1 Evaluation of Prion Reduction Filters with a Highly Sensitive Cell Culture-Based Infectivity Assay...

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Evaluation of Prion Reduction Filters with a Highly Sensitive Cell Evaluation of Prion Reduction Filters with a Highly Sensitive Cell Culture-Based Infectivity AssayCulture-Based Infectivity Assay

Presented By Samuel Coker, PhD

Senior Technical Director Pall LifeScience R&DFDA-TSEAC Meeting on October 28-29, 2010

FDA Meeting, October 28-29, 2010

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Background

The clearance of prion infectivity from biologic fluids with prion removal devices is usually quantified by: The use surrogate marker of infection in in vitro

assays such as: Enzyme-linked Immunosorbent assay (ELISA). SDS-PAGE Western blot. Conformational dependent immunoassay (CDI).

Bioassays based on intracerebral inoculation of hamsters or mice.


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Disadvantages of current methods

Prion infectivity may accumulate in the absence of detectable levels of PrPSc;

Levels of PrPSc do not necessarily correlate with infectivity; Current bioassays using mice and hamsters are slow,

cumbersome, involve the use of hundreds of hamsters for example, and are extremely expensive with a typical endogenous infectivity study costing as much as $250,000 to $500,000 with a single study with a duration of 500 to 600 days. Therefore, the development of a reliable and highly sensitive

cell culture-based infectivity assay may greatly accelerate the evaluation of new prion removal devices.


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Objective of study

The main objective of this present study was to evaluate the use of a a highly sensitive cell culture based infectivity assay to evaluate the effectiveness of the following prototypes of leukocyte and prion reduction filters in removing prion infectivity from 300 mL of red cell concentrates (RCC): Leukotrap affinity prion reduction filter (10 layer variant-PRM3) Leukocyte and prion reduction filter (22 layer variant-PRM3) Leukocyte and prion reduction filter (22 layer variant-PRM6) Leukocyte and prion reduction filter (22 layer variant-PRM7) Leukocyte-reduction filter (BPF4)


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Filter design and configuration

All the filters contained essentially the same prion binding chemistry on polyester fibrous media.

All the prion reduction filters are also capable of removing leukocytes.

PRM3, PRM6 and PRM7 have the same base polyester media but with different hydrophilic and hydrophobic properties which may or may not enhance prion removal.

BPF4 contained polyester fibers with surface chemistry for binding leukocytes.


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Leukocyte-reduction component of the prion reduction filter

Mechanical trapping or sieving based on the structure of the fibers (fiber diameter, pore size, or distribution etc.)

Activation of leukocytes to enhance binding to the polyester fibers,

Indirectly through interaction with platelets resulting in the formation of cell aggregates that are then removed through sieving.


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Materials and Methods

Preparation of 10%(wt/vol) Mouse Brain HomogenateMice brain homogenate from mice infected with the Rocky Mountain Laboratory (RML) scrapie strain were prepared according to the standard protocol of Professor Weissmann's laboratory at SCRIPPS, FL, USA.

Briefly, mice were first inoculated intracranially with high titer brain homogenate from mice infected with RMLscrapie strain. The animals were sacrificed after about 145 days at an advanced stage of disease, and the brains were removed to prepare 10% suspensions in phosphate buffered saline (PBS), pH 7.4. When this method is used with scrapie infected mouse brain homogenate (MBH), the titer is of the order of about of 108.0- 8.4LD50 units per mL.


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Materials and Methods

Five units of 1-2 day-old ABO compatible nonleukocyte-reduced RCC were purchased directly from an AABB accredited blood bank. All 5 units were transferred into a 2-liter blood bag to create a homogenous pool.

Approximately 10.5mL of infectious MBH-RML were added to about 1570mL of pooled RCC such that the final dilution of the MBH-RM with RCC was 1:150.

The infectious prions were mixed with the pooled RCC . The pooled RCC was then divided into 300 mL aliquots.

