 2monovalent oral
poliovirus vaccine (MOPV) was licensed, and
in 1962, type 3 MOPV was licensed. In 1963,
trivalent OPV (TOPV) was licensed, and
became the vaccine of choice in the United
States and most other countries of the world,
largely replacing the inactivated polio
Based on EP and WHO guidelines following tests are used for the
evaluation (standardization) of OPV and IPV products.
General assays: e.g. free formalin test, Sterility test etc.
Free formalin test: inactivation may lead to the presencer of free
formalinmay not exceed 0.02% which is estimated by colour
development with acetylacetone
Sterility test: vaccines must be bacteriologically and mycologicaly
sterile. In each batch of product the number of containers to be
tested depends on the batch size and the subject of
pharmacopoeial regulation. Membrane filtration is commonly used
for sterility testing.
Specific assays :
Batch release for OPV:
potency test in cell culture: the potency of the live viral
vaccines is estimated by using substrates of living cells.
Dilutions of vaccines are inoculated on tissue culture
monolayers in petriplates and the live count of vaccines are
calculated from the infectivity of dilutions and dilution factor
MAPREC test Molecular biological test, Mutant Analysis by
PCR and Restriction Enzyme Cleavage: The MAPREC assay is
a molecular biological method used to determine the
proportion of a 5 single base mutation at a given point
within the viral RNA. If the calculated value of the 6
mutation at this site is greater than acceptable the vaccine
will fail the MAPREC test.
transgenic mouse neurovirulence test (TgmNVT) TgPVR
mice developed by introducing into genome the human
gene encoding the cellular receptor of poliovirus When
infected with poliovirus, TgPVr mice develop flaccid
paralysis and then death in some cases with histological
lesions in central nervous system similar to those observed
Batch release for IPV:
Rat potency test
An improved ELISA test for determination of potency
of Inactivated Poliovirus Vaccine (IPV) is proposed.
The method is based on the use of IgG purified from
immune rabbit serum conjugated with biotin.
The assay is based on direct ("sandwich") ELISA
scheme, in which antigens are captured on ELISA
plates coated with purified rabbit polyclonal D-
antigen specific IgG raised against wild polioviruses of
three serotypes. D-antigen specificity of the IgG was
at least 10 times higher than to H-antigen (heat-
Potency of inactivated poliovirus vaccine (IPV) is tested either by
injecting experimental animals and then measuring neutralizing
antibodies in their sera or by estimating content of D antigen (complete
viral particles) in the vaccine.
The recent derivation of transgenic mice expressing the human poliovirus
receptor (Tg PVR mice) and susceptible to poliomyelitis allowed us to
attempt to develop a new potency test for IPV based on protection of
mice against lethal challenge with polioviruses of all three antigenic
Mice were vaccinated with IPV and then evaluated for induced immunity
against poliomyelitis by intraperitoneal injection with 25 lethal doses of
wild-type virus. A single injection of monovalent type-3 IPV elicited
protective immunity, while 2 injections of trivalent IPV were required to
protect mice against challenge with the same dose of virus. Both
neutralizing antibody response and protection against challenge were
Tg PVR mouse-protection test may be useful in evaluating existing
potency tests of IPV and in attempts to improve formulations of trivalent
IPV or of IPV combined with other vaccines suitable for simplified
childhood immunization schedules.
1 - contains dead bacteria or weak bacteria
2 - stimulates the body to make antioxidants.
3 - gaining immunity after a period.
4 - remain immune for long.
1 - does not contain bacteria or toxins.
2 - contains anti formed in another animal.
3 - acquired immune immediately.
4 - remain a short time.
The production of a vaccine can be divided in the following
1. Generation of the antigen
The first step in order to produce a vaccine is generating
the antigen that will trigger the immune response. For this
purpose the pathogen’s proteins or DNA need to be grown
and harvested using the following mechanisms:
Viruses are grown on primary cells such as cells from
chicken embryos or using fertilised eggs (e.g. influenza
vaccine) or cell lines that reproduce repeatedly (e.g.
Bacteria are grown in bioreactors which are devices that
use a particular growth medium that optimises the
production of the antigens
Recombinant proteins derived from the pathogen can be
generated either in yeast, bacteria or cell cultures.
2. Release and isolation of the antigen
After the antigen is generated, vaccines are
isolated from the cells, used to generate it. For
weakened or attenuated viruses no further
purification may be required.
The aim of this second step is to release as much
virus or bacteria as possible. To achieve this, the
antigen will be separated from the cells and
isolated from the proteins and other parts of the
growth medium that are still present.
Recombinant proteins need many operations
involving ultrafiltration and column
chromatography for purification before they are
ready for administration.
3. Addition of adjuvants, stabilizers and
Once the antigen is developed the vaccine is
formulated by adding adjuvants, stabilizers, and
The role of the adjuvant is to enhance the immune
response of the antigen.
The stabilizers increase the storage life.
Preservatives allow the use of multi dose vials.
Finally, all components that constitute the final
vaccine are combined and mixed uniformly in a single
vial or syringe.
Once the vaccine is put in recipient vessel (either a
vial or a syringe), it is sealed with sterile stoppers.
Is a formal toxoid prepared from the toxins
produced by corynebacterium diphtheriae
A suitable strain of corynebacterium
diphtheriae is grown on liquid medium at
350C for 7 days
After maximum toxin production, the bulk of
the organisms are separated and filtered and
filtrate is sterilized
Preparation of diphtheria toxoid
i. Formal toxoid (FT) in the diphtheria toxin 0.5%
of formalin is added and the mixture is
incubated at 370C for 3-4 weeks to remove the
ii. Toxin-antitoxin floccules (TAF) suitable quantity
of toxoids and antitoxins are mixed to form the
floccules which contain the good antigenic
Floocules are separated and washed to remove
the contaminations and are resuspended in
saline solution containing a bactericide
iii. Alum precipitated toxoid:
It produces more antibodies than formal toxoid and TAF
Slow absorption of precipitated toxoids from the site of
injection and slow excretion from the body increased its
In the procedure high quality toxoids are treated with charcoal
to remove the colouring matter and filtered to remove the
charcoal and other impurities
Than suitable concentration of alum is added
This reacts with bicarbonate, phosphate and proteins in the
toxoid to produce a precipitate of aluminium hydroxide and
The precipitate is washed and suspended in saline containing
iv. Purified toxoid aluminium phosphate: different
purification steps are involved using magnesium
hydroxide to precipitate colour, ammonium sulphate,
cadimum chloride, and some protiens