iVAX Overview

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iVAX Toolkit – Online Access

Whole (live/killed)

vaccinesSubunit vaccines

Genome-Derived, Epitope Driven (GD-ED)

Vaccines

Better understanding of vaccine MOA

Improve vaccine safety and efficacy

Accelerate Vaccine Design

Design next-gen vaccinesbetter/safer/faster

T cells = Immune System Body Armor

T cell response cannot prevent Infection but . . .

T cell response can arm against Disease

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Vaccine Design Tools and Techniques

Analysis

Validation

Engineering

Strain 1

Strain 3

Strain 2

core genomedispensable genes

strain-specific genespangenome

Comparative Genomics ImpactsVaccine Immunogen Selection

Epitope Cross-Reactivity ImpactsVaccine Immunogen Selection

EpiMatrix

• EpiVax uses EpiMatrix to predict epitopes– matrix based prediction algorithm

• Can predict either class I or class II MHC binding– MHC binding is a prerequisite for immunogenicity

T cell epitopes are linear and directly derived from antigen sequence

Binding is determined by amino acid side chains (R groups) and ‘encoded’ in single letter code

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Mature APC

Protein MHC II Pocket

Peptide Epitope

EpiVax HLA “Supertype” Coverage

• EpiVax tests for binding potential to the most common HLA molecules within each of the “supertypes” shown to the left.

• This allows us to provide results that are representative of >90% of human populations worldwide* without the necessity of testing each haplotype individually.

9* Southwood et. al., Several Common HLA-DR Types Share Largely Overlapping Peptide Binding Repertoires. 1998. Journal of Immunology.

EpiMatrix

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• The EpiMatrix algorithm scores all the 9-mers in a given sequence for binding affinity across a range of common HLA and reports both detailed and aggregated results.

• In protein therapeutics and existing vaccines these tools can predict immunogenicity (efficacy). In the design of vaccines these tools allow for educated selection of pathogen genome sequences to make the most effective, efficient and safe vaccine

Easy easy to deliver as peptidesClustiMer

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

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Identifying the most conserved 9-mers allows for protection against more strains with fewer epitopes

Conservatrix Finds Conserved 9-mers

Conservedepitope

CTRPNNTRK

CTRPNNTRKCTRPNNTRK

CTRPNNTRKCTRPNNTRK

CTRPNNTRK

CTRPNNTRK

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BlastiMer: Epitope Exclusion

In all of our vaccines we eliminate cross-reactive epitopesIn all of our vaccines we eliminate cross-reactive epitopes

SelfSelfForeignForeign

MHC

TCR

New iVAX feature: JanusMatrix Homology Analysis

JanusMatrix is designed to predict the potential for cross reactivity between epitope clusters and the human genome, based on conservation of TCR-facing residues in their putative HLA ligands.

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VLQSSGLSYS( T cell epitope)

FLQDSNLYK(T cell epitope with same TCR face)

Another human protein

A different human protein

A human protein

Source protein

Predicted 9-mer epitope from a source protein

Human protein where cross-reactive epitopes are present

9-mer from human prevalent proteome, 100% TCR face identical to source epitope

Source Protein or large peptide

JanusMatrix Cross-reactivity Networks

All induced Teff response

Example: HCV Vaccine22/23 epitopes vs. human proteome

vs. human proteome vs. human microbiome

Induced Treg response

Example: HCV VaccineOne “special” epitope: cross-reactive

STRAIN 01 Q X S W P K V E Q F W A K H X W N X I S X I Q Y LSTRAIN 02 Q A S W P K V E X F W A K H M W N F I S G I Q Y LSTRAIN 03 Q X S W P K X E Q F W A K H M W N F I S G I Q Y XSTRAIN 04 Q A S W X K V E Q F W A K H M W N F X S X I Q Y LSTRAIN 05 Q X S W P K V E Q F W A K H M W N F I S G I Q Y LSTRAIN 06 Q A S W P K X E Q F W A X H M W N F I S G I Q Y XSTRAIN 07 Q X S W P K V E Q F W A K H M X N F I S G I Q Y LSTRAIN 08 Q A S W X K V E Q F W A K H M W N F I S G I Q Y LSTRAIN 09 Q X S W P K X E Q F W A K H M W N F X S X I X Y XSTRAIN 10 Q A S W P R V E Q F W A K H M W N F I X G I Q Y LSTRAIN 11 Q A S W P K V E Q F W A K H M W N F I S G I Q Y LSTRAIN 12 Q A S W X K V E Q F W A X H M W N F I S G I Q Y XSTRAIN 13 Q A S W P K V E Q F W A K H M W N F I S G I Q Y LSTRAIN 14 Q A S W X K X E Q F W A K H M W N F I S X I Q Y LSTRAIN 15 Q A S W P K V E X F W X K H M W N F I S G I Q Y LSTRAIN 16 Q X S W P K V E Q F W A K H M W N F I X G I Q Y LSTRAIN 17 X A S W X K V E Q F W A K H M W N F I S G I Q Y XSTRAIN 18 Q X S W P K X E Q F W A K H M W N X I S G I Q Y LSTRAIN 19 Q A S W X K V E Q F W A K H M W N F I S X I Q Y LSTRAIN 20 Q A S W P K V E Q F W A X H M W N F I S G I Q Y L

x

F W A K H M W N FW P K V E Q F W A

Q A S W P K V E Q N F I S G I Q Y LM W N F I S G I Q

Q A S W P K V E Q F W A K H M W N F I S G I Q Y L

EpiAssembler Produces Immunogenic Consensus Sequences

HP

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50Epitope Cluster ScoreJunctional Cluster Score

