epivax analysis of ebolavirus zaire ebolavirus - mayinga (the current ebola outbreak strain) 2014

EpiVax Analysis of Ebolavirus:

Zaire Ebolavirus - Mayinga

(the current Ebola outbreak strain)

An immunoinformatics data set for Ebola vaccine

developers

Compiled using EpiVax state-of-the-art tools

For more information see http://bit.ly/EpiVax_Vaccine

http://bit.ly/EpiVax_Vaccine

• Biotech company based in Providence, Rhode Island

• Owned and operated by Annie De Groot and Bill Martin

Who Are We? (EpiVax)

2

EpiVax designs and develops

safer, more effective biologic

products and vaccines

Engaged

Dedicated to improving human

health everywhere

Cutting Edge

Continuously innovating

thought leaders in

immunoinformatics

Trusted

Highly Published

Trusted by majority of large

Pharma companies

2 8/20/2014 EpiVax - Non-confidential

2014 West Africa Ebola Outbreak Rapidly Expanding Lethal Virus Outbreak

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http://www.cdc.gov/vhf/ebola/resources/distribution-map-guinea-outbreak.html

8/20/2014 EpiVax - Non-confidential








• Four countries affected: Sierra Leone, Liberia, Guinea, Nigeria

• As of Aug. 15th 2014:

– Suspected and Confirmed Case Count: >2000

– Suspected Case Deaths: >1000

– Mortality Rate: >50%

• No known cure

• No available vaccine

2014 West Africa Outbreak Statistics

4

http://www.cdc.gov/vhf/ebola/outbreaks/guinea/


http://www.cdc.gov/vhf/ebola/outbreaks/guinea/

How Can We Help?

• iVAX is a key tool in designing effective vaccines in a rational, efficient manner

• Already providing access to iVAX toolkit to our commercial and academic collaborators

• We can rapidly analyze Ebola for optimal vaccine components

• Perhaps this will be helpful for better design of Ebola vaccines, making it available to the scientific public is what we can do to help.

• Any questions – please contact [email protected]


mailto:[email protected]

Why are we releasing findings online:

• EpiVax has unique technology for designing vaccines.

• The technology accurately predicted the high immunogenicity of pandemic H1N1 (only one dose of vaccine needed, most individuals protected by seasonal flu exposure) http://bit.ly/Pandemic_H1N1

• The technology accurately predicted H7N9 low immunogenicity “stealth virus”. http://bit.ly/H7N9_Stealth

• We want to make our contribution to stopping Ebola by making our immunoinformatics analysis of the current virus available world-wide.

EpiVax Contributes to Stopping Ebola


http://bit.ly/Pandemic_H1N1

http://bit.ly/Pandemic_H1N1

http://bit.ly/H7N9_Stealth



iVAX – Quick Facts

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• Comprehensive set of vaccine design tools

• Flexible utilization – vaccine design, antigen engineering, vaccine efficacy analysis

• Applications to assay reagent development and diagnostics (ELISpot assays, e.g.)

• Compatible with modular tool development (ex insertion of tools for animal vaccine design)

