1. Vaccine Development

2. What is a vaccine?
Preparation of dead or attenuated pathogens, or their products, that when
introduced into the body, stimulates the production of protective antibodies
or T-cells without causing the disease.
It is the most important immunological products able to eradicate numerous
infectious diseases like smallpox.
Eradication of diphtheria, tetanus, whooping cough, poliomyelitis, measles
and rubella also achieved where deploy vaccines effectively.
Vaccines for several other purposes
therapy of cancer; prevention of allergies; desensitization of allergic patients;
fertility control; and treatment of addictions.

3. Etymology and background
Edward Jenner who, over 200 years ago, showed that inoculating people with material
from skin lesions caused by cowpox (L. vaccinus, of cows; vacca, cow) protected them
from the highly contagious and frequently fatal disease smallpox
He tested his theory in 1796 by inoculating 8-year-old James Philipps with liquid from
cowpox pustule Subsequent inoculation of the boy with smallpox produced no disease.
Before Jenner’s time in India and China where they performed variolation, used
a live smallpox vaccine to generate immunity
 Variola virus=smallpox virus

4. REQUIREMENTS OF AN IDEAL VACCINE
 Provide long lasting immunity.
 Should induce both humoral and cellular immunity.
 Should not induce autoimmunity or hypersensitivity.
 Vaccines must also be perceived to be safe.
 Require a single dose.
 Given early in life.
 Given through nonparenteral route
 Combined form in order to reduce the number of visits to a doctor or medical center.
 Heat stable and retain activity during transport and storage, especially in tropical climates.
 Should be inexpensive to produce, easy to store and administer.

5. How do vaccines work?
• Following injection, the immunogenic agent is phagocytosed by immature dendritic
cells.
• Processed peptides will be presented on the cell surface as separate MHC I or II:
antigenic fragment complexes.
• Th2, each with a receptor for a separate antigenic fragment will be activated.
• B cells, each with a B-Cell Receptor for a separate antigenic fragment will bind antigens
that drain along lymph channels.
• This will induce B-cell activation, differentiation and proliferation with subsequent
isotype switch (IgM to IgG) and memory B cell formation.

6. Basic Types of vaccines
• Whole organism
Live/attenuated vaccines
Killed/inactivated vaccines
• Purified Macromolecules as Vaccines
Toxoids
Capsular polysaccharides
Recombinant microbial antigens/Surface antigen
• Recombinant vaccines-
Vector Vaccines
DNA vaccines

7. Live/attenuated vaccines
 Live/attenuated vaccines are the successful viral vaccines.
 Prepared from attenuated strains that are almost or completely devoid of
pathogenicity but are still immunogenic.
 Live vaccines more closely mimic an actual infection. They multiply in the human
host and provide continuous antigenic stimulation over a period of time.

8. Methods used for attenuation
 Related virus from another animal – e.g. the use of cowpox to prevent
smallpox
 Development of temperature sensitive mutants
 Administration of pathogenic/attenuated virus by an unnatural route
• the virulence of the virus is often reduced when administered by
an unnatural route

9. Methods used for attenuation…
 Passage of the virus in an "unnatural host" or host cell
• the major vaccines been derived this way
• After repeated passages, the virus is administered to the natural host
• Examples: Polioviruses (in monkey kidney cells) and measles (in chick embryo
fibroblasts)
 Genetic engineering techniques
Attenuate a virus irreversibly by selectively removing genes that are
necessary for virulence. Example: herpesvirus vaccine for pigs (thymidine kinase
gene was removed).

10. Bacteria/virus Method Route
Vibrio Genetically modified Oral
Salmonella Genetically modified Oral
Mycobacterium Prolonged subculture ID
Polio Passage in MK cells Oral
Influenza Temperature sensitive mutant IN
Mesales, Mumps,
Rubella
Passage in fibroblasts cells SC
Chicken pox Human diploid cell cultures SC

11. Advantages:
• Infectious microbes can stimulate generation of memory cellular as well as
humoral immune responses.
• Since these can multiply in the host, fewer quantities must be injected to induce
protection.
• A single administration of vaccine often has a high efficacy in producing long-lived
immunity. Multiple booster doses may not be required.
• Whole microbes stimulate response to antigens in their natural conformation. They
raise immune response to all protective antigens.
• Some live vaccines can be given orally; such vaccines induce mucosal immunity and
IgA synthesis, which gives more protection at the normal site of entry.
• Oral preparations are less expensive than giving injections.
• They can lead to elimination of wild type virus from the community

12. Disadvantages
• May very rarely revert to its virulent form and cause disease.
• Live vaccines cannot be given safely to immunosuppressed individuals.
Administration of live attenuated vaccines to people with impaired immune
function can cause serious illness or death in the vaccine recipient.
• Since they are live and because their activity depends on their viability,
proper storage is critical.
• Spread to contacts of vaccinee who have not consented to be vaccinated. In
some cases, it turns out be an advantage.

13. Killed/Inactivated vaccines
• The term killed generally refers to bacterial vaccines whereas inactivated relates to
viral vaccines
• An inactivated whole organism vaccine uses pathogens which are killed and are no
longer capable of replicating within the host
• The pathogens are inactivated by heat or chemical means while assuring that the
surface antigens are intact
• Inactivated vaccines are generally safe, but are not entirely risk free

14. Microorganism Method Route
Salmonella typhi Heat, Phenol, Acetone SC
Bordetella pertussis Merthiolate IM
Poliomyelitis Formalin IM
Rabies virus Phenol SC
Influenza virus Formalin IM
Hepatitis A Formalin IM
Yersinia pestis Formalin IM

15. Advantages
First, they are safe because they cannot cause the disease they prevent and there is
no possibility of reversion to virulence.
Second, because the vaccine antigens are not actively multiplying, they cannot
spread to unimmunized individuals.
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.
Fourth, all the antigens associated with infection are present and will result in
antibodies being produced against each of them.

