MVA TECHNOLOGY IN THE DEVELOPMENT OF HIGHLY COMPLEXED TB VACCINE - - PowerPoint PPT Presentation

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Immunotherapies for cancer and infectious diseases MVA TECHNOLOGY IN THE DEVELOPMENT OF HIGHLY COMPLEXED TB VACCINE CANDIDATES TBVI Symposium Les Diablerets, 3 February 2016 Stphane Leung-Theung-Long Genevive Inchausp Early results in

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Immunotherapies for cancer and infectious diseases

MVA TECHNOLOGY IN THE DEVELOPMENT OF HIGHLY COMPLEXED TB VACCINE CANDIDATES

TBVI Symposium Les Diablerets, 3 February 2016 Stéphane Leung-Theung-Long Geneviève Inchauspé

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Early results in clinical trials with a MVA-TB vaccine

  • Efficacy results from the phase 2b based on MVA-85A did

not match expectations. Many factors may have played a role:

► vaccine target: new borns are a most difficult population ► vaccine make-up: based on a single antigen ► trial design: vaccine injection too close to BCG prime ► vaccine dose, schedule and routes of administration ► vaccine platform: MVA not potent enough, not

generating the right response MVA remains a competitive platform in the TB field and requires to be further explored

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► MVA belongs to the vaccinia viruses

  • Large ds enveloped DNA viruses (approx. 170 Kbp, 230 genes)

► Highly attenuated strain

  • > 570 passages in chick embryo fibroblasts cells
  • ± 15% of DNA lost compared with VV  space for transgenes
  • lysis of infected cells  increased immunogenicity
  • without production of infective particles

► No safety concerns

  • Developed in Germany, in the 70’s (Dr Anton Mayr)
  • to specially vaccinate subjects at risk for smallpox vaccination (CNS

disorders, allergy, skin diseases, etc.)

  • 150,000 subjects vaccinated against smallpox
  • Many trials in prophylaxis (prime-boost), HIV, Malaria, Ebola, Flu, TB…
  • Few hundreds of patients have received MVA-based therapeutic vaccines

(review by Boukhebza et al., Human Vaccines and Immunotherapeutics, 2012)

Poxvirus MVA (Modified Vaccinia Ankara)

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2: Adult vaccines

prophylactic and post-exposure

1: Pediatric vaccine

prophylactic

Vaccine approaches in the fight against tuberculosis

INFECTION PHASES AND DISEASE OCCURRENCE

3: Immunotherapeutic (P3)

(combination with antibiotics)

3: Therapeutic vaccines

in combination with antibiotics:

Increase/acceleration of cure and/or prevention of rebound or re-infection

Transgene priority

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Therapeutic vaccines

  • Definition : Manipulation of the immune system in an antigen

specific fashion

  • Positive way: enhancement of immunity: cancers, infectious

diseases

  • Negative: attenuation of an immune response: autoimmune

diseases

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  • Add a mechanism of action not and/or poorly used by current

therapies (antivirals, antibiotics) i.e. enroll the host’s immune system to participate in viral/bacterial…. clearance of an already (actively) infected carrier

  • These novel immunotherapeutics should try and capture

major immune features found in resolvers/controllers  Knowledge on immune correlates of control/resolution

  • Avoid exacerbation of diseases

 Add vaccine in already treated patients (early control of replication) Aim of therapeutic vaccines targeting chronic infectious diseases

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Therapeutic vaccines Technologies and Marketed Vaccines

Anti-idiotype vaccines

  • Made of antibodies that see other

antibodies as the antigen and bind to it

  • They stimulate the body to produce

antibodies against tumour cells

Dendritic cell vaccines

  • Absorb and present antigen to

lymphocyte for immune system

  • activation. Patient specific vaccines
  • One marketed therapeutic vaccine in

US: prostate cancer, “Provenge” (DC+ PAP/GM-CSF)

Whole-cell Tumour vaccines

  • Solve the problem of undiscovered

antigen as they expose a large range of tumour

  • Autologous: derived from patient own

tumour; allogenic: prepared for any patient

pDNA vaccines

  • DNA is taken up by the APCs and

instructs them to produce antigens continuously

A whole virus/antigen/adjuvant vaccines

  • Designed to stimulate the immune

system by using individual antigens

  • An adjuvant is combined with the

vaccine, which help boost immune response

  • VZV vaccine for the elderly: prevention
  • f reactivation/attenuation of zoster

Viral vectors

  • Utilize viral vector to transfer DNA of the

tumoral or viral antigen to produce antigen proteins in APCs (poxvirus, adenovirus, …)

