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Vaccine Development
Traditional and Modern
Approaches
Rodney Carbis
Head Vaccine Development
International vaccine institute
1
Purpose of Vaccination
2
Purpose of Vaccination
Protect the individual from disease.
Reduce the severity of disease.
Protect the community.
Eradication of the disease.
Reduce the burden of disease.
3
The value of Vaccines
Vaccines are the most cost-effective
tools for preventing death and
disability from infectious disease
Luis Fermín Tenorio,
the last polio case in
the Americas
Peru, 1991
Ali Maouw Maalin,
last case of smallpox
(Somalia,1977)
4
Vaccine Efficacy
Diphtheria
>87% (industrialized countries)
Tetanus
Pertussis
Oral Polio
Measles
Hepatitis B
Hib
3 doses >95%, 2 doses >80%
80% (wide variation)
>90% (lower in tropical countries)
>90% (after 12 mths) 85% (at 9 mths)
75 – 95%
90%
5
Financial Benefits of Measles Vaccination
Cases averted
Lives saved
Cases of mental
retardation averted
Physicians visits saved
52,107,000
5,210
17,360
26,776,000
Hospital days saved
2,972,000
Net benefit achieved
1963 – 1982
USD 5,120,729,000
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Normal Bacterial Flora
Staphylococcus epidermidis
Normal colon
Bacterial colonization of the body
per gm or cm2
Bifidobacterium bifidum
Bacterial colonization
E.coli
7
Role of Bacterial Toxins in disease
DISEASE
(BACTERIA)
Diphtheria
Corynebacterium diphtheriae
Tetanus
Clostridium tetani
Whooping cough
Bordetella pertussis
Cholera
Vibrio cholerae
TOXIN PRODUCED
Diphtheria toxin
Tetanus toxin
Adenylate cyclase toxin
Pertussis toxin
Cholera enterotoxin
8
Invasion of Cells
Shigella bacterium invading HELA cell
9
Basic concept of vaccines
Deliver to the body some part or all of the
disease organism that IMITATES the pathogen
but is not pathogenic.
– Induce protective immune response.
Polysaccharide
LPS
capsular
Entire organism
• live (attenuated)
• killed
Intracellular proteins
Surface proteins
Toxins
10
New developments in vaccines
Identifying the protective antigen
– Common antigens
– Poorly expressed antigens
Expression of antigen
– Production system
Production of protective antigens in yeast etc.
– Vector
Live attenuated bacteria
Live attenuated viruses
11
New developments in vaccines
Antigen presentation
Virus like particles
Adjuvants
Slow release vehicles
Conjugation
Formulation
Multivalent vaccines
Addition of muco-adhesives for oral delivery
Addition of preservatives and stabilizers (lyophilization)
12
Vaccine manufacture
Antigen Production
Eggs
–
Influenza
Bacterial / Yeast fermentation
–
–
–
Whole organism (e.g. Cholera)
Subunit vaccines (e.g. Capsular
polysaccharide, Tetanus and Diphtheria
toxoid)
Genetically engineered proteins (e.g.
Hepatitis B and HPV vaccines)
Cell culture
–
–
Viral vaccines either whole virus or subunit
Genetically engineered proteins
13
Vaccine Processing
Antigen concentration.
Removal of unwanted foreign components.
–
–
–
Host proteins
Host DNA
Adventitious agents
–
–
–
LPS
Unwanted proteins (not involved in immunity)
Unwanted nucleic acid
–
Adjuvants, stabilizers, preservatives etc
Removal of unnecessary Bacterial or Viral
components.
Change of diluting solution.
Addition of other components.
