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VACCINES & SERA


Vaccines or sera are biological products which act by reinforcing the immunological defense of
the body against foreign agencies (infecting organisms or their toxins).
The agents or products through which immunization is achieved are called immunizing agents.
Active immunization is the process of increasing resistance to infections whereby
microorganisms or products of their activity act as antigens and stimulate certain body cells to
produce antibodies with a specific protective capacity. Biological products comprising vaccines and
toxoids confer active immunity.
Passive immunization results in intermediate protection of short duration is achieved by
antibodies administration. Biological products comprise human immune sera and animal immune
sera.
Vaccines are preparations of antigenic materials, which are administered with the objective of
inducing in the
recipient a specific and active immunity against the infectious microorganisms or toxins produced
by them.
They may contain living or killed microorganisms, bacterial toxoids or antigenic material from
particular parts of the bacteria, rickettsia or virus.
Vaccines may be single component or mixed component vaccines.
Single vaccines are prepared from a single species of microorganism.
Mixed or compound vaccines are prepared from two or more species.
Simple vaccines containing only one strain of a species are univalent and those containing two
or more strains of the same species are called polyvalent.
Vaccines may be classified into five types
1. Live attenuated vaccines: consists of live bacteria or viruses which have been rendered
avirulent. Eg. BCG
vaccine, smallpox vaccine.
2. Killed vaccine: these are suspensions of bacteria or of viruses that have been killed by heat or
by disinfectants such as phenol or formaldehyde. The best known killed vaccines are pertussis,
cholera, plague, influenza and rabies vaccine.
3. Toxoid vaccine: Toxoids may be defined as modified toxins, detoxified by the use of moderate
heat and chemical treatment so that their antigenic properties are retained. Toxoids are toxins
whose toxicity has been removed.
Eg. Tetanus toxoid, staphylococcus toxoid.
4. Bacterial cell component vaccine: Some bacterial vaccines do not contain whole bacterial cells
but contain
components of bacterial cells. Such vaccines induce a response that is more specific and effective.
Eg. Haemophilus influenzae Type B Vaccine, Neisseria meningitidis Type A and C vaccine
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5. Viral subunit vaccines: the influenza vaccines are prepared by treating intact influenza virus
particles from
embryonated hens eggs, infected with influenza virus with surface active agent. The virus particles
are disrupted
and release the two viral subunits, haemagglutinin and neuraminidase that are required in the
vaccine. Another
example of viral subunit vaccine is hepatitis vaccine.

PREPARATION OF VACCINES FROM BACTERIA (GENERAL METHOD)


The seed lot system:
The starting stage for the preparation of all microbial vaccines is the isolation of suitable
microbial strains.
Microbial strains are mainly isolated from human infections and in some cases have required
elaborate laboratory manipulation and selection.
Once a suitable strain is available, a sizeable culture is prepared and distributed in a large
number of ampoules and then stored at —70°C or freeze dried. This culture is called 'seed lot'.
The seed is then used to make one or more batches of vaccine production.
If it is found satisfactory, then it is tested for efficacy and safety in clinical trials. Satisfactory
results in the clinical trials validate the seed lot and it is used for production of vaccines.
Production of bacteria and bacterial components for bacterial vaccines:
Bacteria and bacterial components needed for the manufacture of bacterial vaccines are readily
prepared by
fermentation by using different laboratory media
Fermentation: the production of bacterial vaccine batch begins with the recovery of the bacterial
seed lot stored at -70oC. The recovered bacteria are first cultivated through one or more passages
in production medium.
Fermentation process is carefully regulated and monitored for temperature, pH and oxidation-
reduction potential.

Process of bacterial harvest:


The harvest is s complex mixture of bacterial cells and metabolic products. The bacterial
harvesting depends on the nature of the component that is required and may involve one or more
of the following procedures
1. Killing
2. Separation
3. Fractionation
4. Detoxification
5. Adsorption
6. conjugation
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1. Killing:
Killing is the process in which live bacteria in the culture is killed.
Heat or formalin are required to kill the cells of Bordetella pertussis (whooping cough vaccine).
Phenol is
commonly used to kill Vibrio cholerae (cholera vaccine) and Salmonella typhi (typhoid vaccine).

2. Separation:
Separation is the process by which bacterial cells are separated from the fermentation medium.
Centrifugation or precipitation is commonly used for the separation of cells from the culture
fluid.

In the case of vaccines prepared from cells, the fluid is discarded and the cells are
resuspended in a salinesolution. If the vaccines are prepared from a constituent of the fluid, then
the cells are discarded.

3. Fractionation:
The process by which components are extracted from bacterial cells or from the medium is
called fractionation.
The antigens of Pseudomonas aeruginosa are extracted with water.
The polysacchride antigens of Neisseria meningitides are separated from the cells by treatment
with hexadecyltrimethyl ammonium bromide and those of S. pneumonia with ethanol.
The purity can be further improved by resolubilisation and precipitation.

