Vaccine ppt.pptx

5/19/2023 1
Immunological products
Vaccines
Admassu Assen (B.pharm, M.sc)
Lecturer
Mekelle university
College of health sciences
School of pharmacy, pharmaceutics unit and research team
5/19/2023 1

Outline
•Introduction to vaccines
•Convectional vs recombinant vaccines
•Types of vaccines
•Production of Vaccines by rDNA Techniques
•Adjuvant and delivery systems for vaccines
•Handling of vaccines
5/19/2023 2

Introduction to vaccines
• “With the possible exception of clean water,
 vaccines were the single most important contributing factor
to improvements in global health in the twentieth century.”
History of vaccine
• The concept of vaccination was introduced in the late 18th century
by Edward Jenner
• in 1798 he critically observed that milkmaids who incidentally
acquired and developed ‘cowpox’ became ultimately immune to
‘small pox’.
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• Edward Jenner, began to notice a similarity between smallpox
and cowpox.
• Experiment
Deliberately infected with cowpox and allowed to recover
He then injected him with the smallpox virus and observed
if the boy can suffer from smallpox.
Result: The boy didn’t suffered from smallpox.
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Introduction….

• he used cowpox virus as a vaccine to protect humans against
smallpox virus infections.
• This process of deliberately infecting oneself with cowpox to
avoid the more severe form of the disease became popular
around the world and was known as 'vaccination'.
• The term vaccine derives from Edward Jenner’s 1796 use of the
term cow pox (Latin: variolæ vaccinæ, adapted from the Latin
vaccīn-us, from ‘vacca’ cow),
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Introduction….

• Louis Pasteur made another spectacular and revolutionary
advancement by developing the technique of ‘attenuation’
 the vaccine against rabies, (late 1800’s)
• By the end of the 1920s,
 vaccines for diphtheria, tetanus, pertussis (whooping
cough)
 tuberculosis (BCG) were all available.
5/19/2023 6
Introduction….

• Most of the major vaccines used today resulted from the
explosive growth of biomedical research after WWII.
• Enormous advances were made both in biological knowledge,
and in the capabilities of research tools including computers and
microscopes.
• The development of efficient vaccines has resulted
• marked decrease in morbidity and mortality from vaccine
preventable diseases around the world.
5/19/2023 7
Introduction….

Purpose of Vaccines
1. prophylactic
 e.g. to prevent or ameliorate the effects of a future
infection by any natural or "wild" pathogen
• Immunization programmes:- have served to reduce dramatically
the incidence of many killer/disabling diseases, such as
smallpox, polio and tuberculosis.
2. Therapeutic
 e.g. vaccines against cancer are also being investigated.
5/19/2023 8
Introduction….

• Immunization programmes: not only of poorer world regions, but
also amongst the most affluent nations.
• An estimated 500 000 adults die annually in the USA from
conditions that could have been prevented by vaccination.
 include pneumococcal pneumonia, influenza and hepatitis B.
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Introduction….

Mechanism
• Vaccination seeks to exploit the natural defense mechanisms
conferred upon us by our immune system.
both the humoral and cell-mediated arms of the immune
system are activated.
5/19/2023 10
Introduction….

Types of vaccine
• Vaccines can broadly be classified into two:
 Traditional vaccine
 recombinant vaccine
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A. Traditional Vaccines
• The term ‘traditional’ refers to those vaccines whose
development pre-dated the advent of rDNA technology.
• Traditional vaccine preparations have largely been targeted
against
 Viral
 bacterial pathogens and bacterial toxins
 lesser extent, parasitic agents such as malaria.
5/19/2023 12
Types of vaccine…..