Preparation of Red Cell Concentrates


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Experimental design

BPF4 BPF4 BPF4 BPF4

Pr-Filter Pr-Filter Pr-Filter Pr-FilterPr-Filter

#1 #2 #3 #4 #5

Pool of 5 units of RCC (1570mL). 11mL of 10%(wt/vol) mice brain homogenate

RML scrapie strain (MBH-RML). 1:150 Dilution

Prion & LeukocyteReduced RCC

Leukocyte-ReducedRCC

300mL RCCContaining MBH

A Unit (300-320 mL) of RCC


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MATERIALS AND METHODS Standard Scrapie Cell Assay (SSCA)

The SSCA is: Based on the isolation of a cell line (Cath-a

differentiated cells, CAD5; Scripps, FL) that is highly susceptible to RML scrapie strain;

A method for identifying and quantifying prion-infected cells.


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Procedure for SSCA

1. 5000 CAD5 cells in reduced serum medium (Opti-MEM) were dispensed into 96 well tissue culture plates;

2. The cells were allowed to attach to the plates over night in humidified CO2 incubator;

3. The attached cells were exposed to either serial dilutions of MBH-RML (1:5, 1:10, and 1:30) or the test samples (Samples 1-5) and then incubated for 4 days in a humidified CO2 incubator and allowed to grow to confluence.

4. After 4 days, the cells were split 1:10 and then seeded again onto tissue culture plates.

5. After the third split, 20,000 cells of each sample were filtered onto membranes of a 96-well plate (AcroRead, Pall LifeScience);

6. The cells were lysed and treated with proteinase K to eliminate normal PrPC;7. PrPSc infected cells were identified by an Enzymed-Linked Immunosorbent

Assay (ELISA) using monoclonal antibody (D18) and alkaline phosphatase –linked anti-IgG antiserum

8. The infected cells (PrPSc positive cells) were counted using an automated imaging system. The settings of the imaging system was optimized to give maximal ratio of positive cells relative to negative cells

9. The data are expressed as the number of infected cells per 20,000 CAD5 cells.


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Flow diagram of SSCA procedure

Step 1: Expose susceptible CAD5 cellsto brain homogenate or red cell suspensions containing infectious prions

Step 2: Serially propagate cellsand seed ELISPOT

Step 4: Colorimetric Detection

Step5: Analysis – Zeiss KS Elispot Automated Imaging

Step 3: Digest & Denature


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Representative well of an SSCA plate as imaged with the Zeiss KS Elispot system: Infected spots on 20,000 CAD5 cells.


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Spiking study to determine the inhibitory effects of test samples

To confirm that any observed reductions in infectivity were not due to components in the test samples that were inhibitory to the cell line, aliquots of postfiltration samples at different dilutions (1:5,1:10,1:30 and 1:90) were mixed with a predetermined amount of MBH-RML. 1mL of test sample was added to 10µL of 108.75 LD50 /

mL MBH-RML, and 0.145mL of the suspension was added to 5000 CAD5 cells

The proportion of infected cells at the different dilutions of the test samples was determined as previously described.


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Copyright 2008 TSRI

Figure 1A Standard curve of serial dilutions of MBH-RML in the presence and absence of Inhibitor (Pentosan Polysulfate) of infection


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Standard calibration curve of SSCA Figure 1 Dose response of CAD5 cells to different concentrations of RML infectious prions. Each

data point represents the mean standard deviation of 6 replicates.

0 1 2 3 4 50

200

400

600

800

1000

1200

1400

Log (LD50 Units Per mL of Sample)

Pri

on

Infe

ctiv

ity

Sp

ots

Per

20,

000

CA

D5

Cel

ls


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Determination of inhibitory effects of RCC on SSCAFigure 2 Determination of the Inhibitory effects of postfiltrationRCC on prion infectivity. Each bar represents the mean standard deviation of 6 replicates.

