Antigen in

Vaccine Development

Widya Asmara
Figure 14-23 part 1 of 2
Figure 14-23 part 2 of 2
 Type of vaccine’s antigen
• Whole Organisms
– Killed/Inactivated
– Modified Live/Attenuated
• Conventional
• Genetic
• Purified Fractions/Subunits
– Conventional Subunits
– Genetically Engineered Subunits
– Naked DNA
• Vectored Vaccines
– Bacterial
– Viral
 CLASSICAL VACCINE
• In activated / killed vaccine:
– Whole cell / virus particle
• Attenuated strain :
– naturally occuring
– after passages
– Mutagenesis
• Sub unit vaccine
– LPS , protein , peptide, toxoid
 Whole agent vaccines -- Killed
by heat or chemical (formaldehyde)

epitopes

epitopes

i.e.: Inactivated polio vaccine (Salk), Influenza (Classic)

 Attenuation of viruses by passage
through non-human cells
1. Pathogenic virus isolated from
patient, grown in human cells
2. Infect monkey cells with
1 2 cultured virus
3. Virus acquires many mutations
that allow it to grow well in
monkey cells
4. Mutations make the virus
unable to grow well in human
cells
 Vaccine candidate

3 4
 Subunit vaccines

• Single antigen or mixture of antigens

• Safer (cannot reproduce)
• However, often less effective than whole
agent vaccines
• Can be costly
• Always require boosters
 GE vaccines
Attenuation by recombinant DNA technology
Subunit vaccines
Recombinant vaccines
Live vaccines:
GE viruses / RG virus and GE virus-vector vaccines
GE bacteria
Naked DNA vaccines
Edible vaccines produced by GE plants
 Sub-unit vaccine
• Antigen prepared conventionally:
– Culture of WT bacteria/virus
– Isolation & purification of specific antigen
• Antigen prepared by DNA recombinant:

Transformed into
bacteria/yeast
Expressed as heterolog protein
Purified & prepared as vaccine
antigen
Cloned into plasmid
 Attenuation by Recombinant DNA

1. Select pathogenic strain as vaccine candidate

2. Isolate virulence gene (gene cloning)
3. Site-directed mutagenesis to inactivate
4. Transformed inactivated gene into WTstrain
reciprocal recombination
5. Select attenuated mutant-strain
6. Live stable attenuated strain
Homologous / reciprocal recombination
Attenuation of viruses by recombinant DNA techniques
Figure 14-25 part 1 of 2
 Deletion Based Attenuation Salmonella
Strains

• Salmonella strains cause many diseases

– Enteric fever, infant death, typhoid fever, food
poisoning
• Attenuated strains constructed by deletion of
biosynthetic, regulatory and/or virulence genes (100-fold
reduction in virulence)
– Many double deletions used to make reversion less
likely
– Useful for human and animal inoculation
 Deletion-attenuated Salmonella Strains

• Salmonella attenuating mutations

– ∆aroA - first defined, attenuating mutation
• Lesion in aromatic biosynthetic pathway; In vivo
requirement for p-aminobenzoate (precursor to
folate)
• Combination with other aro deletions:
∆aroA∆aroD, ∆aroA∆aroC
• Loss capability to grow without aromatic AA
 Deletion-attenuated Salmonella Strains
• ∆aroA mutants
– ∆aroA mutants derived for a number of other Salmonella
• S. typhi - humans
• S. enteritidis - chickens
• S. typhimurium - sheep, pig
– ΔaroA mutants of originally calf virulent isolates of S.
typhimurium and S. dublin were safe and protective in calves
(>1010 CFU, oral)
– S. typhimurium ∆aroA∆aroD safe and effective in 7 day-old
calves (>1010 CFU, oral)
 Deletion Attenuation Bacterial Strains
Metabolic mutants dependent either on aromatic
copounds (aro-mutants) or pur. Mutans
•Myron Levine & Bruce Stoker  S.typhi strain 541 Ty ~
mutation on aroA and purA
•Immune response disappointing
•Steven Chatfield : mutation in aroA and aroC or aroA and
aroC or aroC and aroD
•Ty2  S.typhi aroC, aroD  highly immunogenic
tolerated by mammalian
•Because para-aminobenzoat & other aromatic compound
are limited in mammalian tissue
Deleted Genes and Their Function in the Development of
Attenuated Strains of Salmonella spp.
 Deletion-attenuated Non-Salmonella
• ∆aro mutants
– Non-Salmonella spp. mutants
• Shigella flexneri - S&E in monkeys; S in humans
• Aeromonas salmonicida - S&E in salmon and trout
• P. multocida and Yersinia enterocolitica - S&E in
mice
Cholera Toxin