• Future application:
 understanding of the molecular mechanisms underlying
additional human diseases suggests several novel applications
of vaccines to treat/prevent diseases such as
 Autoimmune conditions
 cancer
5/19/2023 13
Traditional Vaccines

Traditional Vaccines
• There are 4 main types of traditional vaccines
1. Live attenuated vaccines
– Bacteria
– Viruses
2. inactivated vaccines
– Bacteria
– Viruses
3. Toxoids
4. Pathogen-derived antigens
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1. Live attenuated vaccines
• made up of living virus or bacteria that have been modified
through a process attenuation
• Attenuation (bacterial or viral) represents the process of
elimination or greatly reducing the virulence of a pathogen
Mechanisms
1. Chemical treatment or heat
2. Growing under adverse conditions or Propagation in unnatural
host (usually by repeated culturing).
5/19/2023 15
Traditional Vaccines…

• An attenuated bacterial vaccine is represented by BCG.
• BCG is a strain of tuberculae bacillus that fails to cause
tuberculosis,
• but retains much of the antigenicity of the pathogen.
• Many of the more prominent vaccine preparations in current
medical use consist of attenuated viral particles.
5/19/2023 16

• Most of the viral vaccines currently in use
 Measles/mumps/rubella (MMR) and
 Mumps vaccines consist of live attenuated strains of
Paramyxovirus parotitidis
 Varicella zoster (VZV) vaccines
• Live attenuated vaccines retain their ability to infect and
replicate in the vaccinated host.
 In other words, a good vaccine retains immunogenic
activity without eliciting host pathogenic responses.
5/19/2023 17

NB: A live attenuated vaccine creates a good immune response and
often provides lifelong immunity with only one or two doses.
• Because , antigens are presented as replication competent
bacteria or viruses, both
 humoral and
 cell-mediated immune responses are initiated
5/19/2023 18

advantages associated with live vaccines:
1) cheap to produce because the inoculum dose is relatively less
2) require fewer inoculations
3) do not require adjuvants;
4) elicit both humoral and cell-mediated immune responses; and
5) can be inoculated by the natural route of infection.
5/19/2023 19

disadvantages associated with live vaccines :
1) usually less stable than inactivated vaccines and may require
refrigeration for storage;
2) some of these vaccines under certain situations may revert to
virulent form in the host and thereby lead to clinical disease;
This is known to happen only with live (oral) polio vaccine
3) they may not be recommended for immune-suppressed,
immature, older, or pregnant hosts;
4) they may have a low level of residual virulence; and
5) they may be contaminated with other adventitious organisms.
5/19/2023 20

2. Inactivated vaccines
• Inactivated (killed) pathogenic organisms are used
• do not contain live virus or bacteria
• the simplest way to produce vaccines
 provided the organisms can be cultured easily.
• Therefore, this method is often first tested to develop a potential
vaccine.
5/19/2023 21

• stimulate a weaker immune response than live vaccines and
always require several doses to provide a protective immune
response
• Immunity can also diminish over time and a booster shot is
required to maintain immunity
• fail to elicit effective mucosal and cell-mediated immune
responses
• provide limited protection against mucosal & intracellular
pathogens
5/19/2023 22

Advantage
• it does not require attenuated strains of the pathogen, which
could be difficult to generate for many pathogens.
• Economical
• Less chance of being reverted
5/19/2023 23

Shortcomings
 The process could inactivate not only the organism but also
destroy the antigen conformation
 Incomplete inactivation (e.g., virion aggregation resulting in
poor exposure to the inactivating agent) could result in disease
transmission or severe complications. Now, the methods used
to detect residual infectivity are more stringent
 Because of the non-replicating nature of inactivated vaccines,
multiple immunizations with relatively high doses are required.
5/19/2023 24

methods of inactivating pathogenic organisms;
• treatment with
 chemicals (formalin, formaldehyde, or propiolactate),
 heat
 -irradiation
Example: Virus: Hepatitis A vaccine
Viral particles are propagated in human fibroblasts.
It consists of a formaldehyde-inactivated preparation of the
HM 175 strain of hepatitis A virus.
 Polio vaccine (IPV), influenza, and Japanese encephalitis virus
vaccines
Example: Bacterial vaccines, against pertussis, plague, cholera, and
typhoid
5/19/2023 25

3. Toxiod vaccine
• When the cause of an illness is the bacteria releasing a toxin
that affects the body
• Scientists formulate a vaccine from just the deactivated toxin
(called toxiod), rather than the whole bacteria.
• a toxiod vaccine can provide protection.
• A toxin can be inactivated by treating them with formalin, a
solution of formaldehyde and sterilized water.
5/19/2023 26

