DNA VACCINES FOR BIODEFENSE

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Lucas French
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1 DNA VACCINES FOR BIODEFENSE Lesley Dupuy, Ph.D. Principal Investigator-Contractor Molecular Virology Department Virology Division ATTRIBUTES OF DNA VACCINES Advantages Over Conventional Methods Easily Manufactured Quickly produced in response to emerging threats Established and approved manufacturing procedures Favorable Safety Profile Replication defective Not transmissible No Pre-existing Vector Immunity Flexible Platform Delivered by various methods Easily combined to form multivalent vaccines

2 Quick Tim e and a Quick Tim e and a DNA VACCINES AT USAMRIID Vaccines for Various Biodefense Threats Alphavirus Venezuelan equine encephalitis virus (VEEV) Eastern equine encephalitis virus (EEEV) Western equine encephalitis virus (WEEV) Arenavirus Lassa virus (LASV) Bacillus anthracis Bunyavirus Hantaan virus (HTNV) Puumala virus (PUUV) Rift Valley fever virus (RVFV) Crimean Congo hemorrhagic fever virus (CCHFV) Filovirus Ebola virus (EBOV) Marburg virus (MARV) Flavivirus Tick-borne encephalitis virus (TBEV) Poxvirus Smallpox virus (VARV) Monkeypox virus (MPXV) Vaccinia virus (VACV) DNA VACCINES AT USAMRIID Multivalent DNA Vaccine Combinations Combination of Genes from One Pathogen Poxvirus Combination of Genes from Diverse Pathogens VEEV, EBOV, MARV, B. anthracis TBEV, HNTV, RVFV, CCHFV Combination of Genes from Similar Pathogens VEEV, EEEV, WEEV HTNV, PUUV 2

3 Quick Tim e and a Quick Tim e and a DELIVERY OF DNA VACCINES Technologies Evaluated at USAMRIID Particle-Mediated Epidermal Delivery (Gene gun) Skin Dermabrasion Microneedle Injection Transcutaneous Chemical Liquid Jet Injection Cationic Lipid Electroporation ENCEPHALITIC ALPHAVIRUSES Venezuelan Equine Encephalitis Virus (VEEV) Eastern Equine Encephalitis Virus (EEEV) Western Equine Encephalitis Virus (WEEV) Disease in Humans Fever, headache, myalgia, occasional encephalitis Case Fatality Rates VEEV % EEEV 3-7% WEEV 8-5% Category B Select Agents Ease of production Considerable stability High infectivity in aerosols 3

4 Quick Tim e and a Quick Tim e and a ENCEPHALITIC ALPHAVIRUSES Existing IND Vaccines Live-attenuated VEEV Vaccine (TC-83) Durable protective immune response High non-response rate (~2%) Reactogenic (~25%) Some reversion to virulence Formalin-inactivated VEEV, EEEV, WEEV Vaccines Well-tolerated Require frequent boosting Poor aerosol protection ALPHAVIRUS DNA VACCINES Administration by Gene Gun pwrg777 26S Structural Protein Genes C E3 E2 6K E PowderMed XR- 4

(TDS) EP Technology ELECTROPORATION DELIVERY OF DNA Increased Plasmid Uptake and Antigen

5 Quick Tim e and a ALPHAVIRUS DNA VACCINES Electroporation of Optimized Constructs pwrg777 Codon-optimized 26S Structural Protein Genes 66 kda 45 kda E2 E Ichor TriGrid Delivery System (TDS) EP Technology ELECTROPORATION DELIVERY OF DNA Increased Plasmid Uptake and Antigen Expression Electrode Insertion & DNA Administration Electrical Field Application Antigen Expression in Transfected Tissue 5

6 log Mean Titer VEEV DNA IMMUNOGENICITY Total IgG Antibodies by ELISA N = Day 2 Day 42 Day ug Empty Vector 25 ug 5 ug ug 25 ug 5 ug ug TC-83 VEEV DNA WT VEEV DNA CO VEEV DNA IMMUNOGENICITY Neutralizing Antibodies by PRNT N = 6 log Mean PRNT Day 2 Day 42 Day ug Empty Vector VEEV DNA WT 25 ug 5 ug ug 25 ug 5 ug ug TC-83 VEEV DNA CO 6

