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New Strategies Towards Vaccine Design
Challenging HIV
Ralf WagnerUniv. Prof. for Medical Microbiology and Gene Therapy, University of
Regensburg
immunogen designmolecular virology
ex vivo analysisbasic in vivo / mice
preclinic / rhesus macaquesclinic / phase 1/2/3
•envelope proteins: Env•structural proteins: Gag, Pol•regulatory proteins: Tat, Rev, Nef•accessory proteins: Vpr
Targets of immune response
+ +
Peptides DNAViral Vectors
Bacterial VectorsPseudovirions
Adjuvants (CpG, PEI, MF59)Proteins
Delivery / Combination of Multivalent HIV/AIDS Vaccine Strategies
?
rAd, NYVAC
• neutralizing AB, V1/V2, IgG/IgA• CD4+ T cell response• CD8+ CTLs• broad, cross clade, • high quality, polyfunctional• mucosal immunity• long term memory• feasable immunization regimen
Immune response
•A (Africa) •B (Europe, N & S-America)•C (Asia, Africa …)•E (Asia)•F (Africa)•G (South America, Africa)
HIV Clades
Starting point
Subcloning pCR-script
China
India
Asia
CC
CC C
C
BB
B
B
B
B
Prevalence
Incidence
Virus population
Regional clustering
Host genetic background
Sequence analysis of C (CN54)
LTRLTR
gagpol
vif
vpr
vpuenv
rev
tat
tev
nef
Knowledge on molecular epidemiology
antigens regional strains
mosaic antigens next generationmosaics
CHIVAC EU FP5 / 6Shao Yiming, Hans Wolf
Knowledge on specific targets/ epitopes- Control of virus replication
MHRmajor homology region
HLA-B14-restrictedCTL-epitope (aa298-306)
p1p2
p24 CAp17 MA
0 1 2 3 4 5 6 7 8 9 kb
LTR
tev
gagpol
vif
vprvpu
env
revtat
nef
p6p7
D R F Y K T L R A
LTR
(35) (1) (22) (8)
298 299 300 301 302 303 305304 306
020406080
100
020406080
100
HLA-B14CTL-Epitope
conservative
non-conservative
Infectivity
Infectivity
% s
pec.
Lys
is%
spe
c. L
ysis
D R F Y K T L R A
E K Y F R S/ A V K G
W Y R Q I F H Y F
Focus IR to conserved proteins and epitopes limits immunological escapeTrue for both T and B cell responses
DFG
CRM1ran
REV
RRE
degradation
wt - gag
cis active repressor sequenzes
UTR
SDREV
RRE
splicing- machinery
?wt-gag
AA Human HIV-1Ala GCC GCAArg AGG AGAAsn AAC AATCys TGC TGTGln CAG CAAGlu GAG GAAGly GGC GGA
- - - - + + + +
UTR
-wtg
ag-R
RE
gag-
RR
E
UTR
-wtg
ag-R
RE
C C C CN N NN
Northernconstitutive export
syn-gag
syng
ag
Knowledge on virus replication (I)
BMBF
Risk for RecombinationRNA Packaging
Increased safety Increased yieldsEnhanced immunogenicity
• protein production • DNA vaccines• viral DNA delivery (SFV)• bacterial DNA delivery (Listeria)• adenoviral vectors• herpesviral vectors (EHV)• lentiviral vectors
• easy expression of late lentiviral genes • RNA nuclear export 50x• no Rev, no RRE• no 5‘-UTR, no Y
HIV Vaccines
2
constitutive nc export
syn-gag
CRM1ran
REV
?
RRE
degradation
wt - gag
cis active repressor sequenzes
UTR
SDREV
RRE
splicing machinery
?
