Development of a Recombinant Subunit Dengue Vaccine Flavivirus Vaccination Fondation Mérieux December 8, 2010 Beth-Ann Coller
Recombinant Subunit Dengue Vaccine Recombinant Envelope Protein Vaccine Hawaii Biotech originally developed subunit vaccine based on carboxy truncated envelope protein 80E Recently acquired by Merck. Drosophila S2 insect cell culture expression system is used to produce dengue subunits No live virus, potential to achieve balanced tetravalent immunity without interference Crystal structure of Merck DEN2-80E protein Steve Harrison, Harvard University 2
Recombinant Subunit Dengue Vaccine Overview of Drosophila S2 Expression System Drosophila Expression Vector phbi-20d Gene of Interest Drosophila S2 Cells Native-like Protein Structure Protein Secreted into Culture Medium 3
Recombinant Subunit Dengue Vaccine Rationale for Envelope Protein Based Subunit Vaccine Candidate: Functional regions of Envelope are associated with binding to cell receptors and fusion Envelope important in protective immune response key target for neutralizing Ab Envelope also contributes as cell mediated immune target (CD4 and CD8) Balance of multivalent immunity may be manipulated by varying antigen doses no viral interference No prm protein in purified product minimize risk of enhancement linked to anti-prm antibodies 4
Dengue Vaccine Formulation Development Formulation Development HBI has tested dengue and WN vaccines with variety of adjuvants in preclinical models. Saponin-based adjuvants and MPL/Alum in combination with DEN antigens induce potent neutralizing responses and protective efficacy in primates Access to potent adjuvants and regulatory challenges led to focus in short term on alum-based adjuvants Alum based formulations immunogenic and efficacious if properly formulated and administered. Focus on alum-based vaccines for initial clinical trial. Other adjuvants may offer significant advantages in long term (durability, dose sparing, magnitude and broadness of immune response) 5
Dengue Vaccine Preclinical Studies Non-Human Primate Studies Monovalent DEN-80E studies 1 - Rhesus macaques DEN2-80E + ISCOMATRIX adjuvant 2 - Rhesus macaques DEN2-80E + GSK adjuvants Putnak et al., 2005 3 - Rhesus macaques DEN1-80E + alum adjuvant stand alone and prime boost with LAV Tetravalent DEN-80E studies 1 - Rhesus macaques ISCOMATRIX Adjuvant + DEN2 NS1 pilot study 2 - Rhesus macaques ISCOMATRIX adjuvant +/- DEN2 NS1 study large safety, immunogenicity and efficacy study 6
Dengue Vaccine Preclinical Studies Monovalent DEN2-80E + GSK adjuvants Rhesus Macaques Immunogenicity and Efficacy 2 doses Challenge 2 months post dose 2 Vaccine Formulation PRNT 50 GMT Viremia (Cell culture) Anamnestic Response 5 μg 80%E + AS04-OH 175 2/3 Yes 20 μg 80%E + AS04-OH 541 0/3 Yes 5 μg 80%E + AS04-PO 114 2/3 Yes 1 μg 80%E + AS05 850 0/3 Yes 5 μg 80%E + AS05 1068 0/3 Yes 5 μg 80%E + AS08 1676 1/3 Yes 7
Dengue Vaccine Preclinical Studies Safety/Immunogenicity/Protection of Tetravalent 80E + NS1 Vaccine in Primates Pilot Study Group Antigen formulation Vaccine Dosing Days Number of Animals 1 3 µg each serotype 80E + 0.3 µg NS1 10 µg Adjuvant A + 10 µg Adjuvant B 2 3 µg each serotype 80E + 0.3 µg NS1 50 µg Adjuvant A + 50 µg Adjuvant B 3 1 µg each serotype 80E + 0.1 µg NS1 60 µg of ISCOMATRIX Adjuvant 4 5 µg each serotype 80E + 0.5 µg NS1 60 µg of ISCOMATRIX Adjuvant 0, 28, 67, 102 2 0, 28, 67, 102 2 0, 28, 67, 102 2 0, 28, 67, 102 2 4 Doses - Primary endpoint Safety Safety Profile OK Immunogenicity assessed following each dose Challenge approximately 5 months post dose 4 with DENV2 or DENV4 8