  • One therapeutic vaccine in China:

AdenoP53 in Head and Neck cancers Source: Arrowhead, Capgemini Life Sciences Team Analysis

A diversity of therapeutic vaccine technologies have emerged and it is yet unclear which platforms will prevail

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THE CHECK POINT MODEL FOR HOST IMMUNITY AGAINST TB Immunotherapy could restore key checkpoints

Schön et al., J Internal Medicine, 2013

Loss of protective T cell responses ie loss of functional CD4/ CD8 responses, hampered cytolytic functions, hampered innate immunity, increase T-regs, pD1, IL10, Inflammation MVA inducing cellular-based immunity: Priming de novo poly-functional and multi-antigenic CD4+ and CD8+ T cells capable to exhert effector functions at site of infection Re-boost innate immunity Ideally once inflammation is first (in part) controlled by antibiotics

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Active Latent Resuscitation High plasticity of the MVA has allowed to generate highly complexed candidates

MVA / ACT-LAT-RES

Modified Vaccinia Ankara virus (MVA)

Multi-phase antigens covering all phases of infection (active, resuscitation, latent)

Phases of infection 17 Mtb antigens evaluated

Transgene: Development of multiphasic vectorized TB vaccines

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Antigens

Bioinformatic

  • Exhaustive data basis on known

epitopes

  • MHC Binding/epitope

predictions (class I and II)

Biochemistry

  • Known structure and homologs
  • Prediction of Stability and

difficulty of expression

Data mining

  • Immunogenicity
  • Protection
  • Biological properties

Design and selection of immunogenic sequences (fusions) Construction and ranking of fusions

Construction, in vitro (expression) and in vivo (DNA vaccine) testing

Construction and in vitro ranking of the vaccine candidates – genetic stability Testing and ranking of the vaccine candidates in in vivo efficacy experiments Lead vaccine candidate

General Approach

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Example of antigenic Fusion Design

All fusions are blocks1+2 or blocks1+3 or blocks1+2+3 Example: 3 fusions in MVATG18377: Fusion 11: RpfB-Dhyb*-Ag85B*-TB10.4-ESAT6 Fusion 13: SS-Rv2029*-Rv2626-Rv1733*-Rv0111* Fusion 5: SS-Rv0569-Rv1813*-Rv3407-Rv3478-Rv1807-TM

* Antigen mutated and/or truncated

Antigens with described fold : Ag85B*, Rv2029*, Rv2626, Rv0569, RpfB-Dhyb* Fold unknown or problematic: Rv1813*, Rv3407, Rv3478, Rv1807, ESAT6, TB10.4 Membrane anchorage (signal

  • seq. added):

Rv1733*, Rv0111* or added TM

Block 1 Block 2 (optional) Block 3 (optional)

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Active (3) / Resusc. (2) Latent (4) Latent (5) Active (2) / Latent (2) Latent (5) Active (1) / Resusc. (2) / Latent (2) Active Active Active Latent Resusc. Latent Active Active Active Active

Latent-2A- Active Active-X- Active Active-X- Active Resusc. Latent-2A- Latent Latent-X- Active Active-X- Active Active-X- Active Resusc. Latent-X- Latent 2A cleavage peptides + Linker Linker

Active (1)/ Resusc. (2) / Latent (2)

Large Fusions Individual Ag

  • r

Short fusions

Examples of derived MVA-TB vectors

Active (3)/

  • Resusc. (2)

Latent (2) Active (3)/

  • Resusc. (2)

Active (1) / Latent (1)

Large and short fusions

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  • ●●●●●●●● 13

D0 Immunization (s.c, 107 pfu/mouse) D7 D9 D14 …………

  • Mouse strains:
  • BALB/c (H-2d)
  • C57Bl/6 (H-2b)
  • C3H/HeN (H-2k)
  • HLA-A2 (I-Ab, HLA-A2)
  • Readouts:
  • ELISpot IFNg (production by splenocytes activated in vitro by peptides)
  • In vivo CTL
  • ICS CD4/CD8; polyfunctionality
  • Antibodies

Mouse

Typical early assessment of immunogenicity in mice

(Leung-Theung-Long et al., PLoS One, Nov 2015 + unpublished)

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Immunogenicity of MVA candidate vaccines in BALB/c mice

Illustration of IFN-γ responses specific of 14 antigens (3 expression cassettes)

Broad and significant responses observed (Rv1813, Rv3407, Rv3478 and Rv1807) with the MVA including SS and TM domains in fusion sequence.

