Immunity

Natural Acquired (added during life time)

(constitutional makeup)
Species Active Passive(rapidly lost)
Race
Individuals
Naturally Artificial
stimulated
After Sub clinical • by toxoids or
• suspension of microorganism
clinical infection
infection

Naturally acquired Artificially produced

(injection of antibodies
( mother) containing preparation)
Artificially produce passive immunity

This is effected by injection of antibody produced in animals, usually horse.

Antibody containing preparation are called antisera , sera and official product
include preparation containing-
a. Antitoxic antibodies: neutralizes exotixin
e.g. diphtheria antitoxins; Tetanus antitoxins.

b. Anti bacterial antibody: neutralized endotoxin e.g. Laptospira antiserum.

c. Anti viral antibody: combat viruses. e.g. rabies antiserum

 Differences between antigen and antibody containing
preparations
Antigen containing prep. Antibody containing prep.
Stimulate active immunity Give passive immunity
Patient produces antibody Patient receives antibodies
Immunity develops slowly works rapidly

Long lasting and effective Temporary effect

Used for long term prophylaxis Short term prophylaxis and

PREVENTION therapeutics
 Official vaccines
Diphtheria
Toxoid Staphylococcus Suspension of
Tetanus microorganisms

Rikettsial Viral
Bacterial

Killed Attenuated
BCG
Cholera
Pertusis Killed
Plague
Typhus
TAB
TABC

Killed Attenuated
Influenza; Measles; Poliomyelitis; Rabies Poliomyelitis ; Measles ; Yellow fever;
Small pox
 Antigen containing preparation (Vaccines)

Vaccine contain either toxins of microbes or the microorganism itself, their

toxicity reduce to such level that they do not produce any harm to host other then
just to give stimulation for the development of antibody by the host defense
system.
Antigens are- toxin , toxoids, endotoxins, whole attenuated cells

Toxoid: Toxin + Formalin -- Decrease the toxicity of toxin without affecting

its antigenicity. formalin act on the toxic group
without affecting the antigenically active sites.
Diphtheria

Diphtheria is an acute, toxin-mediated disease caused by the bacterium

Corynebacterium diphtheriae.
 The toxin is responsible for the major complications of
myocarditis and neuritis and can also cause low platelet counts
(thrombocytopenia) and protein in the urine (proteinuria).

 The overall case-fatality rate for diphtheria is 5%–10%.

Diphtheria Vaccine
Preparation :
C. diphtheriae is grown in liquid media excluding the
broth from horse muscles because toxin from horse
muscles may sensitize recipients.
Once the toxin production reach to satisfactory level bulk
organisms are removed on paper pulp( to prevent clogging with filter pads
and ceramic filter) and filtered. Then, filtrate containing toxin are

subjected to convert into toxoids

Incubated at 37 oC
Toxin + formalin Formol toxoids
2-3 weeks

Formol toxoids without purification namely called anatoxin have

excellent antigenic activity but may cause severe senstivity
reactions due to presence of broth constituents and metabolic
products.
 Preparation of other form of toxoids

Toxoids – anti toxin floccules ( TAF) Floccules are washed with

Aluminum precipitated toxoids ( APT) purified water until
preparation become colorless
Purified Toxoid aluminium phosphate(PTAP) and resuspended into saline
solution containing
bactericides

Toxoid antitoxin floccules

3 weeks
Toxoid (100 Unites) + Antitoxin (80 Units) Floccules
Supernatant decanted
produce

•Although the product is neutralized with

antibody, they have significant antigen activity
because neutralization treatment neither damage
antigen (toxiods) nor antibody( antitoxin)
The product must be completely free from
contamination of broth constituents and
metabolic products.
 Aluminum precipitated Toxoids (APT)

based on the principle that slow absorption of toxoids from the site of
administration will enhance the antigenic activity.
a.remove coloring matter
Formol toxoids + Charcoal b. Nonspecific impurity ( to
Mixture
prevent reaction)