4. Detoxification:
Detoxification is the process by which toxins are converted to harmless toxoids.
Formalin is used to detoxify the toxins of C. diphtheria and C. tetani.
Detoxification is generally performed in the fermenter.

5. Adsorption:
The mineral adjuvants or carriers are used to increase immunogenicity and decrease toxicity.
Eg. Aluminum hydroxide, aluminum phosphate, calcium phosphate etc.
Diphtherai vaccine, tetanus vaccine are prepared as adsorbed vaccines.

6. Conjugation:
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Conjugation is the linking of a vaccine component (poor immune response), with a vaccine
component that induces a good immune response.
The immunogenicity for infants of Hemophilus influenza type B is greatly enhanced by the
conjugation with diphtheria and tetanus toxoids.

Production of viruses and viral components for viral vaccines:


Virus replicated only in living cells, hence the first viral vaccines against smallpox and rabies
were made in intact mammalian hosts (calves, sheep, rabbits).
Today the only intact host used in advanced production techniques is the developing chick
embryo.
Almost all virus growth is preferably achieved in cell cultures.
Growth of viruses: The chick embryo is the most convenient host for the growth of viruses that
are needed for influenza and yellow fever vaccines. Influenza viruses accumulate in high titre in the
allantoic fluid of the eggs and yellow fever virus accumulates in the nervous systems of the
embryo.

Processing of viral harvests


Different techniques are used for processing of viral materials.
Allantoic fluid is centrifuged to provide a concentrated and partially purified suspension of
influenza virus.
The concentrated suspension is treated with ether or other agents to split the virus into its
components.

Cell fluid provides infected fluids that contain little debris, which can be separated by filtration.
Most viral vaccines are not inactivated because they are made from cultures consisting of live
attenuated virus.
Poliomyelitis vaccine is inactivated with formalin and rabies vaccine with b-propiolactone.
When processing is complete, the bulk materials may be stored (-70oC) until needed for
blending into final vaccine.

Blending
Blending is the process in which various components of a vaccine are mixed to form a final
product.
A single component final bulk is prepared by adding bacterial suspensions or bacterial
components. Eg. BCG vaccine, cholera vaccine, diphtheria vaccine etc.
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Adjuvants such as adsorbents and preservatives may be included in the final bulk.
When viral vaccines are blended, adequate antigenicity and infectivity should be maintained.
Final bulk is added to appropriate containers.
Live attenuated viral vaccines lose potency in the suspension, hence they are stored at low
temperatures with stabilizers. Eg. Poliomyelitis stabilized with magnesium chloride or sucrose.

Filling and drying


Bulk vaccines are dispensed into single dose ampoules or into multidose vials.
Vaccines that are filled as liquids are sealed and capped in the containers.
Vaccines that are dispensed as dry preparations are freeze dried before sealing.

QUALITY CONTROL OF VACCINES


• The quality control of vaccines is intended to provide assurances of efficacy and safety. Quality of
vaccines are checked in two ways: in-process control and final product control

In-process control
In process quality control is the control exercised over starting materials and intermediates.
The quality control of diphtheria and tetanus vaccines requires that the products are tested for
the presence of free toxins.
Adequate infectivity of the virus from the tissue cultures is an indicator of the adequate virus
content of the starting materials and since infectivity is destroyed in the activation process.
In case of tissue culture substrates, to exclude contamination with infectious agents from the
source animal or in the case of human diploid cells, to exclude abnormal cellular characters.
Monkey kidney cell cultures are tested for simian herpes B virus, simian virus 40 and
mycoplasmas.

product control:
Final product control is the quality control exercised by the monographs of a pharmacopoeia
over products in their final containers.
All vaccines are tested for identity, potency and safety.
Combined vaccines are required to pass tests prescribed for each of the separate components.
Identity tests
Potency tests
• Safety tests, namely; Sterility tests, Free formalin tests, Abnormal toxicity testing, Phenol
concentration, Presence of aluminum and calcium
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Identity tests:
The identities of bacterial vaccines can be checked by precipitation and agglutination reactions.
Inactivated viral vaccines are tested by observation of the specific antibody responses in
vaccinated animals and live viral vaccines by neutralization of their cytopathic effects by specific by
specific antisera.

Potency assay:
Vaccines are tested for potency in which the amount of the vaccine that is required to protect
animals from a defined challenge dose of the pathogen.
Variable doses are injected into a specific groups of animals, and the mortality rate is observed.
Vaccines containing live microorganisms are generally tested for potency by counts of their
viable cells. EG. BCG vaccine.
The potency of live viral vaccines is estimated by using substrates of living cells.
Potency of many viral vaccines are also checked by physicochemical or serological techniques.