14
Growth
of
Virus, Yeast
or Bacteria
Eggs
Clarification
Depth
filter
Concentration
+
Purification
Crossflow
filtration
Tools available to develop a process
Chromatography
Ion exchange
Bacterial / Yeast
Fermentation
Cell culture
Crossflow
filtration
Hydrophobic
Affinity
Ultracentrifugation
Centrifuge
Size exclusion
Chemical
Formaldehyde
precipitation
Inactivation
bPL
15
Antigen
presentation
DT
Formulation
CONHNHCO(CH2)4CONHNHOC-
Vi-CPS
CONJUGATES
Fill and Finish
Combining antigen(s)
Combining with
adjuvant
Stabilizers
VIRUS LIKE PARTICLES
ISCOMS
Preservatives
Cryo-protectants
ADJUVANTS
VIROSOMES
16
Types of Vaccines
17
Types of Vaccines
Inactivated toxins
Inactivated whole bacteria or viruses
Live attenuated bacteria or viruses
Subunit vaccines
Genetically engineered proteins
Polysaccharide vaccines
Conjugated vaccines
Recombinant DNA modified organisms
DNA vaccines
18
Inactivated Toxins
Exotoxin
– Gram negative and
gram positive bacteria.
– Heat Labile.
– Protein.
– Secreted by the
bacteria.
Endotoxin
–
–
–
–
Gram negative bacteria.
Heat stable.
Lipopolysaccharide.
Firmly bound to the
bacteria outer
membrane.
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Toxoid
Not a vaccine against the organism.
Vaccine against pathogenic exotoxin.
Tetanus, diphtheria, (pertussis?), anthrax?
– Purify toxin then chemically inactivate (toxoid)
Risk of incomplete inactivation.
TT, DT.
– Genetically modify toxin so non-toxic
CRM (diphtheria), mLT (cholera, ETEC)
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Inactivated Toxins
Tetanus toxin blocks
neurotransmission from nerve
endings inducing muscle
spasm and paralysis
Diphtheria toxin inhibits
protein synthesis resulting in
cell death
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Tetanus
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Tetanus
Clostridium tetani
Gram positive drum stick shaped rod
Not transmitted from person to person
Transmission via contaminated wounds
Found in soil and animal feces
Replicate in low oxygen environment
Release toxin which enters bloodstream
Acts on the nervous system to block
neurotransmitters
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Tetanus
The toxin causes the disease.
The vaccine consists of toxin which is rendered inactive
(toxoid) but retains immunogenicity.
The immune response to the vaccine is directed against
the toxin rather than the bacteria.
Vaccination results in serum antibody production. The
antibody neutralizes toxin.
Three doses of vaccine recommended to raise antibody
levels to protective levels.
Immunity lasts about 10 years.
Vaccination prevents disease but is incapable of disease
eradication.
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Neonatal and Maternal Tetanus
Tetanus in Children has been largely
controlled by vaccination.
In 1999, WHO estimated 290,000 cases of
neonatal tetanus which resulted in 14%
(215,000) of neonatal deaths. 30,000 (5%)
of Maternal deaths were due to tetanus.
Target to eliminate neonatal and maternal
deaths due to tetanus by 2005 by vaccination
of women of child bearing age.
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Diphtheria
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Diphtheria
Corynebacterium diphtheriae.
Gram positive bacillus.
Only produces a powerful exotoxin when
infected with a bacteriophage. (the phage
carries the toxin gene which coverts the bacteria
from non-toxigenic to toxigenic)
Diphtheria is now well controlled by national
immunization programs.
Only receives attention when routine
immunization programs fall.
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Diphtheria
28
Whole Bacteria or
Virus Vaccines
Cholera bacteria
Polio virus
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Whole Killed
Whole pathogen grown and killed
– Heat, chemical modification (formaldehyde,
bPL,..)
– Pertussis, cholera.
– IPV (Inactivated Polio), Influenza, Hepatitis A.
Advantage
– Relatively easy
– Generally safe to administer - no risk of
reversion, infection.
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Whole Killed
Disadvantages
–
–
–
–
–
–
–
–
–
Numerous injections normally required
No or limited cellular immunity
Immunity often shorter-lasting than live vaccines
Reactogenicity from LPS, lipids..
Adjuvant often required (alum, emulsions)
Risk of growing pathogenic organism (eg. polio)
Risk of incomplete inactivation
Inappropriate immune response ? eg RSV ???
