Frédéric Tangy Unité de Génomique Virale et Vaccination

1. Replicating measles-SHIV vaccine induces long term preservation of central memory CD4 cells in the gut of macaques challenged with SHIV89.6P Frédéric Tangy Unité de Génomique Virale et Vaccination

2. Live-attenuated viruses make very efficient vaccines • Smallpox (eradicated) • Polio (95-100% morbidity reduction) • Measles (95-100% morbidity reduction) • Mumps (95-100% morbidity reduction) • Rubella (95-100% morbidity reduction) • Yellow fever (95-100% efficiency) • Varicella(70-80% morbidity reduction) • …. • Live-attenuated SIV protects macaques

3. N P M F H L C V 150-450 nm Measles virus (Morbillivirus, Paramyxoviridae) non-segmented RNA, negative polarity (16 kb) Despite being a preventable disease, measles is still responsible for 20 million cases and 200 000 deaths annually

4. HK-24 HA-28 CE-6 CEF-13 CEF/85 32°C Edmonston Edmonston-Enders Edmonston A Schwarz ∂ N P M F H L T7 T7t C hh V Measles live attenuated vector • One injection : 103 - 104 TCID50 (9-16 months) • Long-lived protective immunity (NAb + CD8 + CD4) • High genetic stability • Safety and efficacy track record in billions of children • Established logistics for production and distribution of MV vaccine worldwide, low cost of production • Mass vaccination needed for foreseeable future (cloned unpassaged Schwarz strain) • EP-02291551.6, filed May 2002 • EP-02291550.8, filed June 2003 • - EP-06292025.1, filed December 2006 • - Combredet et al. 2003, JVI, 77, 11546-554

5. MV-CS-falciparum MV-DV-EDIII eM MV-DV-EDIII eM MV-prME-YFV MV-N1 MV-S-SARS sol MV-sE-WNV MV-S-SARS MV-H5 Recombinant MV vaccine targets To immunize children simultaneously against measles and other diseases • HIV • Arboviruses (DV, WN, YF, CHIKV…) • Acute respiratorydiseases (RSV, PIV-3, SARS, H5N1…) • Malaria

6. MVSchw-Gag (P3) MVSchw-Gag (P1) MVSchw-Gag HIV Vero cells MVSchw 160 140 HIV 120 Gag p42 MV 60 MV3-gp140∆V3 MV3-gp140∆V3 Mv2-gp160∆V3 Mv2-gp160∆V3 MV2-gp160 MV2-gp140 MV2-gp140 MV2-gp160 P2 P5 Mice Macaques 10e5 10e5 10e4 10e4 10e4 10e4 10e3 10e3 10e3 10e3 MV Ab 10e2 10e2 MV Ab 10e2 10e2 10 10 10 10e1 HIV Ab HIV Ab 1 1 1 1 0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 16 18 20 Months Months MV-HIV x2 MV MV-HIV x 2 MV rMV expresses HIVproteins and is immunogenic MVSchw-Env HIV MV-HIV vectors induce HIV-specific CD4/CD8 T cells and antibodies in mice and macaques, even in presence of measles preexisting immunity Lorin et al. 2004, JVI, 78, 146-157 Lorin et al. 2005, Vaccine, 23: 4463-72

7. ∂ HIV-gp160∆V12 HIV-p55 Gag L F H N P M t l T7t C T7 V 200 nm hh 1 µm 2 µm A B C MV-p55Gag MV-p55Gag MV-p55Gag D E F 500 nm 200 nm MV-p55Gag MV-p55Gag/Env MV-p55Gag/Env rMV expresses HIV VLPs Guerbois et al. 2009, Virology, 388,191–203

8. ∂ N P Gag M gp160 F L T7 V T7t hh C Live replicating chimeric MV/HIV virus Live chimeric MV in which the hemmaglutinin (H) and fusion (F) surface proteins are replaced by HIV or SIV gp160 fused with the intracytoplasmic tail of F -> live processing and fusion of HIV gp -> NAb induction and escape to MV preimmunity. In vitro production : HeLa P4-CCR5, CEMX174, MT4, Jurkat, PBMC In vivo testing : SIV251 rhesus macaque

9. MV-SHIV MV-SHIV SHIV A MV MV SHIV B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 Months 250 350 350 Env #6165 #5797 #5986 Gag 300 Gag 300 200 Gag Env Env 250 250 Gag 150 Tat HIV g-IFN Elispots/106 cells Tat 200 200 Tat 150 150 Env Gag 100 Gag 100 100 Env 50 50 50 0 0 0 d0 d15 M1 M2 d0 d15 M1 M2 d0 d15 M1 M2 Macaques immunization and SHIV89.6P challenge • Cynomolgusmacaques vaccinated with a mixture of recombinant MVSchw : • MV-(GagSIV-gp140HIV) + MV-( TatHIV)+ MV-(NefSIV) (106 TCID50each) • - Intra-rectal challenge with 30 AID50 SHIV89.6p