Safety assay:
Viral vaccines have some problems for safety testing as compared to bacterial vaccines.
Killed bacterial vaccines must be completely free from living microbes used in the production
process.
The final product must provide an assurance that all microoganisms have been killed.
Incomplete virus inactivation is detected by inoculation of susceptible tissue cultures and of
susceptible animals.
The cultures are examined for cytopathic effects and the animals for symptoms of disease.

Sterility assay:
All vaccines must be bacteriologically and mycologically sterile.
In each batch of a product the number of containers to be tested depends on the batch size
and is the subject of pharmacopoeial regulations.
Membrane filtration method is commonly used for sterility testing of vaccines.

Free formalin testing


Inactivation of bacterial toxins (by formalin) may lead to the presence of free formalin in the final
product.
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The concentration of free formalin may not exceed 0.02% which is estimated by color
development with Acetylacetone

Abnormal toxicity testing:


This test is used for detection of toxic contaminant in vaccines.
Five mice (approx. 20g) and two guinea pigs (approx. 30g) are inoculated with one human dose
or 1.0 ml(whichever is less) of the test preparation.
All must survive for seven days without sign of illness.

Phenol concentration
Phenol is used as a preservative in different types of vaccines. Its concentration must not
exceed 0.5w/v%.

Presence of aluminum and calcium:


These are added in the vaccines as adjuvants.
Aluminum must not exceed 1.25mg/dose; estimated complexometrically.
Calcium must not exceed 1.3mg/dose; estimated by flame photometry.

BACTERIAL VACCINES AND TOXOIDS


This test is used for detection of toxic contaminant in vaccines.
Five mice (approx. 20g) and two guinea pigs (approx. 30g) are inoculated with one human dose
or 1.0 ml (whichever is less) of the test preparation.
All must survive for seven days without sign of illness.

BCG VACCINE (BACILLUS CALMETTE GUERIN)


This is a suspension of living cells of a strain of Mycobacterium tuberculosis known as BCG
vaccine (Calmette and Guerin were French bacteriologists).
Originally it was given orally, but due to unreliable absorption from gut, the intracutaneous route
is now preferred.
The vaccine is prepared immediately before use by reconstitution from the dried vaccine with a
suitable liquid.

Preparation
The strain grown on a suitable culture media, shows around 20 million colonies (seed lot
system).
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After suitable growth, they are separated by filtration in the form of cake.
The cake is homogenized in a grinding flask and suspended in a sterile liquid medium designed
to preserve the antigenicity and the viability of the vaccine (determined by plate count).
The suspension is transferred into a final sterile container and freeze dried under conditions
designed to prevent microbial contamination and finally sealed.
It is available as white pellets or powder, which when reconstituted, yields an opalescent and
homogenous suspension.
BCG tends to clump very badly when grown in conventional liquid media, and for a number of
years the preparation of a homogenous suspension depended on grinding these clumps with steel
balls using a sterile ball mill.
To overcome this problem a non-ionic surfactant (polyoxyethylene ether) is included in the
growth medium.
This causes the organism to grown throughout the medium instead of clumps or pellets, which
may even make the reconstitution difficult.
This also improves the appearance of the product.
Fluffiness is reduced, and reconstitution is assisted by the inclusion of dextran, which is also
used for drying the cells.
The medium also contains glucose which prevents excessive drying and, in the optimum
concentration, allows retention of optimum amount of moisture.

Storage:
BCG vaccine should be stored in sealed light resistant glass containers at a temperature
between 2 – 8oC. The reconstituted vaccine should be used immediately after preparation.

Dose:
Prophylactic, by intracutaneous injection, as a single dose, 0.1ml.
Nowadays, a new needleless technique of inoculation, involving penetration of the skin by a
high pressure jet of the preparation, is often used for mass vaccination.
Percutaneous vaccine is used in this method.

Use:
BCG vaccine is used as an immunizing agent which provides protection against tuberculosis.

TAB VACCINE (TYPHOID – PARATYPHOID A, B)


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It is a sterile suspension of Salmonella typhi and Salmonella paratyphi A and B.


TABC contains Salmonella paratyphi C in addition to TAB.
Preparation
TAB vaccine is a mixed polyvalent vaccine and is prepared by mixing of simple vaccines of
Salmonella typhi, Salmonella paratyphi A and Salmonella paratyphi B.
These strains are grown in acid digested agar medium and cultivated for 48 hours at 37oC.
These bacterial strains are harvested with a sterile normal saline solution.
Strains are diluted to form 3000 million organisms/ml of Salmonella typhi and 2250 million
organisms/ml of each of Salmonella paratyphi A and B.
All these strains are killed by addition of 0.1% formalin or by heat treatment.
Bacterial strains are incubated at 37oC for 4 days for detoxification and then tested for sterility.

Bacterial strains are mixed together to contain 1000 million organisms of Salmonella typhi and
750 million
organisms of each of Salmonella paratyphi A and B.
The suspension is transferred to final sterile containers and freeze dried.
The sterility and abnormal toxicity is evaluated further.

