Transcript LBERI NIAID Tech call Minutes 110607 final

LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
7 November 2007
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
5797-1
Milestones
#2
Active
Vaccinations of study personnel
#3
Active -
Optimization of bioaerosol methods
#4
Active -
Confirmation of aerosol in vivo in NHP
#7
Inactive -
Schu-4 aerosol LD50 in cynomolgus
model
#8
Inactive -
LVS vaccine protection in Schu-4 infected
monkeys
#9
Inactive -
Development of GLP protocols for vaccine
efficacy studies in primates
#12/13
Active -
Assays for detecting relevant immune
responses in animals and humans
5797-2
Milestone #2 – Vaccinations of Study Personnel




Overall: >50% complete
Group 1:
–
Day 28 titers completed
–
No re-vaccinations needed at this time
Groups 2 and 3:
–
Completing follow-ups
–
No titer data as of yet
Protection is being provided by the vaccination
5797-3
Goals for Milestone #3 – Bioaerosol Development









Characterize the LVS bioaerosol using the Collison nebulizer

Determine optimum medium for aerosol dispersal (protein conc. &
antifoam)

Determine optimum medium for aerosol recovery (AGI)

Determine spray factors at various challenge concentrations

Determine lowest spray concentration & how to quantitate

Determine differences in spray factor for reconstituted, vs. thawed, vs.
fresh
Compare Collison to sparging generator
Compare Collison to micropump generator
Compare Collison to Aeromist generator
Consider additional bioaerosol generators (Aeroeclipse II, ultrasonic generators,
others)
Determine optimum method for LVS bioaerosol generation
Perform bioaerosol studies with Schu4 as described above to determine if LVS data
are predictive
Compile SOP for Schu4 bioaerosol studies
Write Milestone Completion Report
Red: completed
Green: in progress
5797-4
MS#3 – Flow Diagram
MS 3: Bioaerosol Development
Collison Nebulizer
Aeromist
Micropump
Order & receive
instrument
Order & receive
instrument
Order & receive
instrument
Set up instrument
Set up instrument
Set up instrument
Frozen
LVS
Fresh
LVS
Lyophilized
LVS
Frozen
LVS
Fresh
LVS
Frozen
LVS
Fresh
LVS
Down Select for
Schu 4 Generator
Frozen
LVS
Red: completed
Green: in progress
Blue: steps in the milestone
Fresh
LVS
Prepare bioaerosol SOP and
write MS completion report
5797-5
Milestone #3 – Bioaerosol Development
Accomplishments





Completed Collison LVS testing (fresh vs. frozen)
Completed sparging generator LVS testing; decided to halt
testing based on difficult setup and poor spray factors
Completed micropump LVS testing; spray factors with LVS
comparable to Collison
Tested additional technologies (e.g., ultrasonic, Aeroeclipse II,
others) with BG spores; LVS testing not pursued due to difficult
setup, practicality and/or no significant differences vs. Collison
Focus placed on Aeromist generator due to initial testing with
BG spores; completed series of bioaerosols using fresh and
frozen LVS; spray factors better than any generator tested to
date; delivered pressure critical
5797-6
Aeromist Data to Date

The following slides show Target vs. Actual CFU/mL and Actual
CFU/mL vs. Spray Factors for fresh LVS cultures tested to date
5797-7
Aeromist: Target vs. Actual CFU/mL (Fresh)
8.00
Actual CFU/ml (Log10)
7.50
7.00
6.50
6.00
5.50
5.00
4.50
4.00
3.50
3.00
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
Target CFU/ml (Log10)
7/19/2007 (Aeromist)
7/19/2007 (Collison)
8/10/2007
8/17/2007 (Pre; 10psi)
8/17/2007 (Pre; 20psi)
8/17/2007 (Post; 10psi)
8/17/2007 (Post; 15psi)
8/17/2007 (Post; 20psi)
8/17/2007 (Pre; 15psi)
5797-8
Aeromist: Actual CFU/mL vs. Spray Factor (Fresh)
-4.60
0.00
-4.80
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Spray Factor (Log10)
-5.00
-5.20
-5.40
-5.60
-5.80
-6.00
-6.20
-6.40
-6.60
Actual CFU/mL (Log10)
7/19/2007 (Aeromist)
7/19/2007 (Collison)
8/10/2007
8/17/2007 (Pre; 10psi)
8/17/2007 (Pre; 20psi)
8/17/2007 (Post; 10psi)
8/17/2007 (Post; 15psi)
8/17/2007 (Post; 20psi)
8/17/2007 (Pre; 15psi)
5797-9
Aeromist Results: Fresh LVS

Spray factors comparable to and exceeding those seen with the
Collison
–
May be misleading
–
Dependent upon delivered pressure
 Increased drop in viability observed at higher
delivered pressures
 Optimal delivered pressure appears to be around
10 psi


