vaccine lecture by dr wan iryani from uitm

1. PHC504
BIOTECHNOLOGY BASED
PHC504
BIOTECHNOLOGY BASED
THERAPEUTICS II:
VACCINEVACCINE
DR WAN IRYANI WAN ISMAILDR. WAN IRYANI WAN ISMAIL
DEPARTMENT OF PHARMACEUTICAL SCIENCES
FACULTY OF PHARMACY, UITM
FF1, LEVEL 6
03-3258 4718/4841
W_IRYANI@PUNCAKALAM.UITM.EDU.MY
@WIWI2013

2. OUTLINEOUTLINE
• IntroductionIntroduction
• Recap PHC450: Vaccine Immunology
• Traditional Vaccines• Traditional Vaccines
• Modern Vaccines
C St d• Case Study
• Dengue Vaccine
• Types of vaccine• Types of vaccine
• Malaria Vaccine
• Challenges
• Adjuvants
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3. INTRODUCTIONINTRODUCTION
• Top 15 selling vaccines (2012)op 5 se g acc es ( 0 )
• Prevnar 13® – $3.718 billion – Pfizer
• Gardasil® – $1.900 billion – Merck & Co/Sanofli Pasteur MSD
• PENTAct-HIB – $1.522 billion – Sanofli/Sanofli Pasteur MSD
I f i /P di i $1 183 billi b Gl S ithKli• Infanrix/Pediarix – $1.183 billion – by GlaxoSmithKline
• Fluzone – $1.152 billion – by Sanofli/Sanofli Pasteur MSD
• Hepatitis franchise – $986 million – by GlaxoSmithKline
• Varivax – $846 million – by Merck & Co/Sanofli Pasteur MSD
• Menactra – $735 million – by Sanofli/Sanofli Pasteur
• Zostavax – $651 million – by Merck & Co/Sanofli Pasteur
• RotaTeq® – $648 million – by Merck & Co/Sanofli Pasteur
• Synflorix® – $587 million – by GlaxoSmithKliney $ y
• Pneumovax®23 – $580 million – by Merck & Co/Sanofli Pasteur
• Rotarix – $549 million – by GlaxoSmithKline
• Adacel – $469 million – by Sanofli/Sanofli Pasteur MSD
• Prevnar – $399 million – by Pfizer
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Prevnar $399 million by Pfizer

4. INTRODUCTION
Top 5 selling vaccines (2012)
INTRODUCTION
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5. RECAP PHC450:
VACCINE IMMUNOLOGYVACCINE IMMUNOLOGY
• Vaccination is a part of immunization• Vaccination is a part of immunization.
• Immunization is the process of becoming immune.p g
• Immunization has been classified into 2 types:
1) Active
a. Natural
b. Artificial
2) Passive
a. Natural
b Artificialb. Artificial
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6. ImmunizationImmunization
ActiveActive PassivePassive
NaturalNatural ArtificialArtificial NaturalNatural ArtificialArtificial
Natural active immunization = exposure to an infection
Artificial active immunization = vaccination
Natural passive immunization = natural maternal
serum/milk (contain Ab)
Artificial passive immunization = Ab therapyArtificial passive immunization = Ab therapy
(administration of serum)
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7. Human papillomavirus (HPV) female 13 years old
Immunization in Malaysia
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8. RECAP PHC450:
VACCINE IMMUNOLOGY
• Vaccination is one of the most important medication
VACCINE IMMUNOLOGY
p
worldwide for prevention but not to cure disease.
• Vaccination has reduced the burden of illness,
crippling & death from diphtheria, polio & measles.
• Modern pandemic diseases e.g. flu pandemic (H1N1-
H10N7) HIV/AIDS malaria vaccination is one of theH10N7), HIV/AIDS, malaria vaccination is one of the
solutions
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9. RECAP PHC450:
VACCINE IMMUNOLOGY
History of vaccine:
VACCINE IMMUNOLOGY
• Egyptians & Chinese = exposed individuals to powders
formed from the crusts & scales of pockmarks taken
f i di id l i f llfrom individual recovering from smallpox
Edward Jenner (1974) = individual with cowpox are• Edward Jenner (1974) = individual with cowpox are
protected against smallpox
• Robert Koch & Louis Pasteur =developed vaccine
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10. RECAP PHC450:
VACCINE IMMUNOLOGY
Vaccine
VACCINE IMMUNOLOGY
• Vaccine is a suspension of organisms or fractions of
organisms that is used to induce immunity.