Storage:
Store in well closed containers at a temperature between 2 – 8oC.

Dose:
Prophylactic, 0.5ml (subcutaneous), 2 - 3 injections at 2 – 4 weeks interval.
Booster dose may be given every 1 – 2 years.

uses:
TAB or TABC mixed polyvalent vaccine is used in the prophylaxis of enteric infections.
TAB is also mixed with tetanus vaccine and cholera vaccine.

VIRAL VACCINES
Viral vaccines are prepared by using free living animals, fertile eggs and tissue cultures.
Viruses require a living medium for growth unlike bacteria which can grow on non-living media.
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Viruses are responsible for infectious diseases in man, animals and plants.
Important viral diseases are influenza, common cold, measles, mumps, poliomyelitis, smallpox,
yellow fever,
rabies etc.
For preparation of viral vaccines, viruses are usually grown in the chorioallantoic membrane of
incubated fertilevhen eggs or in whole animals.

POLIOMYELITIS VACCINE
Two types
1. Inactivated or salk-formalin vaccine
2. Live oral or sabin vaccine
Inactivated poliomyelitis vaccine is an aqueous suspension of suitable strains of poliomyelitis
virus, type I, II andvIII, grown in suitable cell cultures and inactivated by a suitable method.
Sabin poliomyelitis vaccine is an aqueous suspension of suitable live, attenuated strains of
poliomyelitis virus,vtype I, II and III, grown in suitable cell cultures. It may contain any one of the
three virus types or a mixture of twovor three of them.

POLIOMYELITIS VACCINE (Inactivated or salk-formalin vaccine)


Preparation:
Three types of poliomyelitis virus are grown separately in either suspended or fixed cell cultures
of monkey kidney tissue (Rhesus monkey kidney).
Nerve cells are avoided because these cells have a short life in tissue culture and sometimes
cause a allergic reaction in the brain.
After the virus suspension has been harvested, it is tested to confirm the presence of
poliomyelitis virus, good virus titre and absence of viral, bacterial and fungal contaminants.
It is passed through filters to remove remaining tissue cells and bacterial cells.
The vaccine is inactivated by using formaldehyde (0.01%), under controlled conditions of pH
and temperature with constant stirring.
Inactivation is usually completed in six days but the absence of active virus should be
confirmed.
The suspension is refiltered and after 9 to 12 days, the suspension is retested for absence of
infective virus.
The univalent vaccines are then blended to give the trivalent product and again tested for
sterility and freedom from infective virus.
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Finally formaldehyde is neutralized with sodium metabisulphite and thiomersal is added as a


bactericide.

POLIOMYELITIS VACCINE (live oral or sabin vaccine)


Preparation:
It is manufactured in the same way as salk type vaccine EXCEPT
Attenuated strains, prepared by rapid passages through tissue cultures of monkey kidney cells
are used.
The virus in the final vaccine must nor represent more than three subcultures from a strain that
laboratory and clinical tests have shown to be satisfactory.
There is no inactivation stage.
Final vaccine is free from bacteria, moulds, viruses as well as virulent poliomyelitis virus.
For primary immunization, oral poliovirus vaccine is generally given at birth and then at 6, 10
and 14 weeks.
Booster doses are given between 15 to 18 months.
Oral poliovirus vaccine is the vaccine of choice for active immunization of children because it is
simple to
administer, well accepted, induces systemic as well as intestinal immunity and is highly efficacious.
Simultaneous vaccination of all infants and children up to 5 years age has eradicated the wild
virus in many countries.

RABIES VACCINE

Rabies vaccine is a suspension of a suitable strain of fixed rabies virus grown in suitable
approved cell cultures and inactivated by suitable method.
The vaccine is prepared immediately before use by reconstitution from the dried vaccine with a
suitable sterile liquid.
Preparation:
Rabies vaccine may be prepared by injecting sheep, rabbits, suckling rats and mice or other
suitable animals intracerebrally with the rabies virus.
Those animals which show typical paralysis are sacrificed and brains are harvested by opening
their skulls in specially arranged sterile rooms.
Brains are emulsified to make 10% suspension in saline solutions.
The suspension is inactivated by using phenol or formaldehyde or b-propiolactone.
Brain emulsion is incubated at 37oC for 24 hours and then diluted with equal quantity of normal
saline.
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The final vaccine may contain a suitable preservative and sufficient neutralizers to adjust the
pH to 7.2.
Rabies vaccine complies with the tests for absence of live virus, sterility and abnormal toxicity.
It should be stored in sealed, light resistant containers at a temperature between 2 – 8oC.
Vaccines containing nervous tissue may cause an allergic response in the brain, leading to
nerve deterioration and paralysis.
These reactions are rare when vaccines are prepared in fertile hen or duck eggs.

MMR VACCINE (MEASLES – MUMPS – RUBELLA VACCINE)


Measles-mumps-rubella vaccine is a mixed preparation containing suitable live attenuated
strains of measles virus, mumps virus and rubella virus.
Mortality due to measles is still high in countries due to malnutrition.

