Bioassays 2011: Scientific Approaches and Regulatory Strategies October 31- November 1, 2011-Bethesda, Maryland.

Development of in vitro functional replacement assays for Botulinum toxin and antitoxin preparationsfor Botulinum toxin and antitoxin preparations

Christine Rasetti-Escargueil Bacteriology Division, NIBSC (Health Protection Agency), UK

National Institute for Biological Standards and ControlAssuring the quality of biological medicines

NIBSC: National Institute for Biological Standards and ControlBiological Standards and Control

Research and DevelopmentResearch and Development

International Standards Influenza Resource Centre (IRC)

CJD Resource Centre

Centre for AIDS Reagents VaccinesBiotherapeutics

NIBSCA Centre of Health Protection AgencyBlanche Lane, South Mimms, Potters BarHerts EN6 3QG http:/www.nibsc.ac.uk

Clostridium botulinum

• Gram-positive obligate anaerobic bacillus, spore-forming, producing botulinum toxin, heat sensitive, prefers low acid environments

• Botulism (Latin: “botulus” sausage) consists of 4 naturally occurringBotulism (Latin: botulus , sausage) consists of 4 naturally occurring syndromes: foodborne, wound, infant botulism, and adult intestinal toxemia (inhalational botulism, iatrogenic botulism)

Clinical syndrome of symmetrical cranial nerve weakness followed by• Clinical syndrome of symmetrical cranial nerve weakness followed by descending, symmetric flaccid paralysis of voluntary muscles, which may progress to respiratory failure and death if untreated. Treatment includes meticulous intensive care, mechanical ventilation and administration of antitoxin.

Botulinum toxins

• Seven toxin serotypes A-G: serotypes A, B, and E account for almost all cases of human botulism, types C and D reported in animals mostly.

• Botulinum neurotoxins (BoNTs) consist of a 100 kDa heavy chain (neuronal targeting) and a 50 kDa light chain acting as a zinc-dependent protease that cleaves the components of a membrane fusion complex (syntaxin, SNAP-25 and synaptobrevin).

Sophisticated strategy to enter neurons:

“BoNT is an astonishing modular nanomachine that unites

recognition, trafficking, unfolding, translocation, refolding,

and catalysis in a single entity.”(Montal, 2010)

Botulism Pathogenesis:M lti t h i f ll l i t i tiMultistep mechanism of cellular intoxication

A: Normal neurotransmitter release

•assembly of a synaptic fusion complex (SNARE proteins:assembly of a synaptic fusion complex (SNARE proteins:synaptobrevin, SNAP-25, and syntaxin)

•fusion of the synaptic vesicle containing acetylcholine with theneuronal cell membrane

•acetylcholine is released into the synaptic cleft, bound onmuscle cell receptors inducing contraction

B: Exposure to Botulinum toxin

•Botulinum toxin binds to the neuronal cell membrane and enters the neuron by endocytosis

li ht h i f b t li t i l ifi it th•light chain of botulinum toxin cleaves specific sites on the SNARE proteins

•assembly of the synaptic fusion complex and acetylcholinerelease are impaired

•without acetylcholine release, the muscle is unable to contract.without acetylcholine release, the muscle is unable to contract.

(SNARE indicates soluble NSF-attachment protein receptor; NSF, N-ethylmaleimide-sensitive fusion protein; and SNAP-25, synaptosomal-associatedprotein of 25 kD. JAMA. 2001;285:1059-1070)

Bio-weapon potential

• Extreme potency and lethality, ease of production, ease of transport and needfor prolonged intensive care

• Botulinum toxin classified by the Centers for Disease Control and Prevention (CDC) as one of six highest risk ‘‘Category A’’ agents likely to be deployed during a biological attack (lethal dose:1ng/kg intravenously or 3 ng/kg inhaledduring a biological attack (lethal dose:1ng/kg intravenously or 3 ng/kg inhaled,1 g of crystalline toxin could kill >1 million people).

• Antitoxin only available therapy to date: passive immunisation with equine y py p qantitoxin, vaccine limited supply for laboratory workers or military, protects against types A-E but prohibits future therapeutic use of toxin.

M t li ti t t tl iMouse neutralization tests currently in use

for the development of antitoxin countermeasure.

Therapeutic use of botulinum toxin

Justinus Kerner (godfather of BoNT)

: approved uses

: pending uses

:future uses?