• Toxiods are safe for use in vaccines
• Diphtheria and tetanus vaccines are two commonly used
toxoid-based vaccine preparations.
Method of Preparation
1. diphtheria vaccine
• growth of Corynebacterium diphtheriae
• The toxoid is then prepared by treating the active toxin
produced with formaldehyde.
5/19/2023 27

2. Tetanus vaccine production
• Clostridium tetani is cultured in appropriate media.
• The toxin is recovered and inactivated by formaldehyde
treatment.
• Both of them are marketed as a sterile aqueous-based product.
5/19/2023 28

4. Pathogen-derived antigen
• These vaccines don’t use the entire microbe, but instead are
made from only part of the microbe.
 The ‘part’ that best stimulates the immune system
• consists of one or more immunogenic epitopes, proteins, or
other components of a pathogenic organism
• Immunogenic epitopes can be chemically synthesized and are
known as peptide vaccines
5/19/2023 29

• The pathogen could be disrupted, and one or more
immunogenic proteins such as
• bacterial cell wall proteins; flagella or pili
• viral envelope, capsid, or nucleoproteins can be purified.
5/19/2023 30

• Generally speaking:
• In whole-killed vaccines, the immunogen is weak to arouse
immune response.
• What is the way out?
 formulated with adjuvants, which are designed to enhance
vaccine persistence and induction of immune responses.
Only adjuvant currently approved by FDA for clinical use is
alum, in the form of vaccines complexed with aluminum
hydroxide or aluminum sulfate.
5/19/2023 31

Recombinant Vaccines
Recombinant vector vaccines
Viral vectors
• Immunogenic foreign epitopes can be expressed on the virus
surface by modifying the viral capsid or envelope protein.
• A wide variety of foreign viral antigens has been expressed in
viral vectors
• experimental animals have showed moderate to complete
protection.
• Immunization with such vectors leads to the foreign viral
antigen expression similar to that of natural infection without
causing disease.
5/19/2023 32

• Antigenic peptides are expressed along with MHC class I and
class II antigens
• result in both humoral and cytotoxic T-cell responses.
• Adenovirus & poxvirus-based vectors have a number of
common advantages
1) vector construction is easy
2) relatively high levels of foreign protein expression are easily
attained
3) relatively thermostability
5/19/2023 33
Recombinant Vaccines…

4) they have a large capacity for foreign DNA insertion;
5) vector derivatives are nonpathogenic; and
6) they have a wide host range.
• More than one foreign antigen can be expressed in the same
vector to provide protection against a number of diseases by
inoculation with a single vector.
• Baits containing a live vaccinia-rabies glycoprotein
recombinant virus vaccine are distributed in the rabies
endemic area
5/19/2023 34

• To increase the safety of viral vectors for immune-compromised
hosts and to control their indiscriminate spread
 replication defective viral vectors have been developed.
• Replication-defective adenovirus vectors are generated by
deleting the early region 1 (E1) genes.
5/19/2023 35

Bacterial Vectors
• similar to viral expression vectors, attenuated bacteria can be
developed as vectors for
 foreign gene expression and delivery for the purpose
of multivalent vaccines.
• Immungenic foreign epitopes can be expressed on bacteria
surfaces by modifying
• cell surface proteins
• fimbria or
• flagella
5/19/2023 36

• Because Salmonella and Vibrio colonize in the intestinal tract,
• attenuated strains of these bacteria were developed as
vectors for mucosal delivery
• Various bacterial vectors have been used to express a number of
• bacterial (B. pertussis, S. pneumoniae, Y. pestis),
• viral (herpesvirus, influenza virus, HIV, simian
immunodeficiency virus, and hepatitis B virus),
• parasitic (S. mansoni) antigens.
5/19/2023 37

Subunit vaccines
• number of expression systems including bacteria, yeasts,
mammalian cells, insect cells, and plants are now available for
foreign protein expression.
• High amounts of a foreign protein can be produced in a bacterial
expression system at a low cost
• Because scale-up and downstream processing have been well
worked out,
 they are usually first tested for subunit vaccine
production.
5/19/2023 38