Concept of Vaccine
• Vaccine teaches the immune system by mimicking a
natural infection
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11. RECAP PHC450:
VACCINE IMMUNOLOGY
• Principles of Vaccination
VACCINE IMMUNOLOGY
p
• A vaccine renders the recipient resistant to infection
• During vaccination, a vaccine is injected or given orallyg , j g y
• The host produces antibodies for a particular pathogen
• Upon further exposure the pathogen is activated by the
antibodies & disease state prevented
• Generally to produce a vaccine, the pathogen is grown
in culture & inactivated or nonvirulent forms are used forin culture & inactivated or nonvirulent forms are used for
vaccination
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12. RECAP PHC450:
VACCINE IMMUNOLOGY
• Features that need to consider in designing
VACCINE IMMUNOLOGY
Features that need to consider in designing
and producing an effective vaccine:
1) Not produces danger or severe side effects1) Not produces danger or severe side effects
2) Long lasting
3) Induces the immune responses
4) Minimizes reinfection
5) Cheap
6) Stable for storage, transport & use
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13. RECAP PHC450:
VACCINE IMMUNOLOGYVACCINE IMMUNOLOGY
• Generally, vaccine can be classified into 2
types
1) Classical vaccine
2) Modern vaccine
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14. RECAP PHC450:
VACCINE IMMUNOLOGY
Cl i l V i
VACCINE IMMUNOLOGY
• Classical Vaccine
•= traditional or conventional vaccine
•effectively prevent a number of infectious
worldwide e.g. Smallpox (1970)
•not a product from genetic/chemical
engineering technologies
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15. RECAP PHC450:
VACCINE IMMUNOLOGY
• 3 major classes of classical vaccine
VACCINE IMMUNOLOGY
j
a. Live
b Inactivatedb. Inactivated
c. Subunit
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16. RECAP PHC450:
VACCINE IMMUNOLOGY
• Classical Vaccines Live virus vaccines in
VACCINE IMMUNOLOGY
a. Live
b. Inactivated
Live virus vaccines in
use:
Attenuated (harmless)
Vi
c. Subunit
Virus
• Measles
M mps• Mumps
• Rubella
• Varicella zoster
LIVE CHIMERIC VACCINE
• Varicella zoster
• Yellow fever
• Live influenza• Live influenza
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17. LIVE VACCINE
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18. RECAP PHC450:
VACCINE IMMUNOLOGY
• Classical Vaccines Inactivated (killed) virus
VACCINE IMMUNOLOGY
Classical Vaccines
a. Live
b Inactivated
Inactivated (killed) virus
vaccines in use:
• Inactivated Polio vaccineb. Inactivated
c. Subunit
(IPV)
• Influenza
• Hepatitis A
• Rabies
• Tick-borne encephalitis
• JE virus
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19. RECAP PHC450:
VACCINE IMMUNOLOGY
• Classical Vaccines Subunit/toxoids vaccines
i
VACCINE IMMUNOLOGY
a. Live
b. Inactivated
in use:
• Influenza virus – viral
glycoproteins purified
c. Subunit
glycoproteins purified
from virus culture
• Haemaglutinin (H) andHaemaglutinin (H) and
neuraminidase (N)
• Hepatitis B – viral
l t i d iglycoprotein expressed in
yeast
• surface (S) antigen• surface (S) antigen
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20. EXAMPLE FDA-APPROVED VACCINE
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21. EXAMPLE FDA-APPROVED VACCINE
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22. RECAP PHC450:
VACCINE IMMUNOLOGY
• Limitations of traditional vaccine:
VACCINE IMMUNOLOGY
• Limitations of traditional vaccine:
1) Not all infectious agents can be grown in culture
2) Animal & human cell culture expensive if needed
3) Yield of viruses from cultures can be low
4) Safety precautions for culture live agents
a Insufficient killing/attenuation of agentsa. Insufficient killing/attenuation of agents
b. Reversion of attenuated agents
5) It does not work for all agents
6) It h h t h lf li6) It has shorter shelf-lives
• However, 30 traditional developed vaccines remain inHowever, 30 traditional developed vaccines remain in
the medical use.
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23. RECAP PHC450:
VACCINE IMMUNOLOGY
• Modern Vaccines
VACCINE IMMUNOLOGY
Modern Vaccines
• Limitation of classical vaccines modern vaccine
• Mostly still at the clinical trial
• 6 major types of modern vaccine
a. Recombinant live vector vaccines
b Recombinant subunit vaccinesb. Recombinant subunit vaccines
c. Anti-idiotype vaccines
d. Synthetic peptide-based vaccines
e. DNA vaccines
f. Edible vaccine
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24. MODERN VACCINE
Types Characteristics
Recombinant Live
Vector Vaccines
Genetically attenuated microorganism, live viral or bacterial
vectors
• Viral Vector
• Bacterial
Vector
Recombinant
Subunit Vaccines
Genetically detoxified proteins – proteins expressed in host
cells – recombinant peptide vaccines
Anti-Idiotype
Vaccines
Antigen-mimicking antibodies
Synthetic Peptide- Linear or cyclic peptides – multiple antigen peptideSynthetic Peptide
based Vaccines
Linear or cyclic peptides multiple antigen peptide
DNA Vaccines DNA or mRNA coding for antigen
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Edible Vaccines Genetically transform selected desired genes into plants and
then inducing these altered plants to manufacture the encoded
proteins (transgenic plants)

25. MODERN VACCINE
• Modern vaccine is majorly based on recombinant
MODERN VACCINE
j y
DNA technology.
• Advantages
• Virulence genes are deleted & organism is still able tog g
stimulate an immune response
• Live nonpathogenic strains can carry antigenic
determinants from pathogenic strainsdeterminants from pathogenic strains
• If the agent cannot be maintained in culture, genes of
proteins for antigenic determinants can be cloned &p g
expressed in an alternative host e.g. E. coli.