Control Sample 1 Sample 2 Sample 3 Sample 4 Sample 50

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

Postfiltration Test Samples

Prio

n In

fecti

vit

y P

er 2

0,0

00 C

ells


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Effect of leukoreduction step on prion infectivity

Figure 3 Effects of leukocyte-reduction step on prion infectivity inunits of red cell concentrates. Each bar represents themeanstandard deviation of a minimum of 5 replicates.

Prefiltration BPF4 -1 BPF4-2 BPF4-3 BPF4-40

100

200

300

400

500

600

700

800

900

Leukoreduction Filters

Pri

on

In

fecti

vit

y S

po

ts P

er

20,0

00 C

ells


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Prion infectivity in red cell concentrates before and after filtration with different prion reduction filters

Figure 4 Reduction in prion infectivity in full units of red cellconcentrates with different prototypes of prion reduction filters.Each bar represents the mean standard deviation of 6replicates. Note: NLR = Non-Leukocyte reduced.

Prefiltration LAPRF-1 B1451AQ B1570AI B1570AK B1570AK-NLR0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

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850

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950

1000

LAPRF-1 = 10 layer prion reduction filterB1451AQ = 22 layer prion reduction filterB1570AI = 22 layer prion reduction filterB1570AK = 22 layer prion reduction filterB1570AK-NLR = 22 layer prion reduction filter

*p<0.05

** ** ** **

** Samples with less than 15 spots are shown ashaving less than 1 spot after background subtraction

Prototypes Prion Reduction Filters

Prio

n In

fecti

vit

y S

po

ts P

er 2

0,0

00 C

ells


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Endogenous infectivity studies with different prototypes of prion-reduction filters

Units (500-550mL) of whole blood were collected from scrapie infected hamsters ( a unit of blood was obtained from 500 hamsters) into CPD anticoagulant.

Units of whole scrapie infected blood were centrifuged at 5000g for 30 minutes.

The supernatants were removed and the red cells were resuspended in SAGM additive solution to produce a unit ( 250-350mL) of RCC.

Each unit of RCC was filtered with either 10 or 22 layer variant of prion reduction filters.

50µL of pre and postfiltration RCC were injected intracranially into healthy normal hamsters. The animals were monitored and maintained for 300-500 days. Those that developed clinical symptoms of scrapie were killed and the brain tested for the presence of PrPSc by Western blot assay using 3F4 monoclonal antibody.


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Summary of results of SSCA

All the 22-layer prion reduction filters independent of the initial base chemistry on the polyester fibers (PRM3 vs. PRM7) removed prion infectivity below the limit of detection of the SSCA. Therefore, the important component is the number of layers of the fibers with the prion removal chemistry.

The maximum reduction observed in the present study with the SSCA was ≥ 2.0 log10 LD50/ mL

The 10 layer variant of the prion reduction filter showed some residual infectivity which is significantly higher than the baseline value obtained with uninfected CAD5 cells


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Flow diagram of endogenous infectivity study :scrapie infected RCC were filtered with 10 and 22 layer prion reduction filters

Scrapie Infected HamstersNormal Hamsters

Intracerebral Injection

Hard spin centrifugationRemove supernatant plasma

Add red cell additive solution

Prion reduction

filter

BRAIN10% brain

homogenateBRAIN10% brain

homogenate

10% Scrapie Infected Hamster Brain Homogenate


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Endogenous infectivity study with 10-22 layer prion reduction filters

Figure 5 In vivo scrapie infection in normal hamsters injected with unfiltered and filtered red cellconcentrates

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

12.0

13.0

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15.0

16.0

7/187

3/413

6/43

0/35

6/84

0/84

Treatment Conditions

Nu

mb

er

of

Ham

ste

rs In

fecte

d R

ela

tive t

o t

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To

tal N

um

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ste

rs T

reate

d (

%)


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Endogenous infectivity study with 10 layer prion reduction filter

0 50 100 150 200 250 30095

96

97

98

99

100

Control Group ReceivedUnfiltered RCC (median onset of

scrapie infection = 130 days*)

Treated Group Received Filtered RCC (median onset ofscrapie infection = 230 days*)

Figure 6 Kaplan-Meier Survival Plot: Comparison of the onset of scrapie infection in Normalhamsters after intracerebral injections of filtered (10 Layer variant) and unfiltered RCC from Scrapieinfected hamsters. Note: Treated hamsters = 413; Control hamsters = 187.