• Many of the immunogenic proteins, especially of viral origin,
require secondary modifications that are important for their
antigenicity
• A yeast-expressed hepatitis B virus surface antigen (HbsAg)-based
subunit vaccine is currently in use for humans
• has demonstrated excellent protection against hepatitis B
virus infection.
5/19/2023 39

• mammalian cells are known to process viral glycoproteins to
their functional form by secondary modifications,
• they are one of the means to produce viral antigens
• However, the expression of such proteins in mammalian cells
is usually too low
5/19/2023 40

DNA Vaccines (polynucleotide vaccines or nucleic acid (NA))
• Immunization of mammalian hosts with a plasmid DNA
• The DNA is taken up by cells, and the gene of interest is
expressed
• The cells expressing the foreign antigen are recognized by the
host immune system, leading to humoral and cell-mediated
immune responses.
• Such vaccines appear to have the primary advantages of both
attenuated and inactivated vaccines but without their known
limitations.
5/19/2023 41

• NA vaccines elicit an immune response similar to that obtained
with live attenuated vaccines.
• They also provide safety similar to that of inactivated vaccines,
however, without the obvious side effects of adjuvants or animal-
derived proteins.
5/19/2023 42

5/19/2023 43

Advantage of rDNA technology in vaccine
development
1. It is feasible to produce subcellular or subunit vaccines using
heterologous expression systems, rather than partially purified
components from the native source (i.e., the pathogen itself)
2. Enabled the introduction of specific alterations in vaccines
enhance their safety and efficacy
3. provide a greater margin of safety
Because the pathogen itself is not used as the source material
5/19/2023 44

4. Production of recombinant proteins in bacterial or mammalian
hosts also allows for the possibility of greater yield and purity,
better control of quality, and lower overall costs.
5/19/2023 45
Advantage …..

Disadvantages
 Even if the key component is known, immune responses
against a single target antigen may be insufficient in protecting
against a wide spectrum of subspecies
 Subcellular and subunit vaccines are non-replicative.
similar to inactivated vaccines in their need for repeated
dosing and help with adjuvants (or conjugation to proteins)
to enhance their responses
5/19/2023 46

Adjuvant
• Administration of many vaccines on their own stimulates a poor
host immunological response. This is particularly true of the more
recently developed subunit vaccines.
we need adjuvant
• Adjuvants
 are compounds that, when administered in combination with
antigens, enhance the immune response to those antigens
5/19/2023 47

Importance of adjuvants
• They elicit an earlier, more potent and longer-lasting,
immunological reaction against co-administered antigen.
• often facilitate administration of reduced quantities of antigen
to achieve an adequate immunological response.
Economic savings, as vaccines (particularly subunit
vaccines) are far more expensive to produce than the
adjuvant.
5/19/2023 48

• This enhanced immunogenicity can be measured as
• an increase of antigen-specific antibody levels in serum
and/or mucosal secretions,
• a response against an increased number of epitopes,
• an increase of cell-mediated immune responses, or
• a combination thereof.
• The mechanisms are not completely understood, but they include
• Immunostimulation
• altered processing of antigens
• sustained release of antigens (depot effect)
5/19/2023 49
Adjuvant…

1. immunostimulation
• The immune system can be divided into
• adaptive immune system,
• comprising of B and T lymphocytes,
• innate immune system, which includes
• neutrophils, macrophages, dendritic cells…
• The innate immune system plays a critical role in the activation
of the adaptive immune system
5/19/2023 50
Adjuvant…

• Dendritic cells are antigen presenting cells that integrate the
signals from the innate immune system and activate T-cells and
possibly B-cells.
• T-cells have antigen-specific receptors that recognize peptides
displayed by MHC I and MHC II molecules
• Dendritic cells express both MHC I and MHC II and, on activation
• The signals that activate dendritic cells include
1. microbial molecules
• dendritic cells have pattern-recognition receptors that can
recognize molecules that are expressed by pathogens
5/19/2023 51
Adjuvant…