Can not focus immunity on protective antigen.
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Inactivated Whole Cell Cholera Vaccine
Manufacturing Process
Fermentation
SBL Vaccine
Cholera toxin
Inactivation
Concentration
And
Washing
B subunit
Bulk
Monovalent
Formulation
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Subunit
Purify a protein or proteins from pathogen
– Eliminate reactogenic contaminants (eg LPS)
– Selective presentation of 'protective' antigens
Pertussis
– Pertussis toxin + filamentous haemagglutinin + pertactin
(no LPS)
Influenza (subunit)
– Mainly haemaglutinin + neuraminidase
Disadvantages
– Requires growing the pathogen and purifying
protective (antigens) subunits.
33
Pertussis
34
Pertussis
Bordetella pertussis.
Gram negative coccobacillus.
Extremely contagious infecting almost all
susceptible close contacts.
WHO estimated in 1999 that pertussis was
still responsible for about 300,000 deaths
mainly in sub-Saharan Africa.
40 million cases annually.
35
Pertussis Vaccines
Whole cell vaccine.
– Whole bacterial cells which are rendered non toxic
using formalin.
– Problem with this vaccine was its reactogenicity.
Acellular pertussis vaccines.
– Contain combinations of purified antigens.
– In the USA, acellular vaccine was introduced for
booster dosing, but in Jan 2000 it was recommended
for all doses.
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Bordetella pertussis
Pertussis toxin
Pertactin
Filamentous haemagglutinin
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Pertussis components and
their role in protection
Pertussis toxin: exotoxin secreted during
bacterial growth. Binds to receptors on
respiratory tract cells.
PT is a major virulence factor and is toxic to
mammalian cells.
Antibody to PT protects mice to lethal challenge.
Antibodies to PT are elicited after both
vaccination and natural infection.
PT is a toxin so must be toxoided for use in a
vaccine.
38
Pertussis components
Filamentous Haemagglutinin: a cell wall
component implicated in attachment to the
respiratory epithelium.
Antibody to FHA protect mice from lethal
respiratory but not intracerebral challenge.
Antibody to FHA is produced as a result of
infection or after vaccination.
Some studies have shown a correlation of
antibody titre to FHA with protection whereas
other studies have not.
39
Pertussis components
Pertacin: a 69 kD outer membrane protein
(OMP).
Protects neonatal mice against respiratory
challenge.
Antibody to Pertactin is produced as a result
of infection or after vaccination with either
whole cell or acellular vaccines.
Antibodies against Pertactin are implicated in
protection against pertussis.
40
Pertussis
Other components being investigated for
their role in toxicity and infection.
– adenylate cyclase
– endotoxin
– tracheal cytotoxin
– heat labile toxin
– agglutinogens
41
Polysaccharide
Many bacteria produce a strain-specific capsular
polysaccharide on their surface.
Antibody to these antigens are protective.
– Streptococcus pneumoniae, Haemophilus Type B,
Meningitis A,C,W,Y (not B!) Typhoid (Vi).
Can be easily purified.
Immunogenic in older children / adults.
But poorly immunogenic in infants
T-cell independent responses
Short lived
Low antibody responses
42
Different forms of Polysaccharide
LPS
Capsular polysaccharide
43
Conjugate
Vaccines
44
Polysaccharide conjugates
45
Haemophilus b
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Haemophilus
47
Haemophilus
Gram negative rods.
Capsulated or non-encapsulated
Capsules are polysaccharide.
Six distinct serological types (a–f) of
encapsulated H. influenzae.
95% of all serious infections attributed to
type b (poly ribosyl-ribitol phosphate PRP)
Most Hib colonization of the upper respiratory
tract are asymptomatic. Disease arises when
the bacteria moves into the blood.
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Haemophilus b
Disease Burden (WHO estimates)
– >500,000 children die from Hib pneumonia
per year.
– >500,000 children die from Hib meningitis
per year.
