new strategies towards vaccine design challenging hiv ralf wagner univ. prof. for medical...

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


#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


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


DNA Prime Induces Polyfunctional and Balanced CD4 and CD8 T-Cell Responses


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



7wksPost 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


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


Boost

Week 502 wks

Post 2nd


Boost

Week 577 wks

Post 2nd

NYVAC-C (IM)+ 3 NYVAC-C/Protein

Boost

Week 2824 wksPost 2nd


Boost

Week 502 wks

Post 2nd


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


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


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


Week

Mean

AU

C

C.con

0 20 40 6010

100

1000

10000


Week

Mea

n A

UC

00MSA

0 20 40 6010

100

1000

10000


WeekM

ean

AU

C

1086 trimer

0 20 40 6010

100

1000

10000


Week

Mea

n A

UC

gp120 TV1

0 20 40 6010

100

1000

10000


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


Week

IgA

An

tib

od

y (M

ean

MF

I)

V2 Epitope (gp70 v1/v2)

0 20 40 6010

100

1000

10000


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

new strategies towards vaccine design challenging hiv ralf wagner univ. prof. for medical...

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