• Interactive web access any time

• Involved training program and technical service


http://bit.ly/EpiPubs

8

iVAX Toolkit – Commercial



iVAX Toolkit – Academic

9

10

Vaccine Design Tools and Techniques

Epitope/Antigen Analysis and Discovery

Vaccine Construct Design/ Optimization

Lab Validation

10

JanusMatrix: Analyze T cell epitope cross-reactivity

TB Epitope

Discovery

Epitope

Validation

Construct

Design

Immuno-

genicity

Tularemia Epitope

Discovery

Epitope

Validation

Construct

Design

Immuno-

genicity

Animal Model

Validation

Smallpox Epitope

Discovery

Epitope

Validation

Construct

Design

Animal Model

Validation

HCV Epitope

Discovery

Epitope

Validation

Construct

Design

Animal Model

Validation

H. pylori Epitope

Discovery

Epitope

Validation

Construct

Design

Animal Model

Validation

Animal Model

Validation

Immuno-

genicity

Immuno-

genicity

Immuno-

genicity

EpiVax Vaccine Design Pipeline

HIV Epitope

Discovery

Epitope

Validation

Construct

Design

Immuno-

genicity

Animal Model

Validation

Influenza Epitope

Discovery

Epitope

Validation

Construct

Design

Immuno-

genicity

Animal Model

Validation

VEEV/EEV Epitope

Discovery

Epitope

Validation

Construct

Design

Immuno-

genicity

Animal Model

Validation

Burkholderia Epitope

Discovery

Epitope

Validation

Construct

Design

Immuno-

genicity

Animal Model

Validation

Selected publications:

12

Publications related to Vaccines 2013-2014

• Lu He, Anne S De Groot, Andres H Gutierrez, William D Martin, Lenny Moise and Chris Bailey-Kellogg.

Integrated assessment of predicted MHC binding and cross-conservation with self reveals patterns of

viral camouflage. BMC Bioinformatics 2014, 15 (Suppl 4): S1 doi:10.1186/1471-2105-15-S4-S1.

http://bit.ly/Viral_Camouflage_2014.

• Zhang S, Desrosiers J, Aponte-Pieras JR, Dasilva K, Fast LD, Terry F, Martin WD, De Groot AS, Moise L,

Moss SF. Human Immune Responses to H. pylori HLA Class II Epitopes Identified by Immunoinformatic

Methods, PLoS One. 2014 Apr 16;9(4):e94974. doi: 10.1371/journal.pone.0094974. eCollection 2014. PMID:

24740005 [PubMed - in process]. http://bit.ly/HPylori_PLOS_One_2014

• De Groot AS, Moise L, Liu R, Gutierrez AH, Terry F, Koita OA, Ross TM, Martin W. Cross-conservation of T-

cell epitopes: now even more relevant to (H7N9) influenza vaccine design. Hum Vaccin Immunother.

2014 Feb;10(2):256-62. doi: 10.4161/hv.28135. Epub 2014 Feb 13. PMID: 24525618 [PubMed - in process].


• Moise L, Gutierrez AH, Bailey-Kellogg C, Terry F, Leng Q, Abdel Hady KM, Verberkmoes N, Sztein MB,

Losikoff P, Martin WD, Rothman A, De Groot AS. The two-faced T cell epitope: Examining the host-

microbe interface with JanusMatrix. Hum Vaccin Immunother. 2013 Apr 12;9(7). PubMed PMID 23584251.

• De Groot AS, Ardito M., Terry, F. Levitz L., Ross T., Moise L., Martin B. Low immunogenicity predicted for

emerging avian-origin H7N9: Implication for influenza vaccine design. Hum Vaccin Immunother. Volume

9, Issue 5 May 2013:950–956 http://dx.doi.org/10.4161/hv.24939 http://bit.ly/H7N9_2013.

http://bit.ly/H7N9_HVandI.

• Moise L, Terry F, Ardito M, Tassone R, Latimer H, Boyle C, Martin WD. Universal H1N1 influenza vaccine

development: Identification of consensus class II hemagglutinin and neuraminidase epitopes derived

from strains circulating between 1980 and 2011. Human Vaccines & Immunotherapeutics 2013; 9:1598 -

1607; PMID: 23846304; http://dx.doi.org/10.4161/hv.25598 8/20/2014 EpiVax - Non-confidential

http://bit.ly/Viral_Camouflage_2014

http://bit.ly/HPylori_PLOS_One_2014


http://dx.doi.org/10.4161/hv.24939

http://bit.ly/H7N9_2013



Selected publications:

13

• Wei R, Xu L, Zhang N, Zhu K, Yang J, Yang C, Deng C, Zhu Z, De Groot AS, Altmeyer R, Zeng M, Leng Q.

