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Finding a safe, efficacious and stable vaccine in the ricin protein fold
Leonard A. SmithSenior Research Scientist (ST)
Medical Countermeasures TechnologyUS Army Medical Research Institute of Infectious Diseases
Fort Detrick, Maryland, USA
Leonard A. SmithSenior Research Scientist (ST)
Medical Countermeasures TechnologyUS Army Medical Research Institute of Infectious Diseases
Fort Detrick, Maryland, USA
4th International Conference on Vaccines and VaccinationValencia, Spain
24 September 2014
4th International Conference on Vaccines and VaccinationValencia, Spain
24 September 2014
UNITED STATES ARMY MEDICAL RESEARCH INSTITUTE OF INFECTIOUS DISEASES
USAMRIIDUSAMRIID“Biodefense solutions to protect our nation”
Castor beans are an important agricultural commodity….
From: Thai Castor Oil Industries
Commercial uses:• Paints, dyes, varnishes• Lubricant• Purgative/laxative• Source of sebacic acid, used in the production of nylon• Animal food supplement• Fertilizer
One million tons of castor beans processed annually
Waste mash from this is ~5% ricin (easily extracted with low technology)
Ricin ToxinDepurination of A4324 (28S rRNA) disrupts binding of elongation factor (EF2) to the 60S subunit and stops protein synthesis
Literature values: kcat~ 1,800 min-1 Km ~ 0.1 µM aerosol LD50 ~ 3-5 µg/kg mouse (death ~ 60 hr)
– Tertiary structure of native ricin solved at 2.5 Å resolution (Robertus et al., Proteins 10: 251, 1991; 10:241, 1991)
– Recombinant A-chain refined to 1.8 Å resolution (Weston et al., J. Mol. Biol. 244: 410, 1994)
– A-chain co-crystallized with small ligands (Robertus et al., J. Mol. Biol. 227: 1136, 1992; J. Mol. Biol. 266: 1043, 1997)
– A-chain mutants (Robertus et al., Protein Eng. 5: 775, 1992; Biochemistry 35: 11098, 1996)
(267 a.a.)
(262 a.a.)
DA 66,000
N-glycosidase
Survival Antigen Dose¶ (Alive/Total) Mean Time to Death
Intraperitoneal Injection of 10 LD50S of Ricin Toxin
rRTA 10 µg 10/10*,ns > 14 daysdgRTA 10 µg 10/10*,ns > 14 days
PBS 0.1 ml 0/10 Between 6 & 20 hrs
Aerosol Whole Body Exposure to Between 5 & 10 LD50S of Ricin Toxin
rRTA 10 µg 10/10*,ns > 14 daysdgRTA 10 µg 10/10*,ns > 14 days
PBS 0.1 ml 0/10 3.70 ± 0.15 days__________________________________________________________________________¶ - Three intramuscular injections at 0, 4 and 8 weeks* - Significantly different (p < 0.05) from PBS controls by Fisher’s Exact Testns - Not significantly different (p > 0.05) from dgRTA by Fisher’s Exact Test
Survival and Mean Time to Death of Mice Vaccinated with rRTA Against IP Injection
or Aerosol Exposure to Ricin Toxin
Domain-based Design Concept Behind Current Lead Candidate Ricin Toxin A Subunit
Olson et al. (2004) Finding a New Vaccine in the Ricin Protein Fold. PEDS 17, 391-397
Purification Process for RVEcTM
Fermentation Cell Pellet
MicrofluidizationCentrifugationDilution
Capture SP-Sepharose FFStep
Dilution to LowerConductivity
Negative Q-Sepharose FFStep
Polish SP-Sepharose HPStep
ConcentrationDiafiltration by
UFDF
Pool according toSCX-HPLC
Bulking of PDS
Reducing SDS-PAGE of RVEcTM from 4 Bench-scale Runs
188 kDa
62 kDa 49 kDa 38 kDa
28 kDa
17 kDa 14 kDa
6 kDa
3 kDa
Lanes:
1: SeeBlue Plus 2 Stds;
2: PDS-007 30 days 4oC
3: PDS-007 -80oC
4: PDS-008 20 days 4oC
5: PDS-008 -80oC
6: PDS-009 10 days 4oC
7: PDS-009 -80oC
8: PDS-010 2 days 4oC
9: PDS-010 -80oC
Load per lane 5 mg
Storage Temp. oC4 -80 4 -80 4 -80 4 -80
1 2 3 4 5 6 7 8 9
007 008 009 010Run number RVEc-PDS-
Purity of RVEcTM PDS Based on Size Exclusion-HPLC
1: Buffer blank; 2: PDS-006; 3: PDS-007; 4: PDS-008; 5: PDS-009; 6: PDS-010
All the samples showed > 99% monomer.