10. MV 2 x MV-SHIV Sacrifice C 0 5 61 - 5 Months Immunity is induced for years 5.5 years Overnight stimulation with specific peptides g-IFN Elispots > 2 x background ICS and FACS analysis confirmation

11. Viral load at challenge Ab resp at challenge 1,E+09 120 Vaccinated 1,E+08 Controls 100 1,E+07 1,E+06 80 SHIV viral load 1,E+05 Vaccinated Anti-HIV gp120 Ab titer (%) 60 1,E+04 1,E+03 40 Controls 1,E+02 20 1,E+01 1,E+00 0 0 1 2 3 4 5 6 7 0 15 30 45 60 75 90 105 120 Months post challenge Days post challenge Neut Ab resp at challenge Cellular resp at challenge 10000 4500 Vaccinated 4000 Vaccinated 3500 1000 Controls Controls 3000 Gag SFC/million cells 50% neutralization titer 2500 100 2000 1500 10 1000 500 1 1 5 7 14 30 50 0 Days post challenge Months post vaccination 0 100 200 300 400 500 Days post challenge Vaccinated animals control SHIV acute replication

12. 2500 10e8 2000 10e7 1500 Controls 1000 10e6 500 10e5 Vaccinated SHIV viral load copies/ml 0 0 6 12 20 42 60 10e4 Days post BCG inoculation 10e3 0 6 12 20 42 60 10e2 2000 10 1500 1 1000 0 10 20 30 40 50 60 70 Days post BCG inoculation 500 0 Days post BCG inoculation Vaccinated animals control SHIV reactivation HIV specificIFNgElispots/ million cells Three years post-challenge, SHIV replication was reactivated by inoculating high-dose BCG intravenously (107pfu). Proliferation of SHIV-specific T cells was higher in vaccinated animals who controlled viral reactivation. BCG (PPD)-specificIFNgElispots/million cells

13. MV-SHIV SHIV BCG Sacrifice 0 2 3 4 5 - 1 1 Years 4 10 3 10 2 10 1 10 0 10 Vaccinated Controls Unchallenged CD4 memory cells are preserved in the gut Five years after challenge, macaques were sacrificed and T lymphocytes populations were analyzed in blood, lymphoid organs and in the gut. Although no difference was observed in the level of circulating CD4 cells between groups, the CD4 central memory lymphocytes were clearly preserved in the gut of vaccinees. 4 10 CD4 Central memory CD4 3 CD28 10 FL4-H: CD4 APC Naïve FL1-H: CD28 FITC 2 CD8 10 Effector memory 1 10 0 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 FL2-H: CD95 PE FL3-H: CD3 PerCP-Cy5.5 CD3 CD95 100 100 p = 0,043 80 Same observation in intra-epithelial lymphocytes (IEL) from jejunum, ileon and colon 80 60 60 % CD4 CM 40 40 p = 0.048 20 20 0 0 IEL Duodenum LPL Duodenum

14. 4.0 HIV CD4+CD154+ HIV CD8+ 1.5 3.5 3.0 1.0 2.5 % Cytokine positive cells % Cytokine positive cells 2.0 1.5 0.5 1.0 0.5 0.0 0.0 112 112 14 28 42 56 84 56 14 28 42 84 0 0 Time (days) Time (days) ∂ HIV-F4 N P M F H L MV-F4 T7 T7t C hh V RMVHIV Demonstrating safety and immunogenicity of recombinant MV-HIV vector in adult HIV-uninfected volunteers GSK-Bio (Rixensart), Institut Pasteur (Paris), St George’s Hospital Medical School (London), CEVAC (Ghent), CIC Cochin-Pasteur (Paris), NIBSC (South Mims) • Identification of a suitable dose of recombinant vaccine • Demonstration of an acceptable reactogenicity profile • Characterization of potential virus shedding • Evaluation of HIV-specific responses raised in volunteers with MV preimmunity • Vector generated • Preclinical mouse and macaque immunogenicity • Clinical GMP lot produced, stability, potency • Toxcicity, biodistribution, shedding in macaques • PhIclinical trial 2010

15. Toxicity, biodistribution, shedding of MV-F4 • Cynomolgus macaques (MV-F4 im, Rouvaxim, MV-F4 iv, PBS im) • Clinical observation • 48 organs, 8 fluids analyzed at different times post vaccination • Histopathology, qRT-PCR (MV and HIV), infectivity assay • No toxicity observed, no temperature elevation • Biodistribution to lymphoid organs • Shedding of RNA sequences (d10) • No shedding of infectious virus • Toxicity validated for phase I trial Phase I, dose-escalation and safety study • 24 adults HIV naïve volunteers (MV serology threshold) • No physical containment (biological containment) • Toxicity, biodistribution, shedding, CMI and Ab immunogenicity

16. Acknowledgements Michèle Février Chantal Combredet Valérie Labrousse Mathilde Guerbois Mariana Mesel Claude Ruffié CPR, Bruno Hurtrel Xavier Montagutelli CEA, Roger Legrand Viral Genomics and Vaccination Unit