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26. RECAP PHC450:
VACCINE IMMUNOLOGY
Modern Vaccines Recombinant live vector
vaccines:
VACCINE IMMUNOLOGY
a. Recombinant live
vector vaccines
b. Recombinant
vaccines:
• Certain gene product from
virulent pathogen to put
into a vector e.g.
subunit vaccines
a. Anti-idiotype vaccines
b S th ti tid
g
attenuated virus: vaccinia
virus, avipox virus,
alphaviruses, adenovirus,
attenuated bacteria: Sb. Synthetic peptide-
based vaccines
c. DNA vaccines
attenuated bacteria: S.
typhii, M. bovis
• still in clinical trials
d. Edible vaccine
still in clinical trials
• e.g. Smallpox
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27. RECOMBINANT LIVE VECTOR
VACCINES
• New generation vaccines = genetically improved life• New generation vaccines = genetically improved life
attenuated vector vaccines
• The desired gene coding for target antigens of the virulent
pathogen is cloned into a vector (attenuated bacteria or virus).
• The vector is infected (or administered orally) to the
person/animal.
• The vector slowly replicates inside the inoculated individual &
it serves as a source of the antigen
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it serves as a source of the antigen.
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28. RECOMBINANT LIVE VECTOR
VACCINES
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31. RECAP PHC450:
VACCINE IMMUNOLOGY
Modern Vaccines
Recombinant subunit
VACCINE IMMUNOLOGY
a. Recombinant live
vector vaccines
b Recombinant
vector vaccines:
• Specific protein
s b nitsb. Recombinant
subunit vaccines
c. Anti-idiotype
subunits
till i li i l t i l
c. Anti idiotype
vaccines
d. Synthetic peptide-
• still in clinical trials
based vaccines
e. DNA vaccines
• e.g. Hepatitis B
f. Edible vaccine
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32. RECOMBINANT SUBUNIT
• = genetically improved subunit vaccines
VACCINES
• = genetically improved subunit vaccines
• it is a vaccine produced from specific protein subunits of ap p p
virus & thus having less risk of adverse reactions than whole
virus vaccines.
• Antibodies usually bind to surface proteins of the pathogen
or proteins generated after the disruption of the pathogen.
• Binding of antibodies to these proteins will stimulate an
immune response
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immune response.
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33. RECOMBINANT SUBUNIT
VACCINESVACCINES
ADVANTAGES DISADVANTAGESADVANTAGES
• No risk of pathogenicity
DISADVANTAGES
• Multiple doses typically
require
• Define composition
Vario s deli er s stems
• Adjuvants needed
• Various delivery systems
• Simplified large-scalep g
production
• Further engineering possible
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• Further engineering possible
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34. RECAP PHC450:
VACCINE IMMUNOLOGY
Modern Vaccines
Anti-idiotype vaccine:
VACCINE IMMUNOLOGY
a. Recombinant live
vector vaccines
b Recombinant
yp
• Antibody-mimicking
vaccines to bind to
ifi ll tb. Recombinant
subunit vaccines
c. Anti-idiotype
specific cell receptor
H i i B
c. Anti idiotype
vaccines
d. Synthetic peptide-
• e.g. Hepatitis B
based vaccines
e. DNA vaccines
f. Edible vaccine
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35. ANTI-IDIOTYPE VACCINE
• The ability of anti-idiotype antibodies to mimic foreign antigens
has led to their development as vaccines to induce immunityhas led to their development as vaccines to induce immunity
against viruses, bacteria & protozoa in experimental animals.
• Anti-idiotypic vaccines comprise antibodies that have three
dimensional immunogenic regions, designated idiotopes, that
consist of protein sequences that bind to cell receptors.
• Idiotopes are aggregated into idiotypes specific of their target
tiantigen.
• Have many potential uses as viral vaccines particularly when
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Have many potential uses as viral vaccines, particularly when
the antigen is difficult to grow or hazardous.
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37. RECAP PHC450:
VACCINE IMMUNOLOGY
Modern Vaccines
Synthetic peptide-based
VACCINE IMMUNOLOGY
a. Recombinant live
vector vaccines
b R bi t
Sy t et c pept de based
vaccines:
• Design of vaccine
b. Recombinant
subunit vaccines
c Anti-idiotype
consisting of selected
epitopes = similar to T
cell & B cell epitopesc. Anti-idiotype
vaccines
d. Synthetic peptide-
p p
• e.g. Foot & mouthy p p
based vaccines
e. DNA vaccines
e.g. Foot & mouth
diseases
f. Edible vaccine
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38. SYNTHETIC PEPTIDE-BASED
VACCINE
• It allows for the design of vaccines consisting of
VACCINE
• It allows for the design of vaccines consisting of
selected epitopes free from irrelevant or unwanted
structures.
• Large amounts of linear peptides resembling Tg g
cell or B cell epitopes can be prepared by
automated methods.
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39. SYNTHETIC PEPTIDE-BASED
VACCINE
ADVANTAGES DISADVANTAGES
VACCINE
ADVANTAGES
• Antigens are precisely
defined & are free from
unnecessary components
DISADVANTAGES
• Synthetic peptides do not
readily stimulate T-cells.
unnecessary components
associated with the side
effect. • Not applicable to all
viruses e g Polio virus
• Stable & cheap to
manufacture
viruses e.g. Polio virus
which has 2 antigenic sites.
