Fundamentals of

Immunology:
Manufacturing and
quality control of
Immunological
products
 Immunity

Natural – part of Acquired-added

constitutional make-up during life time

Active (developed Passive (produced

and lost slowly) and lost quickly)

Naturally acquired Artificially stimulated Naturally acquired Artificially produced

After clinical
infection

By injection of By injection of
antigen containing antibody
After sub-clinical From the mother
preparation (vaccine) containing
infection
preparation (sera or
immunoglobulin)
Immunological products comprise a group of pharmaceutical
preparations of varied composition but with a common
pharmacological purpose: the modification of the immune
status of the recipient, either to provide immunity to
infectious disease, or in the case of in vivo diagnostics, to
provoke an indication of immune status usually signifying
previous exposure to the sensitizing agent.

The immunological products that are currently available are

of the following types: vaccines; in vivo diagnostics; immune
sera; human immunoglobulins; mono-clonal antibodies; and
antibody-targeted therapeutics and diagnostics.
 Introduction
Vaccines are perhaps most widely used and effective products
encountered in medical sciences. At around the turn of the last century
it was discovered that particular microbes gave rise to specific diseases
and this link led to the development of vaccines.

Immunity is the state of protection against infectious disease conferred

either through an immune response generated by immunization or
previous infection or by other non-immunological factors.

Artificial immunity is induced by immunization. This is achieved by

giving a vaccine (active immunization) or immunoglobulin (passive
immunization).

The term vaccination means “protection from the causative infection”

through artificial administration of that specific antigen into the body.
Fig. 3 | The generation of an immune response to a vaccine. The immune
response following immunization with a conventional protein antigen. The
vaccine is injected into muscle and the protein antigen is taken up by dendritic
cells, which are activated through pattern recognition receptors (PRRs) by
danger signals in the adjuvant, and then trafficked to the draining lymph node.
Here, the presentation of peptides of the vaccine protein antigen by MHC
molecules on the dendritic cell activates T cells through their T cell receptor
(TCR). In combination with signalling (by soluble antigen) through the B cell
receptor (BCR), the T cells drive B cell development in the lymph node. Here, the
T cell-dependent B cell development results in maturation of the antibody
response to increase antibody affinity and induce different antibody isotypes. The
production of short-lived plasma cells, which actively secrete antibodies specific
for the vaccine protein, produces a rapid rise in serum antibody levels over the
next 2 weeks. Memory B cells are also produced, which mediate immune
memory. Long-lived plasma cells that can continue to produce antibodies for
decades travel to reside in bone marrow niches. CD8+ memory T cells can
proliferate rapidly when they encounter a pathogen, and CD8+ effector T cells
are important for the elimination of infected cells.
Active immunity
This is the stimulation of the immune mechanism to produce
antibodies by giving an antigen as a vaccine. Such vaccines
may be:
Live attenuated viruses (rubella, measles, oral polio, mumps) or
bacteria (BCG)
Inactivated viruses (parenteral polio, hepatitis A) or parts of the
virus (pneumococcal vaccine, influenza)
Inactivated bacterial toxins (diphtheria and tetanus)
Genetically engineered (Hepatitis B vaccine)
 Different types of Bacterial and Viral vaccines

Types Bacterial Vaccines Viral Vaccines

Live- attenuated BCG Vaccination Measles Vaccination
Vaccines Typhoid Vaccination (oral) Mumps Vaccination
Cholera Vaccination (oral) Rubella Vaccination
Oral Polio Vaccination
Yellow fever Vaccination
Varicella Vaccination
(chickenpox)
Rotavirus Vaccination
Japanese Encephalitis
Vaccination
Toxoids Diphtheria Vaccination
Tetanus Vaccination
Genetically Hepatitis B Vaccination
engineered Corona virus
 Different types of Bacterial and Viral vaccines
Types Bacterial Vaccines
Inactivated Pertussis Vaccination
Vaccines Cholera Vaccination (oral,
combined with recombinant
B-subunit)
Anthrax Vaccination
Plague Vaccination
Polysaccharide H. influenzae B Vaccination
extracts of virus Meningococcal A and C
Vaccination
Pneumococcal Vaccination
Typhoid Vaccination
Viral Vaccines
Influenza Vaccination
Hepatitis A Vaccination
Injectable Polio
Vaccination (Salk)
Rabies Vaccination
Tick-borne Encephalitis
Vaccination
Japanese Encephalitis
Vaccination
 Active Antigenic Products

Active immunity refers to the process of exposing the body to an

antigen to generate an adaptive immune response:

- the response takes days/weeks to develop but may be long lasting—

even lifelong. Active immunity is usually classified as natural or
acquired.
- Wild infection for example with hepatitis A virus (HAV) and
subsequent recovery gives rise to a natural active immune response
usually leading to lifelong protection.
- In a similar manner, administration of two doses of hepatitis A vaccine
generates an acquired active immune response leading to long-lasting
(possibly lifelong) protection. Hepatitis A vaccine has only been
licensed since the late 1980s so that follow-up studies of duration of
protection are limited to <25 years—hence, the preceding caveat about
duration of protection.
 Vaccine types
The majority of population of any country can be expected to have
been immunized against diphtheria, tetanus, BCG, whooping cough
and polio. Depending on their age and gender, they may also have had
measles, mumps, rubella, Haemophilus influenzae type b (Hib)
and Neisseria meningitidis type C (Men C).

