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”

Inflorescence

Mature ovary & seeds

R. communisplant parts

Variation among seeds

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

31.0

21.5

M 1 2 3

SDS-PAGE of Recombinant RTA

rRTA

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

28% 15%

Fraction of Interfacial Surface Area Due to Hydrophobic Residues

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

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

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.