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Rational Vaccine Design Against Complex
Pathogens in the OMICS era
Denise L. Doolan
INFECTIOUS & PARASITIC DISEASES

Responsible for 1/3 of all deaths worldwide

15 million deaths per year (WHO Global Health Estimates 2002-2012)

68% deaths in children < 5 years old = 5 million deaths (Black et al. Lancet 2010; Liu, Lancet 2012)
VACCINES
• Efficient and cost-effective intervention for the
control and eradication of disease, and the
prevention of morbidity and mortality worldwide
• No other modality has had such a major effect
on reducing mortality and improving public
health, except for water sanitation
(World Health Organization, 2006)
• Vaccinology is the only science that has
eradicated an infectious disease (Smallpox, 8 May 1980)
THE VACCINE PIPELINE
The relative size of each bubble corresponds to the number of lives affected by widespread use of the vaccine
World Health Organization. 2006. Eighteen months of immunization and vaccine R & D. www.who.int/vaccine_research/about/gvrf/Kieny_presentation.pdf
NO ANTI-PARASITE VACCINES (HUMANS)...
Tremendous burden of
disease worldwide:
- mortality
- morbidity
- socioeconomic impact
ALMOST NO VACCINE AGAINST ANY CHRONIC DISEASE ...
malaria
schistosomiasis
hydatid disease
hookworm
tuberculosis
HIV
hepatitis
cholera
NO VACCINE AGAINST ANY CHRONIC DISEASE
CAUSED BY A COMPLEX PATHOGEN ...
• pathogens have adapted to long-term coexistence
with the human immune system
• evolved sophisticated immune evasion strategies
• distinct phases of their life cycles
• express hundreds or thousands of potential
antigenic targets
MALARIA
5 billion clinical episodes /yr
300-500 million cases morbidity /yr
10-20 million cases severe disease /yr
~1 million deaths/yr
1 death /15-30 sec Socioeconomic burden
No licensed vaccine
Drug resistance 
Insecticide resistance 
MODELS DEMOSTRATING THE FEASIBILITY OF DEVELOPING A VACCINE
 Irradiated sporozoite immunization
peptide
MHC
-2 microglobulin
Liver stage
(Pre-Erythrocytic stage)
CD4+ and CD8+ T- cells
Infected Hepatocyte
– Antigens: infected hepatocyte
– Mechanism: CD4+ and CD8+ T cells
Naturally acquired immunity
antibody
antigen
merozoite
Infected Erythrocyte
– Antigens: blood stage (merozoite & iRBC)
– Mechanism: antibodies and CD4+ T cells
Blood stage
(Erythrocytic stage)
Antibodies and CD4+ T-cells
MALARIA VACCINE DEVELOPMENT CHALLENGES
•
Immunological complexity and genetic variability of
parasite
–
–
•
multi-stage lifecycle, 23Mb genome, ~5300 proteins
antigenic polymorphism, allelic variation, immune evasion
Limited and arbitrary list of candidate vaccine targets
- < 0.5% of the proteome evaluated as potential vaccine component
(CSP, TRAP, MSP1, AMA1)
•
Limited knowledge of mechanisms of protective immunity
- T cell, antibody
•
Absence of algorithms that predict antigen reactivity and
ability to elicit protective immune response
GLOBAL PROJECT ACTIVITY (MALARIA SUBUNIT VACCINES)
Field Trials
Pre-erythrocytic
Blood Stage
Whole Organism
Sexual Stage
Combination
Preclinical
Development
Single 11site
Project
Phase 1/non
endemic
Phase 1/
challenge
Phase
1/endemic
Phase 2/endemic
http://www.who.int/vaccine_research/links/Rainbow/en/index.html (Vasee Moorthy) (2010)
Phase 3
RTS,S
~ 40 preclinical studies
~ $800 million development cost
Phase 3 trial >$100 million
< 30% efficacy ….
WHOLE PARASITE APPROACHES
RADIATION ATTENUATED SPOROZOITES
GENETICALLY ATTENUATED PARASITES
CHEMICALLY ATTENUATED PARASITES
TRADITIONAL VACCINE TECHNOLOGIES
VACCINE
Almost all current licensed vaccines based on traditional technologies (Jenner, 1796)
> 200 years ago ....