Filter

Formed corresponding aluminum Alum : this will react with

hydroxide and aluminum phosphate
precipitate: these will act as carrier for
the toxoids
bicarbonate; phosphate and
Filtrate
proteins impurities of
toxoids
-higher antibody level ( two dose)
-Free from horse horse protein (improved purity)
- Adult or young children- local reaction due to
carrier

Precipitate is washed and resuspended in saline

solution containing suitable bactericides.
Purified Toxoid Aluminium Phosphate (PTAP)

Difference from APT: it is purer form, its purification carried out by:

a. Using semi synthetic media in preparation of toxin

b. Completely purify the toxoids by using magnesium hydroxide to

precipitate color, phosphate and some proteins

Combined prophylactics:
Diphtheria + Tetanus + Pertussis + Poliomyelitis
Ministry of health- recommended

Summary: If given separately- large number of

Make preparation complete free from toxicity injection, attendance and burden
Eliminate impurities, involve which is burden for children,
Maintain antigenicity at high level.
doctors, nurse, record keeper
To monitor for any adverse effect observe from new developments.
 Tetanus is an acute, often fatal, disease caused by an
exotoxin produced by the bacterium Clostridium
tetani. But prevented by immunization with tetanus
toxoid.
It is characterized by generalized rigidity and
convulsive spasms of skeletal muscles.
The muscle stiffness usually involves the jaw
(lockjaw)and neck and then becomes generalized.
 TETANUS TOXOID
• Tetanus toxoid consists of a formaldehyde-treated
toxin.
• There are two types of toxoid available —
adsorbed (aluminum salt precipitated)toxoid and
fluid toxoid.
• Although the rates of sero conversion are about
equal, the adsorbed toxoid is preferred because
the antitoxin response reaches higher titers and is
longer lasting than that following the fluid toxoid.
Clostridium tetani culture is grown in a peptone-based medium and detoxified
with formaldehyde.
The detoxified material is then purified by serial ammonium sulfate
fractionation, followed by sterile filtration, and the toxoid is adsorbed to
aluminum potassium sulfate (alum).
The adsorbed toxoid is diluted with physiological saline solution (0.85%)and
thimerosal (a mercury derivative) is added to a final concentration of 1:10,000.
Each 0.5 mL dose is formulated to contain 5 Lf (flocculation units)of tetanus
toxoid and not more than 0.25 mg of aluminum.
The residual formaldehyde content, by assay, is less than 0.02%.
 Suspension of Microorganisms
Microorganisms are – bacteria , rickettsia or viruses- live or dead
 Unlike toxoids they are either simple (e.g. Plague- Pasteurella
pestise) or mixture of two or more simple vaccine together ( e. g. TAB
and TABC).
Further
Univalent- from single strains e.g. yellow fever- 17D strains
Polyvalent- More than one strains
e.g. Cholera- Vibrio cholerae :Inaba + Ogawa
Poliomyelities- Poliomyelities type I + II + III
Typhoid – S. typhi, S. paratyphi A and B strains
Variation in Microorganism:
Occur due to the loss of antigens from the cell surface of the microorganisms.

Specification from Pharmacopoeia: ( to ensure strain of proper antigenic properties)

1.Selection of appropriate strains e.g.
Cholera – Smooth strains two main serological types
 TAB- S. typhi, S. paratyphi A and S. paratyphi B.
Plague- Capsulated form of Pasturella pestis
 Typhus – Virulent Ricketsia
Influenza – strains that are non- infective but retains it antigenic properties.
 Yellow fever- 17D.
2. The antigens that must be present

Cholera- all strains must contain heat stable antigen in addition to O

for both type
TAB- O and H antigens and also Vi antigens.