Easy setup
Cost effective
5797-10
Aeromist Data to Date

The following slides show Target vs. Actual CFU/mL and Actual
CFU/mL vs. Spray Factors for frozen LVS cultures tested to date
5797-11
Aeromist: Target vs. Actual CFU/mL (Frozen)
8.00
Actual CFU/ml (Log10)
7.50
7.00
6.50
6.00
5.50
5.00
4.50
4.00
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
Target CFU/ml (Log10)
4/13/2007
6/21/2007
6/26/2007
8/8/2007
8/16/2007 (Pre; 10psi)
8/16/2007 (Pre; 15psi)
8/16/2007 (Pre; 20psi)
8/16/2007 (Post; 10psi)
8/16/2007 (Post; 15psi)
8/16/2007 (Post; 20psi)
8/30/2007 (Collison, Pre; 20psi)
8/30/2007 (Collison, Post; 20psi)
10/26/2007 (Pre; 5psi)
10/26/2007 (Post; 5psi)
5797-12
Aeromist: Actual CFU/mL vs. Spray Factor (Frozen)
-5.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
Spray Factor (Log10)
-5.50
-6.00
-6.50
-7.00
-7.50
-8.00
Actual CFU/ml (Log10)
4/13/2007
6/21/2007
6/26/2007
8/8/2007
8/16/2007 (Pre; 10psi)
8/16/2007 (Pre; 15psi)
8/16/2007 (Pre; 20psi)
8/16/2007 (Post; 10psi)
8/16/2007 (Post; 15psi)
8/16/2007 (Post; 20psi)
8/30/2007 (Collison, Pre; 20psi)
8/30/2007 (Collison, Post; 20psi)
10/26/2007 (Pre; 5psi)
10/26/2007 (Post; 5psi)
5797-13
Aeromist Results: Frozen LVS

Spray factors comparable to and exceeding those seen with the
Collison
–
Efficacy lower than with fresh LVS
–
Again, may be misleading due to delivered pressures
 Increased drop in viability observed at higher
delivered pressures
 Optimal delivered pressure appears to be around
10 psi
 5 psi too low
5797-14
Milestone #4 – Confirmation of Aerosol in vivo in NHP

Continuing husbandry of vaccinated animals
–

Nearing 1 year post-vaccination mark
Currently scheduling ABSL3 move-in dates and aerosol
exposures
–
To follow mouse pathogenicity study
–
Dependant on Biobubble availability
 Initiate study early January 2008
 Tentative completion date: end of January/early
February 2008
5797-15
Milestone #2 – Vaccinations of Personnel
Plans for this month

Continue regular personnel checks and follow-up on any revaccinations that may be required
5797-16
Milestone #3 – Bioaerosol Development
Plans for this month

Initiate testing with Schu4
–
Primary focus on fresh Schu4 cultures
–
Focus to be on Collison and Aeromist
 Collison operated under normal operating
conditions (~20-30 psi delivered)
 Aeromist testing at 7.5 and 10 psi
–

Also plan to slightly increase suspension
volume in generator
Pathogenicity study with mice using LVS and Schu4
–
Collison (normal operating conditions)
–
Aeromist (parameters TBD based on additional practice
bioaerosols)
5797-17
Milestone #4 – Confirmation of Aerosol in vivo in NHP
Plans for this month



Confirm dates for ABSL3 move-in and Schu4 exposure
Decide upon dose and aerosol technology (dependant upon
mouse pathogenicity and MS#3)
Prepare exposure protocol
5797-18
Microbiology Quality Assurance

Routine Maintenance
–
Autoclave



–
Checked weekly
Monthly PM
Shaker incubator

–
As needed repairs
CO2 and regular incubators

–
Monthly PM (preventive maintenance) checks
Eye wash stations

–
Weekly bioindicator checks
As needed PM
Freezers

Annual defrost
5797-19
Microbiology Quality Assurance (cont.)


Quality control performed on all prepared media
–
Culture plates
–
Broth
–
Dilution blanks
Certificates of Analysis received and filed for purchased media
5797-20
Microbiology Quality Assurance (cont.)

Temperature Monitoring
–
Freezers

–
Refrigerators

–
Continuous w/ alarm
Incubators

–
Continuous w/ alarm
Continuous w/ alarm
Water baths

Recorded during use
5797-21
Microbiology Quality Assurance (cont.)