The “Vaccinia Hut”
1796 – Smallpox
Dr Edward Jenner vaccinating James Phipps with cowpox
2015
GENOMICS, PROTEOMICS & TRANSCRIPTOMICS
www.commons.wikimedia.org, biol.lf1.cuni.cz , www.kmle.co.kr
PLASMODIUM - LARGE COMPLEX PARASITE
VACCINE
P. falciparum
23.3 Mb
5,403 genes
P. vivax
26.8 Mb
5,433 genes
P. knowlesi
23.5 Mb
5,188 genes
P. yoelii
23.1 Mb
5,878 genes
Carlton et al. Nature 455:757-763
IMMUNOMICS
• bridges the disciplines of genomics and proteomics via the immune system
• focuses on elucidating the set of antigens that interact with the host immune
system and the mechanisms involved in these interactions
• distinct from reverse vaccinology, which aims to identify the complete
repertoire of antigens that an organism is capable of expressing on its surface
• Use specimens from exposed/immunized humans or animals to identify key
antigens using biologically relevant criteria
(immune reactivity, biological activity, functional significance)
– Antibody screening : sera/plasma
– Cellular screening
: PBMCs
Vaccine antigen discovery using biologically relevant selection criteria
PROTEOME-WIDE SCREENING APPROACHES
• Antibody-based screen
to identify the repertoire of Plasmodium
antigens targeted by antibody responses
• T cell epitope-based screen
to identify the repertoire of Plasmodium
antigens targeted by T cell responses
antibody
antigen
merozoite
infected
erythrocyte
Protein microarray
IFN-g ELIspot
PROTEIN MICROARRAYS
Target genome
Homologous Recombination
PCR Fragment (5’ 3’ adapter sequences)
High
Data Analysis
Low
Linear T7 vector
Day 1:
PCR & Transformation
Day 2:
Plasmid DNA miniprep
& Protein Expression
Quality Control
Scanning
(anti-Tag Ab)
Probing
Day 3: Chip Printing
PROTEIN MICROARRAY BASED ANTIBODY SCREENING
Angela Trieu
2320
proteins
PROTEIN MICROARRAYS IDENTIFY MANY NEW IMMUNOREACTIVE ANTIGENS
Malaria exposed (PNG)
Naive
Top 100
- MSP1
~ 30% protein fragments seropositive
differentially recognized btw malaria
exposed/infected vs naive (padj<0.05)
Novel antigens >> well
characterized historical antigens
- MSP2
- AMA1
- CSP (rank 911)
- TRAP (rank 1051)
PROTEIN INTERACTION NETWORKS OF THE TOP 100 Pf PROTEINS
HIGHLY REACTIVE FOR ANTIBODY RESPONSES
Gene Functional Classification tool available in DAVID Bioinformatics Resource
80%
70%
60%
50%
40%
30%
Cumulative Signal Intensity
Average signal intensity
Ave signal intensity
#
40000
20000
5000
100%
Protected
Frequency
90%
Not
Protected
20%
0%
PFE0060w
PFL2140c
Not Protected
35000
30000
25000
#
0.5
15000
0.4
10000
0
Protected
100000
Not protected
80000
***
60000
40000
20000
10%
0
120000
90%
PF08_0034
PF13_0201
PFC0210c
PF11_0344
PF11_0404
PFD0485w
PFB0150c
PFL2140c
PFL2505c
PFL1620w
PF10_0211 e2s1
PF10_0211 e1s3
PFI0925w
PF13_0222
PF14_0051
PFE0060w
MAL13P1.22
PF08_0054
PFE1085w
PFB0285c
MAL13P1.22
PF13_0222
PFL2505c
PF13_0201
PF11_0404
PFE1085w
PFC0210c
Antigens
PF08_0054
PF08_0034
16 signature antigens
PF11_0344
PFB0150c
PF10_0211
PF10_0211
PFL1620w
PFD0485w
PF14_0051
PFB0285c
PFI0925w
PF08_0034
PF13_0201
PFC0210c
PF11_0344
PF11_0404
PFD0485w
PFB0150c
PFL2140c
PFL2505c
PFL1620w
PF10_0211 e2s1
PF10_0211 e1s3
PFI0925w
PF13_0222
PF14_0051
PFE0060w
MAL13P1.22
PF08_0054
Antigens
Protected
AUC value
45000
10000
8000
6000
4000
