BLI Studies of Anthrax Toxin and Development of Diagnostic … · 2020-02-22 · • Anthrax is...

National Center for Emerging and Zoonotic Infectious Diseases

Division of Scientific Resources / Scientific Products and Support Branch

Presenter

Jason Goldstein PhDImmunochemistry Team Leader

Scientific Products and Support Branch

BLI Studies of Anthrax Toxin and Development of Diagnostic Antibodies

• Anthrax is caused by infection with Bacillus anthracis, a spore‐forming, rod‐shaped bacterium

• Spores are highly resistant to environmental conditions and gain entry via open wound (cutaneous) or ingestion (gastrointestinal disease or inhalation anthrax) which can progress into systemic infection leading to shock, respiratory failure and death

• In the U.S. bioterrorism attacks of 2001, inhalation anthrax had a 45% fatality rate despite antibiotic treatment and aggressive supportive care of the patients

• Development of a safe and effective vaccine for anthrax is vital to the health and safety of those at risk of exposure and an essential component of any bioterrorism defense strategy

• Need exists for rapid point‐of‐care diagnostic enabling early diagnosis and treatment

• Toxins are key targets and anti‐toxin mAbs can serve as diagnostic and therapeutic tools for those at risk of acquiring disease

Anthrax Toxin Model

Maldonado‐Arocho et al. (2009). "Anthrax Toxin". Microbial Toxins: Current Research and Future Trends.

•Microbiological•PCR based•Immunofluorescent (DFA; IHC)•Immunoassay (ELISA; MSD; Luminex)•Indirect Enzymatic Assay•Diagnostic POC device

Diagnostics

Begin with blocked Ab‐coated magnetic beads

Incubate LF in plasma (matrix) with beads (1h)

Wash beads to remove matrix (4 min)

Incubate beads with buffer and substrate(2‐20h)

Analyze supernatant by mass spectrometry

Mass‐Spec Lethal Factor Assay

<4 h Total Time for 1stResponse

Detection and Quantification of Anthrax Lethal Factor in Serum by Mass SpectrometryAnne E. Boyer et.al. Anal. Chem. 2007, 79, 8463‐8470

*1 ng LF

1000 1400 1800 2200 2600 3000

Mass (m/z)

7357.0

0

100

% In

tens

ity

1232.5

1589.8 2804.2*

No Toxin

1000 3000

8030.6

0

100%

Inte

nsity

1402.1

2804.2

Detection of Lethal Factor ActivityMass/chargeLF

SKARRKKVYPYPXENFPPSTARPT

SKARRKKVYPYPXENFPPSTARPT

2804.21232.5

1589.8

Detection and Quantification of Anthrax Lethal Factor in Serum by Mass SpectrometryAnne E. Boyer et.al. Anal. Chem. 2007, 79, 8463‐8470

Lethal Factor

• LF is a zinc‐dependent endoprotease known to target the amino‐terminus of the mitogen‐activated protein kinase kinase (MAPKK) family of response regulators

• 4 domains with specific functions

• 90kDa highly immunogenic in mice

• Immunization BalbC (10ug IV)

• Sp2 fusion and HAT selection stable parent hybridomas

White Light ImageM12002 LFG2 Parent 4C2

Automated Clone Selection

Anti‐IgG‐FITC ImageM12002 LFG2 Parent 4C2

Automated Clone Selection

Screen of anti‐LF Hybridoma SupsLOADr‐LF‐biotin

ASSOChybridomasup (1‐7)

DISSOCPBS

ASSOChybridomasup (8‐15)

DISSOCPBS

BLI Screen for anti‐LF mAb

SA sensor‐15ug/ml LF‐undiluted media

4C2:1C1

LFG2:4B10

4C2:1B11D1:1A5

3D1:1B2

2C6:1C3

3E4:1G9 anti‐PA3C3:1C6

1F4

1C8

1G6

2A6

4C2:1C1

4C2:1B1

2C6:1C3

3D1:1F4

3F3:2A6

1D1:1G63C3:1C8

3E4:1G9 anti‐PA

Panel of 10 Novel anti‐LF mAbs

4C2:1C1

4C2:B1

0

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0 200 400 600 800 1000 1200 1400 1600 1800 2000

