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Gene Olinger, USAMRIID, Fort Detrick USA, presents at the ProImmune Antigen Characterization and Biomarker Discovery Summit, January 2011. ...

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

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  • 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
    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%)
  • 3. Structural Proteins of Ebola Virus
    Organization of the Ebola Virus Genome
    NP Major nucleocapsid protein
    VP35 Phosphoprotein, IFN antagonist
    VP40 Membrane-associated matrix protein
    GP Transmembrane glycoprotein
    sGP Secreted glycoprotein
    VP30 Ribonucleoprotein associated (minor)
    VP24 Membrane-associated protein (minor)
    L RNA-dependent RNA polymerase
    • Single-strand negative sense
    • 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.
    ribonucleoprotein complex
    (NP, VP35, VP30, L)
    and matrix (VP40, VP24)
    diameter 80 nm
    avg. length 665 nm
  • 6. GP on viral surface:
    - GP as binding moiety
    - cellular proteins also incorporated?
    Progeny virions:
    - particle formation at lipid rafts
    - typically made 12 - 48 hours after cell infection
    “dispatched” GP
    (GP1, and cleaved anchorless forms)
    (truncated, frame-shifted GP from non-edited GP gene; EBOV, not MARV)
    • C-type lectins, DC-SIGN (DC, M)
    • 7. TREMs (M , neutrophils)
    • 8. asialoglycoprotein receptor (hepatocytes)
    • 9. folate receptor-
    • 10. TLR, other?
    cell surface GP
    New genomes
    nucleoprotein complex
    Stepwise proteolysis of GP
    - involves endosomal cathepsins, pH
    • partial removal of GP1(mucin-like region)
    • 11. exposure of N-terminal binding domain
    • 12. fusion, release of RNA
    viral mRNAs, new proteins
    Inhibition of Interferon / production
    -  IRF-3
    Inhibition of Interferon // responsiveness
    -  PY-STAT1
  • 13. Filovirus Vaccine Candidates (USAMRIID)- Good News
    DNA-based systems
    Virus Like Particles (VLP)
    Vector-based systems
    VEE replicon
    Adenovirus vector (NIH & Genphar)
    Live attenuated platform(s)
    VSV (deleted VSV GP gene)
  • 14. CBM.VAXV.03.10.RD.PP.004
    Filovirus Vaccine Induced Immunity & Virus Induced Immunopathology.
    Gene Olinger, USAMRIID
    Objective:Characterize the innate and adaptive immune responses in Filovirus infection that result in immune pathology and elucidate the role of the innate and adaptive immune responses in vaccinated animals. Develop assays that predict protection in vaccinated macaques that can be applicable tohuman vaccine trials.
    Description of Effort: The immune dysregulation induced by virus infection and the correlates of protective immune in vaccinated macaques are unclear. Proposal will focus understanding protective immunity for filoviruses & developing assays that predict protection. These immunoassays will be useful for all vaccine platforms being tested.
    Monocytes/DC cells
    CD 4 T cells
    Plasma B cells
    CD 8 T cells
    T Regs
    NK cells
    Major Goals/Milestones:
    FY10: Begin “Good” & “Bad” vaccination to learn about innate and adaptive immunity following vaccination. Develop new assays that will have predictive power in animal models of disease (guinea pigs and macaque).
    FY10-11: Define protective responses, innate, cellular and/or humoral. Develop assays that predict protection.
    FY011: Apply knowledge gained in understanding immune dysfunction to restore innate and adaptive immune responses. Validate assays with blinded vaccinated macaque samples.
    Contact Information:
    Gene Garrard Olinger, Jr., Ph.D., MBA
    1425 Porter Street, Virology Division, Frederick, MD 21702
    (301) 619-8581, Gene.olinger@us.army.mil
    Benefits of Proposed Technology:
    • Understanding of immunity during disease & after vaccination; a correlate of protective immunity.
    • 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
    Dendritic Cells
  • 19. Animal Models for Filoviruses
    Mouse, Guinea Pig, Nonhuman Primate (NHP)
    • Critical link that may be difficult to achieve with animal data
  • Mouse Adapted Ebola Model
    Murine (Inbred) Model
    • Well-characterized immune system
    • 20. Availability of immunological reagents
    • 21. Access to inbred mouse strains
    • Bray et al, J. Infectious Diseases, 1998, Sep. 178: 651-61
    • 22. Ebola Zaire 76 adapted by sequential passage in mice,
    plaque pick with 100% lethality
    • Challenge Dose: 10 to 1000 PFU (~30,000 LD50)
    • 23. Ruffled fur observed from day 5 onward (Balb/c)
    - Viremias on day 7 as high as 6 log10 PFU/ml
    • LD50 < 1 PFU in adult mice (BALB/c) by i.p. route
    • 24. Death on day 6-11
    • 25. Lethal to NHP
    Morbidity (weight Loss), MTD, Viral titers in treated, challenged mice
    Plaque assay or real-time PCR
  • 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
  • 27.