3. Time of Harvesting
BCG- within fortnight
Plague- time of maximum capsulation
Measles vaccine- not more then
a 10 passage strains from seeds.
Killed bacterial suspension
General approach for preparation of killed bacterial suspension

Preparation of bacterial cells:

Each strain is checked for the freedom from variation and absence of
contaminating organisms and then it is inoculated into a liquid media
and incubate under suitable condition one to three days.

At the end of incubation the cells are washed and centrifuged to remove
agar and other broth constituents from the cells by repeatedly washing
until complete freedom from broth constituents and then resuspended in
saline solution.
Sterilization of Bacterial suspensions

Sterilization of cell suspension is carried out either by heat or using

bactericides or a combination of both.

a. By heat: Low temperature is used to avoid the loss of antigenicity;

usually at 56oC for 1 hr. This method is not reliable for spores.

b. By bactericides: When heat treatment affects the antigenicity of the

preparation then chemicals are used, for e.g.0.5% formalin is used to kill
the cell suspension of plague and pertusis, phenol for cholera.
Preparations
• Cholera vaccines from Vibrio cholerae
• Whooping cough vaccines from Bordetella pertusis
• Plague vaccines from Pasturella pestis
• Typhoid Vaccines ( TAB and TABC) from S. typhi and partyphi A &
B and S. typhi A, B and C respectively.
 Living Bacterial cell suspensions:
BCG Vaccines:
Contain live attenuated cell suspension of M. tuberculosis.
Preparation:
For preparation of live cell suspension a strain of bacteria is used. For
attenuation the strain is grown on ox bile media, with hope that animal
variety will have much less virulence than man ; thus can be safe.

Method used for preparation of dead and living cells are same except
that for living preparation
a. No sterilization stages.
b. The viability of the cell must be maintained.
c. Standardization on the basis of viable counts.
Before use, the strains are thoroughly checked for their antigenicity and
freedom from pathogenicity. It is then grown in a liquid broth media for
not more than 14 days and then organisms are separated by
centrifugation, washed and suspended in vehicles and checked for
viability.

Liquid preparations are replaced by freeze dried products for two basic
reasons:
1. Rapid deterioration of product even when stored under ideal condition
2. Because of its short life, control test, including vital test for
virulence can not be completed.
Advantages of freeze drying :
i. Retain its potency at least a year while stored under similar
conditions
ii. All the quality control test can be performed easily.
 Antibody containing preparation:

Diphtheria antitoxin
Botulinium antitoxin
Gas- gangrene antitoxin
Staphylococcus antitoxin
Tetanus antitoxin

Antitoxins are antibodies that are used to protect the body by the process of passive
immunization. The antibodies are produced in a donor animal by active immunization, and
after a period of several weeks to months, the donor's blood is collected and the
immunoglobulins are separated into specific components that can be used to provide
immunity to the recipient.
Diphtheria Antitoxin

Diphtheria Antitoxin is a preparation containing the specific globulin having

specific activity of neutralizing the toxin formed by Corynebacterium
diphtheria. It is obtained by purification of hyper-immune serum/plasma of
healthy equines.

Diphtheria antitoxin, produced in horses, was first used in the United States
in 1891. It is no longer indicated for prophylaxis of contacts of diphtheria
patients, only for the treatment of diphtheria.

Antitoxin will not neutralize toxin that is already fixed to tissues, but it will
neutralize circulating (unbound) toxin and will prevent progression of
disease. The patient must be tested for sensitivity before antitoxin is given.
Preparation of Diphtheria antitoxin

1. Animal Selection and Care

Careful consideration should be given to the appropriateness of the

species and strain chosen.

Factors to be considered :
1) the quantity of Ab or antiserum required (larger animals should be
considered when larger quantities of Ab are required);

2) the phylogenetic relationship between the species from which the

protein antigen originated and the species used to raise the Ab;

3) the intended use of the antibodies

Horses preferred for production of antibody:

 They are large enough thus considerable volume of blood can be drawn
without any ill-effect

Horses are easy to handle, simplicity in administering the injection and

withdrawal of blood.

