1,472 open reading frames from 4 vaccinia and 3 variola virus genomes were computationally screened for conserved Class I MHC epitopes using EpiMatrix (see Methods for details). First, each protein sequence was parsed into 9-mer and 10-mer sequences, each overlapping the next by 8 or 9 amino acids, respectively, for a total of 369,394 9-mers and 367,922 10-mers. Using Conservatrix to discover unique, identical peptides conserved across all vaccinia and variola strains, we narrowed down the Class I smallpox immunome to 27,158 9-mers and 26,287 10-mers. Each of these peptides was then scored for Class I HLA motif matches to the A*0101, A*0201, A*0301, A*2402, B*0702 and B*4403 alleles. More than 1000 EpiMatrix hits (Z-score > 1.64; top 5% of scores) per allele were discovered (data not shown). The top 100 hits for each allele were subject to a BLAST search against the human genome to exclude epitopes that may be recognized as self (with a cutoff of no more than 7 identities in a 9-mer sequence), and the top 40 A2 and 20 B7 peptides in a list of ascending human homology were selected for experimental validation (Figures 2 and 3). Class II HLA Two strategies to identify conserved and immunogenic Class II MHC epitopes were pursued to maximize the likelihood of discovering protective vaccine immunogens. First, an ORF-by-ORF sequence comparison was performed with the Copenhagen vaccinia strain selected as the standard for alignment because it contains the most ORFs of all the strains under consideration. 107 of the 262 ORFs in Vaccinia Copenhagen had matching ORFs in all six alternate strains with at least 80% identity within the first 200 amino acids. These ORFs in Vaccinia Copenhagen were computationally screened using EpiMatrix and ClustiMer to identify epitope dense regions containing sequences predicted to bind multiple Class II HLA alleles (DRB1*0101, *0301, *0401, *0701, *1101, *1301 and *1501). 272 epitope clusters were identified, each bearing at least 90% sequence identity across all seven strains and a cluster score of 15 or above. The sequences were then analyzed by the BLAST algorithm for human homology. Epitope clusters were ranked first by lowest human homology with no more than 7 matches in a 9-mer frame accepted, and then by cluster score. The top 24 epitope clusters were selected for in vitro confirmation (Figure 4). In addition, a 25 th cluster was selected for maximal potential immunogenicity, regardless of human homology. In a second, separate computational screen, ORFs excluded from the investigation above were analyzed using Conservatrix to find identical 9-mers in at least six strains, where minimally three were vaccinia-derived and two variola. 5,781 peptides were discovered, each then scored for binding affinity to a panel of 8 HLA Class II alleles (see above) using EpiMatrix. 786 unique 9-mers were EpiMatrix hits and subsequently input into the EpiAssembler algorithm to identify sets of overlapping, conserved and promiscuous epitopes, termed immunogenic consensus sequence” (ICS) T helper epitopes. 74 ICS with cluster scores greater than 15 were identified and analyzed for human homology using BLAST. Epitope clusters were ranked first by lowest human homology, as above, and then by cluster score. The top 25 ICS were selected for in vitro validation (Figure 4).
1. Faster Vaccines for Biodefense February 18, 2010 www.epivax.com
3. <ul><li>High throughput computing </li></ul><ul><li>Immunoinformatics </li></ul><ul><li>Vaccine design algorithms </li></ul><ul><li>DNA synthesis, scale up, preparation of delivery device </li></ul><ul><li>Delivery via iontophoresis / skin patch </li></ul><ul><li>Animal safety/tox/immunogenicity/validation </li></ul><ul><li>Deployment by mail/pharmacy distribution systems </li></ul>Mandate – Faster, Safer Vaccines Confidential The mandate – Develop vaccines for WMD – pathogen unknown, immediately Does this capacity exist? Yes. The components: This can be prebuilt!! Rapid deployment when genome sequence is in hand
4. Infectious Disease-trained M.D. University of Chicago (1983), Internal Medicine (NEMC 1986). NIH NIAID / NCI: Additional training in parasitology, immunoinformatics and vaccinology (1986-89). CEO of EpiVax since May 1998. $26M in NIH and foundation research funding. Awarded $13M U19 award for vaccine design from the NIH in July 2009 . Associate Professor at Brown University Medical School 1992- present; Professor URI, Director I’Cubed, 2009 –present. Says who? Annie De Groot M.D. CEO, EpiVax
5. EpiVax Management Team http://www.epivax.com/team Confidential Dr. Anne De Groot CEO/CSO Coordinates and directs strategy, business development and scientific programs. William Martin CIO Develops and sustains the infrastructure, informatics, business process, and IT systems in place at EpiVax. Dr. Janet Buhlmann Director of Molecular Immunology Supervises and coordinates a portfolio of scientific projects involving Tregitope technology. Dr. Leonard Moise Scientific Director of Vaccine Research Directly responsible for scientific strategy and laboratory management of all biodefense and NTD vaccine programs.