2
3
1
wt-gag
Knowledge to specify gene design
RNA- and Codon Optimization
1st Generation (EuroVacc):
T Cell VaccineBroad,
PolyfunctionalMimic LTNP Profile
EU FP 5, 6
Sequence analysis of C (CN54)
LTRLTR
gag
pol
vif
vpr
vpuenv
rev
tat
tev
nef
Clade Chassis Status Developer / Manufacturer
GagPolNef C (CN54) DNA-C GMP, B, T, I, Fill. UREG / CobraEnv C (CN54) DNA-C GMP, B, T, I, Fill. UREG / Cobra
GagPolNef / Env C (CN54) NYVAC-C GMP, B, T, I, Fill. Sanofi PasteurGagPolNef / Env C (CN54) MVA-C GMP, B, T, I, Fill Esteban GagPolNef / Env C (CN54) vTT-C GMP, B, T, I, Fill Y. Shao / S-CDC
GagPolNef / Env B NYVAC-B GMP, B, T, I, Fill. Sanofi PasteurGagPolNef / Env B MVA-B GMP, B, T, I, Fill. Esteban
Gene Design, Protein Design and Vectors
Pr RT-N C N RT-C RTMyr- (A) ΔFS Pr - (DN) scNef
Gag
Env gp120
160 kDa readthrough polyprotein, cytoplasmic, not processed
soluble secreted monomeric gp120
RNA and codon optimized
Clinical Analysis - Responders (IFN+ T cells)
Higher percentage of responders in DNA-C prime / NYVAC-C boost group (>90%)compared to NYVAC-C group (<40%)
Durability DNA-C / NYVAC-C >> NYVAC-C
0
20
40
60
80
100
W0 W5 W20 W24 W26 W28 W48
Per
cen
tag
e o
f re
spon
ders DNA C + NYVAC C
NYVAC C alone
P < 0.001
P = 0.019
P = 0.001P < 0.001
DNA-C NYVAC-C
Study design: 2 x 20 HIV negative volunteersLondon (MRC; J.Weber, S. McCormack), Lausanne (CHUV; G. Pantaleo)
Study design: 2 x 20 HIV negative volunteersLondon (MRC; J.Weber, S. McCormack), Lausanne (CHUV; G. Pantaleo)
Gated on CD4 T cells
A
Neg
Env
IL-2
IFN-γ
TN
F-
IFN-γ
0.02% 0%
0%
0.04% 0.07%
0.03%
0% 0%
0% CD
4CFSE
Volunteer # EU11, DNA C + NYVAC C
Gated on CD8 T cells
IL-2
IFN-γ
TN
F-
IFN-γ
0.03% 0.20
0.03%
0.01% 0%
0%
0.07%
0.22
0%
0% CD
8
CFSE
Neg
Env
0.01%
1.94%
0.34%
6.6%0.11%
0.09
0.01%
CD
10
7a
IFN-γ
B
1.21% 0.08%
0.17
1.10% 0%
0%
Volunteer # EU11, DNA C + NYVAC C
T cell responses are broad (mean 4,2 epitopes) and polyfunctional
CD4 and CD8 responses are polyfunctional(IL-2, IFN, TNF)
… mimicking profile seen in LTNPs
Clinical Analysis - Responders (IFN+ T cells)
#SF
C/1
0e6
PB
MC
Env Gag
Pol Nef
Human
0
1000
500
0
1000
500
0 20 24 26 28 484 72 0 20 24 26 28 484 72
0 20 24 26 28 484 720 20 24 26 28 484 720 20 24 26 28 484 728 40 0 20 24 26 28 484 728 40
0 20 24 26 28 484 728 400 20 24 26 28 484 728 400
1000
500
0
1000
500
0 20 24 26 28 484 72 0 20 24 26 28 484 72
0 20 24 26 28 484 720 20 24 26 28 484 720 20 24 26 28 484 728 40 0 20 24 26 28 484 728 40
0 20 24 26 28 484 728 400 20 24 26 28 484 728 40
Env Gag
Pol Nef
Rhesus
Magnitude of Env >> Gag Specific T Cell Responses
Clinical Analysis - Responders (IFN+ T cells)
Re-Design I:
T Cell Immunogens
Immunogen Formulation
Poxviral Vector (Chassis)
TVDC
NYVAC-CDNA-C
?
MHC-IIMHC-I
CTL T
No co-stimulatory signalsNo MHC II presentationNo T cell help
NYVAC-CDNA-C
?