• Easily purified with HPLC
t h i
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techniques
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40. RECAP PHC450:
VACCINE IMMUNOLOGY
Modern Vaccines
a Recombinant live
DNA vaccines:
VACCINE IMMUNOLOGY
a. Recombinant live
vector vaccines
b. Recombinant
• DNA plasmid containing
the antigen gene
subunit vaccines
c. Anti-idiotype
vaccines
• Immunization with DNA
plasmid by
–Injectionvaccines
d. Synthetic peptide-
based vaccines
Injection
–Gene gun
till i li i l t i l
e. DNA vaccines
f. Edible vaccine
• still in clinical trials
• e.g. HIVe.g. HIV
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41. DNA VACCINE
• The antigen-encoding sequence, obtained directly from a DNA
virus or by reverse transcriptase from an RNA virus, is inserted
DNA VACCINE
virus or by reverse transcriptase from an RNA virus, is inserted
into a plasmid between a strong promoter & a poly(A) signal.
Th l id i li t d i b t i l ll & i th ifi d f• The plasmid is replicated in bacterial cells & is then purified for
use as a vaccine.
• Administration of the vaccine can be by injection into muscle or
by using a gene gun that delivers DNA-coated gold beads directly
into skin cells.
• Have promising results but need to confirm it will not trigger an
anti-autoimmune disease & not create cancer-causing mutations
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by insertion into host genome.
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43. DNA Vaccine
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44. DNA VACCINE
ADVANTAGES DISADVANTAGES
DNA VACCINE
• Induction of long term immune
response
• Formation of anti-nucleic
acid antibodies possible
• Induction of both humoral &
cellular immune response
• Integration of host vaccine
DNA into host genome
• Possibility of contructing
multiple epitope in plasmids
• Concept restricted to peptide
& protein antigens
• Heat stable
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• Ease of large scale production
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45. RECAP PHC450:
VACCINE IMMUNOLOGY
Modern Vaccines
R bi t li
Edible vaccines:
VACCINE IMMUNOLOGY
a. Recombinant live
vector vaccines
b Recombinant
• Involved gene
products
b. Recombinant
subunit vaccines
c. Anti-idiotype • still in clinical trialsc t d otype
vaccines
d. Synthetic peptide- • e.g. cholera
based vaccines
e. DNA vaccines
f. Edible vaccine
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46. EDIBLE VACCINE
1997 The first p blished h man trial
EDIBLE VACCINE
• 1997: The first published human trial
• Studies completed & provided proof of
successes in animal & human
• But, still many issues need to beBut, still many issues need to be
addressed
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47. EDIBLE VACCINE
ADVANTAGES DISADVANTAGES
EDIBLE VACCINE
ADVANTAGES
• Ease of immunization
DISADVANTAGES
• Low expression levels of
antigens in transgenic
• Purification is not necessary
antigens in transgenic
plants
• Built-in protection of
antigens
• Level & quality of antigens
cannot be controlled
• Can be produced in large
• Environmental hazards
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49. RECAP PHC450:
VACCINE IMMUNOLOGY
Mechanism of accination imm nolog
VACCINE IMMUNOLOGY
• Mechanism of vaccination immunology
• Establish resistance to virus/pathogenic organism
by evoking an immune response:by evoking an immune response:
1 Give host a foreign organism/protein in non1. Give host a foreign organism/protein in non-
infectious form
2. Antibodies are generated & bind to the foreign
protein
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52. RECAP PHC450:
VACCINE IMMUNOLOGY
• Vaccine Precautions
VACCINE IMMUNOLOGY
• Vaccine Precautions
• Site of administration of antigen
• Intradermal subcutaneous intramuscular oral• Intradermal, subcutaneous, intramuscular, oral
• Hazards
• Especially vaccine from attenuated agents =
reversion to virulence, cause disease in the
immunocompromised patients pregnant womenimmunocompromised patients, pregnant women
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53. DEVELOPMENT OF VACCINE
Small-
pox
Vaccine
by
Ed d
BCQ
Vaccine
Tetanus
Toxoid
Polio
Vaccine
(Injection)
Oral
Polio
Vaccine
MMR
Vaccine
HiB
Vaccine
DNA
Vaccine
& other
vaccines
mostlyEdward
Jenner
1796
1921 1924
(Injection)
1955
Vaccine
1962
1971 1990
mostly
under
develop
ment
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54. VACCINATION IN MALAYSIA
DPT
1960
BCQ
1961
OPV
1972
Measles
1982
Rubella
1988
Hep B
19891960 1961 1972 1982 1988 1989
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55. VACCINATION IN MALAYSIA
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Incidence of Measles in Malaysia

56. CASE STUDY
Deng e Vaccine• Dengue Vaccine
• Malaria Vaccine
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57. DENGUE
• Dengue disease is becoming one of the most important
DENGUE
g g p
emerging vector-borne viral diseases globally.
• 50 million dengue infection cases worldwide in 2012.
• 500 000 cases of severe dengue & 20 000 deaths per year• 500,000 cases of severe dengue & 20,000 deaths per year
• Until August 2014, 65,672 cases in Malaysia (263% increase
compared to 2013) & 148 deaths in Malaysia-stated on 8th October
2014 (95% increase compared to 2013)2014 (95% increase compared to 2013).
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58. DENGUE VIRUSDENGUE VIRUS
• Dengue virus (DENV) = a member of the Flavivirus genus of theg ( ) g
Flaviviriade family.
4 ti i ll di ti t t (DENV1 4) b d• 4 antigenically distinct serotypes (DENV1-4) based on
neutralization assay.
• It is transmitted to humans mainly by Aedes aegypti & A.
albopictus mosquitoes.
• The prevalence of dengue disease is high especially in the Asia-
Pacific region and the Americas.