• B subunit for binding to membrane

receptor site
• A2 peptide acts as connector
between B and A1 subunits
• A1 peptide is the enzymatically
active component which modifies
target G protein, locking adenylate
cyclase in on position in intestinal
mucosal cells
Deletion-attenuated Toxin Gene

•Clone toxin gene

•Cut out internal
segment
•Ligate using linker
•Conjugate into wt
cell
•Recombination
•Chromosomal gene
now attenuated by
deletion
Attenuated Leishmania

• Protozoan parasite
• Traditionally attenuated strains
persist in host (without symptoms)
for extended periods
• Traditional attenuated strains have
undesirable property of reversion.
• Delete DHFR/Thymidylate synthase
• This strain persists only for a few
days in host but provides immunity
Attenuated L. major Immunity

• Inoculate with attenuated L.

major
• Challenge with wt L. major
• Few/small lesions in previously
inoculated mice
– even very sensitive host
strains
 Single gene (subunit) in expression vector
(virus vectored vaccine)
•Isolate the protective antigen gene from the
pathogens

• Insert the gene into viral vector’s genome (i.e. pox,

vaccinia virus) ~ canary pox infects human cells but does
not replicate

•Vaccinate with live virus

• Better presentation
• Humoral & CTL response
Vectored vaccines
Recombinant live
vectored vaccine
 vectored vaccine
Diisolasi fragmen gen yang diinginkan,
dipasang dalam vektor rekombinan
Virus lain (ND,
IBD, ILT, dsb.)

HVT / pox virus

used as vectored vaccine

antigen: co-transfection
HVT-IBD
HVT-ND
HVT-ILT
HVT-ND+IBD

HVT-vectored vaccines can be

administered sc / in-ovo

Pox V / HVT
expressing other
virus proteins
Prospect of multivalent vectored vaccine

Two pathogen-
antigen in one
vectored construct

Combine important
epitopes from two or
more pathogens
Conventional Influenza Virus Vaccine Strain

PB2
PB2
PB1
PA
X PB1
PA
HA HA
NA NA
NP NP
M M
NS NS
PB2
PB1
Attenuated PA New
Donor Master HA Virulent
Strain
NA
NP Antigenic
Attenuated Vaccine M Variant
Strain: Coat of Virulent NS
strain with Virulence Strain
Characteristics of
Attenuated Strain
 Reverse genetic vaccine
• i.e.: influenza vaccine,
• Isolation of viral RNA
• Synthesize cDNA of each gene segment
• Clone them into special plasmids
• If needed: do site directed mutagenesis to alter virulence
sequence
• Transfect into cell culture (i.e.:MDCK)
• Let the gene fragments to rearrange into a new virus
• Prepare them for vaccine
Generation of H5N1 vaccine with modified HA
using plasmid-based reverse genetics

N1 NA PB2 PB1 PA HA

Mod. H5 NP NA M NS
HP avian HA PR8 h.g.
virus donor
RERRRKKR RETR Bi-directional plasmids
expressing both
mRNA and vRNA

Reassortant Transfect
Modified Vero cells
H5N1 Vaccine
 DNA VACCINE ?
GENE FOR AN IMMUNOGEN

INSERT GENE INTO AN EXPRESSION PLASMID

TRANSFORM BACTERIAL CELLS,

GROW BACTERIA ,
PURIFY PLASMID DNA

IMMUNIZE WITH
IMMUNOGEN EXPRESSING PLASMID
 DNA Vaccine
• Create a recombinant plasmid containing a gene encoding a specific antigen.
• Engineered the sequence within bacteria, but enabling it to be expressed in
human
• Introduce it into humans & Let the human cells produce the antigen
• Present it to B-cell & T-cells  Provoke immune response  humoral & cellular
immunity
DNA Vaccine Mechanisms
 DNA vaccines Vs Traditional
vaccines
DNA vaccines Traditional vaccines
 Uses only the DNA  Uses weakened or
from infectious killed form of
organisms. infectious organism.
 Avoids the risk of using  Creates possible risk
actual infectious of the vaccine being
organism. fatal.
 Provide both Humoral
& Cell mediated  Provide primarily
immunity Humoral immunity
 Refrigeration is not  Usually requires
required Refrigeration.
 ADVANTAGES
 Elicit both Humoral & cell mediated
immunity
 Focused on Antigen of interest
 Long term immunity
 Refrigeration is not required
 Stable for storage
 Plasmid with multiple genes provide
immunity against many diseases in one
booster
Malaria: Plasmodium Life Cycle
Pre-erythrocytic stage,
that is the stage that
takes place shortly after
being bitten by an infected
mosquito up to and
including the liver stage.