– 90% of cases are in children under 5 with
peak incidence 6 – 11 months.
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Hib Vaccines
Unconjugated vaccines:
First Hib vaccines consisted of purified PRP.
Immunogenic in children >2 years
Immune response in younger children was poor.
The response to carbohydrate antigens is T-cell
independent. Young children lack the immune
system maturity required to mount such a
response.
50
Hib Vaccines
Conjugated vaccines:
Covalently linking PRP to a protein carrier results
in T-cell recognition of PRP.
Recruitment of T-helper cells allows production
of antibodies to PRP and importantly induces
immunological memory.
This type of response is fully developed at a
young age.
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Typhoid Vaccine
Vi Capsular Polysaccharide
O
COOH
O
Fermentation
AcO
AcN
O
Inactivation
Vi Capsular
Polysaccharide
Purification
Sterile
Filtration
Formulation
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Vi Conjugate vaccine
Vaccine against Typhoid Fever
Exoprotein A (rEPA)
C=O
NHNHCO(CH2)4CONHNH
Spacer
C=O
Vi
Capsular Polysaccharide
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Live Attenuated
Most common type of vaccine.
OPV (oral polio), measles, mumps, rubella, yellow-fever,
rotavirus, influenza, smallpox,
BCG, typhoid, anthrax,
Live pathogen selected or genetically modified
causes no disease or only mild disease.
Derived from non-human source (eg cows, monkeys)
Selected mild strain from humans
Passaged (tissue adapted) strain from humans
Genetically modified (limited ability to replicate)
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Live Attenuated
Advantages:
–
–
–
–
Mimics natural infection
Humoral and cellular response.
Immunological memory.
Generally cheap.
Disadvantages:
–
–
–
–
–
Not suitable for all organisms
May revert to a virulent form
Circulating antibody may interfere with response
Immunity to non-protective antigens
Safety in immuno-suppressed individuals?
55
Live Attenuated Vaccines
Typhoid bacteria
Measles virus
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Measles
57
Burden of Measles
Measles remains one of the major
childhood killers.
WHO estimates 30 million cases of
measles per annum.
875,000 children die from measles
annually.
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Measles
Caused by Rubeola virus
– Genus Morbillivirus
– Family Paramyxoviridae
Six structural proteins
– 3 complexed to the viral RNA
– Matrix protein associated with inner layer of lipid
membrane
– 2 viral membrane proteins
Fusion protein – fusion with host cell
H protein – adhesion of virus to host cell
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Immune response to Measles
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Immune response to Measles
Antibody response.
– Serum IgM
– Serum IgG
– Serum IgA and secretory IgAs.
Re-exposure induces a strong secondary
IgG response and prevents clinical
disease.
61
Immune response to Measles
Cell Mediated Response
– Sensitization of T – lymphocytes.
– Production of cytotoxic T- cells.
Cellular response is important in recovery
from infection. People deficient in this
response are at increased risk of death.
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Measles Vaccine
Killed Inactivated vaccine was abandoned
in 1967 as it did not provide complete
protection.
An attenuated vaccine was also being
developed and first licensed in 1963. This
was found to be too reactogenic for
routine use.
Further attenuation produced an
acceptable vaccine.
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Attenuation of Measles
EDMONSTON B STRAIN
24 passages in primary kidney cells
28 passages in human amnion cells
Passages in chick embryo cells
Vaccine immunogenic but too reactive.
SCHWARZ
85 additional passages in chick embryo cells
Vaccine 90-95% effective, low reactogenicity.
At least 20 years immunity.
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Polio Virus
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Polio Virus
An enterovirus belonging to the family
picornavirus.
Contains RNA surrounded by icosahedral
protein capsid.
Contains no lipid and is acid stable so can
pass through the stomach.
Three antigenically distinct types, 1, 2,
and 3. Vaccines contain all three types.
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Oral Vaccination
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Response to Polio Vaccination
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OPV vs IPV
Oral Polio Vaccine (OPV)
Advantages
– Results in production of secretory IgA. Important in
protecting the individual. Reduces the spread of wild
polio.