100 200 300 400 500 600 700 sfc/106 cells

** * ** * **

MVATG18377

  • Rv2029-Rv2626-Rv1733-Rv0111 +

RpfB-Dhyb-Ag85B-TB10.4-ESAT6 +

  • Rv0569-Rv1813-Rv3407-Rv3478-Rv1807-

MVATG18379

  • Rv2029-Rv2626-Rv1733-Rv0111 +

RpfB-Dhyb-Ag85B-TB10.4-ESAT6 + Rv0569-Rv1813-Rv3407-Rv3478-Rv1807 MVATGN33.1

Medium Irr pept P1 Rv1813 P2 P3 Rv3478 P4 P1 P2 P3 P4 Rv1807 Rv0569 Rv3407

Medians of each group U Mann Whitney test * : p < 0.05 ** : p < 0.01

  • ---- : cut-off

P: peptide pool

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IMMUNOGENICITY OF MVA CANDIDATE VACCINES IN HLA-A2 MICE

ILLUSTRATION OF IFN-g RESPONSES SPECIFIC OF 4 ANTIGENS (1 EXPRESSION CASSETTE)

400 800 1200 1600 2000 sfc/106 cells

MVATGN33.1 MVATG18376

  • Rv2029-Rv2626-Rv1733-Rv0111 +
  • RpfB-Dhyb-Ag85B-TB10.4-ESAT6-

+

  • Rv0569-Rv1813-Rv3407-Rv3478-Rv1807-

Medium Irr pept RpfB-Dhyb P1 P2

P3

P4 Ag85B P1 P2 P3 TB10.4 ESAT6

** ** ** ** ** ** ** *

Broad and significant responses observed with the MVA in HLA-A2 transgenic mice. Following CD4 T cell depletion, significant IFNγ response was still detected for antigens such as RpfB-RpfD fusion protein.

Medians of each group U Mann Whitney test * : p < 0.05 ** : p < 0.01

  • ---- : cut-off

P: peptide pool

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Rv2029

% of specific lysis

P1 P2 P3 P4 20 40 60

* * * Rv1807

% of specific lysis

P1 P2 P3 P4 20 40 60

* * * ESAT-6

% of specific lysis

20 40 60

* TB10.4

% of specific lysis

20 40 60

Ag85B

% of specific lysis

P1 P2 P3 20 40 60

* RpfB-Dhyb

% of specific lysis

P1 P2 P3 P4 20 40 60

* * Rv3478

% of specific lysis

P1 P2 P3 P4 20 40 60

Rv1733

% of specific lysis

P1 P2 20 40 60

* Rv2626

% of specific lysis

P1 P2 20 40 60

* * Rv1813

% of specific lysis

20 40 60

* Rv3407

% of specific lysis

20 40 60

Rv0569

% of specific lysis

20 40 60

* Rv0111

% of specific lysis

P1 P2 P3 P4 20 40 60

* *

MVA-TB immunization can trigger antigen-specific cytotoxic activity in mice

The 14 Ag MVATG18377 candidate induced cytotoxic activity specific of 11 out of 14 Mtb antigens following two injections in BALB/c mice.

in vivo CTL assay

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Immunogenicity studies in non-human primates (naïve animals)

PBMC

  • Substantial MVA-TB immunogenicity demonstrated in primates.
  • Multiple antigens /multiple epitopes targeted.

MVATG18377 (14 Ag) 108 pfu, i.m.

MVATG18377 Weeks 8 18 2 10 20 27 29 MVATG18377 MVATG18377 31

†

spots/106 cells

2 10 18 20 27 29 31 2 10 18 20 27 29 31 2 10 18 20 27 29 31 200 400 600 800 1000 1200 1400 1600 1800 2000

ESAT-6 Rv3478 Rv3407 Rv2626 Rv2029 Ag85B Rv1813 Rv1807 Rv1733 Rv0569 TB10.4 Rv0111 RpfB-Dhyb R632 R634 R818 Weeks Primate ID

†, data not available

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VACCINES ANTIGENS # fusions # Ag MVATG18639

Rv2626/Ag85B - CFP10/ESAT6 - TB10.4/Rv0287 - RpfB/D - Rv1813/Rv3407

5 10 MVATG18598

Rv2626/2A/Ag85B - CFP10/ESAT6 - TB10.4/Rv0287 - RpfB/D - Rv1813/2A/Rv3407

5 10 MVATG18633

Ag85B - ESAT6 - RpfB/D - Rv2626 - Rv1813

5 6 MVATG18690

RpfB/D/Ag85B/TB10.4/ESAT6 - Rv2626/Rv3407

2 7 MVATG18692

RpfB/D/Ag85B/TB10.4/ESAT6 - Rv3478/2A/Rv1733

2 7 MVATG18827

Rv2029/TB10.4/ESAT6/Rv0111 - SS-RpfB/D

2 6

Active – Resuscitation – Latent Heterodimeric partners SS: signal sequence 2A: auto-cleavage peptide