MHC-IIMHC-I
CTL
TH
DC
CTL
TH
?
100 nm
Pr55Gag
cellmembrane
translation
VLP
particlerelease
membranetargeting
Pr55Gag
200 nm
Myr+ (AG) +FS
Pr RT-N C N RT-C RTscNefGag Pr - (DN)
Re-Design – T cell Immunogens
Myr+ (G) ΔFS
Gag
RT-N C N RT-C RTscNef
Envgp140
gp140gp140
encodedprotein
PTVDC (VRC8400-Vector)
EuroVac (Cobra-Vector)
Pr RT-N C N RT-C RTMyr- (A) ΔFS Pr - (DN) scNef
Gag
Env gp120
Gag VLP
gp140trimericsecreted
monomer
no VLP
soluble, cytoplasmic
Re-Design – T Cell Immunogens & Plasmid
Myr+ (AG) +FS
Pr RT-N C N RT-C RTscNefGag Pr - (DN) Gag 95%
PolNef 5%VLP
Pr
features ExpressionGag PolNef Env
+++ + -
+++ - -
- ++ -
- - +++
+ + -
- - +++
Balb/C, DNA
1 2 3 4 5 6 7
0
100
200
300
400
500
600
700irrel. pept.
Gag
Pol1
Env1
CD
8+ I
FN
+ /
3x1
04 C
D8
+
1 mock
2 GPN (Myr-,FS-)
3 GPN (Myr-,FS-) + gp120
4 GPN (Myr+,FS+)
5 GPN (Myr+,FS+) + gp120
6 Gag (Myr+) + PN
7 Gag (Myr+) + PN + gp120
Assembly and release as Pr55-Gag VLP supports
the induction of Gag specific, CD8+ T cells
Re-Design – T Cell Immunogens & Plasmid
Balb/C, DNA
1 2 3 4 5 6 7
0
100
200
300
400
500
600
700irrel. pept.
Gag
Pol1
Env1
CD
8+ I
FN
+ /
3x1
04 C
D8
+
1 mock
2 GPN (Myr-,FS-)
3 GPN (Myr-,FS-) + gp120
4 GPN (Myr+,FS+)
5 GPN (Myr+,FS+) + gp120
6 Gag (Myr+) + PN
7 Gag (Myr+) + PN + gp120
Mixture of 3 DNAs, 2 legs
Co-administration of Env togeher with Gag
(Pol) reduces Gag specific T cell responses
Re-Design – Immunogen Formulation
Balb/C, DNA
1 2 3 4 5 6 7
0
100
200
300
400
500
600
700irrel. pept.
Gag
Pol1
Env1
CD
8+ I
FN
+ /
3x1
04 C
D8
+
9
left : rightleg
Spatial separation / ratio modification (formulation) leads to balanced and high level T cell response
modify ratio
8
Re-Design – Immunogen Formulation
Trivalent DNA
Re-introduction of -1 ribosomal frameshift leads to ~ 20x increased Gag expression, NOT on cost of PolNef
Gag alone expresses equally well Level of Gag expression and VLP formation crucial to
level of induced T cell responses Co-administration of Env suppresses Gag specific T cell
responses
Recommendations:
DNA: 3 plasmids encoding Gag, PolNef and Env optimize molar ratios (trivalent)
NYVAC: 2 viruses expressing GagPolNef and Env (bivalent)
Conclusions Immunogens - Formulation
A BC NMK F E P DHJLGO I
aprox. 15Kb
Fragmented genes Intact genes
NYVAC
aprox. 10KbNYVACKC
Re-Design – Poxviral Vector Design
Growth in human primary keratinocytes Highly attenuated (much less pathogenic compared to NYCBH) No effect on DCs maturation Potentiation of antigen direct and cross-presentation Induction of higher levels of trangene expression Induction of longer persistence of transgene expression Stimulation of antigen-specific memory responses