PRNT50 Titer PRNT50 Titer Dengue Vaccine Preclinical Studies Anti-DEN-1 Neutralizing Ab Response Neutralizing Ab Responses in Monkeys 10000 1000 100 V3J HJC V2G AC70 AA37 10 FTH T206 AJ14 1 Anti-DEN-2 Neutralizing Ab Response 0 28 67 102 130 Study Day 10000 1000 100 10 1 0 28 67 102 130 Study Day V3J HJC V2G AC70 AA37 FTH T206 AJ14 Dark blue: 3 µg each 80E, 0.3 µg NS1, 10 µg each Adjuvant A & Adjuvant B Yellow: 3 µg each 80E, 0.3 µg NS1, 50 µg each Adjuvant A & Adjuvant B Red: 1 µg each 80E, 0.1 µg NS1, 60 µg ISCOMATRIX Adjuvant Blue: 5 µg each 80E, 0.5 µg NS1, 60 µg ISCOMATRIX Adjuvant 9
PRNT50 Titer PRNT50 Titer Dengue Vaccine Preclinical Studies Anti-DEN-3 Neutralizing Ab Response Neutralizing Ab Responses in Monkeys 1000 100 10 V3J HJC V2G AC70 AA37 FTH T206 AJ14 Anti-DEN-4 Neutralizing Ab Response 1 0 28 67 102 130 Study Day 1000 100 10 1 0 28 67 102 130 Study Day V3J HJC V2G AC70 AA37 FTH T206 AJ14 Dark blue: 3 µg each 80E, 0.3 µg NS1, 10 µg each Adjuvant A & Adjuvant B Yellow: 3 µg each 80E, 0.3 µg NS1, 50 µg each Adjuvant A & Adjuvant B Red: 1 µg each 80E, 0.1 µg NS1, 60 µg ISCOMATRIX Adjuvant Blue: 5 µg each 80E, 0.5 µg NS1, 60 µg ISCOMATRIX Adjuvant 10
Dengue Vaccine Preclinical Studies PRNT50 Titer Challenge DENV-2 Challenge Anti-DEN-2 Virus Neutralizing Response 100000 10000 1000 100 10 V3J AC70 FTH AJ14 Protection X 1 0 28 67 102 130 278 322 Study Day 11
Dengue Vaccine Preclinical Studies PRNT50 Titer Challenge DENV-4 Challenge Anti-DEN-4 Virus Neutralizing Response 10000 Protection 1000 100 10 HJC V2G AA37 T206 X X 1 0 28 67 102 130 278 322 Study Day 12
Dengue Vaccine Preclinical Studies Monovalent DEN1-80E + Alhydrogel Rhesus Macaques Recombinant alone or as heterologous primeboost with LAV 3 doses administered @ 0, 1, 2 months Challenge 2 months post dose 3 with wild type DEN1 (WP74) Immunogenicity and Efficacy Group Dose 1 Dose 2 Dose 3 1 LAV DEN1-80E DEN1-80E 2 DEN1-80E DEN1-80E LAV 3 DEN1-80E DEN1-80E DEN1-80E 4 Placebo Placebo Placebo 13
PRNT 50 GMT Dengue Vaccine Preclinical Studies Monovalent DEN1-80E + Alhydrogel Safety No adverse observations Immunogenicity Virus neutralizing antibody 10000 1000 100 10 80E/LAV LAV/80E 80E/80E Placebo 1 Day 0 Day 28 Day 56 Day 84 Day112 Day140 14
Dengue Vaccine Preclinical Studies Monovalent DEN1-80E + Alhydrogel Efficacy - Post Challenge Viremia Results Group Viremia # Animals (mean # days) 1 - LAV/DEN1/DEN1 0/3 2 - DEN1/DEN1/LAV 0/3 3 - DEN1/DEN1/DEN1 0/3 4 - Alhydrogel Placebo 3/3 (4.33) 15
Dengue Vaccine Preclinical Studies Tetravalent DEN-80E + ISCOMATRIX Adjuvant Rhesus Macaques +/- DEN2-NS1 3 doses @ 0, 2, 4 months 3 groups of 12 monkeys each challenge 5 months post dose 3 3 monkeys challenged with each serotype Immunogenicity and Efficacy Formulation DEN 80%E DEN-2 NS1 ISCOMATRIX A 1 µg each 80%E - 60 µg B 1 µg each 80%E 0.1 µg 60 µg Placebo - - 60 µg 16
Dengue Vaccine Preclinical Studies Tetravalent DEN-80E + ISCOMATRIX Adjuvant Safety - no adverse local reactions or impact on clinical chemistry/hematology parameters Post dose 3 PRNT 50 titers - Representative results Virus Serotype PRNT DEN-1 DEN-2 DEN-3 DEN-4 GMTs 672 947 574 280 17
