UNITED STATES ARMY MEDICAL RESEARCH INSTITUTE OF INFECTIOUS DISEASES
USAMRIID
“Biodefense solutions to protect our nation”
Finding a safe, efficacious and stable
vaccine in the ricin protein fold
Leonard A. Smith
Senior Research Scientist (ST)
Medical Countermeasures Technology
US Army Medical Research Institute of Infectious Diseases
Fort Detrick, Maryland, USA
4th International Conference on Vaccines and Vaccination
Valencia, Spain
24 September 2014
R. communis
plant parts
Mature ovary & seeds
Inflorescence
Variation among seeds
Castor beans are an important
agricultural commodity….
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)
From: Thai Castor Oil Industries
Ricin Toxin
Depurination of A4324 (28S rRNA)
disrupts binding of elongation factor
(EF2) to the 60S subunit and stops
protein synthesis
(267 a.a.)
N-glycosidase
DA 66,000
(262 a.a.)
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)
SDS-PAGE of Recombinant RTA
M
31.0
21.5
1
2
3
rRTA
Survival and Mean Time to Death of Mice
Vaccinated with rRTA Against IP Injection
or Aerosol Exposure to Ricin Toxin
Survival
Antigen
Dose¶
(Alive/Total)
Mean Time to Death
Intraperitoneal Injection of 10 LD50S of Ricin Toxin
rRTA
10 µg
10/10*,ns
> 14 days
dgRTA
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 days
dgRTA
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 Test
ns - Not significantly different (p > 0.05) from dgRTA by Fisher’s Exact Test
Fraction of Interfacial Surface Area
Due to Hydrophobic Residues
28%
15%
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
Bulking of PDS
Microfluidization
Centrifugation
Dilution
Capture SP-Sepharose FF
Step
Dilution to
Lower
Conductivity
Negative Q-Sepharose FF
Step
Concentration
Diafiltration by
UFDF
Pool
according to
SCX-HPLC
Polish SP-Sepharose HP
Step
Reducing SDS-PAGE of RVEcTM from
4 Bench-scale Runs
Lanes:
1
2
3
4
5 6
7
8
9
188 kDa
62 kDa
49 kDa
38 kDa
Storage Temp. oC
4 -80 4 -80 4 -80 4 -80
1: SeeBlue Plus 2 Stds;
2: PDS-007 30 days 4oC
3: PDS-007 -80oC
4: PDS-008 20 days 4oC
28 kDa
5: PDS-008 -80oC
17 kDa
14 kDa
6: PDS-009 10 days 4oC
7: PDS-009 -80oC
6 kDa
3 kDa
007
008 009
010
Run number RVEc-PDS-
8: PDS-010 2 days 4oC
9: PDS-010 -80oC
Load per lane 5 mg
Purity of RVEcTM PDS Based on
Size Exclusion-HPLC
Dimer
peak
1: Buffer blank; 2: PDS-006; 3: PDS-007; 4: PDS-008; 5: PDS-009; 6: PDS-010
All the samples showed > 99% monomer.
% Control
Cell-Free Translation Assay
of RVEcTM Vaccine
100
90
80
70
60
50
40
30
20
10
0
rRTA
RTALoop
RTA198
.2-4 .5-4 .1-3 .2-3 .5-3 .1-2 .2-2 .4-2 .01 .02 .03
Concentration (mM)
.06 .13
.25
.50
1
2
4
8
16
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
Inhaled Dose
(Mean ± SEM)
Mean-TimeSurvival
To-Death (h)
(alive/total) (Mean ± SEM)
Serum Anti-Ricin Log +Titers
(Geometric Mean ± SEM)
ELISA IgGc
Neutralization
Assayc
230.4
4.06 ± 0.04e, f
2.67 ± 0.04e
5/5d
-
3.65 ± 0.00e
2.72 ± 0.11e
3.8 ± 0.4
5/5d
-
3.27 ± 0.10e
2.68 ± 0.18e
36.5 ± 4.5
6.1 ± 0.7
3/7
56.4 ± 7.7
2.79 ± 0.11e
2.16 ± 0.31e
42.8 ± 7.6
7.2 ± 1.2
0/4
45.6 ± 4.2
0
0
Months after the
Last Vaccinationa
µ/kg
LD50
1 Month
45.4 ± 3.3
7.6 ± 0.6
7/8d
3 Months
55.9 ± 4.9
9.3 ± 0.8
6 Months
22.6 ± 2.4
12 Months
Succinate Buffer +
0.2% AlhydrogelTMb
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 mg).
 To evaluate immunogenicity of the
RVEcTM in escalating doses (20, 50, and
100 mg).
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.
Demographics
GENDER
AGE
RACE
20 µg/ml
Group
(N=10)
50 µg/ml
Group
(N=10)
100 µg/ml
Group
(N=10)
All
Subjects
(N=30)
Male
7 (70%)
6 (60%)
7 (70%)
20 (67%)
Female
3 (30%)
4 (40%)
3 (30%)
10 (33%)
Median
33.5
34.0
31.0
32.5
Min-Max
(25-47)
(25-49)
(23-45)
(23-49)
9 (90%)
29 (97%)
1 (10%)
1 (3%)
Caucasian/White
10 (100%) 10 (100%)
AfricanAmerican/Black
ETHNICITY Hispanic/Latino
1 (10%)
1 (10%)
Not
Hispanic/Latino
9 (90%)
9 (90%)
2 (7%)
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)
Dose 1
10
10
10
30
Dose 2
10
10
0
20
Dose 3
10
10
0
20
DOSES
GIVEN
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%)
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%)
LIFE-THREATENING
Related Local 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 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%)
The following Local AEs were reported by 1 subject (3%) each:
Arthralgia
Lymphadenopathy
Axillary pain
Injection site hematoma
Injection site papule
Joint range of motion decreased
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
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
ELISA GMT
TNA GMT
Booster ELISA
Booster TNA
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
Acknowledgements
USAMRIID
University of Nebraska
USAMMDA
Dr. Phillip R. Pittman
ScottJohnson
Mike Meagher
Sharon Maloid
COL Fernando B. Guerena
Terri Deal
LTC Robert Rivard
MAJ Matthew Chambers
JSTO-CBD
Denise Clizbe
Erin Reichert
Sarah Norris
Dan Wolfe
Carolyn Mentzer
Ralph Tammariello
CBMS
Changhong Lindsey
Janice Rusnak
Wendy Giles
Chris Dorsey
Edward Brown
Supported by the Joint Science and Technology Office for
Chemical Biological Defense, Defense Threat Reduction Agency.
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.