Dimerpeak
Cell-Free Translation Assay of RVEcTM Vaccine
0
10 20
30
40
50 60
70
80 90
100
.2-4 .5-4 .1-3 .2-3 .5-3 .1-2 .2-2 .4-2 .01 .02 .03 .06 .13 .25 .50 1 2 4 8 16
Concentration (mM)
% C
on
tro
l
rRTA
RTALoop
RTA198
Summary of survival, mean-time to death, and preexposure serum anti-ricin titers in African green monkeys vaccinated with three doses of RVEcTM and challenged
1, 3, 6 and 12 months after the last vaccination with a lethal dose of aerosolized ricin by head-only exposure
Months after the Last Vaccinationa
Inhaled Dose(Mean ± SEM)
Survival(alive/total)
Mean-Time-To-Death (h)
(Mean ± SEM)
Serum Anti-Ricin Log +Titers(Geometric Mean ± SEM)
µ/kg LD50 ELISA IgGcNeutralization
Assayc
1 Month 45.4 ± 3.3 7.6 ± 0.6 7/8d 230.4 4.06 ± 0.04e, f 2.67 ± 0.04e
3 Months 55.9 ± 4.9 9.3 ± 0.8 5/5d - 3.65 ± 0.00e 2.72 ± 0.11e
6 Months 22.6 ± 2.4 3.8 ± 0.4 5/5d - 3.27 ± 0.10e 2.68 ± 0.18e
12 Months 36.5 ± 4.5 6.1 ± 0.7 3/7 56.4 ± 7.7 2.79 ± 0.11e 2.16 ± 0.31e
Succinate Buffer + 0.2% AlhydrogelTMb
42.8 ± 7.6 7.2 ± 1.2 0/4 45.6 ± 4.2 0 0
a – Three intramuscular injections of RVEcTM (80 µg/dose) at 0, 4, & 8 weeks; b – One control NHP was challenged with aerosolized ricin at each time period; c - Variation among column means is significantly (p < 0.0001) greater than expected by chance by One-way ANOVA; d - Significantly different (p < 0.01) from succinate buffer controls by Fisher’s Exact test; e - Significantly different (p < 0.01) from succinate buffer controls Tukey-Kramer Multiple Comparison test; f - Significantly different (p < 0.01) from 12-months by Tukey-Kramer Multiple Comparison test.
Phase 1 Clinical Trial
Objectives:
To evaluate the safety of the RVEcTM in escalating doses (20, 50, and 100 g).
To evaluate immunogenicity of the RVEcTM in escalating doses (20, 50, and 100 g).
Objectives:
To evaluate the safety of the RVEcTM in escalating doses (20, 50, and 100 g).
To evaluate immunogenicity of the RVEcTM in escalating doses (20, 50, and 100 g).
Phase 1 Clinical Trial Open-label, uncontrolled, escalating
multiple-dose vaccine study.
Group 1 (20 μg) and Group 2 (50 μg) received 3 doses at 4-week intervals.
Group 3 received a single dose of 100 µg.
10 subjects per group.
To ensure the safety of the subjects, dose administration proceeded in a staggered fashion.
Open-label, uncontrolled, escalating multiple-dose vaccine study.
Group 1 (20 μg) and Group 2 (50 μg) received 3 doses at 4-week intervals.
Group 3 received a single dose of 100 µg.
10 subjects per group.
To ensure the safety of the subjects, dose administration proceeded in a staggered fashion.
Demographics20 µg/ml Group(N=10)
50 µg/ml Group(N=10)
100 µg/ml Group(N=10)
All Subjects(N=30)
GENDER Male 7 (70%) 6 (60%) 7 (70%) 20 (67%)
Female 3 (30%) 4 (40%) 3 (30%) 10 (33%)
AGE Median 33.5 34.0 31.0 32.5
Min-Max (25-47) (25-49) (23-45) (23-49)
RACE Caucasian/White 10 (100%) 10 (100%) 9 (90%) 29 (97%)
African-American/Black
1 (10%) 1 (3%)
ETHNICITY Hispanic/Latino 1 (10%) 1 (10%) 2 (7%)
Not Hispanic/Latino
9 (90%) 9 (90%) 10 (100%) 28 (93%)
Dose Groups
20 µg/ml Group(N=10)
50 µg/ml Group(N=10)
100 µg/ml Group(N=10)
All Subjects(N=30)
DOSES GIVEN
Dose 1 10 10 10 30
Dose 2 10 10 0 20
Dose 3 10 10 0 20
Related Systemic Adverse Events
20 µg/ml Group(N=10)
50 µg/ml Group(N=10)
100 µg/ml Group(N=10)
All Subjects(N=30)
ANY SYSTEMIC AE 7 (70%) 10 (100%) 7 (70%) 24 (80%)
MILD 6 (60%) 9 (90%) 5 (50%) 20 (67%)
MODERATE 3 (30%) 5 (50%) 3 (30%) 11 (37%)
SEVERE 1 (10%) 2 (20%) 1 (10%) 4 (13%)
LIFE-THREATENING 0 0 2 (20%) 2 (7%)
Headache 4 (40%) 5 (50%) 1 (10%) 10 (30%)
Nausea 1 (10%) 2 (20%) 2 (20%) 5 (17%)
Haematuria 2 (20%) 1 (10%) 0 3 (10%)
Related Local Adverse Events20 µg/ml Group(N=10)
50 µg/ml Group(N=10)
100 µg/ml Group(N=10)
All Subjects(N=30)
ANY LOCAL AE 7 (70%) 8 (80%) 2 (20%) 17 (57%)
MILD 7 (70%) 8 (80%) 2 (20%) 17 (57%)