These different commercially available vaccines can be classified into

one of four types depending on the nature of the vaccine antigens—
i) live attenuated,
ii) killed inactivated,
iii) toxoid and
iv) subunit.
Subunit vaccines can be further subdivided into those where the
antigen is produced using recombinant DNA technology and those
based on normal bacteriological growth processes.
 Live Attenuated Vaccine

There are several approaches to attenuating a viral

pathogen for use in humans. One involves growing the virus
in a foreign host—for example, measles virus is cultivated in
chick egg fibroblasts—viral replication in such circumstances
results in the appearance of a number of mutant types.

Those mutants with enhanced virulence for the foreign host

are then selected as potential vaccine strains since they
generally show reduced virulence for the human host and
this is a particularly useful approach for RNA viruses
(including CORONA viruses) which have a high mutation
rate.
Live attenuated vaccines that might be used in the occupational
setting include measles, mumps, rubella and chickenpox.

Using measles as an example, the vaccine is injected deep

sc/im where virions enter various cell types using receptor-
mediated endocytosis. Within the cytosol, proteolytic
degradation of viral proteins occurs; the peptides produced are
then loaded onto major histocompatibility complex type I
molecules and the complex is displayed on the cell surface.

Circulating cytotoxic T cells (Tc) with the appropriate high-

specificity TCRs are able to recognize the complex and release
cytokines that instruct the (infected) cell to undergo programmed
suicide (apoptosis). It appears that some Tc become memory
cells but the basis of this is incompletely understood.
Killed/inactivated vaccines
The term killed generally refers to bacterial vaccines, whereas
inactivated relates to viral vaccines.
Typhoid was one of the first killed vaccines to be produced and was
used among the British troops at the end of the 19th century.
Polio and hepatitis A are currently the principal inactivated vaccines
used in many countries, whole cell pertussis vaccine continues to be
the most widely used killed vaccine.

The adaptive immune response to a killed/inactivated vaccine is very

similar to a toxoid vaccine with the exception that the antibody
response generated is directed against a much broader range of
antigens.
Hepatitis A is an example of an inactivated vaccine that might
be used by occupational health practitioners. It is a formalin
inactivated, cell culture adapted, strain of HAV; vaccination
generates neutralizing antibodies and protective efficacy is in
excess of 90%.

Additionally, staff working with children who are not toilet trained
or in residential situations where hygiene standards are poor
may also be offered vaccination.

Primary immunization with a booster between 6 and 12 months

after the first should provide a minimum 25 years protection.
 Disadvantages of Killed/inactivated
vaccines
1. They usually require several doses because the microbes are unable to
multiply in the host and so one dose does not give a strong signal to
the adaptive immune system; approaches to overcome this include the
use of several doses and giving the vaccine with an adjuvant.
2. Local reactions at the vaccine site are more common—this is often due
to the adjuvant.
3. Using killed microbes for vaccines is inefficient because some of the
antibodies will be produced against parts of the pathogen that play no
role in causing disease.
4. Some of the antigens contained within the vaccine, particularly proteins
on the surface, may actually down-regulate the body's adaptive
response—presumably, their presence is an evolutionary development
that helps the pathogen overcome the body's defenses.
 Toxoid vaccines
Certain pathogens cause disease by secreting an exotoxin: these
include tetanus, diphtheria, botulism and cholera—in addition, some
infections, for example pertussis, appear to be partly toxin mediated.

Characteristics of Tetanus:
In tetanus, the principal toxin (termed tetanospasmin) binds to specific
membrane receptors located only on pre-synaptic motor nerve cells.
Subsequent internalization and migration of this toxin to the central
nervous system blocks the metabolism of glutamate which is essential
for the normal functioning of GABA (gama amino butyric acid)-ergic
neurons.

As GABAergic neurons are inhibitory for motor neurons, their non-

functioning results in excess activity in motor neurons with the muscles
supplied by these nerves contracting more frequently than normal
giving rise to muscle spasms which are a characteristic feature of
tetanus.
 Preparation of Tetanus Toxoid

Tetanus toxoid vaccine is manufactured by growing a highly

toxigenic strain of Clostridium tetani in a semi-synthetic medium.
Bacterial growth and subsequent lysis release the toxin into the
supernatant and formaldehyde treatment converts the toxin to a
toxoid by altering particular amino acids and inducing minor
molecular conformational changes.

Ultra-filtration then removes unnecessary proteins left as a

residual from the manufacturing process to produce the final
product.