7 log Mean Titer Mean SFU/ 6 Cells VEEV DNA IMMUNOGENICITY Total IgG Antibodies by ELISA N = ** ***** Day 2 Day EP +EP 5 ug VEEV DNA CO **p <., *****p <. VEEV DNA IMMUNOGENICITY Cellular Responses by IFN- ELISPOT N = 6 No Peptide B-Gal 75 VEEV E VEEV E2 5 * 25 5 ug Empty Vector 5 ug VEEV DNA CO *p <.5 7

8 Percent survival VEEV DNA PROTECTION Aerosol VEEV Challenge Survival N = Days Postchallenge 5 ug VEEV DNA CO TC-83 5 ug Empty Vector Aerosol challenge:, LD 5 VEEV DNA IMMUNOGENICITY Neutralizing Antibody Responses by PRNT N = 4 log Mean PRNT ug VEEV DNA CO 5 ug VEEV DNA CO 5 ug Empty Vector Days Post-st Vaccination 8

9 Quick Tim e and a Mean Temperature Elevation ( C) Mean Viremia (PFU/ml) VEEV AEROSOL CHALLENGE Serum Viremia by Plaque Assay N = Days Postchallenge 5 ug Empty Vector 5 ug VEEV DNA CO 5 ug VEEV DNA CO Aerosol challenge: ~3 ED 5 VEEV AEROSOL CHALLENGE Fever Responses by Telemetry N = 4 **** 5 ug Empty Vector 2 5 ug VEEV DNA * CO 5 ug VEEV DNA CO * Days Postchallenge Aerosol challenge: ~3 ED 5 *p <.5, ****p <. 9

10 Qu ic k Tim e and a Qu ic k Tim e and a VEEV DNA IN RABBITS Neutralizing Antibody Responses by PRNT N = 5 log Mean PRNT ug VEEV DNA CO Days Post-st Vaccination COMBINED ALPHAVIRUS DNA Neutralizing Antibody Responses by PRNT N = 5 Log Mean PRNT ug VEEV 5 ug each V/E/W 5 ug EEEV 5 ug each V/E/W 5 ug WEEV 5 ug each V/E/W

11 Qu ic k Tim e and a ALPHAVIRUS DNA VACCINES Summary and Conclusions Codon-optimized Alphavirus DNA vaccines are potent when delivered by electroporation. Robust humoral and cellular immune responses Durable neutralizing antibody responses Potential to achieve protective immune responses at low doses and with few vaccinations Immune responses to codon-optimized Alphavirus DNA vaccines not significantly altered when delivered by electroporation as a trivalent combination. FILOVIRUS VIRAL HEMORRHAGIC FEVERS Ebola Virus (EBOV) and Marburg Virus (MARV) Negative-sense, single-stranded RNA viruses Epidemiology Transmission by close contact (blood or body fluids) Natural host unknown (suspected fruit bats) Clinical Features Incubation period: 4-2 days Abrupt onset of nonspecific symptoms Impaired liver function Bleeding and dysregulated coagulation (clotting) Death/shock 6-9 days after onset High case fatality rates (4-9%) Category A Select Agents Ebola virus species Zaire Sudan Ivory Coast Reston Bundibugyo

12 Qu ic k Tim e and a Qu ic k Tim e and a MULTI-EPITOPE VACCINES Genome-Derived Epitope-Driven Approach Highly accurate tools available for in silico identification of Class I/II T-cell epitopes Advantages to this approach for vaccine development: Effective: Focus immune response on epitopes essential for protection Safe: Elimination of epitopes against conserved self antigens Broad: Selection of epitopes conserved across multiple strains or subtypes Fast: Protective vaccines developed in 24 months MULTI-EPITOPE VACCINES EpiMatrix T-cell Epitope Predictions Input Sequences: Zaire Ebola Virus (ZEBOV): Glycoprotein (GP) Nucleocapsid protein (NP) Sudan Ebola Virus (SEBOV): Glycoprotein (GP) Nucleocapsid protein (NP) Venezuelan equine encephalitis virus Subtype IAB (Strain Trinidad donkey) 26S structural gene ORF 2

Qu ic k Tim e and a Qu ic k Tim e and a MULTI-EPITOPE VACCINES EpiMatrix T-cell Epitope Predictions Epivax in silico class II binding prediction Cluster with multiple predicted epitopes in ZEBOV GP