    • Experimental design:
    • 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*.
    • 34. Possible to run blood counts & other**.
    Filovirus Guinea Pig Models
    • Hartley
    • 35. USAMRIID Strain 13
  • Nonhuman Primate Filovirus Models:Macaques
    Most nonhuman primates are naturally susceptible to human isolates of both Ebola and Marburg viruses
    For Ebola virus, rhesus macaques are routinely used for therapeutic studies
    Challenge dose is 1,000 pfu
    Given by intramuscular injection
    Current Ebola virus (Zaire) stock is 100% lethal in Rhesus macaques in our studies (n=17)
    Current Sudan virus (Boniface) stock is 100% lethal in Rhesus (n=6)
  • Development of Disease in NHP
    Onset of viremia and recovery of virus from target organs
    Asymptomatic or Preclinical Disease
    Days post infection
    Cyno 0 1 2 3 4 5 6
    Rhesus 0 1 2 3 4 5 6 7 8 - 10
    Day 2
    Day 3
    Day 4
    Day 5
    Day 6
  • 37. Filovirus VRP Vaccine Efficacy Studies: Experimental Design
    bleed dates
    -28* 0 3 5 7 10 14 21 28
    Rhesus Macaque
    Vaccinate animals with VRP
    Virus exposure; 1000PFU
    End of study
    Efficacy read-outs:
    • Viremia, plaque assay and real-time PCR
    • 38. Survival and mean time-to-death
    • 39. Body temperature ; weight changes
    • 40. Hematology, liver enzymes, serum cytokine responses
    n = 3 + 1 control
    *if multiple doses of vaccine administered, challenge occurs 28 days after final vaccination.
  • 41. Protection of VRP-vaccinated CynomolgusMacaques from Lethal Challenge with Marburg Virus (Musoke Isolate)a
    GP 3/3* 0/3 -
    NP 2/3 3/3 8
    GP+NP 3/3* 0/3 -
    Flu HA 0/3 3/3 9, 9, 10
    aCynomolgus Macaques were challenged with 1000 PFU MBGV (Musoke) subcutaneously. Surviving animals remained healthy 11 months post challenge.
    b Denotes the antigen delivered by VEE replicon (3 doses, 107 FFU/dose, 1 month intervals)
    c All (and only) animals that displayed signs of illness were demonstrably viremic
    * p = 0.05
    • Hevey, et. al., Virology, 1998: 1st demonstration of protective filovirus vaccine!
  • Protection against Ci67, Ravn & Musoke Virus after Immunization with Ci67 VRP (guinea pigs)
    a Guinea pigs were challenged with 1000 PFU MBGV (Ci67) IP.
    b Denotes dose of VEE replicon FFU/dose, control replicon used at 108 FFU/ml
    Guinea pigs groups consist of 3 males and 3 females for at total of 6.
  • 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
    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?
    Adoptive transfer into
    unvaccinated mouse
    Lethal Challenge
    Vaccinate Mice
  • 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
    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- -
    CD44 -
    INF- -
    CD44 +
    Spleen cells + Overlapping Peptides
    5 hr
    CD 44
    *Gate on CD8 cells
    Intracellular Cytokine Staining (ICC)
    CD8+, CD44+, INF-+
  • 52. Individual Screen of A Positive Peptide Pool for VP24 (Balb/c)
    No peptide
    0.09 %
    Peptide # 34
    0.19 %
    Peptide # 43
    3.34 %
    Peptide # 31
    Peptide # 32
    24 %
    0.13 %
    Peptide # 36
    Peptide # 35
    Peptide # 33
    Anti-INF- PE
    Peptide # 45
    Peptide # 44
    Peptide # 42
    0.10 %
    Anti-CD44 FITC
  • 53. Cr51
    Positive Peptide = Potential CTL Epitope
    Positive peptide
    CD8 CTL
    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
    CD8 CTL
    Functional assay chromium release
  • 54. Ex-vivo 7d restim 51Cr Adoptive Transfer
    Anti-INF- PE
    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
    Negative Pool
    Lassa Peptide
  • 58. HLA A.201 Epitope Mapping
    Method 1 Method 2
    15-mer Overlapping GP
    Algorithm Predicted Epitopes
    T2 HLA A.201 (MHC-class I)
    Stabilization Assay
    ProImmune Reveal and Prove? Spelling
    9-mer peptides
    MHC I
    T2 cell + Peptide
    Background Negative Positive
  • 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.
  • 65. United States Army Institute for Infectious Diseases (USAMRIID)Virology Division
    • Dr. Hensley’s group
    • 66. Dr. Lee’s group
    • 67. Vet Med Staff
    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.
    Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the U.S. Army.
  • 69. Questions/Discussion