Myr+ (AG) +FS
Pr RT-N C N RT-C RTscNefGag Pr - (DN)
Envgp140
gp140gp140
bivalent2 viruses
Collaboration Bert Jacobs, Karen Kibler
Re-Design II:
Assess immunogenicity of next generation immunogens in NHP
„Optimal“ immunization schedule
TVDC
NHP Study Objectives:
TVDC
To evaluate the immunogenicity of: 2nd generation immunogens, DNA and NYVAC replication competent NYVAC-KC and replication
deficient NYVAC clade C TV1 gp120 (formulated with MF59) in
combination with DNA/NYVAC regimens
To compare scarification vs. i.m. route of administration (NYVAC-KC)
To evaluate the effects of DNA priming on T-cell and antibody responses
2nd Generation DNA and NYVAC constructs
Trivalent DNA-C: Env ZM96, Gag ZM96, Pol-Nef CN54
Bivalent NYVAC-C: Env ZM96, chimeric Gag ZM96/Pol-Nef CN54
Bivalent NYVAC-C-KC: Env ZM96, chimeric Gag ZM96/Pol-Nef CN54
All constructs RNA- and Codon optimized (expression yields, vector stability)
Re-Design II – NHP Study Immunogens:
NYVAC-C-KC (IM)
NYVAC-C (IM) NYVAC-C + Protein (IM)
0 4 128 20 24 28 32 36
DNA-C (IM)
NYVAC-C-KC (Scar) Protein
NYVAC-C-KC (IM)
NYVAC-C (IM)
ICSELISpot
48
Ab
Re-Design – Opt Clinical Study Protocol (Rhesus)
Protein
Protein
NYVAC-C + Protein (IM)
N8
8
8
8
8
G1
2
3
4
5
0 4 128 20 24 28 32 36 48
0 4 128 20 24 28 32 36 48
DN
A p
rime
grou
psN
o D
NA
gro
ups
Immunogenicity of DNA
priming (post 3 DNA
injections, w12) vs. NYVAC
alone (post 2 injection, w8) 4
weeks after last injection
Re-Design – Opt Clinical Study Protocol (Rhesus)
DNA Prime Induces Polyfunctional and Balanced CD4 and CD8 T-Cell Responses
Re-Design – Opt Clinical Study Protocol (Rhesus)
AUP444. T-Cell Responses Following DNA Prime/NYVAC Boost
501050
2050
3050
4050
5050
6050
9050
Group 1 – NYVAC-C-KC
2 wksPost 1stNYVAC
Week 22
NYVAC-C KC: replication competent in human cells
SF
Us
per
106 B
lood
Mon
onu
cle
ar C
ells
IFN- ELISpot
8050
7050
Week 50 Week 57 Week 22 Week 50 Week 57
P54
0P
566
P53
7P
555
P53
2P
534
P54
7P
549
P56
8P
544
P53
6P
552
P54
6P
558
P56
2P
564
P53
9P
554
P56
7P
545
P55
7P
559
P56
3P
548
16 wksPost 2ndProteinBoost
2 wksPost 3rdProteinBoost
Week 48 Week 50 Week 57
Group 2 – NYVAC-C-KC IM Group 3 – NYVAC-C IM
NYVAC-C KC: replication defectivein human cells
2 wksPost 1stNYVAC
16 wksPost 2ndProteinBoost
2 wksPost 3rdProteinBoost
7wksPost 3rdProteinBoost
16 wksPost 2ndProteinBoost
2 wksPost 3rdProteinBoost
P53
9P
554
P56
7P
545
P55
7P
559
P56
3P
548
P53
9P
554
P56
7P
545
P55
7P
559
P56
3P
548
P53
9P
554
P56
7P
545
P55
7P
559
P56
3P
548
Week 222 wks
Post 1stNYVAC
P56
8P
544
P53
6P
552
P54
6P
558
P56
2P
564
P56