Food and Drug gAdministration, Allergan, Ronny Gal, Sanford C. Bernstein, 2009

Expanding clinical indications and cosmetic applications lead to exponential increase of in vivo potency testing

Review: Hanchanale et al. Urologia Internationalis : 2010;85:125–130,

Potency test of Botulinum toxin A for injection: Monograph 2113 of European Pharmacopoeia 7 0injection: Monograph 2113 of European Pharmacopoeia 7.0

• The potency of the final reconstituted product is determined by a lethality assay in mice (mouse LD50 assay) : Intraperitoneal injection of escalating doses into groups of mice mice monitored over 72 or 96 hours post injection until deathof mice, mice monitored over 72 or 96 hours post-injection until death.

• Potency expressed in terms of the LD50 in mice (defined as the amount of toxin that kills 50% of mice) or expressed relative to the reference preparation (calibrated in LD50).

This method is considered inhumane due to its lethal endpoint and the large number of animals needed

Bulk purified toxin Final bulk Final lotSeed lot

> 100,000 foldDilution

Toxin Purification

Proof of active toxin!

Adler et al.2010

Potency test! (Specific activity)

Potency test!

Replacement bioassaysMain requirements: accuracy, sensitivity, specificity, reproducibility,Main requirements: accuracy, sensitivity, specificity, reproducibility, robustness and transferability to quality controlled settings

Examples  of assay types Endpoint measurement

Throughput speed

Advantages‐disadvantages Sensitivity References

C ll f (I i )Cell free (In vitro)FluorescenceELISAEnzyme cleavage assaySurface plasmon resonanceMass spectrometry

SNARE cleavage Medium to high

Low

Quantitative, high sensitivity, adaptable for high throughput but high false positive rate, multiple steps required

Not suited for HTS (high throughput screening)

7nM to 15pM1‐10LD50 (5pg/ml‐2ng/ml)0.01LD50/ml (40fg/ml)5pgml0.2 mouse LD50

Dong et al.2004, Hines et al.2008

Sharma 2011, Ferreira 2004

Ekong 1997, Sesardic 1999

Ferracci 2005, Marconi 2008

Boyer 2005, Kalb 2006

screening)

Cell basedPrimary cellsPC12 cells with FRET read‐outChick embryo neurons

SNARE cleavage Medium  Quantitative, high sensitivity, cost effective, challenging to adapt for HTS

1‐10pM 50nM10nM

Sheridan 2005, Keller 2004

Dong 2004

Stahl 2007, Nuss 2010

Neurones from mouse embryonic stem cellsDifferentiated human neuronal cells Neurotransmitter 

release

0.1LD50 (0.1pM)30‐100pM

Pellett 2011

Rasetti‐Escargueil 2011

Tissue based (ex vivo)Nerve‐evoked muscle twitch 50% paralysis Low Quantitative high sensitivity results 1LD (0 028pM) Sheridan 1999 Adler 1995Nerve‐evoked muscle twitch 50% paralysis 

timeLow Quantitative, high sensitivity, results

within 2 hours, neutralizing antibodies quantification, not suited for HTS, animals and specific skills required

1LD50 (0.028pM) Sheridan 1999, Adler 1995, 

Rasetti‐Escargueil  2009,2011

Whole animal (in‐vivo)Mouse bioassay Lethality or time  Low Quantitative, high sensitivity, no HTS,  1LD50 (10pg/ml) Sesardic 1994, 1996

.Local paralysis assay to death

Degree of paralysis

high cost, animal welfare concerns,functional assays allowing testing of therapeutics

0.1LD50 (1pg/ml)

Botulinum Project (2006-2009)Home Office Counter Terrorism and Intelligence Directorate UKHome Office, Counter Terrorism and Intelligence Directorate, UK

Objective: Development of in vitro potency assays for botulinum toxins andanti-toxin antibody preparations

• Optimisation and development of mouse nerve diaphragm assay for botulinum AOptimisation and development of mouse nerve diaphragm assay for botulinum A,

B and E toxins

N di h t ti f t A B d E tit i i ith• Nerve diaphragm testing of type A, B and E antitoxins, comparison with mouse

bioassay

• To develop neuronal cell based model and establish neurotoxicity marker for

type A toxin on differentiated cells

Hemidiaphragm assayOptimisation of mouse nerve diaphragm assay forOptimisation of mouse nerve diaphragm assay for botulinum A, B and E toxins

195

220

me

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Type A toxin

50%

Par

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is ti

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• Improved reproducibility using in-bred Balb/c mice: very low Chart Window

(g)

