Gene Olinger, USAMRIID, Fort Detrick USA, presents at the ProImmune Antigen Characterization and Biomarker Discovery Summit, January 2011. Protective Immune Reponses to Ebola Virus

1. Protective Immune Responses to Ebola virus: Identification of novel HLA-A*0201-restricted CD8+ T-cell epitopes on Ebola Zaire glycoprotein. Presented by: Gene Garrard Olinger, Jr. Ph.D., MBA Virology Division Unclassified /FOUO 16 Oct 2007

2. Filovirus Viral Hemorrhagic Fevers Ebola and Marburg First case 1976, Zaire Negative sense, enveloped, ssRNA virus, filamentous morphology Epidemiology Natural host is unknown Transmission associated with close contact (blood or body fluids) Clinical Features Incubation period: 4-21 days Abrupt onset of nonspecific symptoms Liver function impaired Bleeding & dysregulated coagulation (clotting) Death/shock 6-9 days after onset Case fatality rates high (40-90%)

4. RNA of ~19 kilobases- Produces 7 mRNAs upon infection

5. Viral Glyoprotein (GP) Targeted by Most Vaccine Platforms Glycoprotein (GP) spikes exist as trimers. Structure is incompletely described: crystallography is available only for a portion of EBOV base, GP2 . viral envelope: only GP ectodomain (GP1 and most of GP2) is known to be exposed on exterior surface Virus-associated host cell proteins (specifically or nonspecifically incorporated into virions) have neither been described nor excluded. interior ribonucleoprotein complex (NP, VP35, VP30, L) and matrix (VP40, VP24) diameter 80 nm avg. length 665 nm

7. TREMs (M , neutrophils)

8. asialoglycoprotein receptor (hepatocytes)

9. folate receptor-

11. exposure of N-terminal binding domain

12. fusion, release of RNAviral mRNAs, new proteins Replicase/transcriptase 3’ 5’ NP VP35 VP40 VP30 VP24 L GP Inhibition of Interferon / production -  IRF-3 Inhibition of Interferon // responsiveness -  PY-STAT1

13. Filovirus Vaccine Candidates (USAMRIID)- Good News DNA-based systems Subunit Virus Like Particles (VLP) Vector-based systems VEE replicon Adenovirus vector (NIH & Genphar) Live attenuated platform(s) VSV (deleted VSV GP gene) Safety Potency

15. Correlate/Surrogate of Immunity Assay for Phase I and Phase II vaccine studies.

16. Novel assays that are logistically and economically feasible for Warfighter vaccination and protection.Key Challenges: Immune responses in naive and vaccinated macaques are poorly understood. Innovation will be necessary to find novel methods to assay protective immune responses. Maturity of Technology: TRL 2-3

17. Venezuelan Equine Encephalitis Replicon (VRP) Expressing Filovirus Glycoprotein Genes

18. USAMRIIDVEE RepliconFilovirus Vaccine Development Goals Evaluate VEE replicon (virus-like replicon particles - VRP) vaccine Define dose and route Define antigens necessary for efficacy Glycoprotein (GP) or combination vaccine with six Ebola Zaire proteins Required antigen components Understand protective immune responses Antibody responses Pre- and postexposure T cell responses Correlate of immunity  human assays What are the Protective Adaptive Immune Responses? B Cell NK Cell Antibody Macrophage CTL Dendritic Cells CD4 CD8

20. Availability of immunological reagents

24. Death on day 6-11

25. Lethal to NHPMorbidity (weight Loss), MTD, Viral titers in treated, challenged mice Plaque assay or real-time PCR BALB/c

26. Mutations in Mouse Adapted Ebola Model Nucleotide Amino acid Nucleotide Protein Zaire ’76 Mouse-adapted Changed 683 NP A G S to G 2425 NP T C none 3163 VP35 C T A to V 5219 VP40 T C none 6231 GP T C none 6774 GP T C none 9563 intergenic A G none 10343/44 intergenic A insertion none 10493 VP24 C T T to I 14380 L T C F to L 16174 L A G I to V 16755 L T G none **From Wilson, J., Kondig, J., Kuehne, A., Hart, M.K., GenBank accession number AF499101.

28. Challenge dose ~1,000 pfu

29. Use Hartley guinea pigs

30. Either sex, ~500 g, purchased from Charles River

31. Expect untreated, control Ig-treated controls to die within 7-14 days of challenge

32. Systemic (IP) administration of Mabs.

33. Morbidity (weight loss & Clinical Score), MTD, viremia on various days*.

38. Survival and mean time-to-death

39. Body temperature ; weight changes

40. Hematology, liver enzymes, serum cytokine responsesCynomolgusMacaque n = 3 + 1 control *if multiple doses of vaccine administered, challenge occurs 28 days after final vaccination.