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59. DENGUE VIRUS
• Diameter: 40 – 50 nm sphere (size: 1/5 to 1/3 of a normal erythrocyte),
i h d b di t i l t bl (Gi R k )
DENGUE VIRUS
mosaic-shaped bodies, stain purple to blue (Giemsa Romanowsky).
• Dengue is a lipid-enveloped positive-sense, single-stranded RNA virus
• The RNA genome of DENV is ± 10.7 kb in length & encodes 3 structural
proteins: capsid protein (C), precursor membrane/membrane protein (PrM/M)
& envelope protein (E)& envelope protein (E).
• Besides the structural proteins, there are 7 nonstructural proteins (NS)
which are associated with viral replication & disease pathogenesiswhich are associated with viral replication & disease pathogenesis.
•The coding of the viral proteins is organized in the genome as C-prM-E-NS1-
NS2A-NS2B-NS3-NS4A-NS4B-NS5NS2A NS2B NS3 NS4A NS4B NS5
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60. DENGUE VIRUS
• Envelope glycoproteins are associated with binding to host
DENGUE VIRUS
p g y p g
receptors, agglutination of erythrocytes, development of
neutralizing antibody, and immune response.
• Each subtype of dengue is 65% genetically related to one
another.
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61. DENGUE VIRUS PATHOGENESISDENGUE VIRUS PATHOGENESIS
• The complicated pathogenesis of dengue hemorrhagic
fever/dengue shock syndrome (DHF/DSS) is not fully resolved.
• Challenge: genotypic differences (DENV1-4), especially DENV 2 & 3g g yp ( ), p y
with mutations in the E & NS3 proteins virulence
• The critical phase of dengue disease is not observed at the peak of• The critical phase of dengue disease is not observed at the peak of
viremia but rather when the viral burden has started to decline.
Thi h l d t th ti th t i ( d ti• This has led to the suggestion that immune responses (adaptive
immune responses, inflammatory mediators & autoimmunity) are not
only responsible for virus clearance but also contribute to
pathogenesis.
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62. DENGUE VACCINEDENGUE VACCINE
• Currently, there are no antiviral or other specific therapeutic
modalities or effective vaccines to treat or prevent the disease.
• Supportive care with vigilant monitoring is the principle form of
management.
• The current candidate vaccines:
1. Live attenuated virus vaccine (tetravalent formulations)( )
2. Live chimeric virus vaccine (based on no.1)
3. Inactivated virus vaccines
4. Live recombinant, DNA & subunit vaccines
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63. DENGUE VACCINEDENGUE VACCINE
• Major aim of dengue vaccine: neutralize all 4 serotypes of DNAj g yp
virus (DENV1-4) simultaneously & equally (= balance immune
response) tetravalent vaccine formulation
• Issue in dengue vaccine development:
• exhibited good immunogenicity & safety profiles in tested animals
but not in human clinical trials
• Imbalanced immune response (antibody production & cellular
immune responses)
• Effectiveness & efficacy issues to evaluate the overall
performance of a candidate vaccine
• Lack of known correlates of protection (asymptomatic persons,p ( y p p ,
small % of infected subject severe clinical symptoms
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64. 1. LIVE ATTENUATED VIRUS VACCINES
• inexpensive
• contain weakened viruses that still can induce adaptive immune
responses to both structural & nonstructural proteins
• the replication of live attenuated viruses should be sufficiently
restricted to avoid pathological effects e.g. 17D strain of YFVp g g
• tetravalent dengue vaccine = the ideal vaccine should provide
balanced immunity to all four dengue serotypesbalanced immunity to all four dengue serotypes.
• problems: unequal immunogenicity of four serotypes in theg y y
tetravalent formulations.
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65. 1. LIVE ATTENUATED VIRUS VACCINES
• A modern approach is based on site-directed mutagenesis of the viral
t tt tigenome to cause attenuation.
• A deletion of 30 nucleotides (∆30) in the 3’-untranslated region of DENV4
was first demonstrated to attenuate DENV4 = DENV4 ∆30 Phase 1 Clinicalwas first demonstrated to attenuate DENV4 = DENV4 ∆30 Phase 1 Clinical
Evaluation
• Success for DENV1 & DENV4 but not for DENV2 & DENV3• Success for DENV1 & DENV4 but not for DENV2 & DENV3
• Alternative: Chimeric strategy for DENV2 & DENV3 was designed using
∆30DENV4 as genetic backbone for DENV2 & DENV3 = DENV2/4∆30 &∆30DENV4 as genetic backbone for DENV2 & DENV3 = DENV2/4∆30 &
DENV3/4∆30
• Monovalent DENV vaccines (DEN1∆30 DENV2/4∆30 & DENV3/4∆30 &Monovalent DENV vaccines (DEN1∆30, DENV2/4∆30 & DENV3/4∆30 &
DEN1∆30) Phase 1 Clinical Evaluation
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66. 1. LIVE ATTENUATED VIRUS VACCINES
Crystal structure of E106 Fab in complex with DENV-1 E DIII.
