Pre-clinical Development of a Nasal Adenovirus-Based Vaccine Against Ebola Virus (CRTI 06-0218RD) Public Security S&T Symposium 2009
Mar 26, 2015
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