MSD

Cou

nts

ng/mL Anti‐LF mAB

MSD Analysis

4C2:1C1

4C2:1C1*

4C2:1B1

NIH LF 9

3D1:1F2

1D1:1G1

2C6:1F4

LFG2:4B10

NIH LF 6

LFG2:3D10

3C3:1D3

PAG1 3C6

r‐LF Coated 100 ng/well

From Conrad Quinn MVBDB/DBD/NCIRD

Primary mAbassociation







2C6:1B4 mAbassociation

2C6:1F4 mAbassociation

3C3:1A3 mAbassociation

2C6:1H4 mAbassociation

2C6:1BF5 mAbassociation

3C3:1D3 mAbassociation

3C3:1A1 mAbassociation

Epitope Binning with High Affinity mAb Clones

SA sensor‐15ug/ml b‐r LF‐300nM purified IgG

Response to LF 4C2:1C1 4C2:1B1 3C3:1F5 1D1:1A5 2C6:1C3 3D1:1B2 3D1:1C2

4C2:1C1 2.035 0.2903 0.1981 0.4615 0.4064 0.3304 0.3218 0.20914C2:1B1 1.8202 0.3788 0.2777 0.4969 0.4361 0.3624 0.3635 0.2713C3:1F5 0.5113 1.5511 1.3692 0.2643 0.4439 0.3634 0.3626 0.2651D1:1A5 1.3584 1.3192 1.3127 0.5042 0.2598 0.3541 0.3617 0.25142C6:1C3 0.5816 1.5962 1.4197 0.5703 0.4898 0.2414 0.2377 0.15023D1:1B2 0.5973 1.6132 1.4967 0.5779 0.5188 0.254 0.2656 0.15223D1:1C2 0.5504 1.5259 1.3973 0.5481 0.4829 0.2487 0.2547 0.1512LFG2‐4B10 1.6967 1.3832 1.4946 0.5418 0.2598 0.3952 0.3748 0.2749

2C6:1B4 2C6:1F4 2C6:1H4 2C6:1F5 3C3:1C6 3C3:1A1 3C3:1A32C6:1B4 0.634 0.1734 0.2533 0.177 0.2116 0.4747 0.4374 0.56672C6:1F4 0.6966 0.1826 0.2562 0.1753 0.2249 0.5101 0.4641 0.58692C6:1H4 0.5438 0.2183 0.3404 0.2327 0.2817 0.5077 0.472 0.58152C6:1F5 0.5961 0.1836 0.2685 0.1844 0.2355 0.4744 0.4292 0.56743C3:1C6 0.5435 0.3128 0.5724 0.362 0.4493 0.276 0.2363 0.30523C3:1A1 0.4914 0.2888 0.5733 0.353 0.425 0.2804 0.2467 0.32043C3:1A3 0.5125 0.2662 0.5473 0.3306 0.4047 0.2215 0.2091 0.2676LFG2‐4B10 1.6444 0.3262 0.5676 0.3732 0.4247 0.447 0.3972 0.4972

Primary mAb Secondary mAb

Epitope Binning Chart

Lethal Toxin (LTx)

•LTx toxin of B. anthracis are binary combinations of protective antigen (PA) with lethal factor (LF)

•Protective Antigen (PA) is the cell binding component of toxin and is responsible for bringing the catalytic LF into host cells

•PA63 forms an oligomer comprising up to seven molecules of PA63 and is responsible for toxin internalization

