Pre-clinical Development of a Nasal Adenovirus-Based Vaccine Against Ebola Virus

(CRTI 06-0218RD)

Public Security S&T Symposium 2009

PROJECT BACKGROUND

• An ongoing NIH sponsored phase 1 clinical trial is evaluating Adenovirus-based vaccine to stimulate significant immune responses against Ebola virus.

• Goal: Pre-clinical development of a nasal adenovirus-based vaccine against Ebola virus.

• We have developed an optimized adenovirus-based Ebola vaccine that can stimulate both mucosal and systemic immune responses following nasal immunization.

• The first objective is to develop a formulated pre-clinical grade optimized AdHu5 EBOV vaccine.

• The second objective is to advance the knowledge on immune correlates of protection against EBOV in NHPs.

Different Vaccine StrategiesDifferent Vaccine Strategies

– DNADNA– VacciniaVaccinia– VEE repliconVEE replicon

– VLPs VLPs (Warfield et al. 2007)(Warfield et al. 2007)

– HPIV3 HPIV3 – VSVVSV– AdenovirusAdenovirus

Have successfully protected NHP

(Bukreyev et al., 2007)

(Jones et al., 2005)

(Sullivan et al., 2000)

Objective

• To optimize expression of the ZEBOV glycoprotein from an adenoviral vector and compare it to the CMV driven EboGP Ad vaccine vector.

Improving the expression cassette

Codon optimization Improved CAG promoter

FIX MMI: Computer control

Image created by Robert Voyer, BRI-ACTDSP, JAN. 1998

Ad5-GFP-Q production in 60L- Ad5-GFP-Q production in 60L- BioreactorBioreactor

Adenovirus Purification

DNAse treatmentCentrifuge/ conditioning

Adenovirus Production

PERMEATE(Waste)

Cell lysis

LIQUIDHarvest

UltrafiltrationConcentration

Filtration

SOLID

Anion-ExchangeChromatography

Fractogel EMD-DEAE

Size ExclusionChromatographySephacryl S-400

Purified Adenovirus

RETENTATEConcentratedAdenovirus

Expression Ebola ZGP

Recombinant Adenovirus

HEK 293

Ad-CMV/ZGP

Ad-CAG/OptZGP

Survival and weight loss 28 days post-Survival and weight loss 28 days post-vaccinationvaccination

Vaccine Concentration (IFU/Mouse)

Survival (Percentage)

Weight Loss (Percentage)

Ad-CMVZGP

1 x 107 100 0

Ad-CMVZGP

1 x 106 100 0

Ad-CMVZGP

1 x 105 40 17

Ad-CAGoptZGP

1 x 106 100 0

Ad-CAGoptZGP

1 x 105 100 0

Ad-CAGoptZGP

1 x 104 100 0

Control na1 0 >25

Mice were challenged with a lethal dose (LD50 = 1000) of mouse-adapted ZEBOV

Survival of mice 30 min post challengeSurvival of mice 30 min post challenge

Percentage weight loss in post-exposure Percentage weight loss in post-exposure treatmenttreatment

Immune response 6 days post-Immune response 6 days post-vaccinationvaccination

Formulations Tested

1 Saline, vehicle control (pH 7.4)2 Ad-ZGP in PBS (pH 7.4)3 Ad-ZGP + Base formulation

(Sucrose (10mg/ml), Mannitol (40mg/ml) and Pluronic F68 (0.001%))4 Ad-ZGP + Base formulation + Selenium (1ug/ml) 5 Ad-ZGP + Base formulation + beta-cyclodextrin (0.5%)6 Ad-ZGP + tertiary amine beta-cyclodextrin (5%)7 Ad-ZGP + chitosan (1%)8 Ad-ZGP + Base formulation + chitosan (1%)9 PEGylated Ad-ZGP in PBS (pH 7.4)10 PEGylated Ad-ZGP + Base formulation

Effect of Formulation on Transduction of Calu-3 cells

Frequency of IFN Positive Cells 10 Days After Vaccination (ELISPOT)

Neutralizing Antibody Levels Against Zaire Ebola GP 16 Days After Vaccination

Survival Upon Challenge with Mouse Adapted Ebola

Promising Preparations

Unsuccessful Preparations

Guinea pigsInitial Treatment Ad-lacZ I.M. Ad-lacZ I.M. Ad-lacZ I.N. Ad-lacZ I.N. Nil Nil

Dose (Total Infectious Particles) 2.5 x 1011 2.5 x 1011 2.5 x 1011 2.5 x 1011 n/a n/a

Serum Neutralizing Antibodies Detected cde 122 ± 52

153.9 ± 124.2 8.9 ± 7.7 22.2 ± 27.8 n/a n/a

Nasal Lavage Neutralizing Antibodies Detected cde 0 0 0 0 n/a n/a

Secondary Vaccination f

Ad-CAGoptZGP

I.N.

Ad-CAGoptZGP

I.M.

Ad-CAGoptZG

P I.N.

Ad-CAGoptZG

P I.M. Ad-lacZ I.M. PBS

Dose (Total Infectious Particles) 1 x 1010 1 x 1010 1 x 1010 1 x 1010 1 x 1010 n/a

Serum Neutralizing Antibodies Detected i 20 ± 13 23 ± 8 40 ± 35 20 ± 20 0 n/a

Nasal Lavage Neutralizing Antibodies Detected i 0 13 ± 12 7 ± 12 27 ± 12 0 n/a

             

Maximum Weight Loss (%) 1 ± 1.9 1 ± 2.8 1 ± 1.8 3 ± 2.4 >20 >20

Percent Survival 100% 100% 100% 100% 0% 0%

PROJECT STATUS AND ACCOMPLISHMENTS

• Significant progress steps related to milestones– Comparative evaluation of the first and second

generation Ad-Ebo vaccines in mice and guinea pigs.

• Key Technical Accomplishments, Innovations, Inventions– 100-fold improvement in protective dose, post-exposure

protection and overcoming pre-existing immunity.

• Potential linkages to projects not defined in the Project Charter.– Gene optimization may be beneficial to other vaccine

platform such as VSV.

PROJECT SUMMARY AND CONCLUSIONS• The optimized vaccine also fully protected mice against a lethal

challenge with ZEBOV at a dose 100 times lower than the minimal dose required to achieve full protection with the first generation vaccine.

• Complete survival was also achieved 30 minutes post-exposure although weight loss was observed.

• Higher number of IFN-γ, TNF-α and IL-2 positive CD8 T cells and NAB were detected from splenocytes of Ad-CAGoptZGP immunized mice 6 days post-vaccination when compared to AdCMVZGP immunized mice.

• Several promising formulations have been identified. Mice vaccinated by the nasal route with these preparations have similar or slightly improved immune responses against Ebola and can survive challenge.

• Published in: PLoS ONE, 2009;4(4):e5308. Epub 2009 Apr 23.

PROJECT REVIEW COMMITTEE• Core Members:• Project Champion: Dr. Harvey Artsob• Project Manager: Dr. Gary Kobinger• Portfolio Manager: Norm Yanofsky• NRC Management Representative: Dr. Amine Kamen• University of Texas Management Representative: Dr.

Maria Croyle• Associate Members:• Procurement Lead: Daniele Cole / Doris Bruneau• Deputy Project Manager: Dr. Jim Strong

• Recording Secretary: Doris Bruneau

Routes of immunization: protection in mice against 200 LD50 of MA-Ebola virus

Intramuscular (I.M.)

(%)

Intranasal (I.N.)

(%)

Oral

(%)

Vehicle 0 0 0

AdHu5-ZGP 100 100 100

01000AdHu5-ZGP + human Ig