– IgAs has been observed to persist for 5 – 6 years
Disadvantage
– Genetic instability of Type 3 strain rarely results
vaccine induced poliomyelitis.
– Transmission of vaccine strains to unvaccinated
individuals.
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OPV vs IPV
Inactivated Polio Vaccine
– Safe and effective.
– Lack of production of mucosal IgAs results in
failure to eliminate intestinal re-infection and
fecal excretion.
– Recommended for countries where polio is no
longer endemic.
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Recombinant
Protein produced by genetic engineering
Express protective antigen in safe easy-togrow organism
– Hepatitis B (HBsAg expressed in yeast)
– HPV (papilloma L1 expressed in yeast)
VIRUS LIKE PARTICLES
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Recombinant
Advantage
– Safe. Growth in non pathogenic yeast cells
– Easier – in case of difficult to grow viruses like
hepB, HPV.
Disadvantages:
– Need to identify protective antigen/s
– Obtaining antigen in 'correct' conformation
– Usually poorly immunogenic alone
– Poor CMI – requires adjuvant.
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Hepatitis B
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Hepatitis B
74
Hepatitis B
More than 2 billion people alive have at some
time been infected with Hepatitis B virus.
350,000 remain chronically infected carriers.
Every year there are
– 4 million acute cases
– 1 million deaths
Carriers
– 25% of children (<7 years) become carriers.
– 10% of older children and adults become carriers.
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Aims of Hep B Vaccination
Vaccination aimed at reduction of:
– Clinical disease.
– HBV transmission.
– Chronic hepatitis and its associated liver
damage and liver cancer.
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Hepatitis B
During infection large amounts of HBsAg
are produced.
Only small amounts of HBsAg combine
with the cores to form whole virus.
Remainder is released into the blood
stream as spherical particles or filaments.
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Immune Response to Infection
Antibody to HBsAg indicates clinical
recovery and is associated with immunity
to Hepatitis B.
Antibody to HBcAg is also produced and is
first to appear after infection.
– Present in blood of acute and chronic subjects
as well as those recovered.
– This antibody does not neutralize virus.
78
Plasma derived Vaccines
79
Plasma derived Vaccines
Safe and Effective.
Rely on supplies of infected plasma.
Long production cycles.
Fears of contamination with other blood
borne diseases.
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Recombinant Vaccines
E.coli
S. cerevisiae
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Recombinant Vaccine
82
Recombinant Vaccine
The gene coding for HBsAg was
discovered in 1970.
The gene has been inserted into a yeast
cell.
As the yeast cell grows it produces large
amounts of HBsAg.
The HBsAg is extracted and purified then
incorporated into the vaccine.
83
Recombinant Vaccine
Advantages of the recombinant vaccines.
– Produced more quickly.
– In larger quantities.
– Free from infectious virus particles.
84
Recombinant DNA modified organisms
Live Vectors
Cloning of genetic material from one
organism into another.
The non virulent parent organism
expresses the antigens of the cloned
genetic material.
A vaccine would elicit a response against
the introduced antigen as well as the
original organism.
85
Recombinant DNA modified organisms
Vaccinia virus
expressing papilloma
virus antigens on its
surface.
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DNA Vaccines
Involves the injection of naked DNA
coding for one or more genes.
The gene is grafted onto another piece of
DNA which acts as a vector.
Injected into muscle tissue, once in the
cell the gene prompts the cell to produce
antigen.
The immune system then mounts an
immune response.
87
DNA Vaccines
Viral protein
mRNA
Antibody-producing
cell
Nucleus
Injected DNA
Class1 MHC
coding for a
specific antigen
Cytotoxic T-lymphocyte
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DNA Vaccines
Clinical trials to date with naked DNA
vaccines have not proved to be that
successful
DNA vaccines may be useful as a priming
dose in prime-boost regimes due to their
ability to induce cell mediated immune
responses.
89
Thank you
90