Equipment Calibrations
–
Pipettors

–
Balances

–
NIST traceable – use 2 years and replace
Temperature Probes – all incubators

–
Annual calibration and when used (June 2007)
Thermometers

–
Annual calibration and when used (February 2007)
Annual calibration (September 2007)
BSC

Annual recertification (July 2007)
5797-22
Aerosol Quality Assurance






Yearly recertification of the Class III biosafety cabinets (gloveboxes) in
1027 and 1028
Daily eyewash checks
Yearly calibrations of:
–
Bios flowmeter
–
TSI APS
–
Alicat Mass Flow Controllers
Daily calibration of AGIs
Daily calibration of aerosol generators
LabVIEW Program validated and reviewed annually
5797-23
IS and Data Management Quality Assurance

Data backup
–


All data on ABSL3 server backed up daily
Microbiology data recorded and maintained in workbook format
–
Reviewed regularly
–
Scanned out of ABSL3 regularly
Aerosol data recorded and stored via LabVIEW
–
Data sheets scanned out after each bioaerosol day
5797-24
Milestone #12/13 – Immune Responses in Animals
and Humans
Immunoassay Development and Comparisons in Animal Models
Choose PBMC
Purification Method
Choose PBMC
Freezing Method
Method chosen:
Purdue ListServ
Testing 3 protocols:
Cerus, CTL, Lyons
Phenotype Blood
and PBMCs
Test whether method
results in loss of B
cells
Red: completed
Green: in progress
Blue: steps in the milestone
Proliferation
assay:
Works for
Con A and
LVS
Develop
Immunoassay
methodologies
IFNg
ELISPOT
Plasma
IgG
ELISA
Plasma
IgA
ELISA
IFNg
Intracellular
Staining
5797-25
Update on Test of Freezing PBMCs



Goal: To select the freezing protocol which provides the best recovery
of cell viability and function when thawed
We originally compared 3 protocols which differed slightly in % and
type of serum used: Cerus, CTL and Lyons
–
Cerus: Frozen in 80% FBS/20% DMSO at 5 x 106/ml
–
CTL: Frozen in 90% human A/B serum/10% DMSO at 10 x 106/ml
–
Lyons: Frozen in Gibco Recovery Cell Culture Freezing Media
(contains optimal ratio of fetal bovine serum:bovine serum and
10% DMSO) at 5 – 10 x 106/ml; also, thawed in presence of
DNAse and left in 37o incubator for 30 – 60 minutes before use
We have stopped testing the Lyons protocol as it results in poor viable
cell yield upon thawing
5797-26
Summary of Freeze/Thaw using CERUS Protocol
Expt
Day post
Vacc.
# NHPs
Avg.
Cell
Avg. Recovery of
Con A Prolif.
Avg. Recovery
of LVSffProlif.
Avg. Recovery
of
LVShkProlif.
Recov
ery
TUL 8 (11/16/06)*
0 (ID)
3
60%
> 100%
NA
NA
TUL 9 (12/17/06)
28 (SC)
2
60%
53%
39.5%
33%
TUL 11
(3/26/07)**
117 (SC)
1
108%
64.4%
>100%
>100%
TUL 14 (6/11/07)*
203 (ID)
3
83.4%
64.4%
>100%
>100%
TUL 15 (6/12/07)
195 (SC)
1
82.6%
35.3%
9.7%
14.2%
TUL 16 (7/16/07)*
238 (ID)
1
63.5%
>100%
72.4%
>100%
TUL 17 (7/24/07)*
237 (SC)
3
68.9%
>100%
98.4%
99.4%
TUL 18
(8/23/07)**
278 (ID)
1
7.5%
NA
70.2%
>100%
*Background proliferation of frozen cells greater than fresh cells
**Proliferation to LVS not great even using fresh cells
5797-27
Summary of Freeze/Thaw using CTL Protocol
Expt.
Day post
Vaccinat
ion
# NHPs
Avg. Cell Recovery
Avg. Recovery Avg. Recovery
of Con A
of
Prolif.
LVSffProlif.
Avg. Recovery
of
LVShkProlif.
TUL 8 (12/18/06)
28 (ID)
2
70%
> 100%
NA
NA
TUL 11
(3/26/07)**
117 (SC)
1
38.5%
>100%
>100%
>100%
TUL 12 (4/9/07)
140 (ID)
1
54.4%
NA
98.0%
NA
TUL 14
(6/11/07*)
203 (ID)
3
89.6%
>100%
>100%
>100%
TUL 15 (6/12/07)
195 (SC)
1
75%
74.2%
12.7%
7.0%
TUL 16
(7/16/07)*
238 (ID)
2
69.6%
>100%
96.7%
>100%
TUL 17
(7/24/07)*
237 (SC)
2
69.4%
>100%
>100%
56%
TUL 18
(8/23/07)**
278 (ID)
1
10%
NA
25.7%
49.2%
*Background proliferation of frozen cells greater than fresh cells
**Proliferation to LVS not great even using fresh cells
5797-28
Comparison of Freeze/Thaw Protocols
Freeze/Thaw
Protocol
Avg. Cell
Recovery
Avg. Recovery
of Con A
Prolif.
Avg. Recovery
of LVSffProlif.
Avg. Recovery
of LVShkProlif.
CERUS
66.7%
73.9%
70%
78.1%
CTL
59.6%
95.7%
76.2%
68.7%
Interpretation: The two protocols are relatively equivalent in yielding viable
cells that proliferate in response to mitogen and antigen; however, the
CERUS protocol is more straightforward and less cumbersome to execute.
Therefore, we propose to move forward with the CERUS protocol as our
standard protocol.
5797-29
Update on IFNg ELISPOT detection from LVSvaccinated NHPs