8P
544
P53
6P
552
P54
6P
558
P56
2P
564
P56
8P
544
P53
6P
552
P54
6P
558
P56
2P
564
Week 487 wks
Post 3rdProteinBoost
P54
0P
566
P53
7P
555
P53
2P
534
P54
7P
549
P54
0P
566
P53
7P
555
P53
2P
534
P54
7P
549
P54
0P
566
P53
7P
555
P53
2P
534
P54
7P
549
7 wksPost 3rdProteinBoost
Week 48
10050
11050
12050
13050
14050
15050
D3/N-KC(im)/3P
D3/N(im)/3P
D3/N-KC(scar)/3P
T-Cell Responses After NYVAC+gp120 Immunization
50
625
1200
1775
2350
2925
3500
4075
5800
Group 4 – NYVAC-C-KC
7 wksPost 2nd
NYVAC-C-KC (IM)+ 3 NYVAC-C-KC/Protein
Boost
Week 28
Group 5 – NYVAC-C
NYVAC-C KC: replication competent in human cells
SF
Us
per
106 B
lood
Mon
onu
cle
ar C
ells
NYVAC-C: replication defective in human cells
IFN- ELISpot
5225
4650
P53
8P
543
P53
0P
565
P54
1P
531
P53
5P
533
P56
0P
569
P55
3P
542
P56
1P
556
P55
0P
551
24 wksPost 2nd
NYVAC-C-KC (IM)+ 2 NYVAC-C-KC/Protein
Boost
Week 502 wks
Post 2nd
NYVAC-C-KC (IM)+ 3 NYVAC-C-KC/Protein
Boost
Week 577 wks
Post 2nd
NYVAC-C (IM)+ 3 NYVAC-C/Protein
Boost
Week 2824 wksPost 2nd
NYVAC-C (IM)+ 2 NYVAC-C/Protein
Boost
Week 502 wks
Post 2nd
NYVAC-C (IM)+ 3 NYVAC-C/Protein
Boost
Week 57
P56
0P
569
P55
3P
542
P56
1P
556
P55
0P
551
P56
0P
569
P55
3P
542
P56
1P
556
P55
0P
551
P56
0P
569
P55
3P
542
P56
1P
556
P55
0P
551
4 wksPost 2nd
NYVAC-C (IM)+ 2 NYVAC-C/Protein
Boost
Week 48
P53
8P
543
P53
0P
565
P54
1P
531
P53
5P
533
P53
8P
543
P53
0P
565
P54
1P
531
P53
5P
533
P53
8P
543
P53
0P
565
P54
1P
531
P53
5P
533
4 wksPost 2nd
NYVAC-C-KC (IM)+ 2 NYVAC-C-KC/Protein
Boost
Week 48
Nkc2NkcP3
PrimaryImmunization
Boost N2NP3Primary
ImmunizationBoost
Key time points: Groups #1-3 (DNA prime) at week 22Groups #4,5 (no DNA) at week 28
Value Exact P 1.Group 1 vs. Group 2 vs. Group 3 0.972.Group 4 vs. Group 5 0.0033.Group 4 vs. Groups1-3 <0.001
Group Comparison of IFN- ELISpot Responses by Wilcoxon Rank Sum Test
CD4+ and CD8+ T Cell Function Groups
G 2 T+ + ++ + -+ - ++ - -- + +-+ --- +
G 1
-3G
4,
5G
1-3
G 4
, 5
Breadth of T-Cell Responses in DNA/NYVA-KC/gp120 Group #2
Breadth of T-Cell Responses in NYVAC-KC/gp120 Group #4
No significant differences in the magnitude of vaccine-induced T-cell responses between the DNA prime groups
Significant differences in the magnitude between:
DNA prime and no DNA groups and
NYVAC and NYVAC-KC in the no DNA prime groups
DNA prime groups induced more polyfunctional CD4 and CD8 T-cell responses
DNA prime groups had dominant Gag/Pol while no DNA groups predominant Env T-cell responses
Conclusions I – T-Cell Response
Is the Profile of T-Cell
Response and Immunization Regimen
(DNA Prime vs. No DNA) Associated
to Better Antibody Response?