Sporozoites
Pre-erythrocytic
Stage
Liver Stage
 Pre-erythrocytic Stage Vaccines
• How they work:
– Generates Ab response against sporozoites and
prevents them from invading the liver
– Prevents intra-hepatic multiplication by killing
parasite-infected hepatocytes
• Intended Use:
– Ideal for travelers - protects against malaria
infection
 Malaria: Plasmodium Life Cycle

Asexual
Erythrocytic Blood Stage
Stage
Merozoites
 Asexual Erythrocytic Stage Vaccines
• How they work:
– Elicit antibodies that will inactivate merozoites
and/or target malarial Ag expressed on RBC
surface
– Inhibit development of parasite in RBCs
• Intended Use:
– Morbidity reduction in endemic countries
 Malaria: Plasmodium Life Cycle

Sexual
Stage

Gametocytes
 Sexual Stage Vaccines
• How they work:
– Induces Ab against sexual stage Ag
– Prevents development of infectious
sporozoites in salivary glands of mosquitoes
– Prevent or decrease transmission of parasite
to new hosts
• Intended Use:
– Decreased malaria transmission
Vaccines stimulate immune memory

•Killed virus vaccine requires

multiple doses (booster shots)
to adequately stimulate a
protective immune response

•Live virus vaccines replicate in

the host.
•No requirement for boosters.
antibodies formation
T cell dependent and independent B cell responses

2 signal model: engagement of antigen receptor (BCR,

“signal 1”) is not sufficient to activate B cell. Also need
co-stimulatory signal (“signal 2”).
Signal 1
From antigen

Signal 2
From Th
Signal 3
From Th
 Mekanisme pembentukan antibodi (vaksinasi)
Killed bacteria (pemberian 1)

Reseptor sel B Phagocytosis oleh APCs :

Macrophage, dendritic,
Proliferasi &
diferensiasi sel B Phagolysosom
Epitop peptida
Plasma
IgM cell
Aktivasi sel Th MHC2-epitop
sitokin

Plasma cell,
class switch
IgG; better affinity antibody
Memory B cell

netralisasi, opsonisasi, No infection

Killed bacteria (pemberian 2/booster) aktivasi komplemen
 Mekanisme imunitas seluler (CMI)
Vaksin live attenuated
(bakteri intra seluler, virus),
Vectored vaccines

Multiplikasi intra sel, Diproses dalam Epitop peptida

sintesis protein proteosom
antigen

Cytokine
from Th Aktivasi sel Tc MHC1 - epitop

Activated T Memory T cell

cell
Designing antigen
Vaccine Development
Approval Process
Discovery

Phase 1

Phase 2

Phase 3

Phase 4
 Phase 1
Following submission of IND a candidate vaccine is tested in
a small number (10-30) healthy human adult volunteers who
are not at risk for the disease in question.
Main goal is safety and to a lesser extent immunogenicity
involving different doses and different immunization
schedules.
Phase 1 studies can be done in academic or biotech settings
but need institutional Investigative Review Board approval
Including informed consent to protect volunteer rights
GMP, GCP, & GLP must be used throughout
 Phase 2
• Involves a larger number of volunteers (50-500), usually a
mixture of low-risk and higher risk individuals from the
population where phase 3 studies will be done. Informed
consent must be obtained from all participants.
• Main goal is to generate safety data as well as information
for refining dosage and immunization schedule, vaccine
formulations and lot consistency
• May also provide preliminary data relative to efficacy
• If challenge studies are to be included, they must not
include vulnerable populations
• Trials usually take 18-24 months because of screening and
enrolling large numbers of participants
 Phase 3
The randomized, controlled clinical trial aimed at
providing scientific evidence about the clinical
performance of vaccines.
Involves 1000s of participants from high risk
populations where individuals, having given informed
consent, are randomized into test and control groups
to be given vaccine or placebo in a blinded study to
avoid bias.
The goal of phase 3 trials is to determine vaccine
safety and efficacy in a large study population sample
leading to licensure application
 Phase 4
• Phase 4 trials are done after a vaccine has been
shown to work and has been granted a licence.
So they are looking at vaccines that are already
available for doctors to prescribe, rather than new
vaccines that are still being developed.
• The main reasons pharmaceutical companies run
phase 4 trials are to find out more about the side
effects and safety of the vaccine, what the long
term risks and benefits are, and how well the
vaccine works when it is used more widely than in
clinical trials.
Steps in Vaccine Development
Drug / Vaccine Discovery

Clinical Trial Authorization

New Vaccine Submission

Authorization

Market Authorization