6. EpiVax: Four Core Strengths http://www.epivax.com/services Confidential EpiVax Services Epitope Mapping HLA Binding T cell assays In vivo assays (HLA Tg mice) Fee for Service EpiVax Vaccines Grant funded R & D Excellent proof of principle Grant Funded (SBIR, R21, R01; >26M in funding since 1998) EpiVax 2nd Generation Therapeutics Select targets Funded Res. Or Joint Devt Develop molecule and license Sponsored Research / Joint Development Immuno-modulation “ Epi-13” Tregitopes In preclinical development- may be large market - allergy, autoimmunity Options available for selected “Field of Use”
7. The Traditional Approach to Vaccines Effects of whole virus/bacteria unpredictable-contradictory; Process lengthy, prone to process issues, regulatory delays Cross- reactive with Self or other Pathogens T reg epitopes? Skew immune response?
10. Vaccine Design Tools and Techniques http://www.epivax.com/platform Confidential <ul><li>We use a comprehensive suite of tools and techniques for screening and deimmunizing therapeutics </li></ul><ul><li>EpiMatrix (CTL/ T Helper) </li></ul><ul><li>ClustiMer (Promiscuous Epitopes) </li></ul><ul><li>Immunogenicity Scale (Ranking Proteins) </li></ul><ul><li>OptiMatrix (Pinpoint Deimmunization) </li></ul><ul><li>HLA Binding Assays (Class I/Class II) </li></ul><ul><li>Ex-vivo immunoassays (ELISpot, ELISA, Searchlight) </li></ul><ul><li>HLA Transgenic Mice (Class II) </li></ul>
11. Genome-Derived, Epitope-Driven Vaccine Approach: Confidential Emergency Use Authorization may obviate need for these lengthy pre-clinical tests In Silico EpiMatrix / ClustiMer / OptiMatrix [class I and class II alleles] Conservatrix / BlastiMer/. EpiAssembler/ VaccineCAD In Vitro HLA binding assay ELISpot - ELISA - Multiplex ELISA - FACS - T regulatory T cell profiling In Vector DNA prime/peptide (pseudoprotein boost) vaccines Vaccine delivery / formulation optimization / detolerizing delivery agents In Vivo HLA DR3, DR4 transgenic mice HLA class I transgenic mice Vaccination, Comparative studies
12. Proposed “Cassette” Approach to Faster Vaccines “ On Demand” PREPARED IN ADVANCE ! Step 1 Step 2 Step 3
13. Design can be done “on the fly” literally within 24 hours What the proposed product? An ‘on demand’ vaccine In Silico – Discover Minimum Essential Units of Pathogens Driving Immune Response Safe: Eliminate all cross-reactive entities and toxicity in silico Concatenate in “chain” of information using “VaccineCAD” Rapid insertion into DNA Plasmid; fast manufacturing scale up; Electroporation through skin (iontophoresis) on pre-manufactured micro needle patches
14. Components of supply chain that would be prepared in advance
15. Proposed Partners for Faster Vaccines Pilot Project
17. 1. Downloaded Y16780(V. Minor), X69198 (V. Majo), L22579 Bangladesh and U94848 (Ankara), M35027 (Copenhagen, AF095689 (Xian tan), AY243312 (WR) Genomes from GenBankå 2. Identified highly conserved sequences - two methods (ICS and straight conservation) 3. 4. Identified potential Class II T cell clusters as well as A2, A24, B7, B44 epitopes 4. Analyzed Epitopes for homology to human sequences using BLAST Algorithm 5. Down selected best candidates AF095689 (Xian tan) M35027 (Copenhagen) U94848 (Ankara) L22579 Bangladesh AY243312 (WR) Y16780 (V. Minor) X69198 (V. Major)
18. Genome-derived epitope driven vaccine Construct Design / Assembly DNA insert Intended Protein Product: Many epitopes strung together in a “String-of-Beads” Reverse Translation: Determines the DNA sequence necessary to code for the intended protein. This DNA is assembled for insertion into an expression vector. Protein product (folded) DNA Vector