4

5

05/10/2006 13:27:57.296

Control Balb/c mouse

0 50 100 150 200 25020

45Type B toxin

Type E toxin

Toxin concentration (LD50/ml)

5

variability of 50% paralysis times whatever the dose or toxin serotype

• 7 times more sensitive to type A toxin and 7–12 times more sensitive to type E toxin relative to type B toxin

40 B lb/ i i d t d f ll d

Balb/c mouse12LD50/ml Type A toxin

Channel 3 (g

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Chart Window

Channel 3 (g) 4

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09/01/2007 13:32:58.546

• <40 Balb/c mice required to produce a full dose response curve for any toxin serotype (<6 mice per dose): refinement

• Mouse nerve–diaphragm: functional in vitro modelproviding accurate, sensitive and reproducible assessmentof toxin activity

MF1 mouse12LD50/ml Type A toxin

C

0

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16:40 33:20 50:00 1:06:40 1:23:20 1:40:00 1:56:40 2:13:20 2:30:00 2:46:4

Chart Window

Channel 3 (g)

2

3

4

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09/11/2006 13:01:03.875

of toxin activityRasetti-Escargueil, C., Jones, R. G. A., Liu, Y., Sesardic, D., 2009. Measurement of botulinum types A, B and Eneurotoxicity using the phrenic nerve-hemidiaphragm: improved precision with in-bred mice. Toxicon 53, 503-511

0

1

16:40 33:20 50:00 1:06:40 1:23:20 1:40:00 1:56:40 2:13:20 2:30:00 2:46:40

Hemidiaphragm assayNerve diaphragm testing of type A, B and E antitoxins, comparison with mouse bioassay

1000ρ =0.997

ml

10000ρ = 0.991

ml

Type B antitoxinsType A antitoxins

1

10

100

1000ρ

diap

hrag

m I

U/m

100

1000

ρ

diap

hrag

m I

U/m

0.01 0.1 1.0 10 100 1000 100000.01

0.1

1

In vivo IU/ml

Hem

id

1 10 100 1000 100001

10

In vivo IU/ml

Hem

id

1000

10000ρ =0.964

m IU

/ml

Type E antitoxins•Titrations curves established with NIBSC international standard reference Botulinum antitoxins A, B and E.

10

100

Hem

idia

phra

gm •Neutralising potency estimates in IU/ml of Botulinum antitoxin standards, human polyvalent antiserum and polyvalent test materials were obtained using mouse ex vivo hemidiaphragm and compared with in vivo paralysis assay or lethality assayC R tti E il* Y Li P Ri b R G A J D S di Ph i h idi h hi hl

1 10 100 1000 100001

In vivo IU/ml

H C. Rasetti-Escargueil*, Y. Liu, P. Rigsby, R.G.A. Jones, D. Sesardic, Phrenic nerve-hemidiaphragm as a highly sensitive replacement assay for determination of functional botulinum toxin antibodies, Toxicon 57 (2011) 1008–1016.

Summary-Hemidiaphragm assayy p g y

• Number of animals needed to produce statistically useful data in the hemidiaphragm assayis much lower than in the mouse lethality or paralysis bioassay:

mouse bioassay: 31-48 mice for one test sample + 32-48 mice for the reference sample(CV:11%)

hemidiaphragm assay: 30 Balb/c mice to produce a full titration curve (CV: 3% to 13%)hemidiaphragm assay: 30 Balb/c mice to produce a full titration curve (CV: 3% to 13%).Only 4 to 7 mice necessary to assess neutralising potency of each antitoxin sample.

• Excellent agreement between the two assay systems for each toxin serotype: almostperfect concordance for type A antitoxins, substantial concordance for type B and type Ep yp , yp ypantitoxins.

• The hemidiaphragm assay involves all functional domains of BoNT, differentiates betweenmajor BoNT serotypes (A, B, D and E), is reliable with diverse sample matrices providedthat activity is >0.2 IU/ml. Used at NIBSC for antitoxin potency, pending regulatoryadoption by product validation.

->Hemidiapragm method is a suitable highly sensitive, accurate, specific and robustreplacement or refinement to the mo se lethalit bioassareplacement or refinement to the mouse lethality bioassay

Cell based assay developmentTo develop neuronal cell based model and establishTo develop neuronal cell based model and establish neurotoxicity marker for type A toxin on differentiated cells

• Need for the development of cell based functional assays for the testing of new generation antitoxins and replace mouse lethality bioassay

• Cell based assays are retaining the three main stages of toxin mode of action in vivo (receptor binding, translocation, enzyme activity), minimum accepted unit that can cover all the steps of BoNT mechanism of toxicity

• Have potential to offer entire replacement of animals at all stages of production process including production of active bulk toxin. Immortalised cell line desirable in QC setting, easy to maintain, expandable.