2. host cell components that are expressed and/or released
by cells when they undergo stress and pathologic cell
death (necrosis).
• danger signals
• It is likely that the primary mechanism by which adjuvants
stimulate the immune response is by
• direct or indirect signaling through pattern-recognition
and danger signal receptors
5/19/2023 52
Adjuvant…

2. Altered Processing of Antigens ( reading assignment)
3. Sustained release of antigens
• The slow and continued release of antigens has been
postulated to induce a strong immune response
• through continued activation of the immune system.
• This may contribute to the adjuvant effect of aluminum-based
adjuvants and mineral oils.
• E.g use of poly PLGA microspheres
5/19/2023 53
Adjuvant…

• An ideal adjuvant should display several specific characteristics
• safety (no unacceptable local/systemic responses);
• elicit protective immunity, even against weak immunogens;
• be non-pyrogenic;
• be chemically defined (facilitates consistent manufacture
and QC testing);
• be effective in infants/young children;
• yield stable formulation with antigen;
• be biodegradable;
• be non-immunogenic itself.
5/19/2023 54
Adjuvant…

Types of Adjuvants
1. Mineral based adjuvants
2. Oil-based emulsion adjuvants
3. Bacteria/bacterial products as adjuvants
5/19/2023 55

Mineral-based adjuvants
• A number of mineral-based substances display an adjuvant
effect
• Although calcium phosphate, calcium chloride and salts of
various metals (e.g. zinc sulphate and cerium nitrate) display
some effect
• Aluminium-based substances are by far the most potent
5/19/2023 56
Types of Adjuvants…

Aluminum
• Aluminum adjuvants in human vaccines are either
• aluminum hydroxyphosphate (aluminum phosphate) or
• aluminum oxyhydroxide (aluminum hydroxide)
• Aluminum-based vaccines are prepared
• by adsorption of antigen to commercial aluminum hydroxide
or aluminum phosphate gels or
• by mixing antigen with alum (potassium aluminum sulfate),
resulting in precipitation.
5/19/2023 57

• Aluminum adjuvants are universally used
 in diptheria–tetanus–pertussis (DTP) vaccines and
 in most hepatitis B vaccines and have an excellent safety
record
Mode of action of Mineral adjuvants
• The major mode of action
• depot formation at the site of injection.
• The aluminium compounds are also capable of activating
complement
• This can lead to a local inflammatory response, with consequent
attraction of immunocompetent cells to the site of action.
5/19/2023 58

Draw back of Aluminium based adjuvants
 They tend to effectively stimulate only the humoral arm of the
immune response.
 They cannot be frozen or lyophylized, as either process
promotes destruction of their gel-based structure.
 aluminium-based products display poor or no adjuvanticity
when combined with some antigens (e.g. typhoid or
Haemophilus influenzae type b capsular polysaccharides).
5/19/2023 59

• To date,
• aluminum salts,
• MF59, (w/o emulsion comprising squalene, Tween 80 & Span
85) and
• Virosomes (viral hemagglutinin & membrane-derived
phospholipids)
• are the only adjuvants which have been used in
licensed products
5/19/2023 60

Delivery of Vaccines
Parenteral & mucosal route
• The success of vaccination depends primarily on the method of
presenting the antigen to the host immune system
• delivered by parenteral
• intravenous, intramuscular, intraperitoneal, intradermal,
and subcutaneous administration,
• intranasal, oral, and transdermal delivery have also been
effective.
• In some cases, vaccination through mucosal routes
resulted in better responses
5/19/2023 61

1. Parenteral Route
• remains the immunization method of choice for most antigens
• it provides more effective immune response than do any
other routes of vaccination
• Parenteral vaccination is difficult in the developing countries
where medical care is not well-established.
• Vaccination of a large number of subjects using
hypodermic needles, which is a highly labor-intensive
procedure, requiring healthcare personnel, is not practical
5/19/2023 62
Delivery of Vaccines…