Vaccine-Induced Antibody Response
Neutralizing antibodies
ADCC
Binding antibodies (total IgG)
Plasma Env IgA antibodies
Plasma IgG gp70 V1V2 antibodies
Montefiori Lab
Neutralizing Antibody Titers All for TZM.bl cellsKruskal-Wallis Rank Sum Test
HIV Isolate
BaL.26
2
3
4
5
Lo
g1
0 T
iter
MW965.26 SF162.LS SS1196.1Bx08.16 MN.3 TV1.21
1
Groups 1-3-DNA Prime Week 36(after 2nd protein)
Groups 4,5-No DNA Week 26(after 2nd NYVAC/protein)
ADCC Activity in DNA/NYVAC-KC/gp120 (Gr.#2) Vs. DNA/NYVAC/gp120 (Gr.#3)
4,000
3,000
2,000
1,000
0
Tite
r
Wk 22 Wk 30 Wk 36 Wk 22 Wk 30 Wk 36
Group 2 Group 3
ADCC Activity in NYVAC-KC/gp120 (Gr.#4) Vs. NYVAC/gp120 (Gr.#5)
25,000
20,000
15,000
10,000
5,000
0
Tite
r
Wk 14 Wk 26 Wk 14 Wk 26
Group 4 Group 5
Wk22: after 3 DNA / 1 NYVACWk32: after 3 DNA / 1 NYVAC / 1 proteinWk32: after 3 DNA / 1 NYVAC / 2 protein
Wk 14: after 2 NYVAC / 1 NYVAC + proteinWk 26: after 2 NYVAC / 2 NYVAC + protein
Cross-Clade Plasma IgG Antibody TitersVaccine Strain Env gp120 (TV1)
0 10 20 30 4010
100
1000
10000
100000
Group 1Group 2Group 3Group 4Group 5
Primary Clade C Env (1086 trimer)
0 10 20 30 4010
100
1000
10000
100000
Clade A Consensus Env (A1.con)
0 10 20 30 4010
100
1000
10000
100000
Clade B Consensus Env (B.con)
0 10 20 30 4010
100
1000
10000
100000
Clade C Consensus Env (C.con)
0 10 20 30 4010
100
1000
10000
100000
Group M Consensus Env (ConS)
0 10 20 30 4010
100
1000
10000
100000
IgG
Tite
r (M
ean
AU
C)
Weeks
Groups 1-3-DNA PrimeGroups 4,5-No DNA
Plasma IgG gp70 V1/V2 Antibody Titers
100000
10000
1000
100
10
1Wk 0 Wk 14 Wk 22 Wk 26 Wk 30 Wk 36
IgG
an
tibo
dy
(Tite
r)
Groups 1-3-DNA PrimeGroups 4,5-No DNA
NYVAC-KC and NYVAC plus gp120 groups compared to DNA prime groups showed:
Greater NAb activity against Tier 1 viruses and SHIVs (TZM.bl assay)
Greater ADCC activity
Earlier and greater cross-clade IgG antibody titers (more than 6 Mo after 1st immunization)
Earlier, but comparable plasma Env IgA and IgG V1/V2 responses
Greater magnitude in T-cell responses do not necessarily translate in better NAb responses
Conclusions II – Antibody Response (prior to week 48 boost)
What Are the Changes in the Profile of
Antibody Responses After
- Protein (Grs. #1-3) and
- NYVAC+Protein (Grs. #4/5) Boost
at Week 48 ?