• Combines with LF which can trimerize form lethal toxin (LTx) complex >700kDa

LTx

FreePA63

Free LF

0

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MSD

Cou

nts

ng/mL Anti‐LF mAb

MSD Analysis

PAG1 3C6

4C2:1C1

4C2:1C1*

4C2:1B1

1D1:1G1

LFG2 4B10

LFG2:3D10

NIH LF 6

3D1:1F2

2C6:1F4

NIH LF 9

3C3:1D3

r‐LTx Coated 100 ng/wellFrom Conrad Quinn MVBDB/DBD/NCIRD

0

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MSD

Cou

nts

ng/mL Anti‐LF mAb

LTx Coated

4C2:1C1

3D1:1F2

2C6:1F4

3C3:1D3

0

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500000

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MSD

Cou

nts

ng/mL Anti‐LF mAB

LF Coated

4C2:1C1

3D1:1F2

2C6:1F4

3C3:1D3


4C2:1C1

3D1:1F2

2C6:1F43D1:1F23C3:1D3

Antibody Response to LTx Complex by BLI

LTx Toxin Build

PA63 LF

mAb Properties for Diagnostic

1. Sensitivity: mAb recognition of LF antigen at low levels

2. Immunoprecipitation (IP): LF component recognized in LTx by mAb‐bound to magnetic protein‐G beads for enzymatic analysis

3. Enhance Proteolytic Activity: Enhancement of LF catalytic activity throughmAb/epitope Interaction

4. Specificity: Detection LF epitope w/wo LTx confirmation

Immunoblot Detection Predicts Linear Epitope for anti‐LFG2:4B10 mAb

LFG2:4B10

LFG2:3D10

LFG2:4B10 Increases Rate of Catalysis for LF

From Anne Boyer CCB/DLS/NCEH

mAb Epitope Maps to Accessible Loop Proximal to LF Catalytic Pocket

Peptide Loading25ug/ml

mAb LFG2:4B10 Kon mAb LFG2:4B10 Koff

PBSbaseline

LF predicted sequence (7aa)

non‐specific peptide

Epitope PhageDisplay Peptide (7aa)

Epitope Peptide Binding to anti‐LFG2:4B10 mAb

Multivalancy Monoclonal LF Toxin Capture

LTx

FreePA63

Free LF

PA‐2mAb

LFmAb

PA‐1mAb


Anthrax Toxin Detection POC Diagnostic

Label Free (BLI) Study of LF Toxin Complex

LOADmAb anti‐PA13E4:1G9

B

ASSOCPA63

DISSOCPA63 ASSOC

LF DISSOC

LF

ASSOCanti‐LFLFG2:4B10

DISSOC

SATURATIONanti‐LF

LFG2:4B10

ASSOCanti‐PA23C6:2E9

DISSOC

Titration of anti‐LF mAb

500nM

166nM

LTx Build

56nM18nM6nM

ASSOCanti‐LFLFG2:4B10

DISSOC

Sensor Location Sensor Info

Baseline Loc.

Assoc. (Sample) Loc. Sample ID Dissoc. Loc. Conc. (nM) Response KD (M) kon(1/Ms) kon Error kdis(1/s) kdis Error Rmax Rmax Error kobs(1/s) Req

Req/Rmax(%) Full X^2 Full R^2

A7 AVR1162‐biotin A7 AVR1674 A8 500 0.4466 4.79E‐09 2.92E+04 1.51E+03 1.40E‐04 3.99E‐05 0.4073 0.0046 1.48E‐02 0.4034 99 0.075145 0.829905

B7 AVR1162‐biotin B7 AVR1674 B8 166 0.2577 1.40E‐09 1.14E+05 3.14E+03 1.60E‐04 1.95E‐05 0.2553 0.0013 1.91E‐02 0.2532 99.2 0.007403 0.95758

C7 AVR1162‐biotin C7 AVR1674 C8 56 0.2156 3.43E‐10 1.65E+05 4.79E+03 5.65E‐05 2.42E‐05 0.2291 0.002 9.30E‐03 0.2278 99.4 0.007753 0.969468

D7 AVR1162‐biotin D7 AVR1674 D8 18 0.1048 1.69E‐09 1.88E+05 1.26E+04 3.18E‐04 3.84E‐05 0.1319 0.0052 3.70E‐03 0.1205 91.4 0.003061 0.959953E7 AVR1162‐biotin E7 AVR1674 E8 6 0.0022 5.91E‐07 1.09E+04 1.40E+06 6.43E‐03 3.62E‐03 0.5062 64.6666 6.49E‐03 0.0051 1 0.003588 0.22081

F7 AVR1162‐biotin F7 AVR1674 F8 56 0.187 2.00E‐09 1.71E+05 1.75E+03 3.42E‐04 8.80E‐06 0.1948 0.0006 9.92E‐03 0.188 96.5 0.000623 0.99599

G7 AVR1162‐biotin G7 AVR1674 G8 0 ‐0.0131H7 AVR1162‐biotin H7 AVR1674 H8 0 ‐0.0281


Baseline Loc.