42. Efficacy of VRP-MARV Against Various Agents and Routes of Exposure a = Cynomolgus macaques used in this study. Rhesus macaques used in all other studies b = Similar protection levels achieved with either one or two doses of VRP c = all studies included control animals vaccinated with irrelevant antigen; no control animals survived

43. 20 Development of a Phase I Clinical Trail Product- AlphaVax NIH Grant # 5 UO1 AI53876-4 leveraged with JSTO Funded intramural program. Marburg GP (Ci67), proprietary cGMP compliant vector. Characterized and optimized VRP for suitability for manufacturing: Sequence (replicon plasmid DNA) Optimize VRP yields Adventitious agent assays Release Criteria Pre-pilot lot completed Before final Marburg strain down-selection concluded, a pilot GMP lot of Musoke GP replicon VRP was completed Formal (GLP) toxicology study in rabbits conducted with Musoke GP VRP No toxicity issues identified Pilot lot of Ci67 GP replicon produced (1/3 scale of cGMP run) Technical transfer to AlphaVax Lenoir facility for cGMP production. cGMP product produced and stored at AlphaVax. Ready for pre-clinical and phase I studies.

44. Ebola Vaccine Product HISTORY VRP-ZEBOV GP/NP or GP alone protects guinea pigs. Pushko, et al. J Virol2001. VRP-ZEBOV GP/NP failed to protect at 107 FFU Geisbert et al., Emerg Infect Disease 2002. New approaches taken (multiple proteins/Higher doses) Vaccine is efficacious in NHP. Two vaccine components- expressing Zaire ebolavirus or Sudan ebolavirus glycoprotein. Mouse, guinea pig and NHP data

45. Efficacy of VRP-ZEBOV & VRP-SEBOV Against Various Routes of Exposure a = all studies included control animals vaccinated with irrelevant antigen; no control animals survived b = animals received 2 doses of VRP. Single dose was not uniformly protective (2 out of 3 for EBOVZ and 0 out of 3 for EBOVS

46. Does the vaccine protect & mechanism? Cells Serum (Antibody) Adoptive transfer into unvaccinated mouse Lethal Challenge Vaccinate Mice Protected

47. Antibody Component of Protection Does vaccine induced antibody response provide protection?

48. Survival of Mice Vaccinated to Ebola and Identification of Protective Immune Responses * Survivors/Total (%) Protection Replicon BALB/c C57Bl/6 Sera Transfer GP 9/10 (90%) 10/10 (100%) 32/40 NP 10/10 (100%) 15/16 (93%) 1/40 VP24 15/15 (100%) 0/40 (0%)** 0/20 VP30 25/25 (100%) 25/25 (100%) 0/20 VP35 38/40 (95%) 40/40 (100%) 0/20 VP40 15/15 (100%) 16/20 (80%) 0/20 Lassa N 0/30 (0%) 0/30 (0%) 0/40 * Optimized Vaccine platform ** No protection observed in C57Bl/6 mice vaccinated with VP24 expressing VRP. Protection data improved based on conservative replicon titer estimation.

49. Properties of Ebola GP Monoclonal Antibodies MAb Specificity % Survival Days given Isotype 1 13F6 Zaire GP1 (401-417) 90-100% -1,+1 G2a ATQVEQHHRRTDNDSTA 2 6D8 Zaire GP1 (389-405)90-100% -1,+1 G2aKLDISEATQVE 50-60% +2 3 12B5 Zaire GP1 (477-493) 60-80% -1,+1 G1 GKLGLITNTIAGVAGLI 4 13C6 Zaire, IC, Sudan GP1, sGP conformational 90-100% -1,+1,+2 G2a 5 6D3 Zaire, IC GP1, sGP conformational 80-100% -1,+1,+2 G2a Reference: Wilson J, Hevey M, Bakken, R, Guest S, Bray M, Schmaljohn A, and Hart MK. 2000. Science 287: 1664-1666. Linear Epitope Confirmational Epitope

50. Does the cellular response provide protection for Ebola proteins? GP – portion of protective response is antibody mediated. Other studies have shown Cytotoxic T lymphocyte (CTL) responses Potential CTL component of protection NP, VP24, VP30, VP35, VP40? No protection when serum is transferred CD8 CTL?

51. Method for Identifying Cellular ResponsesIntracellular Cytokine Staining (ICC) Flow Cytometry FACS INF- + CD44 - INF- + CD44 + Vaccinate Mice INF- INF- - CD44 - INF- - CD44 + Spleen cells + Overlapping Peptides 5 hr CD 44 CD8 CD44 *Gate on CD8 cells Intracellular Cytokine Staining (ICC) CD8+, CD44+, INF-+ INF- Negative Positive

52. Individual Screen of A Positive Peptide Pool for VP24 (Balb/c) No peptide 0.09 % Peptide # 34 0.19 % Peptide # 43 3.34 % PMA/Ionomycin Peptide # 31 Peptide # 32 24 % 0.13 % 0.52% Peptide # 36 Peptide # 35 Peptide # 33 0.13% 0.10% 0.38% Anti-INF- PE Peptide # 45 Peptide # 44 Peptide # 42 0.10 % 0.17% 0.62% Anti-CD44 FITC