We are attempting to optimize the ELISPOT assay that detects
IFNg secretion by LVS-stimulated PBMCs from previously
vaccinated NHPs
Thus far we have determined that 200,000 cells/well, 15 mg/ml
coating antibody and 1 mg/ml detection antibody is optimal
We have also determined that high RBC contamination increases
spot detection in all wells, including unstimulated wells
How does the freeze/thaw process affect LVS-stimulated IFNg
secretion?
Are there any differences when comparing ID- vs. SC-vaccinated
NHPs?
5797-30
100
90
80
Media
LVS hk Hi
LVS ff Hi
70
60
50
40
30
None, SC
None, ID
CTL, SC
CTL, ID
NT
Cerus, SC
20
10
0
Cerus, ID
Cell Mean for IFNg Spots
Both CERUS and CTL Freeze/Thaw protocols appear
to spare IFNg secretion
TUL 16 an 17 data shown; 1 – 2 NHPs each; 200,000 cells/well
5797-31
Day 278, SC
Day 278, ID
Day 237, SC
Day 237, ID
Day 238, SC
Day 238, ID
Day 195, SC
20
10
0
Day 195, ID
70
60
50
40
30
Media
LVS hk Hi
LVS ff Hi
Day 203, SC
100
90
80
Day 203, ID
Cell Mean for IFNg Spots
PBMCs from SC vaccinated NHPs may secrete more
IFNg after LVS hk-stimulation than ID vaccinated
NHPs
5797-32
PBMCs from Naïve NHPs show some proliferative
response to HK-LVS
Cell Mean for RLU normal
3000000
2500000
2000000
A00896
A00908
A00937
A04274
A04344
1500000
A04367
1000000
500000
0
Media
LVS hk Hi
LVS ff Hi
5797-33
PBMCs from Naïve NHPs secrete IFNg in response to
LVS
Cell Mean for IFNg Spots
160
140
120
A04274
A04344
A04367
100
80
60
40
20
0
Media
LVS hk Hi
LVS ff Hi
Note: Most responsive to FF-LVS, whereas the
proliferative response was more sensitive to HK-LVS
5797-34
Update on IgA anti-LVS ELISA




We were having trouble detecting IgA anti-LVS in the plasma of
vaccinated NHPs
It was unclear whether the Goat anti-monkey-IgA-HRP detection
antibody was working
Tested the detection antibody by coating ELISA plates with
human IgA – it was capable of detecting human IgA
Interpretation: NHP plasma does not contain IgA anti-LVS
antibodies
5797-35
MS 12/13: Plans for the next month


Test IFNg secretions from ID and SC vaccinated NHPs on the
same day
Test more naïve NHPs to see if they respond to LVS by
proliferation or IFNg secretion
5797-36
QA in Wilder Lab
Item
QA Performed by:
Scheduled for testing
Last Tested
Balances
Contractor to LRRI
Yearly
6/7/07
Pipettmen
Contractor to LRRI
Yearly
2/21/07
BioSafety Cabinet
Contractor to LRRI
Yearly
1/17/07
Fume Hood
Contractor to LRRI
Yearly
7/10/07
-80 Freezer
LRRI surveillance
Daily
11/5/07
CO2 Incubator
Lab Technician
Monthly
10/12/07
Eye Wash
Lab Technician
Weekly
10/30/07
5797-37
Action Items




Trevor- Changes to milestone completion or start dates will be
included in the LBERI Monthly Technical report due 11/7/07 to UNM.
Julie: will report the stimulation index in the monthly report for the
comparison of the Cerus and CTL protocols for recovering
proliferative activity post freezing.
Julie: In the monthly report, LBERI will present the ID vs SC data on
fresh cells by grouping the ID together and grouping the SC together,
and by removing the “no data’ available columns, per Freyja’s
suggestion
Julie and Trevor: will send the written abstracts to Barbara by Wed
11/7 or early on Thurs 11/8, so Vicki and Rick can review and return
the abstracts to LBERI before the 11/15/07 deadline.
5797-38