The toxoid is physico-chemically similar to the native toxin thus

inducing cross-reacting antibodies but the changes induced by
formaldehyde treatment render it non-toxigenic.
Toxoid = a chemically modified toxin from a pathogenic microorganism, which is no longer toxic
but is still antigenic and can be used as a vaccine.
Preparation of Diphtheria Toxoid
Diphtheria toxin is a protein exotoxin produced by the bacterium
Corynebacterium diphtheriae. It is produced as a single
polypeptide that is readily spliced to form two subunits linked by
a disulphide bond, Fragment A and Fragment B.

Methods of preparing diphtheria toxoid (DT) are well known in

the art. For instance, DT may be produced by purification of the
toxin from a culture of Corynebacterium diphtheriae followed by
chemical detoxification, or may be made by purification of a
recombinant, or genetically detoxified analogue of the toxin.

Corynebacterium diphtheriae is cultured under aerobic

conditions. Rappuoli et al (Biotechnology,1985, p 161-163) suggest that
pO2 should be regulated at 25% by aerating with a mixture of air
and oxygen which is automatically regulated to maintain the
desired pO2
Manufacture of Diphtheria toxoid

A strain of C. diphtheriae that is known to produce large

amounts of toxin is grown in a liquid medium conducive to
toxin production.

After appropriate incubation, sterilization is achieved by

centrifugation and filtration.

After ascertainment of potency, the filtrate is incubated with

formalin for conversion of toxin to toxoid. The product is then
further purified and concentrated to achieve the necessary
dosage. It is adsorbed onto an aluminum salt, usually
aluminum hydroxide or aluminum phosphate.
 Preparation of Pertussis Toxoid

According to the preparation methods, there is

employed a culture supernatant of a Bordetella
pertussis phase I strain or a concentrate thereof. The
cultivation of the Bordetella pertussis phase I strain can
be carried out in a suitable manner.
Thus, for example, the strain is cultivated in a liquid
medium (Cohen-Wheeler medium, Stainer & Scholte
medium, etc.) at about 35° to 37° C for about 5 to 7
days.
The supernatant of the resulting culture is collected by
filtration or centrifugation. Either this supernatant fluid
or a concentrate thereof can be used in the subsequent
step of removing its endotoxin.
Preparation of Pertussis Toxoid

The concentrate can be obtained by salting out

which is conventional. Thus, for example, 2 to 5 kg of
ammonium sulfate is added to 10 lit each of the
culture supernatant and, after mixing, the precipitate
formed is collected by an expedient technique such
as filtration or centrifugation.

This precipitate is then dissolved in a suitable amount

of 0.05 M phosphate buffer supplemented with 1 M
sodium chloride, and the supernatant is obtained by
centrifugal sedimentation or the like procedure to give
a concentrated fluid.
Advantages of Toxoid vaccine
There are three principal advantages of toxoid vaccines.
1. First, they are safe because they cannot cause the disease
they prevent and there is no possibility of reversion to
virulence.
2. Second, because the vaccine antigens are not actively
multiplying, they cannot spread to unimmunized individuals.

3. Third, they are usually stable and long lasting as they are less
susceptible to changes in temperature, humidity and light which
can result when vaccines are used out in the community.
 Disadvantages of Toxoid vaccine
Toxoid vaccines have two disadvantages.

1. First, they usually need an adjuvant and require several doses

for the reasons.
2. Second, local reactions at the vaccine site are more common—
this may be due to the adjuvant or a type III (Arthus) reaction—
the latter generally start as redness and indurations at the
injection site several hours after the vaccination and resolve
usually within 48–72 h.
The reaction results from excess antibody at the site
complexing with toxoid molecules and activating complement
by the classical pathway causing an acute local inflammatory
reaction.
 Passive Antigenic products
Passive immunity refers to the process of providing IgG
antibodies to protect against infection; it gives immediate, but
short-lived protection for several weeks to 3 or 4 months at
most.
Passive immunity is usually classified as natural or acquired.
The transfer of maternal tetanus antibody (mainly IgG) across
the placenta provides natural passive immunity for the newborn
baby for several weeks/months until such antibody is degraded
and lost.
In contrast, acquired passive immunity refers to the process of
obtaining serum from immune individuals, pooling this,
concentrating the immunoglobulin fraction and then injecting it to
protect a susceptible person.
 Commonly used immunoglobulin
preparations (gammaglobulin)
(i) Human Hepatitis B Immunoglobulin:
Human hepatitis B immunoglobulin is presented as two vial
sizes of 200 and 500 IU. Each millilitre contains 10–100 mg/ml
human protein of which at least 95% are gammaglobulins (IgG).

This product is prepared from plasma from screened donors,

selected from the USA. One millilitre contains not <100 IU of
hepatitis B antibody.

Its use occupationally is for the immediate protection of non-

immune health care workers exposed to hepatitis B viruses
(together with an appropriate vaccination programme)