Preparation of live, inactivated, Toxoid and subunit vaccines

1. SHRUTHI.K
DEPT.OF MICROBIOLOGY
PONDICHERRY UNIVERSITY
TRADITIONAL VACCINE
PREPARATION

2. TYPES OF TRADITIONAL VACCINES
• Inactivated vaccine preparation
• Live vaccine preparation
• Toxoid vaccines
• Subunit vaccines

3. INACTIVATED VACCINE PREPARATION
• An inactivated vaccine is one that uses a
dead or killed virus or bacteria to help
your body develop an immune response.
• An inactivated vaccine cannot cause the
particular disease (e.g. flu and others
below) that it is intended to prevent.
• Some inactivated vaccines (e.g. polio
and pertussis) require multiple doses
and periodic boosters for protection to
continue. Live vaccines require only one
dose.

4. How Are Inactivated Vaccines Created?
• The common means to make a pathogen safe for use in a
vaccine is by treatment with heat or chemicals. This kills the
pathogen but still allows it to induce an immune response to at
least some of the antigens contained within the organism.
• Heat inactivation is often unsatisfactory because it causes
extensive denaturation of proteins.
• Chemical inactivation with formaldehyde or various alkylating
agents has been successful.
• (e.g.: Salk Polio vaccine, Leptospirosis (2- and 4-serovar)
vaccines, Some Borrelia burgdorferi (canine Lyme disease)
vaccines,Parenteral Bordetella bronchiseptica vaccine

5. How is Ender’s vaccine prepared ?
• The enabling breakthrough for the vaccine came with
Enders' poliovirus propagation in cell cultures of nonneural
tissue.
• Trivalent killed Salk polio vaccine was prepared using virus
grown in Macaques monkey renal cell cultures and was
licensed in 1955 which faced many problems.
• A new indigenous was contaminating Macaques monkey
polyoma virus, SV40 which was resistant to Salk vaccine.

6. • Discovery of SV40 virus was derived to use kidneys for cell
culture from Macaques monkeys that were not infected with
the then ubiquitous presence of indigenous viruses.
• Renal cells from African Cercopithecus monkey were found
to be highly permissive to viral replication with
cytopathogenic change, and allowed to detect the presence
of hitherto undetectable agents.
• Efforts to overcome the highly variable potency of the killed
vaccine led us to develop a purified poliomyelitis vaccine
but was ultimately discontinued for commercial reasons.

7. Disadvantages:
• Even though the pathogens they contain are killed,
inactivated whole-organism vaccines still carry certain
risks.
• Large quantities of the infectious agent must be handled
prior to inactivation, and those exposed to the process
are at risk of infection
• Inactivated vaccines are generally less immunogenic
and tend not to have an extended duration of immunity
(memory) compared to attenuated vaccines
• Inactivated vaccines often contain an adjuvant intended
to incite local inflammation and enhance the immune
response to the antigen.

8. LIVE VACCINE PREPARATION
• Variolation, first brought to the UK
from Turkey by Lady Mary Wortley
was the first form of live vaccine.
• Edward Jenner inoculated Phipps with
material from a cow infected with
cowpox, which resulted in a lesion
indicating infection, and then seven
weeks later with material from a
smallpox pustule. He was later cured.
• Egg: measles, mumps vaccine,
rubella vaccine, influenza vaccine,
chicken pox, smallpox, oral polio
vaccine.

9. • Attenuation usually involves deletion of essential virulence
factors or mutation of genes encoding metabolic enzymes
whose function is essential for survival outside the
laboratory.
• Attenuated viruses produce infections that are milder than
the illnesses produced by the virulent wild-type
counterparts from which they are derived.
• Attenuated OPV exhibits a different pattern of tropism than
does wild-type poliovirus, since it replicates well in the
gastrointestinal tract but poorly in the central nervous
system (CNS). In contrast, wild-type virus replicates
robustly in both sites.

10. • Attenuated viruses used as vaccines depend for their
efficacy on replication of the agent, which generates
antibody and cellular immunity, as well as innate immune
• IgG and IgA are secreted against OPV and rotavirus.
• Two current live attenuated vaccines are genetic
reassortants: influenza and one of the rotavirus vaccines.
• In the case of influenza, both live and inactivated, the RNA
segments coding for hemagglutin and neuraminidase are
reassorted with RNA segments coding for the six other
viral proteins that are obtained from attenuated strains.
• Thus, the reassortant is attenuated but induces antibody
responses against the two viral surface proteins.

11. Louis pasteur’s work:
• The discovery of the Chicken cholera vaccine by Louis
Pasteur revolutionized work in infectious diseases and can
be considered the birth of immunology.
• The notion of using a weakened form of the disease to
provide immunity was not new, but Pasteur was the first to
take the process to the laboratory, impacting all virologists
who followed after him.
• The microbe, weakened in the lab, had taught the chicken
immune system to fight the infection without causing any
serious harm to the chicken. This type of vaccine is called
a live, attenuated vaccine.