Elevated antigen-specific Th2 type response is associated with the poor prognosis of hand, foot and

mouth disease. Virus Res. 2013 Jul 22. doi:pii: S0168-1702(13)00231-1. 10.1016/j.virusres.2013.07.009.

• Leonard Moise, Ryan Tassone, Howard Latimer, Frances Terry, Lauren Levitz, John P. Haran, Ted M. Ross,

Christine Boyle, William D. Martin, Anne S. De Groot. Immunization with Cross-conserved H1N1 Influenza

CD4+ T Cell Epitopes Lowers Viral Burden in HLA DR3 Transgenic Mice. Hum Vaccin Immunother. 2013

Sep 17;9(10). http://bit.ly/H1N1_DR3_2014

• Moise L., Marcello, A, Tassone, R., Cousens, L., Martin, W., De Groot, A.S., Building Better Biotherapeutics

and Vaccines by Design: EpiVax, Inc., an Immunology Company. Rhode Island Medical Journal, February

2013. rimed.org/rimedicaljournal/2013-02/2013-02-19-bio-epivax.pdf

• He Y, Cao Z, De Groot AS, Brusic V, Schönbach C, Petrovsky N. Computational vaccinology and the

ICoVax 2012 workshop. BMC Bioinformatics. 2013;14 Suppl 4:I1.doi: 10.1186/1471-2105-14-S4-I1. Epub

2013 Mar 8. PubMed PMID: 23514034; PubMed Central PMCID: PMC3599086.

• De Groot AS, Einck L., Moise L, Chambers M., Ballantyne J., Malone RW, Ardito M, Martin W. Making

Vaccines “On Demand”: A Potential Solution for Emerging Pathogens and Biodefense? Human

Vaccines and Immunotherapy. Accepted for publication June 27, 2013. http://bit.ly/FastVax

• Leonard Moise, Frances Terry, Andres H. Gutierrez, Ryan Tassone, Phyllis Losikoff, Stephen H. Gregory,

Chris Bailey-Kellogg, William D. Martin, Anne S. De Groot. Time for T? Immunoinformatics addresses the

challenges of vaccine design for NT and EID. Expert Review of Vaccines, Accepted 3 Aug 2014.

• Opinion. Anne S. De Groot, Lenny Moise, Rui Liu, Andres H. Gutierrez, Ryan Tassone, Chris Bailey-Kellogg,

William Martin. Immune Camouflage: Relevance to Vaccine Design and Human Immunology Submitted 3

Aug 2014, July 2014, Human Vaccines and Immunotherapy.

• Moise L, Terry F, Gutierrez AH, Tassone R, Losikoff P, Gregory SH, Martin WD, De Groot AS. Smarter

vaccine design will circumvent regulatory T cell-mediated evasion in chronic HIV and HCV infection. In:

Why vaccines to HIV, HCV and Malaria have so far failed - challenges to developing vaccines against

immunoregulating pathogens. Accepted August 15 2014. Frontiers in Microbiology. 2014. Editor (Gowans).

http://bit.ly/H1N1_DR3_2014

http://bit.ly/FastVax

http://bit.ly/FastVax

Steps to Vaccine Design

• Define Pathogen (genome, diagnostic test)

• Correlates of Immunity?

• Critical Antigens - one or many?

• Animal Model? Does it predict protection?

• Prototype Vaccine - Preclinical Proof

• Safety and Toxicity, GMP, Stability

• FDA “IND” (Investigational New Drug)

• Clinical trials (Phase I, II, III)

• FDA Approval / Distribution / Access


• Circulating strain: Zaire Ebolavirus

Ebolavirus Circulating strain


Uniprot contains data for 16 Filoviridae strains:

Filoviridae

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Code Strain EBOEC Zaire ebolavirus (strain Eckron-76) EBOG4 Zaire ebolavirus (strain Gabon-94) EBOIC Ivory Coast ebolavirus (strain Cote d'Ivoire-94)