Peptides in Default order in construct HP_IIB

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50Epitope Cluster ScoreJunctional Cluster Score

Peptides in Optimized order in construct HP_IIBE

piM

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VaccineCAD

VaccineCAD Eliminates Introduced Junctional Epitopes

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DNA Vector

DNA insert

Intended Protein Product: Many epitopes strung together in a “String-of-Beads”

Protein product (folded)

Output: Multi-Epitope Gene Design

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

Multiple Delivery Platforms Possible

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iVAX: Case Studies

Vaccine

Immunogenic

Epitopes

Shared

Immunogenic

Epitopes

smallpoxvaccinia

Case Study: Smallpox VaccineVennVax

Immunoinformatics Summary

(18/110) Epitopes derived from structural proteins

(66/110) Epitopes derived from regulatory factors

(26/110) Epitopes derived from Hypothetical and unknown proteins

AF095689 (Xian tan)

M35027

(Copenhagen)

U94848 (Ankara)

AY243312(WR)

Y16780

(V. Minor)

X69198

(V. Major) L2

2579

Bangladesh

2. Identified highly conserved 9-mers

3. Identified promiscuous Class II T cell epitopes and Class I epitopes

4. Eliminated human homologous epitopes

1. Downloaded 7 complete genomes from GenBank.

Moise L et al. Vaccine. 2009;27:6471-9

Immunogenicity Day 56

1. epitope DNA vaccine prime (IM)2. epitope peptide boost (IN)

ImmunizationsDays 0, 14, 28, 42

ChallengeDay 65

Case Study: VennVax Immunizationin HLA DR3 Transgenic Mice

Moise L et al. Vaccine. 2011;29:501-11

• Challenge done at 10X LD50 for Smallpox• Related publication: http://www.ncbi.nlm.nih.gov/pubmed/21055490

100% survival of Vaccinated mice vs. 17% of placebo

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Vaccinated

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DNA DNA boost boost Challenge17%

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VennVax protects againstlethal vaccinia challenge

Moise L et al. Vaccine. 2011;29:501-11

VennVax Protection: Morbidity

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Placebo

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Case Study: VennVax Protection: Antibody Titers

Moise et al. Vaccine. 2011; 29:501-11

VennVax conferred protection without any B cell epitopes

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• 5 prototype vaccines using iVAX with 3 validated in animal models (VennVax, TulyVax, and H.pylori)

• Validation of remaining prototype vaccines (FluVax) currently in progress

Other Vaccine Design Case Studies

gB-2 (EPX Score: -24.56)

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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/Shanghai/1/2013 (H7N9) . . . . . . . .. . . . . . . . -8.11HA A/mallard/Netherlands/09/2005 (H7N7) . . . . . . -8.63

Random Expectation

HA A/mallard/Netherlands/12/2000 (H7N3) .. . . . . .-9.91

HA A/chicken/Italy/13474/1999 (H7N1) . . . . . . . . . -6.23

H7 HAImmunogenic Potential

Immunogenicity of H7N9 HA predicted to be LOWPrevious vaccines poorly immunogenic

H7N9: Prediction of low-immunogenicity

H1N1 (2009)

H7N9 (2013)

T Cell Epitope Content1 HIGH VERY LOW

T Cell Epitope Cross Reactivity2 MEDIUM VERY

LOW

Cross-Reactive Antibody Immunity3 LOW LOW

Predictive protective effect of

vaccination in standard influenza

vaccine

HIGH LOW

IAV T Cell Epitope Responses by CategoryAverage Immune Response N=4 Subjects

In Novartis Phase I trial of H7N9 cell culture vaccine showed only 6% of 400 healthy volunteers achieved a protective response when given two doses of the 15ug un-adjuvanted vaccine

EpiVax predictions

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IVAX makes vaccines:

More Effective: IVAX can be used to design more targeted, immunogenic vaccines or predict the immunogenicity of existing vaccines

Safer: Epitope-driven DNA or Sub-unit vaccines mitigate the risks associated with live, attenuated or VLPs without sacrificing efficacy. Less antigenic load.

Faster: After H7N9 genome was released EpiVax personnel were able to complete a vaccine sequence in less than 48 hours – approx 20 hours total labor.

Accessing the ToolsContact Jason Del Pozzo: bda@epivax.com

Confidential33

PreDeFT: Fee for service in silico immunogenicity analysis. Performed on a protein by protein basis. Pricing based on length of sequence(s).

Limited ISPRI Website: Limited access to EpiVax’ Interactive Protein Screening and Reengineering Interface. Available for set numbers of proteins.

Unlimited ISPRI Website: Unlimited access to EpiVax’ Interactive Protein Screening and Reengineering Interface. Available in three year lease periods.

Fee for Service: HLA Binding Assays, HLA Transgenic Mice, ELISpot Assays.

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