• The results of parenteral vaccination depend on the route of
administration.
• For plasmid DNA vaccines, the highest levels of antibodies
were induced by
• IM and IV injections,
• significant titers were also obtained with sublingual and
intradermal delivery
• Other routes, such as intraperitoneal, subcutaneous, intranasal
inhalation and instillation, intrarectal, intravaginal, ocular, and
oral, did not result in significant immune responses.
5/19/2023 63

2. Mucosal Route
• Vaccination through mucosal routes provides new avenues of
vaccination with a unique advantage of mucosal immunity, that
may not be obtained, through parenteral vaccination.
• Does not involve hypodermic needles
• the total surface area of the mucosal surfaces in the GI,
respiratory, and urogenital tracts where many infectious
pathogens come into contact with the host is huge.
 Thus, preventing infections at the mucosal surface provides
an immunological first line of defense against diseases
5/19/2023 64

Oral vaccination of the various mucosal routes,
• the most preferable mode of vaccination
• its ease of use and low cost of manufacturing
• Furthermore, GIT provides the largest component of the
mucosal immune system
• Oral administration of vaccines has high acceptability, by
avoidance of injection, to individuals of all ages
• an antigen is taken up by M-cells in the Peyer’s patch of the
gut-associated lymphoid tissue
5/19/2023 65

• limitations of oral vaccination is that it does not always induce
sufficient immunity
• GIT is designed to digest proteins and most antigens are
proteins
• systemic uptake of antigens from the GI tract is very poor
• Way out ??
• encapsulation of antigens in microparticles
5/19/2023 66

Intranasal vaccination route
• has received growing interest for non-invasive immunization.
• solution and microsphere formulations tend to show good
immune responses
• Nasal vaccine delivery
• superior to oral delivery in inducing specific IgA and IgG
responses in the URT
• preferable to the oral route for distant mucosal vaccination
that might be used to prevent adhesion of pathogens to
the urogenital tract
• microspheres made of mucoadhesive polymers are preferred
5/19/2023 67

Pulmonary vaccination
• useful in mass vaccination campaigns
• A conventional method of pulmonary delivery of drugs using
metered-dose, propellant-driven, small-particle aerosols was
used to deliver killed whole bacterium vaccines
• Direct gene transfer into the respiratory system can be carried
out for either therapeutic or immunization purposes.
• Cells in the lung can take up and express plasmid DNA
whether it is administered in naked form or formulated with
cationic liposomes.
5/19/2023 68

3. Parenteral and Mucosal Combination Vaccination.
• The combination of mucosal and systemic immunization routes
 e.g., parenteral immunization followed by oral immunization
or vice versa
• generally induces mucosal immune responses that are superior
to immunization by either route alone
5/19/2023 69

Reading Assignment
• Challenges in future vaccine formulations.
• Development of an AIDS vaccine and its challenges.
• Cancer vaccines.
5/19/2023 70

Storage and Handling
• With few exceptions most biologicals are stored in a refrigerator
(2-8 ◦c) and freezing is avoided.
• Expiration depends on products and storage temperature most
having a one year or longer duration before expiry.
• Biologiclas are sensitive to extreme temperatures: exposure to
heat or freezing can decrease their potency and effectiveness.
5/19/2023 71

If damaged products are administered the person may;
• Get little of the intended benefit
• Not be able to build up immunity and get the infection
• Get the disease that the biologic was intended to protect
against
• A major action expected from the pharmacist is to maintain the
‘cold chain’.
5/19/2023 72

• A cold chain is a temperature-controlled supply chain.
• An unbroken cold chain is an uninterrupted series of storage
and distribution activities which maintain a given temperature
range.
• It is used to extend and to help ensure the shelf life of
products such as chemicals, foods and pharmaceutical drugs.
• Packing (keeping) biological products in refrigerators should
allow circulation of cool air.
5/19/2023 73

• Separate refrigerator dedicated to biologicals is preferable to
minimize the frequency of opening of the door.
• WHO recommends opening of refrigerator not to be more than
four times a day.
• Periodic checking of the temperature of the refrigerator and
freezer is advisable for the pharmacist.
• Storing containers of the same vaccine together should be done
to avoid confusion because of look-alike products.
5/19/2023 75

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