A7 AVR1162‐biotin A7 AVR1674 A8 500 0.4466 8.23E‐10 9.78E+04 4.32E+03 8.05E‐05 2.57E‐05 0.3841 0.0026 4.90E‐02 0.3835 99.8 0.240155 0.961715



D7 AVR1162‐biotin D7 AVR1674 D8 18 0.1048 8.23E‐10 9.78E+04 4.32E+03 8.05E‐05 2.57E‐05 0.1744 0.0072 1.84E‐03 0.1668 95.6 0.240155 0.961715

Affinity Constant for anti‐LFG2:4B10 mAb

LOCAL

GLOBAL

KD=0.823 nM

LTx Build

Anti‐LF Binding and Saturation

ASSOCanti‐PAPAG2 3C6:2E9

DISSOC

Titration of anti‐LF mAb

1000nM333nM

111nM

37nM

12nM

4nM

Sensor Location Sensor Info Baseline Loc.



A7 AVR1162‐biotin A11 AVR1046 A12 1000 1.1425 4.26E‐09 3.15E+04 1.79E+03 1.34E‐04 3.22E‐05 1.083 0.0081 3.16E‐02 1.0784 99.6 0.394374 0.779291




E7 AVR1162‐biotin E11 AVR1046 E12 12 0.5084 1.52E‐11 2.81E+05 3.84E+04 4.27E‐06 7.41E‐05 0.5407 0.0418 3.38E‐03 0.54 99.9 0.103913 0.900787


G7 AVR1162‐biotin G11 AVR1046 G12 0 1.2911

H7 AVR1162‐biotin H11 AVR1046 H12 0 ‐0.0117


Baseline Loc.



A7 AVR1162‐biotin A11 AVR1046 A12 1000 1.1425 3.35E‐10 2.01E+05 5.88E+03 6.74E‐05 1.53E‐05 1.0448 0.0055 2.01E‐01 1.0445 100 2.341059 0.981921




E7 AVR1162‐biotin E11 AVR1046 E12 12 0.5084 3.35E‐10 2.01E+05 5.88E+03 6.74E‐05 1.53E‐05 0.719 0.016 2.48E‐03 0.6995 97.3 2.341059 0.981921


Affinity Constant for anti‐PA2 3C6:2E9 mAb

LOCAL

GLOBAL

KD=0.335 nM

Study Conclusions

• Novel Panel of anti‐LF mAbs identified and characterized

• BLI studies using Octet technology were employed in discovery process under several binding orientations

• Distinguish mAb specificity between LF and protein complex LTx

• Data generated from BLI correlated with traditional binding studies

• Stepwise binding and kinetics of antigen‐Ab complex determined with Octet

• Immunodiagnostic tools available for improved detection of early Anthrax infection

For more information please contact Centers for Disease Control and Prevention

1600 Clifton Road NE, Atlanta, GA 30333Telephone, 1‐800‐CDC‐INFO (232‐4636)/TTY: 1‐888‐232‐6348E‐mail: [email protected] Web: www.cdc.gov

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

Immunochemistry Team

National Center for Emerging and Zoonotic Infectious Diseases

Division of Scientific Resources / Scientific Products and Support Branch

Uzma AnsariKiosy LinAmanda LyonsR. Lee PittsXiaoling TangStacey Sweat

Scientific Products and Support Branch

Dennis Bagarozzi Jr.Curtis Taylor

Meningitis and Vaccine Preventable Diseases Branch/NCIRD

Conrad QuinnNazia Kamal

Clinical Chemistry Branch/NCEH

Anne Boyer

BLI Studies of Anthrax Toxin and Development of Diagnostic … · 2020-02-22 · • Anthrax is...

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