3000
2000
1500
1000
800
0
AUC
Serodominance does not correlate with protection
Frequency of recognition (Percentage of individuals
that recognise an antigen)
Not
Protected
PF11_0294
PFI0925w
PFB0285c
PFI0570w
PFC0805w
PF14_0051
PF11_0440
MAL7P1.12e
PFI0240c
PF10_0183
PF11_0486
PFD0485w
PF10_0211
PF14_0648
PFL0555c
PFL1620w
PF10_0211
PF08_0060
PFI1425w
MAL13P1.107
PFB0150c
PF11_0344
PF14_0664
PF11_0353
PFE0060w
PF08_0034
PF14_0626
PF10_0183
PF08_0054
PFL2140c
PF14_0051
PFE1085w
PFC0210c
PF11_0395
PFC0120w
PF14_0454
PF11_0404
PFL0635c
MAL7P1.146
PF10_0143
PF07_0061
PFI0580c
PF10_0320
PFC0440c
PF11_0226
MAL7P1.146
PF14_0722
PF13_0190
PF13_0201
PF07_0089
PFL2505c
PF10_0213
PFD0450c
PFB0895c
PF13_0222
PF11_0395
PF14_0035
PF11_0008
PF14_0419
PFI0265c
MAL8P1.139
MAL7P1.89
MAL13P1.346
PFL0115w
MAL13P1.107
PFI0240c
PFL0440c
PF13_0179
MAL7P1.146
PFE1120w
PF11_0156
MAL13P1.176
PF13_0350
PF11_0106
MAL13P1.22
PFI1170c
MAL7P1.138
PF14_0338
PFC0430w
PF14_0179
PF14_0270
PF07_0016
PFL0445w
PFL2165w
PF14_0461
PF11_0395
Protected
PFE1085w
PFB0285c
Frequency of recognition (Percentage of individuals
that recognise an antigen)
Signal intensity
1
AUC value
0.9
0.8
0.7
0.6
0.3
#
0.2
0
0.1
Antigens
Magnitude
100%
Protected
80%
Not
Protected
70%
60%
50%
40%
30%
20%
10%
0%
Antigens
46 signature antigens
PROTEOME-WIDE SCREENING APPROACHES
• Antibody-based screen
to identify the repertoire of Plasmodium
antigens targeted by antibody responses
• T cell epitope-based screen
to identify the repertoire of Plasmodium
antigens targeted by T cell responses
antibody
antigen
merozoite
infected
erythrocyte
Protein microarray
IFN-g ELIspot
T CELL-BASED SCREENING
Joanne Roddick
5,268 (P. falciparum PROTEOME)
PlasmoDB
PUBMED
Florens et al. (2002) Nature
Le Roch et al. (2004) Gen Res
Hall et al. (2005) Science
Tarun et al. (2008) PNAS
Vaughan et al. (2008) Bioinform
Trieu et al. (2011) MCP
PRE-ERYTHROCYTIC PROTEINS
1450 Proteins
5 PEPTIDES PER 3 CLASS I &
1 CLASS II (A2, A3/A11, A24, DR)
Prediction of MHC-binding
capacity by motif analysis
29,000 Peptides
Pools of 20 synthetic peptide per protein
IFN-ϒ ELIspot (PBMC from P.
falciparum exposed individuals in
PNG)(n=10/Ag)
Comparative genomics and
proteomic analyses
SCREEN POOLS FOR
IMMUNE REACTIVITY
PRIORITIZE IMMUNOREACTIVE
ANTIGENS FOR VALIDATION
T CELL ANTIGEN REACTIVITY
Low reactivity
High reactivity
CSP
T-cell magnitude
STARP
EXP1
Responders
AMA1
SPECT
LSA3
SSP2
T-cell frequency
12 proteins (11%) recognized by 100% subjects
294 proteins (29%) recognized by >50% subjects
56% proteins not recognised (<20% subjects)
PROTEOME-WIDE T CELL SCREENING IDENTIFES MANY NEW
IMMUNOREACTIVE ANTIGENS
N=80 antigens
n=12
100%
recognition
n=24
90%
recognition
(all novel)
0-49 SFC/m
50-99 SFC/m
100-149 SFC/m
150-199 SFC/m
n=44
80%
recognition
>200 SFC/m
Sporozoite expression
All antigens
(in rank order)
MudPIT (protein abundance)
T CELL REACTIVITY DOES NOT CORRELATE WITH EXPRESSION STAGE
70
60
50
40
30
20
10
0
0
200
400
600
Reactivity rank
800
Blood stage expression
Top 36 antigens
(not in rank order)
Ring
Trophozoite
Schizont
1000
1200
PROTEOME-WIDE SCREENING APPROACHES
Carla Proietti
• Antibody-based screen
• T cell epitope-based screen
antibody
antigen
merozoite
infected
erythrocyte
?