In addition there red blood cell readily settle and pack tightly which
facilitates the separation of serum.
First the horse are isolated at least seven days before of immunization.

a.Horses are thoroughly checked for the infectious diseases.

b.They are immunized against tetanus, to which horses are very

susceptible .

c. Their blood is examined for existing antibodies (basal immunity

shortens the time required to reach desired antibody level)
2. Immunization-

After Isolation, gradually increasing amounts of toxoids are injected into the
neck muscle every few days for several months.

The first dose is usually less than 5ml and may end at as much as 600 ml by
the time a satisfactory level of antitoxin titer has been attained.

(Antibody titer indicates the level of the antibodies in a blood sample, defined as
the greatest dilution (lowest concentration) of the blood sample at which an
antibody assay, such as ELISA, still produces a detectable positive result. The
higher the antibody concentration in the blood, the greater the dilution that will
produce a detectable signal).
Blood Collection

Eight liters of blood are withdrawn aseptically from the jugular vein into bottles
containing anticoagulant solution. The bleeding is repeated twice over the
next eight days after which the horse is given ten days rest.

Another short course of antigen is administered to stimulate further antibody

production, and afterwards three bleedings are repeated.

Rest, course of antigen and bleeding repeated until the animal stops
production of sufficient antitoxin titre. Usually 4-5 courses.

Blood is stored in refrigerator until cells have settled.

Plasma is siphoned off aseptically and calcium chloride is added to induce

clotting. Serum is separated by filtration
serum refinement:
Carried out to prevent
a.the hypersensitivity reactions such as anaphylactic shock.
b.Serum sickness:

Purification – Antitoxins are usually beta globulins.

Antibacterial and antiviral antibodies are usually gamma
globulins.
Two method are used for purification:
1.Concentration by fractional precipitation:
Using ammonium sulfate : sufficient ammonium sulfate added to the serum to
achieve one third saturation. Gamma globulin will be precipitated which will be
separated and discarded.
Add more ammonium sulfate to get half saturation. Beta globulin fraction
together with its associated antitoxin, will be slowly precipitated.
The liquid portion contain albumin is separated by a filter press and discarded.
The precipitate is removed from the filter cloths to sheets of cellulose film
which are made into bags and suspended in tanks of chlorinated running
water. Dialysis takes place and ammonium sulphate passes out through the
cellophane and, as it is removed, the antitoxic globulin passes back into
solution.

The process takes from 1-2 days and, the chlorine in the water prevents
microbial contaminants from multiplying and producing pyrogen.

The solution is adjusted to isotonicity with blood plasma. If required,

preservative is added.

Finally, it is passed through pyrogen removing and sterilizing filters.

2. Concentration by proteolytic digestion:
The serum is diluted, pepsin is added and the pH is adjusted to approx. 4.
After incubation at 37°C for two days the following changes occur:
The Albumin is almost completely digested and the products will pass
through a dialysis membrane.
 The gamma – globulin fraction is partly digested to dialyzable compounds
and partly precipitated by the pH.
 The beta-globulin is split into two fragments , only one of which has the
antitoxic activity. Both are colloidal solution and are too large to pass through
a dialyzing membrane.
The liquid is filtered to remove the precipitated gamma – globulin and the
filtrate is subjected to ultrafiltration during which the dialyzable products of
the digestion, inorganic salts, and the bulk of the water are removed.
The concentrate is heated at 55°C for 1 hr after addition of ammonium
sulphate.
The inactive fragment of the beta-globulin is denatured by this treatment and is
precipitated in presence of inorganic salts.
It is filtered off and more ammonium sulphate is added to the filtrate to
precipitate the active fragment.
This is separated, dialyzed, adjusted to isotonicity, and preserved.

The incidence of serum sickness with this product is low (Only 5%).

STORAGE:
2 - 80C in dark and should not be allowed to freeze.