13 Qu ic k Tim e and a Qu ic k Tim e and a MULTI-EPITOPE VACCINES EpiMatrix T-cell Epitope Predictions Epivax in silico class II binding prediction Cluster with multiple predicted epitopes in ZEBOV GP MULTI-EPITOPE VACCINES HLA Class II Epitope Selections Cluster Address EpiMatrix EpiMatrix CLUSTER EpiBars? Input Sequence (w/ FLANKS) Cluster Sequence Hydro-phobicity HITS SCORE (#) (w/o FLANKS) (w/o FLANKS) EBOLA-SUDAN-GP 8-96 VIAFLILAKPKETFLQS Yes (2) EBOLA-SUDAN-GP STTLFKINNNTFVLLD Yes (2) EBOLA-SUDAN-GP TPQFLFQLNDTIHLHQQ Yes (2) EBOLA-SUDAN-GP GTGLSSSQILSSSPTMAPSP Yes (2) EBOLA-SUDAN-GP TEGLMHNQNALVCGLR Yes () EBOLA-SUDAN-GP TQALQLFLRATTELRTYT Yes (2) EBOLA-SUDAN-GP LRTYTILNRKAIDFLLR. 2.5 Yes () EBOLA-SUDAN-NP 8-35 DLDYHKILTAGLSVQQGI Yes () EBOLA-SUDAN-NP 7-87 ADSFLLLLCLHHAYQGDH No EBOLA-SUDAN-NP GHMMVIFRLMRTNFLIKF Yes () EBOLA-SUDAN-NP YAPFARLLNLSGVNNLEHG Yes (2) EBOLA-SUDAN-NP EETYYHLLKTQGPFEA Yes (2) EBOLA-ZAIRE95-GP SIPLGVIHNSTLQVSDVD Yes () EBOLA-ZAIRE95-GP RWGFRSGVPPKVVNY Yes () EBOLA-ZAIRE95-GP NLTYVQLESRFTPQF Yes () EBOLA-ZAIRE95-GP TPQFLLQLNETIYTSGK Yes (2) EBOLA-ZAIRE95-GP TEDHKIMASENSSAMVQ Yes () EBOLA-ZAIRE95-NP 8 - HHAYQGDYKLFLESGAVKY LEG Yes (2) EBOLA-ZAIRE95-NP VKNEVNSFKAALSSLAKHGE Yes (2) EBOLA-ZAIRE95-NP EHSFEEMYRHILRSQGPFDAV Yes (3) VEEV-26S 3-5 TDPFLAMQVQELTRSMANLTF Yes (2) VEEV-26S HDGYVRLQTSSQYGLD Yes (2) VEEV-26S GHGYFLLARCPAGDSI Yes () VEEV-26S ASTWLFCRSRVACLTPY Yes (2) VEEV-26S NTNLVLQRPKAGAIHVP Yes () 3

14 Qu ic k Tim e and a Qu ic k Tim e and a MULTI-EPITOPE VACCINES VaxCad Arrangement of Final String of Beads MULTI-EPITOPE VACCINES VaxCad Arrangement of Final String of Beads 4

15 Qu ic k Tim e and a MULTI-EPITOPE VACCINES Summary and Conclusions Putative Class II T-cell epitopes in the structural proteins of ZEBOV, SEBOV, and VEEV predicted with EpiMatrix Predicted Class II epitopes validated by in vitro HLA binding assays 74% of EpiMatrix predictions met or exceeded in binding assays Optimal order of selected Class II epitopes determined with VaxCad Final Class II multi-epitope DNA vaccine constructed Currently initiating studies to evaluate immunogenicity and protective efficacy of Class II construct in HLA transgenic mice ACKNOWLEDGEMENTS Connie Schmaljohn, Ph.D. Jay Hooper, Ph.D. Daniel Mitchell, Ph.D. Michelle Richards Brian Livingston, Ph.D. Drew Hannaman Barry Ellefsen Lillian Chau Annie DeGroot, Ph.D. Bill Martin Lenny Moise, Ph.D. Jason Del Pozzo Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the U.S. Army. The research described herein was sponsored by the Defense Threat Reduction Agency (DTRA). Research was conducted in compliance with the Animal Welfare Act and other federal statues and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 996. The facility where this research was conducted is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. 5

ADEPT. Accelerated Defense against Emerging Pathogen Threats

ADEPT. Accelerated Defense against Emerging Pathogen Threats ADEPT Accelerated Defense against Emerging Pathogen Threats Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the U.S. Army. Parallel