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
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 2
Poxvirus MVA (Modified Vaccinia Ankara) ► 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) 3
Vaccine approaches in the fight against tuberculosis Transgene priority 3: Therapeutic vaccines in combination with antibiotics: 3: Immunotherapeutic (P3) 1: Pediatric vaccine 2: Adult vaccines Increase/acceleration of cure and/or (combination with antibiotics) prophylactic prophylactic and post-exposure prevention of rebound or re-infection INFECTION PHASES AND DISEASE OCCURRENCE 4
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 5
Aim of therapeutic vaccines targeting chronic infectious diseases 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) 6
A diversity of therapeutic vaccine technologies have emerged and it is yet unclear which platforms will prevail Viral vectors Whole-cell Tumour vaccines • Utilize viral vector to transfer DNA of the • Solve the problem of undiscovered tumoral or viral antigen to produce antigen as they expose a large range of antigen proteins in APCs (poxvirus, tumour adenovirus, …) • Autologous: derived from patient own • One therapeutic vaccine in China: tumour; allogenic: prepared for any AdenoP53 in Head and Neck cancers patient Anti-idiotype vaccines pDNA vaccines • Made of antibodies that see other • DNA is taken up by the APCs and Therapeutic vaccines antibodies as the antigen and bind to it instructs them to produce antigens Technologies • They stimulate the body to produce continuously and Marketed Vaccines antibodies against tumour cells A whole virus/antigen/adjuvant Dendritic cell vaccines vaccines • Absorb and present antigen to • Designed to stimulate the immune lymphocyte for immune system system by using individual antigens activation. Patient specific vaccines • An adjuvant is combined with the • One marketed therapeutic vaccine in vaccine, which help boost immune US: prostate cancer, “ Provenge ” (DC+ response PAP/GM-CSF) • VZV vaccine for the elderly: prevention of reactivation/attenuation of zoster Source: Arrowhead, Capgemini Life Sciences Team Analysis 7
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 8
Transgene: Development of multiphasic vectorized TB vaccines Multi-phase antigens Modified covering MVA / ACT-LAT-RES all phases of Vaccinia Ankara infection (active, virus (MVA) resuscitation, latent) 17 Mtb antigens evaluated Latent Resuscitation Active Phases of infection High plasticity of the MVA has allowed to generate highly complexed candidates 9
General Approach Antigens Biochemistry Data mining Bioinformatic • • • Known structure and homologs Immunogenicity Exhaustive data basis on known • • Prediction of Stability and Protection epitopes • • difficulty of expression Biological properties MHC Binding/epitope predictions (class I and II) 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 10
Example of antigenic Fusion Design Membrane Antigens with Fold unknown or anchorage (signal described fold : problematic: Rv1813*, seq. added): Ag85B*, Rv2029*, Rv3407, Rv3478, Rv1733*, Rv0111* or Rv2626, Rv0569, Rv1807, ESAT6, TB10.4 added TM RpfB-Dhyb* Block 1 Block 2 (optional) Block 3 (optional) 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 11
Examples of derived MVA-TB vectors Active (1)/ Resusc. (2) / Latent (2) Large Active (3) / Resusc. (2) Latent (4) Latent (5) Fusions Active (1) / Resusc. (2) Active (2) / Latent (5) / Latent (2) Latent (2) Active Active Active Latent Resusc. Individual Ag Latent Active Active Active Active or 2A cleavage Latent-2A- Active-X- Active-X- Latent-2A- Short fusions Resusc. peptides + Active Active Active Latent Linker Latent-X- Active-X- Active-X- Latent-X- Resusc. Linker Active Active Active Latent Active (3)/ Latent (2) Resusc. (2) Large and short fusions Active (3)/ Active (1) / Resusc. (2) Latent (1) 12
Typical early assessment of immunogenicity in mice (Leung-Theung-Long et al., PLoS One, Nov 2015 + unpublished) Immunization (s.c, 10 7 pfu/mouse) Mouse D0 D7 D9 D14 ………… • Mouse strains: • BALB/c (H-2 d ) • C57Bl/6 (H-2 b ) • C3H/HeN (H-2 k ) • HLA-A2 (I-A b , HLA-A2) • Readouts: ELISpot IFN g (production by splenocytes activated in vitro by peptides) • • In vivo CTL • ICS CD4/CD8; polyfunctionality • Antibodies ●●●●●●●●● 13 13
Immunogenicity of MVA candidate vaccines in BALB/c mice Illustration of IFN- γ responses specific of 14 antigens (3 expression cassettes) Medians of each group 700 U Mann Whitney test MVATG18377 -Rv2029-Rv2626-Rv1733-Rv0111 + * : p < 0.05 RpfB-Dhyb-Ag85B-TB10.4-ESAT6 + ** : p < 0.01 -Rv0569-Rv1813-Rv3407-Rv3478-Rv1807- 600 ----- : cut-off MVATG18379 -Rv2029-Rv2626-Rv1733-Rv0111 + P: peptide pool RpfB-Dhyb-Ag85B-TB10.4-ESAT6 + Rv0569-Rv1813-Rv3407-Rv3478-Rv1807 500 * MVATGN33.1 sfc/10 6 cells 400 ** ** * ** 300 200 100 0 Medium Irr pept P1 P2 P3 P4 P1 P2 P3 P4 Rv0569 Rv1813 Rv3407 Rv3478 Rv1807 Broad and significant responses observed (Rv1813, Rv3407, Rv3478 and Rv1807) with the MVA including SS and TM domains in fusion sequence. 14
IMMUNOGENICITY OF MVA CANDIDATE VACCINES IN HLA-A2 MICE ILLUSTRATION OF IFN- g RESPONSES SPECIFIC OF 4 ANTIGENS (1 EXPRESSION CASSETTE) Medians of each group U Mann Whitney test 2000 MVATGN33.1 * : p < 0.05 MVATG18376 -Rv2029-Rv2626-Rv1733-Rv0111 + ** : p < 0.01 -RpfB-Dhyb-Ag85B-TB10.4-ESAT6- + ** ----- : cut-off -Rv0569-Rv1813-Rv3407-Rv3478-Rv1807- P: peptide pool 1600 sfc/10 6 cells 1200 ** 800 ** ** ** ** ** * 400 0 Medium Irr pept P1 P2 P3 P4 P1 P2 P3 RpfB-Dhyb Ag85B TB10.4 ESAT6 Broad and significant responses observed with the MVA in HLA-A2 transgenic mice. Following CD4 T cell depletion, significant IFN γ response was still detected for antigens such as RpfB-RpfD fusion protein. 15
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