Preparation:
The viruses are grown in chick embryo cells or in suitable approved cell cultures.
The virus suspensions are harvested at a time appropriate to the strain of virus being used and
suspensions of the same component are pooled.
The clarified preparations of the individual viral components are mixed and a suitable stabilizer
is added.
The vaccine does not contain any added antimicrobial preservative.
The final product is freeze dried to a moisture content shown to be favorable to the stability of
the dried vaccine.
The vaccine is prepared immediately before use by reconstitution from the dried vaccine with
the liquid stated on the label.
The vaccine is prepared by mixing lyophilized measles 5000 TCID50 of Schwarz strain, mumps
5000 TCID50 and rubella 1000 TCID50 or lyophilized measles 5000 TCID50 of Edmonston Zagreb
strain, mumps 5000 TCID50 and rubella 4000 TCID50 per unit dose (0.5 ml).
A single dose is injected (subcutaneous or intramuscular in children older than 12 months for
protection against these three diseases.)

DIPHTHERIA VACCINE OR TOXOID


Diphtheriavaccine or toxoid is a formol toxoid prepared from toxins produced by
Corneybacterium diphtheria.
Preparation:
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A suitable strain of Corneybacterium diphtheria is grown on a liquid medium (dextrose veal


infusion medium) at 35oC for 7 days.
After maximum toxin production, the bulk of the organisms are separated on paper pulp and the
filtrate is sterilized using fibrous pads or ceramic candles.

PREPARATION OF DIPHTHERIA TOXOID:


1. Formol toxoid (FT):
In the diphtheria toxin, 0.5% formalin is added and the mixture is incubated at 37oC for 3 – 4
weeks to remove toxicity.
The toxoid is confirmed for sterility and presence of toxins.
The final preparation is known as Formol Toxoid (FT).
For many years this product was used in its unpurified form.
It was an excellent antigen but it often caused severe reactions.
Different purification techniques are used for the purification of toxoids but it reduces its activity
and stability.

2. Toxin – Antitoxin Floccules (TAF):


If suitable quantity of toxoids (100 units) and antitoxins (80 units) are mixed they form floccules.
These floccules contain good antigenic activity.
These floccules are separated and washed to remove all containants.
Floccules are again resuspended in a saline solution containing bactericide.
This product is known as toxin – antitoxin floccules.

3. Alum precipitated toxoid (APT):


This preparation resulted from the discovery that slow adsorption of precipitated toxoids from
the site of injection and slow excretion from the body led to increased antigenic activity.
High quality formol toxoid is treated with charcoal to remove coloring matter and other
impurities.
The charcoal is separated by filtration and suitable concentration of alum is added.

This reacts with bicarbonate, phosphate and protein impurities in the toxoid to produce a
precipitate containing aluminum hydroxide and phosphate.
Then the precipitate is washed and suspended in saline containing bactericide.
Alum is used to potentiate the effect of antigens (adjuvants).
APT produces more antibodies than FT and TAF.

4. Purified toxoid aluminum phosphate (PTAP):


Purified toxoid aluminum phosphate (PTAP) is a pure toxoid prepared by using a semi-
synthetic medium in the preparation of the toxin.
It is prepared by using different purification techniques, involving the use of magnesium
hydroxide, to precipitate color, phosphate, ammonium sulphate, cadmium chloride and some
proteins.
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TETANUS VACCINE OR TOXOID


Tetanus vaccine or toxoid is prepared from the exotoxin of the anaerobe Chlostridium tetani.
This toxoid is prepared by using veal (calves) infusion peptone medium and maintaining
anaerobic conditions (anaerobic jar).
It is available in the forms of Alum Precipitated Toxoid (APT), Purified Toxoid Aluminum
Phosphate (PTAP) and other forms except Toxoid – Antitoxin Floccules (TAF).

PERTUSIS VACCINE OR WHOOPING COUGH VACCINE


Pertusis vaccine is a sterile suspension of killed Bordetella pertussis.
Preparation:
The Bordetella pertussis culture is maintained on a charcoal agar medium.
Preinoculum of culture is inoculated in Cohen Wheeler liquid medium and incubated for 48
hours at 37oC on a rotary shaker.
The bacteria are harvested and suspended in a saline or other appropriate solution isotonic
with blood.
The bacteria are inactivated using a suitable chemical agent (0.1% formalin) or by heating ate
56oC.
The suspension is stored at a temperature of 2 – 8oC for a period of 2 – 3 months to diminish
its toxicity.
The final suspension is made using a saline or other suitable solution isotonic with blood
containing a suitable antimicrobial preservative.
The opacity is again adjusted to 40,000 million organisms per CC.
The absorbed vaccine is prepared by addition of aluminum phosphate, aluminum hydroxide or
calcium
phosphate.