53. Cr51 Positive Peptide = Potential CTL Epitope CD8+ Positive peptide CD8 CTL CD8+ Binds epitope but unresponsive Lytic CTL Responsive but not lytic Bulk Re-stimulation for 7 days with peptide Adoptive transfer to unvaccinated mice Challenge with Ebola ICC Assay ELISpot + CD8 CTL Functional assay chromium release

54. Ex-vivo 7d restim 51Cr Adoptive Transfer vP24171 20/20 vP24159 19/20 Anti-INF- PE vP24214 17/20 Anti-CD44 FITC

55. CD8 T Cell Responses to Ebola Proteins

56. Summary of Protective Immune Mechanisms Induced in Mice by VEE Replicons Immunogen BALB/c C57Bl/6 GP Ab/CTL(2) Ab/ CTL (1*) NP CTL (1) CTL (3) VP24 CTL (3) None (0) VP30 CTL (3) CTL (2) VP35 CTL (2) CTL (3) VP40 CTL (3) CTL (1) Type of protective response/# of CTL epitopes identified

57. PMA/INO Postchallenge-cynomolgus macaque GP vaccinated No Peptide NP-1 9mer CD69 Negative Pool Lassa Peptide

58. HLA A.201 Epitope Mapping Method 1 Method 2 15-mer Overlapping GP Algorithm Predicted Epitopes Nanomers T2 HLA A.201 (MHC-class I) Stabilization Assay ProImmune Reveal and Prove? Spelling 9-mer peptides + 15-mer MHC I T2 cell + Peptide Background Negative Positive 0.08% 11.60% 18.45%

59. RESULTS REVEALTM MHC peptide Binding Assay: Eighty-one 9–mer custom peptides were generated and assembled with A*0201 and analyzed to determine their level of MHC incorporation. The peptide binding score is shown as a percentage relative to the binding of the pass/fail control. Thirteen peptides had scores greater than the pass/fail control, and two had scores equal to this control. All 15 were considered positives, 14 of which were successfully synthesized for later in-vitro testing.

60. Quick Check Off Rate Analysis: All peptides passing the REVEALTM screen were synthesized as ProVETMpentamers for validation of putative T cell epitopes. Resulting pentamers were incubated and analyzed at 37C to determine peptide-MHC complex stability. Off rates of complexes were measured after 0 h, 2 h, and 24-h of incubation. The percent denaturation is plotted above.

61. The off rates of the peptides in terms of t1/2 half-life values are shown above. The higher the Quick Score the better the epitope. The off rates are plotted above as the t1/2 half-life values. Higher t1/2 values indicate slower off-rates and more stable epitopes

62. Results of the T2-MHC Stabilization Assay %Positive HLA-A*0201 PE on surface of T2 Cells The MHC Stabilization Assay resulted in three strongly positive Ebola GP epitopes: gp17 (GLICGLRQL), gp20 (FLYDRLAST), and gp23 (FLLQLNETI).

63. The results of the MHC Stabilization Assay differed somewhat from the predictions by ProImmune based on the t1/2 values of the 14 9-mer peptides. Gp19 (ILFQRTFSI) was not a positive epitope in the MHC stabilization assay, but was predicted by ProImmune to be the strongest epitope. Our results also indicated that gp17 was the third strongest epitope, but ProImmunes results showed this epitope with a t1/2 value of 14.96 (h) to be just above the pass/fail mark of 16.29 (h). However, our results and ProImmune’s predictions coincided, as gp20 and gp23 were determined to be strong epitopes with both methods. Of the 15-mer overlapping peptides, those that contained the above positive 9-mer peptides sequences (gp17, gp20, and gp23) were consistently positive with the MHC Stabilization Assay. While some other 15-mer peptides that did not contain any of the 14 predicted epitopes by ProImmune resulted in positives, these results could not be consistently reproduced.

64. Conclusions Our studies identified several putative CD8+ T cell eptiopes within the Ebola Zaire glycoprotein. The online and ProImmune binding prediction algorithums were effective at identifying A*02001 HLA binding sequences. While some differences existed between the methods (i.e., strength of binding), each was capable of identifying the putative CD8 epitopes at comparable cost. Future efforts will focus on evaluating the putative epitopes in vivo and determining if the CD8 T cell responses generated by vaccination or following challenge. The functional characteristics of these CD8 responses will be determined.

66. Dr. Lee’s group

67. Vet Med StaffUSAMRIID Julia Biggins Corinne Scully Calli Lear Laura Irene Prugar*, Rebekah M. James ProImmune- Jeremy Fry and staff Funding Acknowledgement The research described herein was sponsored by the Medical Biological Defense Research Program, [0247J098].

68. Animal Use Research was conducted in compliance with the Animal Welfare Act and other federal statutes 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, 1996. The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Disclaimer Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the U.S. Army.

69. Questions/Discussion