(A) The E106 Fab epitope on DENV-1 E DIII is comprised of residues in the A-strand (K307 and K310), the end of the B-strand (K325 and Y326), and
th ti BC (E327 T329 d D330) d DE (K361 d E362) l Th i l b li lik DIII i h i bl ( it i ithe connecting BC (E327, T329, and D330) and DE (K361 and E362) loops. The immunoglobulin-like DIII is shown in blue (epitope regions in
magenta), with the E106 heavy chain in green and light chain in cyan. (B) Heavy chain residue W98 binds in a deep pocket contributed by the aliphatic
groups of side chain DENV-1 E DIII residues K307, K325, and E327 and main chain of Y326, in stereo (top panel). The electron density map is
contoured at 1.1σ. Ball and stick representation of the molecular interactions involving T329 (the residue that escapes neutralization [13] in stereo
(bottom panel). (C) Surface representation of DENV-1 E DIII (top) and E106 Fab (bottom) highlighting residues making direct contacts in the complex
(see Table S1). DENV-1 E DIII residues previously identified by yeast surface display are displayed in magenta [13]. (D) The structural epitope on
DENV-1 DIII is shown in ribbon representation. (E) Sequence of DIII of DENV-1 aligned with that of DENV-2, -3, -4 and WNV highlighting the E106
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DENV 1 DIII is shown in ribbon representation. (E) Sequence of DIII of DENV 1 aligned with that of DENV 2, 3, 4 and WNV highlighting the E106
structural epitope, which is conserved in DENV-1 genotypes but not DENV serotypes or WNV. Identical residues are represented by a dot, deleted
residues by a hash. For comparison, The DIII structural epitopes of WNV E16, DENV-2 1A1D-2, DENV cross-reactive 4E11 and 2H12 MAbs and
DENV-1-E111 contact residues are labeled with green, purple, light purple, orange and cyan asterisks respectively
Edeling MA, Austin SK, Shrestha B, Dowd KA, et al. (2014) Potent Dengue Virus Neutralization by a Therapeutic Antibody with Low Monovalent Affinity Requires Bivalent Engagement. PLoS
Pathog 10(4): e1004072. doi:10.1371/journal.ppat.1004072
http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004072

67. 1. LIVE ATTENUATED VIRUS VACCINES
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Construction of a tetravalent vaccine by Sanofi Pasteur

68. 1. LIVE ATTENUATED VIRUS VACCINES
• The prM and E genes of DEN4∆30 was replaced with those ofp g p
DENV2,[111] and the entire 3′-UTR of DENV3 was replaced with that of
DEN4∆30
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Development of the live tetravalent vaccine by the Laboratory of Infectious Diseases at the National Institutes of Allergy and
Infectious Diseases, NIH. Immune response to dengue virus and prospects for a vaccine', Murphy and Whitehead, 29, 2011

69. 2. LIVE CHIMERIC VIRUS VACCINES2. LIVE CHIMERIC VIRUS VACCINES
• Chimeric live attenuated vaccines are designed by combiningg y g
genes from different sources to create a live attenuated virus.
S i ti t tl t d i i i hi h d i l• Scientists are currently studying vaccines in which dengue viral
genes have been genetically engineered into either a live
attenuated vaccine for yellow fever or an attenuated dengue virus
from a single serotypefrom a single serotype.
• Ideal chimeric live attenuated vaccines should have the sameIdeal chimeric live attenuated vaccines should have the same
characteristics that are described above for live attenuated
vaccines.
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70. 2. LIVE CHIMERIC VIRUS VACCINES2. LIVE CHIMERIC VIRUS VACCINES
• The most advance product by Sanofi Pasteur = ChimeriVax
Dengue tetravalent vaccine (CVD1-4) utilized the licensed YFV
17D vaccine as backbone , each expressing the prM & E genes of
one of the four DENV serotypes
• Pre-clinical studies demonstrated that the tetravalent vaccine is
genetically & phenotypically stable, less neurovirulent than YFD
17D & i i i k17D & immunogenic in monkeys.
• Phase 1 = safe with relatively low viremiaPhase 1 safe with relatively low viremia
• Phase 2 = only 30% effectiveness & efficacies against only
DENV1 3 & 4 serotypes risk of ADEDENV1, 3 & 4 serotypes risk of ADE
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71. 3. INACTIVATED VIRUS VACCINES
• Inactivated vaccines are made of virus particles that have beenp
destroyed
I th i th d ti bl t d i• In the vaccines, the dengue antigens are able to produce an immune
response.
• The inactivated vaccines generally have a high level of safety
because the virus does not replicate.
• They may, however, require booster vaccinations to provide long-
term immunity, and they may be more expensive to produce than live
attenuated vaccines.
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72. 3. INACTIVATED VIRUS VACCINES3. INACTIVATED VIRUS VACCINES
• 2 advantages over live virus vaccinesg
• No possibility of reverting to virulence (safety) &
• Relative ease of inducing balanced immune responses (for
tetravalent vaccines)tetravalent vaccines)
• 2 disadvantages
• Lack of the immunity to NS proteins &
• A requirement of adjuvants for enhancing immunity
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73. 4. LIVE RECOMBINANT, DNA &
SUBUNIT VACCINESSUBUNIT VACCINES
• subunit vaccines are made of dengue proteinsg p
• In the vaccines, the dengue antigens are able to produce an immune
response.
• The subunit vaccines generally have a high level of safety because• The subunit vaccines generally have a high level of safety because
the virus does not replicate.
• They may, however, require booster vaccinations to provide long-
term immunity, and they may be more expensive to produce than live
attenuated vaccines.