DPT VACCINE / TRIPLE VACCINE/DIPHTHERIA – TETANUS – PERTUSIS VACCINE

DPT vaccine is prepared from Diphtheria formol toxoid, tetanus formol toxoid and suspension
of killed Bordetella pertussis.
These three components are mixed in the following proportions to form DPT vaccine.

Pertusis vaccine - 0.5 CC


Diphtheria toxoid - 0.2 CC
Tetanus toxoid - 0.3 CC
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Merthiolate (0.01%) is commonly added as a preservative immediately before mixing and then
the preparations to form DPT vaccine are mixed.
The antigenic properties of DPT vaccine are adversely affected by exposure to the action of
certain antimicrobial preservatives like phenol and some of the quaternary ammonium compounds.

ANTITOXIC SERA / ANTITOXINS


Antisera or immunosera are preparations which contain antibodies.
If the blood of an immune person or animal is withdrawn and allowed to clot, a large number of
antibodies are found in the serum that separates.
A serum may contain antitoxic, antibacterial or antiviral antibodies and these are known as
antitoxic, antibacterial or antiviral serum respectively.
Antitoxic sera are commonly called as antitoxins.
Antitoxic sera are more effective than antibacterial or antiviral sera.

DIPHTHERIA ANTITOXIN
Diphtheria antitoxin is a sterile, non-pyrogenic solution containing the specific antitoxic
antibodies obtained from the serum of healthy horses and have the power of neutralizing the toxin
formed by Corynebacterium diphtheria.
Preparation
The method of preparation is divided in following steps
1. Preparation of toxin
A pure culture of Corynebacterium diphtheria is grown in a suitable culture media at 37oC for 4
– 5 days.
After incubation, 0.5% phenol is added and the culture media is filtered through bacteria proof
filters.
The filtrate (crude toxin) is converted into a toxoid.

2. Selection of horse
Horses are selected for production of Diphtheria antitoxin because they are easy to handle and
readily produce antitoxins.
Considerable quantity of blood can be withdrawn at a time without any ill effects. The horses
selected must be free from diseases.
Horses are kept in an isolated place for 7 days and then carefully examined for infectious
diseases.

3. Active immunization of the horse


Diphtheria toxoid is given to the selected healthy horses for active immunization.
The toxoid is injected into the neck muscles by intramuscular injection.
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The first dose of toxoid is not more than 5ml which is gradually increased daily or after 1 - 2
days for about 2
months and as much as 600 ml is injected for the final dose.

4. Separation of serum for the horse:


After about 10 days of the final dose injection, about eight liters of blood is withdrawn
aseptically into bottles
containing an anticoagulant.
Similarly two more collections each of eight liters are made within next eight days, after which
the horse is given about 15 days’ rest.
Another course of toxoid is repeated in similar doses to stimulate further antibody production.
Blood is collected in three batches each about eight liters.
Further courses of administration of the toxoid and bleeding are continued for 4 – 5 times or till
the animal stops producing satisfactory antitoxins.
After the collection of the blood, it is allowed to clot, to separate the serum.
The serum contains antitoxin along with other proteins, such as beta-globulins, gamma-
globulins and albumins.
Antitoxins are largely associated with beta-globulins.

5. Concentration and refinement


Horse serum contains a high concentration of several other proteins wich may cause
undesirable reactions, such as anaphylactic shock or serum sickness.
So these undesirable proteins are separated by fractional precipitation or by fractional
proteolytic digestion
method
Diphtheria antitoxin has a potency of not less than 1000 IU/ml, in the case of antitoxins
obtained from horse
serum and not less than 500 IU/ml for antitoxins obtained from other animals.

Storage
Diphtheria antitoxin is stored in containers protected from light at a temperature between 2 –
8oC.
It should not be allowed to freeze.
Dose
Diphtheria antitoxin is administered by subcutaneous or intramuscular injection.
For prophylactic use, the dose is not less than 1500 IU and for therapeutic use, the dose is not
less than 50,000 IU.

Use
Passive immunizing agent for diphtheria.
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TETANUS ANTITOXIN
Tetanus antitoxin is a preparation containing antibodies that have the power of specifically
neutralizing the toxins formed by Clostridium tetani.
It is obtained by fractionation from serum of horses or other mammals that have been
immunized against tetanus toxins.
Method of preparation, storage and dose are similar to diphtheria antitoxin.

ANTIVIRAL SERA
These are used in passive immunization against certain viruses.
It acts differently as viruses are intracellular parasites while antibodies cannot penetrate the
cells.
Inactivation of viruses takes place in body fluids or on surfaces at which invasion occurs.
The main source of antiviral antibodies is human serum.
This is because the horse is not susceptible to many viruses against which protection may be
required. Eg.
Measles, rubella, poliomyelitis etc.
However, official antiviral sera (Eg. Rabies antiserum) is prepared in horses.
Rabies antiserum or Antirabies serum (ARS)
Antirabies serum (equine rabies immunoglobulin, ERIG) is a refined, concentrated and
lyophilized serum from horses hyperimmunized by repeated injections of fixed rabies virus.
Horses are injected with dead virus and when a good level of immunity has developed, are
injected with live viruses.
The method of protein purification used to refine the serum are designated to separate the
gamma-globulin
fraction that contains the antiviral antibodies.
Antirabies serum is indicated promptly after suspected exposure and is give simultaneously
with rabies vaccine to non-immunized individuals.