12. Research study:
• Live vaccines against measles, tuberculosis (BCG), polio
(OPV) and smallpox reduce mortality more than explained
by target-disease prevention.
• It was hypothesised that revaccination in presence of prior
immunity enhances beneficial non specific effects.
• It might be beneficial to vaccinate women of fertile age with
live vaccines to increase the amount of maternal antibody.
• It might be beneficial to give a live vaccine together with
antibody against the target disease.
• The hypothesis has generated testable deductions which if
verified could lead to major changes in our understanding of
vaccines and in the organisation of vaccination programmes

15. INFLUENZA VACCINE FOWL CHOLERA

16. Disadvantages:
• Integration of the plasmid harbored by bacterial vaccine
vehicles is a potential hazard.
• The route of administration of the vaccine may also be
important when evaluating hazards. As live bacterial
vaccines is fit for mucosal administration one must
remember that ingestion of foreign DNA does occur.
• Peptides can be absorbed through the mucosa and
some may induce an allergic reaction.
• Vaccination using live bacterial vaccines or exposure to
the natural infections can lead to the formation of auto
reactive antibodies

17. TOXOID VACCINE
• Toxoid proteins are biologically inactivated forms of toxins.
• The most often used toxoid is tetanus toxoid, but
diphtheria-derived toxoids and other proteins are also used
occasionally.
• Tetanus toxoid has 106 amine groups, 10 sulfhydryls, 81
tyrosine residues, and 14 histidines that may participate in
conjugation reactions with hapten molecules.
• Diphtheria toxoid is derived from a protein secreted by
certain strains of Corynebacterium diphtheriae. Its
molecular weight is approximately 63,000 Daltons.

18. • Both protein toxoids can be used to couple haptens
through any of the chemical reactions described in this
section. They generate strong immunological responses in
vivo.
• Toxoid vaccines are made from selected toxins (proteins)
that have been sufficiently attenuated (rendered harmless)
yet are able to induce a humoral (antibody) immune
response.
• Toxoid vaccines tend not to have a duration of immunity
comparable to attenuated viral vaccines; therefore,
multiple sequential initial doses may be required to protect
(especially among very large and small breed dogs).
Revaccination (booster) may be required multiple times in
a single year depending on individual patient risk factors.

19. SUBUNIT VACCINE
• Many of the risks associated with attenuated or killed whole
organism vaccines can be avoided with a strategy that uses
only specific, purified macromolecules derived from the
pathogen.
• The three most common applications of this strategy,
referred to as a subunit vaccine, are inactivated exotoxins or
toxoids, capsular polysaccharides or surface glycoproteins,
and key recombinant protein antigens.
• Diphtheria and tetanus vaccines have been made by
purifying the bacterial exotoxin and then inactivating it with
formaldehyde to form a toxoid.

20. • Vaccination with the toxoid induces antitoxoid antibodies,
which are capable of binding to the toxin and neutralizing its
effects.
• Conditions for the production of toxoid vaccines must be
closely controlled and balanced to avoid excessive
modification of the epitope structure while also
accomplishing complete detoxification
• The virulence of some pathogenic bacteria depends
primarily on the antiphagocytic properties of their hydrophilic
polysaccharide capsule.
• These findings provide the rationale for vaccines consisting
of purified capsular polysaccharides.

21. • Streptococcus pneumoniae- 13 antigenically distinct
capsular polysaccharides.
• Neisseria meningitidis- surface glycoproteins
DISADVANTAGES:
• One limitation of some subunit vaccines, especially
polysaccharide vaccines, is their inability to activate T Helper
cells. Instead, they activate B cells in a thymus- independent
type 2 (TI-2) manner, resulting in IgM production but little
class switching, no affinity maturation, and little, if any,
development of memory cells.

25. REFERENCES
• Vaccines in historic evolution and perspective: a narrative of vaccine
discoveries by Maurice R Hilleman.
• https://www.aaha.org/guidelines/canine_vaccination_guidelines/vaccine
_types.aspx
• https://www.verywellhealth.com/what-is-an-inactivated-vaccine-201081
• https://www.sciencedirect.com/topics/neuroscience/attenuated-vaccine
• Revaccination with Live Attenuated Vaccines Confer Additional Beneficial
Nonspecific Effects on Overall Survival: A Review by Christine S.
Benn,A.B.C, Ane B. Fisker,A.B Hilton C. Whittle,D and Peter Aabya,B
• https://www.vbivaccines.com/wire/louis-pasteur-attenuated-vaccine/
• https://www.slideshare.net/doctormansij/recent-advances-in-vaccinedr-
mansij-biswas
• Owen,J. Punt,J. Stranford,S. Kuby Immunology, seventh edition, 2013,
W. H. Freeman and Company