19. Design the arrangement of the epitopes to minimize the immunogenicity of junctional peptides and focus the immune response to the desired epitopes 1 2 3 Improving Vaccine Design by aligning epitopes : Vaccine-CAD De Groot AS , Marcon L, Bishop EA, Rivera D, Kutzler M, Weiner DB, Martin W. HIV vaccine development by computer assisted design: the GAIA vaccine. Vaccine. 2005
20. Example: Vaccine CAD Confidential
21. Confidential Example: Vaccine CAD – final order
22. Result of Live Aerosol Challenge: 100% survival of GD-ED vaccinated mice vs. 17% of placebo Confidential 100% 0 20 40 60 80 100
23. How to deliver Genome Derived –Epitope Driven Vax DNA – chain of epitopes, or peptide in liposomes ICS-optimized proteins in VLP ICS-optimized whole proteins
24. DNA Vaccines – A Faster Solution <ul><li>Ronald B Moss: Prospects for control of emerging infectious diseases with plasmid DNA vaccines </li></ul><ul><li>Journal of Immune Based Therapies and Vaccines 2009, 7:3 </li></ul><ul><li>http://www.jibtherapies.com/content/7/1/3 </li></ul><ul><li>Experiments almost 20 years ago demonstrated that injections of a sequence of DNA encoding part of a pathogen could stimulate immunity. It was soon realized that "DNA vaccination" had numerous potential advantages over conventional vaccine approaches including inherent safety and a more rapid production time. These and other attributes make DNA vaccines ideal for development against emerging pathogens. Recent advances in optimizing various aspects of DNA vaccination have accelerated this approach from concept to reality in contemporary human trials. </li></ul><ul><li>Although not yet licensed for human use, several DNA vaccines have now been approved for animal health indications. The rapid manufacturing capabilities of DNA vaccines may be particularly important for emerging infectious diseases including the current novel H1N1 Influenza A pandemic, where pre-existing immunity is limited . Because of recent advances in DNA vaccination, this approach has the potential to be a powerful new weapon in protecting against emerging and potentially pandemic human pathogens. </li></ul>
25. If Needed - Validation: In Vivo Model for validation: HLA or humanized transgenic Mice HLA DR3 HLA A2/DR1 HLA DR1 HLA D2 HLA A2 HLA B7
26. EpiVax Vaccine Program Overview Confidential <ul><ul><li>Smallpox – a safer VV vaccine? </li></ul></ul><ul><ul><li>“ MDR” and “XDR” TB . . . (with Sequella) </li></ul></ul><ul><ul><li>Tularemia </li></ul></ul><ul><ul><li>Multipath (Burkholderia/Tularemia </li></ul></ul><ul><ul><li>. . . and with the DoD. . . </li></ul></ul><ul><ul><li>A multi epitope vaccine for Ebola, EE’s, </li></ul></ul><ul><ul><li>and Bacterial pathogens (Tularemia/Burk) </li></ul></ul>
27. Current Collaboration with the DoD Confidential <ul><li>EpiVax is currently funded to design, develop, and test a single T cell epitope based vaccine targeting three bacterial bioterror agents with NMRC (PI Dr. Andrea Keane Meyers). The targets of this program are Francisella tularensis (the causative agent of Tularemia and category A priority pathogen), Yersinia pestis (plague, a category A priority pathogen), and Burkholderia mallei (a category B priority pathogen). Funding for this project would enable and accelerate the EpiVax-NMRC vaccine design and development collaboration. </li></ul><ul><li> </li></ul><ul><li>EpiVax is also designing, developing, and testing a multipathogen vaccine targeting