N=1200 Pf proteins
(25% of complete proteome)
2320 protein fragments
Protein microarray
cell-free protein
expression
N= 1450 Pf proteins
(complete pre–erythrocytic proteome)
29,000 peptides
IFN-g ELIspot
20 peptides per protein
3x HLA class I & 1x HLA class II
FREQUENCY & MAGNITUDE of T cell & Antibody responses
Frequency
0%
10 - 30%
30- 60%
60 - 100%
Outer ring: T cell
Inner ring: Ab
Magnitude
T cell
Ab
NO CORRELATION BETWEEN T CELL & ANTIBODY RESPONSES
... INVERSE CORRELATION ...
T cell targets are not antibody serodominant ...
Ab ANTIGENS ARE POLYMORPHIC; T CELL ARE CONSERVED
3.1–fold difference in SNPs /gene amongst 144 Pf strains
3
AB
T cell
SNPs (Top 100)
2.5
Log(SNPs)
2
1.5
1
0.5
0
0
-0.5
20
40
60
Antigens
80
100
Kruskal-Wallis equality-of-populations rank test p<0.001
DISTINCT GLOBAL BIOCHEMICAL & STRUCTURAL FEATURE
PROFILES BETWEEN Ab TARGETS AND T CELL TARGETS
PCA
RDA
Antibody target
T cell target
T CELL AND ANTIBODY TARGETS CHARACTERIZED
BY 69 BIOCHEMICAL AND STRUCTURAL FEATURES
Genomic and protein attributes
47 features
Protein expression
6 features
Similarity
2 features
Population biology
1 feature
Protein features
13 features
T CELL AND ANTIBODY TARGETS CHARACTERIZED
BY 69 BIOCHEMICAL AND STRUCTURAL FEATURES
Antibody
29 features positively associated
• Exons
• Molecular weight
• TM domains
• PEXEL
• SNPs density
T cell
2 features positively associated
•Q
• Acidic residuals
24 features inversely associated
12 features inversely associated
• Isoelectric point
• Expression in liver stage
• Non polar aliphatic residuals
• Basic residuals
• Signal peptide,
• TM domains
• Secondary sheet, helix
• Aromaticity
CONCLUSIONS
• Immunomic approaches and proteome-wide datasets of antibody or T cell
responses allow for identification of novel antigens targeted by protective immunity
• Antigens recognized by antibodies or T cell responses are broadly distributed over
the Plasmodium proteome (but a large number of antigens are not recognized)
• 30% of proteome recognized by antibodies or T cells
• Not all antigens are equal
• hierachy of antigenicity, immunogenicity & protective capacity
• many novel antigens >> historical vaccine candidates
• distinct antigens sets are preferentially targeted by T cells vs antibodies
• antigens recognized preferentially by antibodies are highly polymorphic while
the most T-cell reactive antigens are conserved
• Molecular & biochemical features can discriminate between Ab & T cell targets
• Novel antigens identified using immunomics are promising vaccine antigens
Carla Proietti
Angela Trieu
Joanne Roddick
Bruno Douradinha
Phil Felgner
Matthew Kayala
Pierre Baldi
Sophie Schussek
Simon Apte
Penny Groves
David Pattinson
Julie Burel
Lea Lekieffre
Karina deSousa
Animal House
Flow Facility
John Sidney
Bjorn Peters
Yohan Kim
Scott Southwood
Xiaowu Liang
Douglas Molina
Angela Yee
Rie Nakajima-Sasaki
Joselyn Pablo
Lutz Krause
Shiahab Hasan
Papua New Guinea
Institute of Medical
Research
Alessandro Sette
University of Bamako
Mali
Ravi Kolla
NIH/NIAID Lab of
Malaria & Vector
Research
Stephen Hoffman
Eric James
Leanne Robinson
Jack Taraika
Danga Mark
Yagaum HRC staff
Danielle Stansic
Ivo Mueller
Peter Siba
Daniel Freilich
Lolita Bebris
Mara Berzins
Thomas Richie
Noelle Patterson
Boubacar Traore
Kassoum Kayentao
Aissata Ongoiba
Safiatou Doumbo
Didier Doumtabec
Younoussou Kone
Peter Crompton
Louis Miller
Susan Pierce
volunteers …