Neutralizing Antibody Titers All for TZM.BL After Boost at Week 48 Kruskal-Wallis Rank Sum Test
ADCC Titers After Boost at Week 48Kruskal-Wallis Rank Sum Test
B.con
0 20 40 6010
100
1000
10000
100000Group 1Group 2Group 3Group 4Group 5
Week
Mea
n A
UC
A1.con
0 20 40 6010
100
1000
10000
100000Group 1Group 2Group 3Group 4Group 5
Week
Mean
AU
C
C.con
0 20 40 6010
100
1000
10000
100000Group 1Group 2Group 3Group 4Group 5
Week
Mea
n A
UC
00MSA
0 20 40 6010
100
1000
10000
100000Group 1Group 2Group 3Group 4Group 5
WeekM
ean
AU
C
1086 trimer
0 20 40 6010
100
1000
10000
100000Group 1Group 2Group 3Group 4Group 5
Week
Mea
n A
UC
gp120 TV1
0 20 40 6010
100
1000
10000
100000Group 1Group 2Group 3Group 4Group 5
Week
Mea
n A
UC
Durable IgG anti-Env Breadth to Week 51
Plasma IgA Responses to TV1 and gp70 V1V2
Vaccine Strain Env gp120 (TV1)
0 20 40 6010
100
1000
10000
100000Group 1Group 2Group 3Group 4Group 5
Week
IgA
An
tib
od
y (M
ean
MF
I)
V2 Epitope (gp70 v1/v2)
0 20 40 6010
100
1000
10000
100000Group 1Group 2Group 3Group 4Group 5
Week
IgA
An
tib
od
y (M
ean
M
FI)
No DNA NYVAC-KC and NYVAC groups (NYVAC+gp120 boost) showed:
- Substantial transient increase in T-cell response
- Stably elevated but no increase in antibody response
DNA groups (only gp120 boost) showed:
- Slight transient increase in T-cell response
- Substantial increase in antibody response
Conclusions III – Vaccine-Induced Responsesat Week 48 boost
DNA NYVAC
T-cell
Antibody
Strong T-cell Polarization Phase
Protein
Weak Induction of Antibody
Protein
Late Increase of Antibody Response
0 1 5 8 1272 Months
100
10
T-Cell Polarizing Vaccine RegimenM
ag
nitu
de
of
Re
spo
nse
NYVAC+
Protein
Strong Antibody Polarization Phase
NYVAC+
Protein
Late Increase of T-cell Response
NYVAC NYVAC+
Protein
T-cell
Antibody
Weak T-cell Polarization Phase
0 1 3 126 Months
100
10
Antibody Polarizing Vaccine RegimenM
ag
nitu
de
of
Re
spo
nse
Future Plans –
Optimization of vaccine regimens
Acceleration of the induction of the antibody response
Acceleration of prime/boost immunization schedule of T-cell polarizing vaccine regimens
Circumvent the interference of potent vaccine-induced T-cell response with the induction of potent antibody response
Development of immunization regimens resulting in a synergistic effect between T-cell and antibody polarizing vaccine regimens
Increase breadth of T and B cell responses
Re-Design III:
Broaden T Cell Responses
Improve B Cell Responses
Reference SequenceMGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTPIVQNIQGQMVHQ
Los Alamos HIVDatabase
2. Structur/Function Conservation (VLP)
3. OptimizationAlign epitopes to reference sequence and find all single point amino acid substitutions
A. Classify all substitutions according to their effect on the functionality of the protein
CLASSIFIER
no effect
negative effect
B. Remove all epitopes whichinduce “harmful” amino acid substitutions
1. Initialization
A. Assign each epitope a numerical value(=score) to assess its importance to bein the optimized immunogene
6
4
3
51
3
s(x)