two viral bioterror agents with USAMRIID (PI Dr. Connie Schmaljohn). The targets of this program are Ebola (a category A priority pathogen) and Venezuelan Equine Encephalitis (VEE, a category B priority pathogen). Funding for this project would enable and accelerate the EpiVax-USAMRIID vaccine design and development collaboration. </li></ul><ul><li>Both vaccines have dual military and civilian, war time and peacetime use. The goal of the combined program (USAMRIID and NMRC) is to develop a single, combined anti-bioterror vaccine. This Department of Defense-biotech collaborative project is consistent with the goals of the BDRD and DTRA chem-bio defense teams to develop vaccines against category A-C viral and bacterial bio-threat agents. </li></ul>
28. New tools for new vaccine challenges <ul><li>Characteristics of current vaccine development processes </li></ul><ul><ul><li>Inefficient </li></ul></ul><ul><ul><li>Expensive </li></ul></ul><ul><ul><li>Time consuming (20+ years to vaccine) </li></ul></ul><ul><ul><li>High risk </li></ul></ul><ul><ul><li>Relatively unchanged for decades </li></ul></ul><ul><li>Vaccine Challenges and Emergent IDs - a new level of risk </li></ul><ul><ul><li>Unprecedented infectious disease threats (population density, global travel, global warming, widespread bioengineering technology and capabilities) </li></ul></ul><ul><ul><li>10+ years from threat to vaccine is unacceptable </li></ul></ul><ul><ul><li>Advances in genomics, microbiology and immunology (including high throughput systems) create an opportunity to re-think current processes </li></ul></ul><ul><li>Why wait? The time to begin “smarter, faster” vaccines is now! </li></ul>Is it time to Get “Smart” about Vaccines?
29. Who uses EpiVax Tools? The U.S. Department of Defense* Roche Amgen Eli Lilly Abbott Boehringer Ingelheim BMS And many, many more. . . Confidential
30. Selected publications: <ul><li>McMurry JA, et al. (TB) Current Molecular Medicine. 2007 Jun; 7(4): 351-68. </li></ul><ul><li>McMurry JA, et al. (Tularemia) Vaccine. 2007 Apr 20; 25(16): 3179-91. </li></ul><ul><li>De Groot AS, Moise L. (Review of tools). Exp. Rev Vaccines. 2007 Apr.: 6 (2): 125-7. </li></ul><ul><li>Knopf, P. , et al. (C3d) Immunology and Cellular Biology. 2008 Mar-Apr;86(3):221-5. </li></ul><ul><li>L. Moise et al. (H. pylori). Human Vaccines 2008, Hum Vaccin. 2007 Dec 8;4(3). </li></ul><ul><li>A. S. De Groot, et al. (HIV) Identification of Immunogenic HLA-B7 “Achilles’ heel” Epitopes Within Highly Conserved Regions of HIV. Vaccine. Vaccine. 2008 Jun 6;26(24):3059-71. </li></ul><ul><li>Gregory SH, Mott S, Phung J, Lee J, Moise L, McMurry JA, Martin W, De Groot AS.Epitope-based vaccination against pneumonic tularemia. Vaccine. 2009;27(39):5299-306. </li></ul><ul><li>Leonard Moise, Julie A. McMurry, Soren Buus, Sharon Frey, William D. Martin and Anne S. De Groot. In Silico-Accelerated Identification of Conserved and Immunogenic Variola/Vaccinia T-Cell Epitopes, Vaccine (David Weiner, special editor). http://dx.doi.org/10.1016/j.vaccine.2009.06.018 Vaccine. 2009 Jun 24. </li></ul><ul><li>McMurry J, Johansson BE, and De Groot AS . A call to cellular & humoral arms: Enlisting cognate T-cell help to develop broad-spectrum vaccines against influenza A. Human Vaccine. 2008;4(2):148-57. </li></ul><ul><li>De Groot AS , Ardito M, McClaine EM, Moise L, Martin W. Immunoinformatic comparison of T-cell epitopes contained in novel swine-origin influenza A (H1N1) virus with epitopes in 2008-09 Conventional Influenza Vaccine. Vaccine. 2009. Aug 3. </li></ul>