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74. 4. LIVE RECOMBINANT, DNA &
SUBUNIT VACCINESSUBUNIT VACCINES
• DENV E protein is used as the major immunogen
• Certain live viral vectors e.g. adenovirus, alphavirus & vaccinia virus
are designed for direct administration to the host & have been
engineered to express DENV E protein for further evaluation as vaccinesengineered to express DENV E protein for further evaluation as vaccines
• Recombinant E proteins expressed from yeast & insect cells have been
used to test for immunogenicity & protective efficacy in animal modelsused to test for immunogenicity & protective efficacy in animal models.
• Alternatives: NS1-based vaccines, E-NS1 protein expressed by E. coli
& prM-E-NS1 proteins encoded by DNA vaccine& prM-E-NS1 proteins encoded by DNA vaccine
• This type of vaccine is relatively simple to produce, but may require
multiple doses to provide immunity Therefore this option may not bemultiple doses to provide immunity. Therefore, this option may not be
practical for widespread vaccinations.
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75. CHALLENGES IN DENGUE VACCINE
DEVELOPMENTDEVELOPMENT
• The complicated pathogenesis of dengueThe complicated pathogenesis of dengue
hemorrhagic fever/dengue shock syndrome (DHF/DSS)
is not fully resolved.
• Challenge: genotypic differences (DENV1-4),
i ll DENV 2 & 3 ith t ti i th E & NS3especially DENV 2 & 3 with mutations in the E & NS3
proteins virulence
• A clinically relevant animal model for dengue
infection and vaccine development is lackingp g
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76. MALARIAMALARIA
• Malaria is the most devasting parasitic disease afflictingg p g
humankind
It lt f i f ti ith t it f th• It results from infection with protozoan parasites of the genus,
Plasmodium (P. falciparum & P. vivax) & is transmitted by female
anophelene mosquitoes
• Of the 3.4 billion people in 108 countries at risk of malaria, 1.2
billion are at high risk of disease.billion are at high risk of disease.
• In 2012, it was estimated that this disease caused 200 death per
day
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77. MALARIAMALARIA
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78. CHALLENGES IN MALARIA VACCINE
DEVELOPMENTDEVELOPMENT
1) Malaria parasites are complex eukaryotes
i d f ti i t t lcomprised of many antigenic targets complex
vaccine need to be designed
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79. CHALLENGES IN MALARIA VACCINE
DEVELOPMENT
2) Strong capacity of Plasmodium parasite to evade
DEVELOPMENT
) g p y p
host’s immune system
3) Technical difficulties & high cost associated with
scaling up production including the dose required to
induce long-lasting protective immunity, transport &g g p y, p
storage
4) Risk of reversion to virulence
5) Selection of adjuvants
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80. MALARIA VACCINEMALARIA VACCINE
• The first successful malaria vaccine trial, based on aThe first successful malaria vaccine trial, based on a
“whole parasite” approach, was conducted in humans
in the 1950s.
• But, it was not economically produced in a large-
l iscale size.
• With biotechnology approach, in 1980s, the focus
shifted to so-called “subunit” approaches.
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81. MALARIA VACCINEMALARIA VACCINE
• Subunit vaccineSubunit vaccine
• Involves individual recombinant parasite proteins
administered as monovalent preparations or combinations of
lti l t i t th ith diff t t & dj tmultiple proteins together with different vectors & adjuvants
that enhance the immune response
• It has been developed based on antigens which are
expressed in almost every stage of the parasite lifecycle:
1 Pre-erythrocytic vaccines1. Pre erythrocytic vaccines
2. Blood stage vaccines
3. Transmission-blocking vaccines
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82. MALARIA VACCINEMALARIA VACCINE
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83. 1. PRE-ERYTHROCYTIC VACCINES1. PRE ERYTHROCYTIC VACCINES
• Aim to prevent infection by targeting the infective stage
(sporozoite).
• Alternatively it can target antigens expressed by liver stageAlternatively, it can target antigens expressed by liver stage
parasites to prevent the emergence of merozoites into the
bloodstream, the stage of infection responsible for the clinical
symptoms of malaria infection.
• Disadvantage:
ti d i l t d d i t l i f ti i l ith• antigen dose inoculated during a natural infection is very low, with
only a small number of sporozoites injected by the vector (~20)
not sufficient dose
• Only one sporozoite needs to escape the vaccine-mediated immuneOnly one sporozoite needs to escape the vaccine-mediated immune
response & invade liver cells for ~10,000 infectious merozoites to be
produced disease
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84. 2. BLOOD STAGE VACCINES2. BLOOD STAGE VACCINES
• The majority of malaria vaccine candidates areThe majority of malaria vaccine candidates are
designed to protect against the blood stage of
infection (prevent the disease) because of all of
the symptoms of malaria occur during this stage.
• One approach is to target merozoite antigens to
prevent red blood cell invasion & reduce the
d it & l f it i th i f t ddensity & prevalence of parasites in the infected
cells reduce malaria transmission
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85. 3. TRANSMISSION-BLOCKING
VACCINESVACCINES
• Aim to target antigens expressed during lifecycleAim to target antigens expressed during lifecycle
stages in the mosquito host e.g. gametocytes or
oocyst antigens.
• Not directly prevent infection or clinical disease
• But can assist elimination efforst to prevent the
onward transmission of infections that may be
imported into an elimination zone.
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86. MALARIA VACCINEMALARIA VACCINE
• In general, development of a vaccine against P.ge e a , de e op e t o a acc e aga st
falciparum is well advanced with 31 promising
antigens identified.