ANTIVENOMS
The incidence of snakebites in different parts of the world and the recognition of the particular
species of greatest medical importance is fundamental to the appropriate design of monospecific
and polyspecific antivenoms in countries and regions. Up-to-date knowledge is therefore highly
relevant to antivenom manufacturers and regulators, especially for the selection of the most
appropriate venoms, or venom mixtures, to be used in the production and quality control of
antivenoms.
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Venom: The toxic secretion of a specialized venom gland which, in the case of snakes, is delivered
through the fangs and provokes deleterious effects. Venoms usually comprise many different
protein components of variable structure and toxicity.

Antivenoms: A purified fraction of immunoglobulins or immunoglobulin fragments fractionated from


the plasma of animals that have been immunized against a snake venom or a snake venom
mixture.

There are variations in venom composition and antigenicity within the geographical range of a
single taxonomic species as well as other causes of intra-species variation. Therefore, pooled
representative samples of venoms should be prepared from snakes of different geographical
origins and ages. Cross-neutralization of venom outside the range of venoms used for
immunization may extend the range of therapeutic applications of some antivenoms. Results of
preclinical potency testing may be used to identify a potential cross-neutralization capacity of
antivenoms, which should subsequently be confirmed by clinical testing in envenomed patients. In
vitro immunological cross-reactivity should not be used as the single basis for recommending
therapeutic use of an antivenom outside the range of venoms used in its production.

Procedure:
The snake is gently removed from its cage with a hook and either placed on a foam rubber pad
before being pinned behind the head or encouraged to crawl into a transparent plastic tube. For
very dangerous species, the use of short-acting general anaesthesia, or moderate cooling (15°C)
during milking can be considered (e.g. inhaled sevofluorane, halothane or even carbon dioxide) as
it reduces the risk of accidents both to the snake and to the snake-handler. For the collection of
venom, the snake’s head is grasped between index finger and thumb, just behind the angle of the
jaw, while the snake’s body is held between the trunk and the arm of the snake handler. By
applying gentle pressure, the snake's jaws are forced open, the fangs exposed and, in the case of
vipers, erected. In the case of large vipers, the dental sheath is retracted when necessary with
clean forceps
.
The fangs are pushed through a plastic/parafilm membrane hooked over the lip of a glass vessel,
and venom is squeezed out. The use of siliconized containers might be considered to minimize
venom attaching to the container surface. While a brief electrical impulse of moderate intensity can
be applied to stimulate venom secretion, this technique is not used or required by most venom
producers, although it may help in avoiding debris in the venom.

Any venom sample contaminated with blood should be rejected. After venom extraction, the fangs
are carefully withdrawn from the collection vessel, while preventing damage to the mouth and
dentition and avoiding the snake’s impaling itself with its own fangs.
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DIAGNOSTIC AGENTS
Diagnostic preparations are used as in vivo diagnostics, when injected intradermally into the
patients.
Diagnostics tests are based upon hypersensitivity reactions and it shows sensitivity to antigens by
the presence of antibodies.
It is also used to assess immunocompetency in individuals with possible cell mediated
immunodeficiency.

THE SCHICK TEST


The Schick test works on the following principle.
“When a dose of toxin is injected intracutaneously and if the patient is immune, the presence of
antibodies will neutralize the toxin. If antitoxins are absent, the toxin produces a local
inflammation.”

1. Schick test toxin


Schick test toxin is the preparation used in the Schick test to determine susceptibility to
diphtheria.
It is prepared from a toxigenic strain of Corynebacterium diphtheria.
The toxin s purified and then diluted with a sterile aqueous buffer solution of pH 7.2 – 7.6.
The preparation is made isotonic to the blood and the test dose is 0.1-0.2ml.
Schick test toxin is stored at temperature of 2 – 8oC

2. Schick control
Schick control is the Schick test toxin that has been heated at a temperature not lower than
70oC and not higher than 85oC for atleast five minutes to destroy the toxin.
It may be used in conjugation with Schick test toxin in order to exclude reactions due to non-
specific substances.
Schick control is prepared from the same batch of Schick test toxin with which it is to be stored
at a temperature of 2 – 8oC.
Left arm Right arm Explanation Inference
(Toxin) (control) Toxin is- Individual is-
Large flushed area No reaction Not neutralized Not immune
No reaction No reaction Neutralized Immune
Large flushed area Small flushed area Not neutralized; Not immune to a
sensitivity pseudo-reactor
to broth constituents
Small flushed area Small flushed area Neutralized; sensitivity Immune and a
to pseudoreactor
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broth constituents