Undifferentiated SH‐SY5Y cells in full medium (cell line originally derived from a human neuroblastoma from the European Collection of Cell Cultures)

Differentiated cells supplemented with retinoic acid for 5 days followed by BDNF in serum‐free medium every other day until fully mature neuronal morphology with a robust network of neuritic processes connecting the cells

Is this cell based assay a functional model?functional model?

Confocal fluorescence of markers of differentiation: MAP-2 proteins(green) at the distal portions of the neurites(g ) p

Presence of specific neuronal markers andkey components of synaptic activity

100μm

100μm100μm

Immunocytochemical detection of the presence of SNAP-25 (a) and synaptophysin (b) in human neuroblastoma SH-SY5Y differentiated cell (Rasetti-Escargueil et al. The Botulinum Journal 2011)

Potassium-evoked [3H]-noradrenaline release in undifferentiated and differentiated SH-SY5Y cellsundifferentiated and differentiated SH SY5Y cells

20

15

e re

leas

e(%

) •Accurate and reproducible quantification of neurotransmitter release (counting protocol reviewed to maximise both efficiency and reproducibility)

5

10

nora

dren

alin

e

•Cell differentiation improved their functionality

Basal Stimulated Basal Stimulated 0

5

[3 H]-n •All three steps of Botulinum toxin

intoxication are involved in the neurotransmitter release assay

Data on the graph are the mean of seven experiments each determined in 9 to 2 replicate for undifferentiated cells and the mean of three separate experiments each determined in 6 to 10 replicates for differentiated cells.

Undifferentiated Differentiated

Dose effects of Botulinum type A toxin:Establish neurotoxicity markersEstablish neurotoxicity markers

24 well plate-42h incubation 96-well Corning® Ultra-web plate-68h incubation

96-well plate format yielded lower variability in neurotransmitter measurements and increased sensitivity to botulinum toxiny

Gangliosides treatment improved cell sensitivity

Inhibition of [3H]-noradrenaline release: Establishing a dose response curve to Botulinum type A g p yptoxin

100IC50 100 M ith t GT1b t t t d

60

80

bitio

n

IC50: 100 pM without GT1b pre-treatment and 35 pM after GT1b pre-treatment (non linear regression model, GraphPadPrism)

20

40 Without GT1b

With GT1b% In

hib

30

Toxin A neutralisation

-12 -11 -10 -9 -80

Toxin A (LogM)

20

% R

elea

seControl Toxin A 300pM Toxin A 300pM +antitoxin

0

10

%

Improving cell line sensitivity further?

Membrane vesicle trafficking

• In neurons that are actively releasing neurotransmitters, FM1-43 becomes internalized within the recycled synaptic vesicles and the nerve terminals brightly stained as a result f i l t ffi ki d i th ti l ti i t lof vesicle trafficking during the stimulation interval (Betz and Bewick, 1992).

Labelling of differentiated SH-SY5Y cells with vesicle recycling marker FM1-43 (SH-SY5Y cells passage 21 on day 17 differentiation)

SH-SY5Y cells passage 21 on day 19 differentiation: nuclei, actin, recombinant botulinum

Inhibition of synaptic vesicle recycling activity: Ti FAXS d FACS l iactivity: Tissue FAXS and FACS analysis

BoNT A blockade of neurotransmitter releaseneurotransmitter releaseconcomitant to reduced vesicle trafficking in differentiated SH-SY5Y cultures

Control Botulinum type A 1nM Botulinum type A 1nM+ standard antitoxincultures

Summary-Cell based assay development

• Reproducible and robust differentiation protocol applied on SH-SY5Y cells generating neuronal cultures stable for 2 to 3 weeks.g g

• Staining of typical neuronal markers, presence of secretory vesicles, active vesicle trafficking and ability to release neurotransmitters.

• Accurate and reproducible quantification of noradrenaline release levels were significantly higher in differentiated cells and correlated with vesicle turnover assessments.

• The suitability of differentiated SH-SY5Y cells for detecting high picomolar levels of Botulinum type A was evidenced using a functional end point relevant to neurotoxicity. y

• Differentiated neuroblastoma cell line SH-SY5Y can provide a suitable basis for the development of cell based assays, easy to standardise but not as sensitive than primary neuronal cellsthan primary neuronal cells.