MODERATE 1 (10%) 0 0 1 (3%)
SEVERE 1 (10%) 0 0 1 (3%)
Injection site pain 7 (70%) 8 (80%) 1 (10%) 16 (53%)
Arthralgia Axillary pain Injection site hematomaInjection site papuleJoint range of motion decreased
Lymphadenopathy
The following Local AEs were reported by 1 subject (3%) each:
Grade 4 AEs Subject 0049, Male, 100 µg/ml Dose Group
– Blood creatine phosphokinase increased (peaking at 3,960: normal range 20-287)
– Onset: 1 day post-vaccination 1
– Duration: 8 days
– Relationship to vaccine: Possible
– Treatment given: Counseling
Subject 0053, Male, 100 µg/ml Dose Group– Blood creatine phosphokinase increased, peaking at 30,718:
normal range 20-287
– Onset: 28 days post-vaccination 1
– Duration: 14 days
– Relationship to vaccine: Possible
– Treatment given: Hospitalization
Subject 0049, Male, 100 µg/ml Dose Group– Blood creatine phosphokinase increased (peaking at 3,960:
normal range 20-287)
– Onset: 1 day post-vaccination 1
– Duration: 8 days
– Relationship to vaccine: Possible
– Treatment given: Counseling
Subject 0053, Male, 100 µg/ml Dose Group– Blood creatine phosphokinase increased, peaking at 30,718:
normal range 20-287
– Onset: 28 days post-vaccination 1
– Duration: 14 days
– Relationship to vaccine: Possible
– Treatment given: Hospitalization
Serious AEs Subject 0023, Female, 20 µg/ml Dose Group
– Arthralgia– Onset: 112 days post-vaccination 3– Duration: 53 days– Relationship to vaccine: Definite– Treatment given: Outpatient surgery
Subject 0053, Male, 100 µg/ml Dose Group– Rhabdomyolysis– Onset: 31 days post-vaccination 1– Duration: 3 days– Relationship to vaccine: Possible– Treatment given: Hospitalization
Subject 0023, Female, 20 µg/ml Dose Group– Arthralgia– Onset: 112 days post-vaccination 3– Duration: 53 days– Relationship to vaccine: Definite– Treatment given: Outpatient surgery
Subject 0053, Male, 100 µg/ml Dose Group– Rhabdomyolysis– Onset: 31 days post-vaccination 1– Duration: 3 days– Relationship to vaccine: Possible– Treatment given: Hospitalization
Conclusion RVEcTM at 20 µg and 50 µg doses are safe
and immunogenic in man
A single RVEcTM booster elicits a robust anamnestic response
The RVEcTM booster appears to be necessary for the production of high levels of neutralizing anti-ricin antibodies
Next study should increase interval between 2nd and 3rd shots
RVEcTM at 20 µg and 50 µg doses are safe and immunogenic in man
A single RVEcTM booster elicits a robust anamnestic response
The RVEcTM booster appears to be necessary for the production of high levels of neutralizing anti-ricin antibodies
Next study should increase interval between 2nd and 3rd shots
USAMRIID
Dr. Phillip R. Pittman
COL Fernando B. Guerena
LTC Robert Rivard
MAJ Matthew Chambers
Denise Clizbe
Sarah Norris
Carolyn Mentzer
Ralph Tammariello
Changhong Lindsey
Wendy Giles
University of Nebraska
ScottJohnson Mike Meagher
USAMMDA
Sharon Maloid
Terri Deal
JSTO-CBD
Erin Reichert
Dan Wolfe
CBMS
Janice Rusnak
Chris Dorsey
Edward Brown
Supported by the Joint Science and Technology Office for Chemical Biological Defense, Defense Threat Reduction Agency.
Acknowledgements
Disclosures and Disclaimers
The views and opinions expressed in this presentation are those of the author(s) and do not reflect official policy or position of the Department of the Army, Department of Defense or the U.S. Government.
Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. USAMRIID is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.
The views and opinions expressed in this presentation are those of the author(s) and do not reflect official policy or position of the Department of the Army, Department of Defense or the U.S. Government.
Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. USAMRIID is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.
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