B) Analyse incompatibilities between epitopes
C. Find a set of compatibleepitopes with maximaltotal score.
D. Return immunogensequence: Freezeall substitutions inreference sequence
E. Remove all used epitopes
Re-Design – T Cell Responses (CutHiVac, Replivax)
Objective: Broaden /focus T cell responses by incorporaing T cell epitopes into Gag and maintain VLP formation (cross presentation)
• Synthetic biology
techniques to generate
different types of Env libraries
(randomized, SeqPer) and
• … to efficiently subclone libs
into pQL13 enabling
• inducible expression of Env;
cotranslational coupling of
selection marker; coupling of
phenotype and genotype
• HT generation of stable cell-
lines (<10E5) by bulk
transfection of Env libraries
cloned into pQL13
• FACS based screening
procedure
• bNMAbs, germ line versions
Genotype
Phenotype
Single integrationsFlp-In stable cell-lines
Antibiotic selection for 25 days
Analysis by
deep
sequencing
Env library
pQL13⑥ QL cloning
① Transfection: pQL13 + helperplasmid
②
„Linked“ cellular Env-library
⑤ Nested PCR
Laser
Cells
④ FACS-sorting
③ bnMAB binding
Re-Design – B Cell Responses (IAVI, Replivax)
Objective: Exploit bnMAbs / germ line versions to select Env‘s with new properties and the potential to induce broad neutralization
Applying Synthetic Biology Principles for Better Vaccines: Challenging HIV
ConstructionCycle(s)
Knowledge
Gene Design - Modelling
GeneOptimizerTM
Protein Design - Modeling
Initialisation Immunogen Design
Master
Los AlamosDatenbank
4
3
5
1
3
s(x)
Epitop-Scoring
Optimization
Immunogen
CD
4
Env
CFSE
Clinical trial: Phase IIa
SpecificationParts, devices
Chassis - Delivery
plasmids,bacteria, viruses Screen
ex vivo / in vivo testing
Molecular EpidemiologyPathogenesis
Viral Replication
„Master Gene“
Benedikt AsbachAlex KlicheJosef KöstlerKathi BöcklJens WildK. KindsmüllerP. Rosenstock
Funding
EU 7th Framework CutHiVac Gates Foundation PTVDC, VDC Gates Foundation RepliVax
IAVI Stabilized new trimers
NIH HIVRAD
BMBF HIVCOMPNET, BioChance
DFG, BFS, Hektor Stiftung
Industry
Sanofi Pasteur Jim Tartaglia
Novartis Susan Barnett
Polymun Dietmar Katinger
GeneArt AG
Univ. Partners
CHUV, Lausanne Guiseppe PantaleoUCL, London Robin WeissUniv. Cambridge UK Jon HeeneyDPZ Göttingen C. Stahl HennigUniv. Leiden Cornelius MeliefCDC Beijing Shao YimingUniv. Oxford, UK Quentin Sattentau
Acknowledgement
Wagner Group: Molecular epidemiology, immunogen development, biochemistry, gene design, DNA vaccine, NYVAC / vector prototypes, ex vivo analysis / preclinic & immunization scheduleWagner Group: Molecular epidemiology, immunogen development, biochemistry, gene design, DNA vaccine, NYVAC / vector prototypes, ex vivo analysis / preclinic & immunization schedule
Acknowledgements (PTVDC)PTVDC Participating Organization Key Scientific Personnel
Centre Hospitalier Universitaire Vaudois (SponsoringOrganization)
G. Pantaleo (PI)
T. Calandra
Fred Hutchinson Cancer Research Center L. Corey
Consejo Superior de Investigaciones Cientificas M. Esteban
Biomedical Primate Research Centre /University of Cambridge J. Heeney
Arizona State University B. Jacobs
Institute of Research in Biomedicine A. Lanzavecchia
University of Washington D. Koelle
University Paris 12, CHU Henri Mondor Y. Levy
Murdoch University S. Mallal/M. John
Leiden University Medical Centre C. Melief
University of Montreal R-P. Sekaly
Imperial College of Science, Technology and Medicine J. Weber
Sanofi Pasteur J. Tartaglia
University of Regensburg R. Wagner
Project Management Song Ding
Thanks
To Bill & Melinda Gates Foundation for its Support in the Development of the DNA/NYVAC Platform
Special thanks to:
Nina Russell, Pervin Anklesaria and Jose Esparza for their support in the development of the PTVDC, PVRD and more recently RepliVax projects
Knowledge to specify target genes / epitopes- Control o virus replication
MHRmajor homology region
HLA-B14-restrictedCTL-epitope (aa298-306)
p1p2
p24 CAp17 MA
0 1 2 3 4 5 6 7 8 9 kb
LTR
tev
gagpol
vif
vprvpu
env
revtat
nef
p6p7
D R F Y K T L R A
LTR
(35) (1) (22) (8)
298 299 300 301 302 303 305304 306
020406080
100
020406080
100
HLA-B14CTL-Epitope
conservative
non-conservative
Infectivity
Infectivity
% s
pec.
Lys
is%
spe
c. L
ysis
D R F Y K T L R A
E K Y F R S/ A V K G
W Y R Q I F H Y F
Focus IR to conserved proteins and epitopes limits immunological escapeTrue for both T and B cell responses
DFG