*Log-rank (Mantel-Cox) Test p =0.01

Time - Onset of Scrapie Infection (Days)

Perc

en

t su

rviv

ing

Aft

er

Receiv

ing

Scra

pie

In

fecte

d R

CC

(%

)


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Endogenous infectivity study with 22 layer prion reduction filterFigure 7 Kaplan-Meier Survival Plot: Comparison of the onset of scrapie infection in

normal hamsters after intracranial injections of filtered (22 Layer variant) andunfiltered RCC from scrapie Infected hamsters. Note: Treated hamsters = 84; Control

hamsters = 84

0 50 100 150 200 250 300 350 400 450 500 550 60090

91

92

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100

Control Group Received Unfiltered RCC

Treated Group Received Filtered RCC

Log-rank (Mantel-Cox) Test p = 0.014

Time - Onset of Scrapie Infection in Hamsters (Days)

Perc

en

t su

rviv

ing

Aft

er

Receiv

ing

Scra

pie

In

fecte

d R

CC

(%

)


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Summary of endogenous infectivity data and their relationships to the results of the SSCA

All the 22-layer prion reduction filters significantly prevented the transmission of scrapie infection into hamsters that received filtered RCC over the lifespan (500-550 days) of the hamsters. In contrast, significant number of hamsters that received unfiltered RCC developed scrapie infection (Figures and 7).

In the study with the 10 layer prion reduction filter, 3/413 (0.74%) of the hamsters that received filtered RCC developed scrapie with a median onset of scrapie infection at 130 days post-treatment compared to 6/187 (3.74%) and median onset of 230 days in the control hamsters that received unfiltered RCC (Figure 6A).

These endogenous infectivity data are in agreement with the in vitro infectivity assay, the SSCA which showed residual prion infectivity with the 10 layer prion filter and none (below limit of detection) with the 22 layer.


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Conclusions

These results demonstrate the utility of the highly sensitive cell culture-based infectivity assay for screening reduction filters.

The use of this type of in vitro infectivity assay will substantially help expedite the screening and discovery of devices aimed at reducing the risk of vCJD disease transmission through blood transfusion.

The use of this infectivity assay will also significantly reduce the cost for developing and evaluating devices for prion clearance.

It is very important that methods for screening potential prion removal chemistries or ligands include an infectivity assay at a very early stage of the screening process to complement other in vitro assays.

Although for the final release of any prion reduction device, it may still be necessary to conduct a limited endogenous infectivity bioassay, the use of SSCA should help improve and greatly expedite the process of screening and developing new devices for prion clearance from biological fluids.


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Acknowledgments

Professor Charles Weissmann (Scripps, FL) Dr. Christopher Baker (Scripps, FL) Dr. Cheryl Demczyk (Scripps, FL) Ms. Fabiola Andrade (Pall Medical Research Lab, NY) Professor Maurizio Pocchiari (Istituto Superiore di Sanita, Rome, Italy) Dr. Franco Cardone (Istituto Superiore di Sanita, Rome, Italy) Dr. Richard Carp (NY Institute for Basic Research, NY) Dr. Richard Kascsak (NY Institute for Basic Research, NY) Ms. Regina Kascsak (NY Institute for Basic Research,NY) Mr. Clifford Meeker ( NY Institute for Basic Research, NY) Dr. Joseph Cervia (Pall Medical, NY) Mr. Allan Ross (Pall Medical, NY) Dr. Stein Holme (Pall Medical, NY) Members of Pall QIRP internal review process (Pall Medical, NY)

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