EBORE Reston ebolavirus (strain Philippines-96)

EBORR Reston ebolavirus (strain Reston-89)

EBORS Reston ebolavirus (strain Siena/Philippine-92) EBOSB Sudan ebolavirus (strain Boniface-76)

EBOSM Sudan ebolavirus (strain Maleo-79) EBOSU Sudan ebolavirus (strain Uganda-00)

EBOZ5 Zaire ebolavirus (strain Kikwit-95)

EBOZM Zaire ebolavirus (strain Mayinga-76) <strain that is analyzed here MABVA Lake Victoria marburgvirus (strain Angola/2005) MABVM Lake Victoria marburgvirus (strain Musoke-80)

MABVO Lake Victoria marburgvirus (strain Ozolin-75) MABVP Lake Victoria marburgvirus (strain Popp-67)

MABVR Lake Victoria marburgvirus (strain Ravn-87)


• Data was obtained from Uniprot

• Filoviridae reference dataset was downloaded by searching the Protein Knowledgebase (UniProtKB) (query: taxonomy:11266 AND reviewed:yes)

• 97 retrieved sequences, by protein:

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iVAX Analysis of Ebola Virus Data Retrieval

Protein Count Protein Count

NP 12 ssGP 5

VP35 10 VP30 10

VP40 11 VP24 11

GP 16 L 11

sGP 11


http://www.uniprot.org/

http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=11266

• RNA virus, member of the Filoviridae family (composed of the Cuevavirus, Ebolavirus, and Marburgvirus genera)

• Ebolavirus contains seven genes:

3’ – leader – NP – VP35 – VP40 – GP/sGP – VP30 – VP24 – L – tailer – 5’

• “Envelope glycoprotein” (GP) is the sole protein expressed at the surface of the virus

• GP is likely to be a good candidate for vaccines

Ebolavirus Structure

8/20/2014 EpiVax - Non-confidential 19

“Immunogenicity Scale”


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The number of T cell epitopes

contained in the sequence of a

protein contributes to its

immunogenicity

Proteins can be compared based on

T cell epitope content per unit

sequence.



gB-2 (EPX Score: -24.56)

- 80 -

- 70 -

- 60 -

- 50 -

- 40 -

- 30 -

- 20 -

- 10 -

- 00 -

- -10 -

- -20 -

- -30 -

- -40 -

- -50 -

- -60 -

- -70 -

- -80 -

Thrombopoietin

Human EPO

Tetanus Toxin

Influenza-HA

Albumin

IgG FC Region

EBV-BKRF3

Fibrinogen-Alpha

Follitropin-Beta

HA A/California/07/2009 (H1N1)

HA A/Victoria/361/2011 (H3N2)

HA A/Texas/50/2012 (H3N2)

HA A/Anhui/1/2013 (H7N9)

HA A/mallard/Netherlands/09/2005 (H7N7)

Random Expectation

HA A/mallard/Netherlands/12/2000 (H7N3)

HA A/chicken/Italy/13474/1999 (H7N1)

H7 HA Immunogenic Potential

New H7N9 Flu is Predicted to be

POORLY IMMUNOGENIC

http://bit.ly/H7N9_HVandI 8/20/2014 EpiVax - Non-confidential

http://bit.ly/H7N9_HVandI



iVAX Analysis of Ebola Virus Class II HLA Binding Analysis – Immunogenicity Scale

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• Immunogenicity scale of Zaire Ebolavirus – Mayinga proteins (with EpiMatrix scores) – Red is high potential, blue is low.

Excellent vaccine

candidate

Poor vaccine

candidate

Theoretical minimum

antigenicity score


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ClustiMer tool finds “hot spots”

DRB1*0101

DRB1*0301

DRB1*0401

DRB1*0701

DRB1*0801

DRB1*1101

DRB1*1301

DRB1*1501

• T cell epitopes are not randomly distributed but instead tend to cluster in specific regions. – These clusters can be very powerful, enabling significant immune responses to low-scoring proteins.