TUBERCULIN TEST
Persons suffering from tuberculosis or who have recovered from an active or latent infection
are extremely
sensitive to a protein constituent of the tubercle bacillus, Mycobacterium tuberculosis.
The tuberculins contain this protein and, therefore, when they are applied to or injected into the
skin of sensitized individuals a local inflammation is set up.
Minute doses are sufficient to cause the reaction.
In general, only people who have never been infected will give a negative result.
The bacterial protein (tuberculin), which may be regarded as a toxin, acts as an antigen which
stimulates the
tissues to produce corresponding antibodies.
These antibodies react strongly with the antigen when they meet it again, causing inflammation,
and even
necrosis, of the cells in the area.
This hypersensitivity reaction, or allergy, appears to be a defense mechanism designed to limit
the spread of
infection.
The mechanism of immunity to tuberculosis is uncertain.
In individuals who are not actually suffering from the disease the presence of tuberculin
sensitivity usually
indicates immunity.
It is the tuberculin negative group, therefore, that can usefully be given BCG vaccine after
which, if the
immunization has been successful the tuberculin reaction should be positive.

OLD TUBERCULIN
Tuberculin was first made by Koch, and as the official preparation is still based on his
preparation it is known as Old tuberculin.
It is the heat-concentrated fluid from a culture of either the human or bovine strain of
Mycobacterium tuberculosis grown in a fluid medium.
Early products contained large amounts of nonspecific, reaction-causing impurities, partly from
the meat broth used for growing the organisms and partly from the metabolism and autolysis of the
latter.
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Unwanted broth constituents are now excluded by the use of a synthetic medium containing 5%
glycerin, dextrose and, as the only sources of nitrogen. asparagine (an amino acid) and ammonium
salts.
After inoculation with the organisms the medium is incubated at 37oC for six weeks or more.
Mycobacterium tuberculosis has a very long generation time of about a day and, therefore,
prolonged incubation is necessary to obtain abundant growth.
The culture is then steamed for an hour.
This kills the organisms and destroys the antigenicity of many of the protein impurities but does
not harm the
tuberculin which is very heat stable.
The preparation is then concentrated to about one-tenth of its original volume, a preservative
bactericide is added, and the solution is sterilized by filtration.

TUBERCULIN PURIFIED PROTEIN DERIVATIVE (TUBERCULIN PPD)


The active protein from Old Tuberculin was isolated by Seibert in the 1930s and is now known
by the above
names or simply as PPD. It is free from broth constituents and metabolic and autolytic products of
the bacteria.
Two types are officially recognized, mammalian and avian. The former is prepared from the human
or bovine
strain of Mycobacterium tuberculosis, the latter from the avian type. After growing the organisms in
a synthetic
medium, as in the preparation of Old Tuberculin, the culture is filtered through paper pulp, to
remove the bacteria, and then subjected to ultra-filtration to remove the glycerin and mineral salts.
To this a protein precipitant (e.g. ammonium sulphate or trichloracetic acid) is added. The
precipitated protein is separated, purified, and made available as a freeze-dried powder,
hypodermic solution tablets, or a concentrated solution.

TUBERCULIN TESTS
1. The Mantoux Test
This is the most precise method. Initially, a very low dose (I unit) is injected intracutaneously in
a volume of 0,1 ml. If the result is negative the dose is increased to 10 units and, if there is still no
reaction, to 100 units. Graded dosage is necessary because of the great difference in tuberculin
sensitivity between individuals. It is necessary to start with a low dose since very sensitive persons
may show severe reactions to the higher concentrations. To avoid repeated testing, the WHO has
used, in some of its surveys, a single dose of 5 units, which explains why the Pharmacopoeia
mentions four strengths.

2. The Heaf Test


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This has become the most popular method in the UK because the method of inoculation is less
painful, simpler, and more reliable than use of a syringe and needle. A spring-release device
makes several intracutaneous punctures of equal depth through a film of tuberculin previously
applied to the skin. PPD is preferred for this lest.

3. The Tine Test


An ethylene-oxide sterilized, disposable unit consisting of a short plastic handle connected to a
stainless-steel disc carrying four tines (teeth) coated with old tuberculin (5 unit strength) is
commercially available. The results compare favorably with those from the Mantoux test. The
method is painless and quick, and syringes, solutions etc. are unnecessary. The results of these
tests are read after 72 hours. A positive reaction consists of a raised indurated area; erythema
without induration is disregarded. Various percutaneous tests have been tried, They involve
application of tuberculin to the surface of the skin in an ointment, jelly, filter paper patch etc., but
they are much less reliable than intracutaneous methods.
Individual Schick Test Tuberculin Test
Immune Negative Positive
Non immune Positive Negative

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