16. Disadvantages:
• Since the microorganisms cannot multiply, a large number are required to
stimulate immunity.
• Periodic boosters must be given to maintain immunity.
• Only humoral immunity can be induced.
• Most killed vaccines have to be injected.
• Some vaccines such as Bordetella pertussis induce ill effects like postvaccinial
encephalomyelitis. Anaphylactic reaction to neomycin or streptomycin may occur in
(Inactivated Polio Vaccine) recipients. Anaphylactic hypersensitivity to eggs may
occur in recipients of influenza vaccine.
• Inactivation, such as by formaldehyde in the case of the Salk vaccine, may alter
antigenicity.
• Presence of some un-inactivated microbes can lead to vaccine-associated disease.

17. SUBUNIT VACCINES:
Subunit vaccines contain purified antigens instead of whole organisms. Such a preparation
consists of only those antigens that elicit protective immunity. Subunit vaccines are
composed of toxoids, subcellular fragments, or surface antigens.
Administration of whole organism, as in case of pertussis was found unfavorable immune
reactions resulting in severe side effects.
The effectiveness of subunit vaccines in increased by giving them in adjuvants. Adjuvants
slow antigen release for a more sustained immune stimulation.

18. Antigen Microorganism Route
Cell wall
polysaccharide
Hemophilus influenzae b
Nesseria meningitides
Streptococcus pneumoniae
Group B Streptococcus
IM
Toxoid Clostridium tetani IM
Membrane proteins Influenza virus IM
Microbial proteins Bordetella pertussis IM

19. Advantages
• They can safely be given to immunosuppressed people
• They are less likely to induce side effects.
Disadvantages
• Antigens may not retain their native conformation, so that antibodies produced against
the subunit may not recognize the same protein on the pathogen surface.
• Isolated protein does not stimulate the immune system as well as a whole organism
vaccine.

20. Recombinant vaccines
 Contains either a protein or a gene encoding a protein that is immunogenic
 The desire genes identified and isolated from a pathogen and expressed in E. coli
or suitable host for production of the proteins
 The proteins then purified and mixed with suitable stabilizers and adjuvants and
used for immunization.
 The vaccines based on recombinant proteins are also called subunit vaccines.
e.g. Malaria vaccine, passed phase II now entering phase III.

21. Examples of expression vectors
1. Genetically engineered microorganisms, e.g., yeast for the expression of hepatitis B
surface antigen (HBsAg) used as vaccine against hepatitis B virus
2. Cultured animal cells, e.g., HBsAg expressed in CHO (Chinese hamster ovary) cell line
and C-127 cell line
3. Transgenic plants, e.g., HBsAg, HIV-l (human immunodeficiency virus-I) epitope (in
experimental stages)
4. Insect larvae; the gene is integrated into a bacculovirus genome, which is used to
infect insect larvae. Often a very high quantity of the recombinant protein is
produced .

23. Vector vaccines
 DNA sequence coding for the foreign gene (SARS-CoV-2) is inserted into the
plasmid vector along with a Chimpanzee Adenovirus promoter sequences.
 The resultant recombination Chimpanzee adenovirus vector is then introduced
into cells to generate a virus that expresses the foreign gene.
 The recombinant virus vaccine can then multiply in infected cells and produce
the antigens against which immunity develops.
 The genes of several viruses can be inserted, so the potential exists for
producing polyvalent live vaccines.

24. …Vector vaccines
• Proteins encoded by these genes are appropriately expressed in vivo with respect to
glycosylation and secretion.
• They are processed for major histocompatibility complex (MHC) presentation by the
infected cells, thus effectively endowing the host with both humoral immunity and cell-
mediated immunity.
• A number of organisms have been used for vector vaccines, including the vaccinia virus,
canarypox virus, attenuated poliovirus, adenoviruses, attenuated strains of Salmonella,
the BCG strain of Mycobacterium bovis, and certain strains of streptococcus that normally
exist in the oral cavity

26. Advantages
• Those vectors that are not only safe but also easy to grow and store can be chosen.
• Antigens which do not elicit protective immunity or which elicit damaging responses can
be eliminated from the vaccine. Example Cholera toxin A can be safely removed from
cholera toxin.
Disadvantages
• Since the genes for the desired antigens must be located, cloned, and expressed
efficiently in the new vector, the cost of production is high.
• When engineered vaccinia virus is used to vaccinate, care must be taken to spare
immunodeficient individuals.

27. Toxoids
A toxoid is a chemically or physically modified toxin that is no longer harmful but
retains immunogenicity
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
Specific physical or chemical modification of the toxins produces a toxoid, which is a
vaccine
Fig: Modification of toxin to toxoid

28. Tetanus Toxoid (TT)
Manufactured by growing a highly toxigenic strain of Clostridium tetani in a
semi-synthetic medium.
Formaldehyde treatment converts the toxin to a toxoid by altering particular
amino acids and inducing minor molecular conformational changes.
The toxoid is physicochemically similar to the native toxin thus inducing cross-
reacting antibodies. But, the changes induced by formaldehyde treatment
render it non-toxigenic

29. Advantages of toxoids
There are three principal advantages:
First, they are safe because they cannot cause the disease they prevent
and there is no possibility of reversion to virulence.
Second, because the vaccine antigens are not actively multiplying, they
cannot spread to unimmunized individuals.
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