• ClustiMer recognizes T cell epitope clusters as polypeptides predicted to bind to an unusually large number of HLA alleles.

• T cell epitope clusters make excellent vaccine candidates:

– compact; relatively easy to deliver as peptides; highly reactive in-vivo 8/20/2014 EpiVax - Non-confidential

26

DNA

Vector

DNA insert

GP protein cartoon with immunogenic epitope clusters in red:

Protein product:

many epitope

clusters as a

“string of beads”

Concept: Multi-Epitope Vaccine Design

GP is the sole protein expressed at the surface of the virus: 676 residues full length

DNA – chain of epitopes, or peptide in liposomes ICS-optimized proteins in VLP ICS-optimized whole proteins

Possible Delivery Platforms

Protein Code # clusters

NP NP_EBOZM 14

VP35 VP35_EBOZM 5

VP40 VP40_EBOZM 7

GP GP_EBOZM 16

sGP SGP_EBOZM 12

ssGP SSGP_EBOZM 10

VP30 VP30_EBOZM 8

VP24 VP24_EBOZM 9

L L_EBOZM 62

iVAX Analysis of Ebola Virus Class II HLA Binding Analysis – Clusters of Promiscuous Epitopes

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Immunogenic clusters found by ClustiMer in Zaire Ebolavirus – Mayinga

< surface protein



8/20/2014 29

EpiMatrix scores of GP immunogenic clusters

EpiVax - Non-confidential

iVAX Analysis of Ebola virus Class II HLA Binding Analysis – Clusters of Promiscuous Epitopes

8/20/2014 30

Removed clusters with no EpiBar or high hydrophobicity (highlighted in red, EpiVax standard approach)



8/20/2014 31

• Immunogenicity scale of clusters found in Zaire Ebolavirus – Mayinga GP protein


iVAX Analysis of Ebola Virus Class II HLA Binding Analysis

8/20/2014 33

• EpiMatrix maps T cell epitopes across Class II HLA DRB1 alleles

• EpiMatrix report of GP, cluster 16


Is this cluster conserved in other Ebola strains?

• Homology search of GP, cluster 16

• Conservation among the Filoviridae family (excluding Zaire Ebolavirus – Mayinga), 15 maximum hits


8/20/2014 34 EpiVax - Non-confidential



• EpiMatrix maps T cell epitopes across HLA alleles


Cluster EPX score

# epitopes TCR-facing residues conservation (1)

Human cross-reactivity (2)

Microbiome cross-reactivity (3)

GP_EBOZM:16 47.6 3 20 % 0 - 4 15 – 43

GP_EBOZM:32 18.95 1 20 % 5 17

GP_EBOZM:85 15.38 1 47 % 0 9

GP_EBOZM:156 17.43 2 60 - 67 % 0 - 2 10 – 19

GP_EBOZM:190 11.22 1 20 % 0 25

GP_EBOZM:215 10.05 1 20 % 1 12

iVAX Analysis of Ebola Virus Class II HLA Binding Analysis – Conservation with Human

8/20/2014 57

The table displays ranges in the case a cluster contains several epitopes.

(1) % of Filoviridae strains with similar TCR-facing residues to the ones in Zaire Ebolavirus – Mayinga epitopes

(2) Number of human sequences with similar TCR-facing residues to the ones in Zaire Ebolavirus – Mayinga epitopes

(3) Number of human microbiome sequences with similar TCR-facing residues to the ones in Zaire Ebolavirus –

Mayinga epitopes

Cross-reactivity analysis of GP clusters (JanusMatrix analysis)


Cluster EPX score

# epitopes TCR-facing residues conservation (1)

Human cross-reactivity (2)

Microbiome cross-reactivity (3)

GP_EBOZM:238 13.8 1 27 % 2 6

GP_EBOZM:245 28.17 2 13 - 20 % 0 - 16 12 – 139

GP_EBOZM:336 15.84 1 20 % 2 12

GP_EBOZM:555 10.5 1 67 % 0 31

GP_EBOZM:566 22.55 2 67 % 3 - 8 76 – 49

GP_EBOZM:579 20.25 1 27 % 2 21

iVAX Analysis of Ebola Virus Class II HLA Binding Analysis – Conservation with Human

8/20/2014 58

The table displays ranges in the case a cluster contains several epitopes.