Dengue Vaccine Preclinical Studies Tetravalent DEN-80E + ISCOMATRIX Adjuvant Efficacy - Post Challenge Viremia Data Group Challenge Virus Viremia # Animals (mean # days) 1 μg each DEN1 0/3 (0) Tetravalent 80E DEN2* 0/3 (0) + ISCOMATRIX Adjuvant DEN3 0/3 (0) DEN4* 0/3 (0) 1 μg each DEN1 1/3 (0.33) Tetravalent 80E DEN2* 0/3 (0) + 0.1 μg NS1 DEN3 0/3 (0) + ISCOMATRIX Adjuvant DEN4* 0/3 (0) DEN1 3/3 (5) ISCOMATRIX DEN2* 3/3 (2.3) Adjuvant Control DEN3 3/3 (3.7) DEN4* 2/3 (1.7) * Amplification on C6/36 prior to plaquing on Vero 18
Dengue Vaccine Preclinical Safety Studies Preclinical Toxicity Testing GLP Rat repeat dose safety study to assess local and systemic toxicity of vaccine antigens alone or formulated with ISCOMATRIX Adjuvant No significant toxicity/reactogenicity found with vaccine antigens or formulated vaccine GLP Rabbit repeat dose safety study to assess local and systemic toxicity of antigens alone or in alum based formulation No toxicity/reactogenicity with any formulation Group Formulation 1 Vehicle Control - PBS 2 Alhydrogel 85 Adjuvant Control 3 Antigen Control Tetravalent 80E + DEN2 NS1 4 DEN1-80E adsorbed on Alhydrogel 85 5 Tetravalent 80E + DEN2 NS1 adsorbed on Alhydrogel 85 19
Dengue Vaccine Preclinical Safety Studies Preclinical Toxicity Testing GLP Rabbit repeat dose safety study to assess local and systemic toxicity of antigens alone or in alum based formulation Immunogenicity results post dose 3 Group Formulation DEN1 ELISA EC 50 GMT 1 Vehicle Control PBS <250 2 Alhydrogel 85 Adjuvant Control <250 3 Antigen Control Tetravalent 80E + DEN2 NS1 5229 4 DEN1-80E adsorbed on Alhydrogel 85 9816* 5 Tetravalent 80E + DEN2 NS1 adsorbed on Alhydrogel 85 9429 * PRNT GMT 236 20
Recombinant Subunit Clinical Study Phase 1 Monovalent Clinical Study COHORT 1 N=8 2 N=8 TREATMENT Low dose DEN1-80E 10 µg + Alhydrogel 1.25 mg Al High dose DEN1-80E 50 µg + Alhydrogel 1.25 mg Al Placebo-controlled, double-blind study 6 active, 2 placebo per cohort Dose escalation low and high doses 3 doses administered at 0, 1, 2 months Primary endpoint safety Immunogenicity evaluation virus neutralizing antibody Last subject, last visit in June 2010. Study still blinded. 21
Recombinant Subunit Clinical Study GMT PRNT50 Titers Virus Neutralizing Antibody Preliminary Results Dose 1 Dose 2 Dose 3 100 Cohort 1 10µg +Alum Cohort 2 50µg +Alum 10 Placebo 1 Week 0 Week 2 Week 4 Week 6 Week 8 Week 10 22
Recombinant Subunit Clinical Study Conclusions: Based on preliminary, unaudited, blinded data - monovalent DEN1-80E vaccine adjuvanted with aluminum hydroxide is immunogenic in healthy, adult, flavivirus-naïve volunteers consistent with preclinical data. Study included a dose escalation with monitoring by a Safety Committee - no safety signals Demonstrates potential of the recombinant proteins. Plans for testing of tetravalent product under development 23
Acknowledgements Saint Louis University Center for Vaccine Development Sarah George Nicole Purcell Carolyn Stefanski Linda Eggemeyer All the Study Volunteers Johns Hopkins University Anna Durbin Janece Lovchik Walter Reed Army Institute for Research Ken Eckels Rick Millward Stephen Thomas David Bradley Amy Dean Robert Putnak University of Texas Medical Branch Robert Tesh CSL Clinical and Statistical Support Debbie Drane Martin Pearse Elenie Bartzokis Steve Honey Gary Stevens Hawaii Biotech, Inc. Jon Ruckle Maile O Connell Tim Martyak Mike Thorne Vidya Pai Robbin Henley David Clements Michele Yelmene Support from: U.S. Department of Defense, National Institutes of Allergy and Infectious Disease, Pediatric Dengue Vaccine Initiative, State of Hawaii, Merck, and Hawaii Biotech Investors 24