MVA-TB genetically stable ie fit for manufacturing

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Therapeutic efficacy studies in mice : Reduction/ Prevention of Rebound and/or Acceleration of control

2 4 6 8 10 20 5 15 CFU per lung (log10) 25 Time after infection (weeks) Antibiotics + antibiotics MVA n x injections Antibiotics + MVA-TB vaccines Mtb (H37Rv)

Initial experiment performed with one MVA-TB genetically stable candidate (10 antigens)

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  • 4

H37Rv challenge 2.5 log10 cfu Mouse BALB/c RHZ 5 days/week, oral gavage Weeks 23 11 RHZ: Rifampin, Isoniazid, Pyrazinamide

  • Mice: BALB/c
  • RHZ-treated group as control
  • MVATG18598 prototype (10 Ag, 107 pfu/s.c. injection)

MVA-TB therapeutic efficacy study in combination with drugs: control of relapse

TB Alliance and Dr Eric Nuermberger (Johns Hopkins University) support

8

CFU evaluation in lung

MVA schedule 1 MVA schedule 3 MVA schedule 2 3

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Median lung CFU count at Week 8 : shortening

  • Safety : Bacterial burden and histology :
  • MVA did not impair RHZ therapy efficacy (no negative interference).
  • Multiple MVA injections did not worsen lung inflammation.
  • Trend to reduced bacterial burden in the MVA-TB groups compared with RHZ group

(not statistically significant)

log10 M. tuberculosis CFU

ed HZ 3x 5x 7x 1.0 1.5 2.0 2.5 6.0 6.5 7.0

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Groups Mice relapsing CFU [median (IQR)] Number Percentage RHZ 10 / 12 83% 2.9 (0.5-3.7) RHZ / MVA schedule 1 (3x) 6 / 12 50% 0.2 (0.0-3.3) RHZ / MVA schedule 2 (5x) 8 / 12 67% 2.5 (0.0-3.8) RHZ / MVA schedule 3 (7x) 7 / 12 58% 2.8 (0.0-3.6)

CFU count at Week 23: relapse

  • MVA schedule 1 (3x) has the most robust impact on relapse – 50% of mice do not

relapse while 83% do in the control group

  • Bacterial burden is lowered (median 3 logs lower)
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Ongoing

  • Therapeutic mouse efficacy studies with all 6 genetically stable candidates MVA-TB in

mice

  • Different kinetics; Different routes; Different positionings (during/post antibiotic

treatment)

  • Prophylactic heterologous prime-boost in non-human primates including a multi-antigen

MVA-TB vaccine

  • Collaboration with GSK and AERAS
  • Supported by AERAS
  • Organisation of potential therapeutic efficacy in Guinea Pig with MVA-TB candidates

with Chinese National Institutes for Food and Drug Control (NIFDC)

  • Pre-requisite for a development in China
  • Development of a cell-line based manufacturing process for large scale production of

MVAs-TB

  • Transgene/Emergent BioSolutions + NIH grant
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Acknowledgements

TRANSGENE Lyon, France

  • Marie Gouanvic
  • Charles-Antoine Coupet
  • Aurélie Ray
  • Clément Levin
  • Audrey Glaize
  • Cécile Bény
  • Emmanuel Tupin
  • Stéphane Leung-Theung-Long
  • Geneviève Inchauspé
  • Romain Micol
  • Valentina Ivanova-Segura
  • Ludovic Dendane

Strasbourg, France

  • Martine Marigliano
  • Jean-Baptiste Marchand
  • Nathalie Silvestre
  • Thierry Menguy
  • Joan Foloppe
  • Doris Schmitt
  • Chantal Hoffmann
  • Murielle Klein
  • Véronique Koerper
  • Sophie Steinbach
  • Fabrice Le Pogam
  • Patricia Kleinpeter
  • Dominique Villeval
  • Sophie Jallat
  • Annick Hoh
  • Anthony Cristillo
  • Maria Cecilia Huaman
  • Philip Seegren
  • Priyanka Dhopeshwarkar

NIH support through grant awarded to Emergent BioSolutions/Transgene subcontractor

  • Eric Nuermberger
  • Paul Converse
  • Sandeep Tyagi
  • Tom Evans
  • Barry Walker
  • Nathalie Cadieux
  • William Reiley