(1) % of Filoviridae strains with similar TCR-facing residues to the ones in Zaire Ebolavirus – Mayinga epitopes

(2) Number of human sequences with similar TCR-facing residues to the ones in Zaire Ebolavirus – Mayinga epitopes

(3) Number of human microbiome sequences with similar TCR-facing residues to the ones in Zaire Ebolavirus –

Mayinga epitopes

Cross-reactivity analysis of GP clusters (JanusMatrix analysis)


• Zaire Ebolavirus – Mayinga surface protein, GP, has a low immunogenic potential at the protein level

• iVAX identified 16 clusters of immunogenicity in the GP sequence

• Several clusters present: – High EpiMatrix scores

– High degree of conservation among Filoviruses

– Low degree of cross-reactivity to human and human microbiome sequences

• iVAX can be used to identify potential targets for vaccine development

iVAX Analysis of Ebola Virus Class II HLA Binding Analysis – Summary


Class I Immunogenicity screening of Zaire Ebolavirus – Mayinga GP protein

• iVAX identified 143 9-mers and 10-mers sequences with increased immunogenic potential in regards to at least one HLA Class I allele (1)

• From these, 80 sequences present limited homology to human sequences (less than 5 hits)

• From these, 59 sequences have at least one positive hit and no negative hit in IEDB

Please contact [email protected] for more information.

iVAX Analysis of Ebola Virus Class I HLA Binding Analysis (for details email [email protected])


(1) HLA-A*0101, HLA-A*0201, HLA-A*0301, HLA-A*2402, HLA-B*0702, HLA-B*4403


• Immunogenicity screening of Zaire Ebolavirus – Mayinga GP protein

iVAX Analysis of Ebola Virus Class I HLA Binding Analysis (for details email [email protected])

8/20/2014 66

143 9-mers and 10-mers with increased immunogenic potential to at least one HLA Class I allele (1)

80 sequences with limited homology to human sequences (less than five hits)

59 sequences with at least one positive hit and no negative hit in IEDB

(1) HLA-A*0101, HLA-A*0201, HLA-A*0301, HLA-A*2402, HLA-B*0702, HLA-B*4403


• iVAX can:

– Rapidly analyze pathogens

– Evaluate immunogenic potential of peptides against HLA Class I and II alleles

– Quickly identify regions of interest for vaccine development

– Screen candidates against human germline sequences

• We hope our analysis and predictions will help researchers around the world in the fight against Ebola

• Any questions – please contact [email protected]

Summary



Vaccine Collaborations

Don Drake

Brian Schanen

Sharon Frey

Mark Buller

Jill Schreiwer

Hardy Kornfeld

Jinhee Lee

Liisa Selin Connie Schmaljohn

Lesley C. Dupuy

Ted Ross

Mark Poznansky

Tim Brauns

Pierre LeBlanc

AI058326

,

AI058376

,

AI078800

,

AI082642 http://bit.ly/EpiPubs 70

Vish Mokashi

Biswajit Biswas

Manon Cox

Indresh Srivastava

Manabu Ato

Yoshi Takahashi

Drew Hannaman


epivax analysis of ebolavirus zaire ebolavirus - mayinga (the current ebola outbreak strain) 2014

Health & Medicine

dose of vaccine

h7n9 influenza vaccine

west africa ebola outbreak

terry f

martin wd

epivax analysis of ebolavirus

effective vaccines

t cell epitopes