• Currently, 27 subunit candidates comprising
different domains & alleles for 22 differentdifferent domains & alleles for 22 different
antigens are being tested in pre-clininal or
clinical trials.
• But, all have different formulations.
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88. ADJUVANTS FOR MALARIA VACCINEADJUVANTS FOR MALARIA VACCINE
• During the last decades, adjuvants have become increasingly
i t t t f th d l t f iimportant components for the development of vaccines.
• They are compounds that enhance & direct specific immune
l ifi d b d th i i i l h i fresponses, classified based on their principal mechanism of
action.
• Major challenge in malaria vaccine development is not
considering the role of other components of the formulation
especially adjuvants that can induce & modulate the immune
response.p
• Several antigen candidates may be had been abandoned based
on the unsatisfactory clinical results obtained because they werey y
inadequately adjuvanted.
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89. ADJUVANTS FOR MALARIA VACCINEADJUVANTS FOR MALARIA VACCINE
• Example of adjuvants:Example of adjuvants:
1. Alum
2 Emulsion2. Emulsion
3. Saponins
4. Virus-Like Particles
5. Toll-Like Receptors
6. Virosomes
7. Viral vectors
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90. ADJUVANTS FOR MALARIA VACCINEADJUVANTS FOR MALARIA VACCINE
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91. ALUMALUM
• It is noncrystalline gel-like forms of aluminum salts & wasy g
the first adjuvant approved for human use around 80 years
ago.
• It is a component of numerous licensed vaccines, such
diphtheria-tetanus-pertussis (DTP), hepatitis A and B virusp p ( ), p
(HAV, HBV), human papilloma virus (HPV), Haemophilus
influenza, and Streptococcus pneumoniae.
• It has the capacity to stimulate strong humoral responses
(Th2). The interaction of Alum with the immune system has( ) e te act o o u t t e u e syste as
not been completely clarified and several mechanisms of
action have been proposed.
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92. ALUMALUM
• First, it was believed that Alum only produced aFirst, it was believed that Alum only produced a
depot effect and thereby a sustained release of
antigen.
• However, several studies have reported a rapid, p p
desorption of this adjuvant from the injection site.
What is clear is that the administration of the
ti i ti l t f f it t bantigen in a particulate form favors its capture by
antigen-presenting cells (APCs)
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93. ALUMALUM
• Alum has also demonstrated its own immunostimulatory
capacity; a direct or indirect (mediated by dangerouscapacity; a direct or indirect (mediated by dangerous
signals such uric acid) ability of Alum to activate NALP3, a
component of the inflammasome complex, has been
d ib ddescribed.
This acti ation leads to caspase 1 acti ation•This activation leads to caspase-1 activation,
proinflammatory cytokine secretion (IL-1 , IL-18, IL-33), and
monocyte migration to lymph nodes (LNs) for their
diff ti ti i t i fl t d d iti ll (DC )differentiation into inflammatory dendritic cells (DCs).
C tl Al i th t d dj t i th li i l•Currently, Alum is the most used adjuvant in the clinical
evaluation of malaria vaccines.
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94. ALUMALUM
• The first malaria vaccine candidate extensivelyThe first malaria vaccine candidate extensively
tested in endemic areas was the Alum adjuvanted
SPf66 multistage antigen.
• Despite these findings, the clinical research withp g ,
these antigens continued following their
reformulation with other adjuvants and/or
bi ti ith th ticombination with other antigens.
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95. SUMMARYSUMMARY
• IntroductionIntroduction
• Recap PHC450: Vaccine Immunology
• Traditional Vaccines• Traditional Vaccines
• Modern Vaccines
• Case Study
• Dengue Vaccine
• Types of vaccine• Types of vaccine
• Malaria Vaccine
• Challenges
• Adjuvants
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96. REFERENCESREFERENCES
• Barry, A. E., Arnott, A.,Strategies for designing and monitoring malaria vaccines targeting
diverse antigens Front Immunol 2014 Jul 28;5:359 doi: 10 3389/fimmu 2014 00359diverse antigens, Front Immunol, 2014 Jul 28;5:359. doi: 10.3389/fimmu.2014.00359.
• Chokephaibulkit, K., Perng, G. C., Challenges for the formulation of a
universal vaccine against dengue Exp Biol Med (Maywood) 2013 May;238(5):566-78 doi:universal vaccine against dengue, Exp Biol Med (Maywood), 2013 May;238(5):566-78. doi:
10.1177/1535370212473703.
• Elena Mata Aiala Salvador Manoli Igartua Rosa Maria Hernandez Jose Luis Pedraz• Elena Mata, Aiala Salvador, Manoli Igartua, Rosa Maria Hernandez, Jose Luis Pedraz,
Malaria Vaccine Adjuvants: Latest Update and Challenges in Preclinical and Clinical
Research, BioMed Research International
Volume 2013 (2013), doi.org/10.1155/2013/282913
•Shu-Wen Wan, Chiou-Feng Lin, Shuying Wang, Yu-Hung Chen, Trai-Ming Yeh,Hsiao-
Sheng Liu, Robert Anderson and Yee-Shin Lin, Current progress in dengue vaccines,
Journal of Biomedical Sience 2013 20:37 doi:10 1186/1423-0127-20-37Journal of Biomedical Sience, 2013, 20:37. doi:10.1186/1423-0127-20-37.
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97. THANK YOU
97