LBERI- UNM Tech Call Minutes: 2/06/07
Prepared by: Barbara Griffith 2/6/07
Sent to LBERI: 2/6/07 for review
Reviewed and Edited by: Bob Sherwood (2/15/07), Julie Wilder (2/6/07)
Distributed to NIAID: 2/15/2007
Present: Bob Sherwood, Barbara Griffith, Julie Wilder, Rick Lyons, Joe Breen
Absent: Kristin DeBord, Freyja Lynn, Ed Barr, Vicki Pierson, Marlene Hammer,
2/6/07 Action Items
Bob- Titer of LVS stock in 10% sucrose will be available next week (~2/12)
Barbara- contact Bob regarding last few edits on LVS growth SOP (emailed Bob on 2/6/07)
Barbara- update MS#3 timeline in MS Project file; revised end date is ~ 4/15/07 (completed
2/13/07)
Bob- 5 sprays scheduled in February to complete Collison and start sparging generator
Julie is contacting the Victor service rep to clarify differences in aperture settings that could
underlie background differences between the Victor at UNM and Victor Light at LBERI.
Julie- multiple followup experiments on MS 12 and MS 13
Julie- will do ConA test again, and if the SC/ID difference disappears, then the next time LRRI
vaccinates, will look at a the phenotype of cells in the blood and PBMCs during the initial weeks
after vaccination.
LBERI Update on Animal Model Development
Milestones
#3
#2
#4
#7
#8
#9
Active InactiveActive Inactive Inactive Inactive -
#12
Active -
#13
Active
Optimization of bioaerosol methods
Vaccinations of study personnel
Validation of aerosol in primates
Schu-4 aerosol LD50 in cynomolgus model
LVS vaccine protection in Schu-4 infected monkeys
Development of GLP protocols for vaccine efficacy
studies in primates
Assays for detecting relevant immune responses in
animals and humans
Compare assays in animal models (sensitivity)
A. Action Items
1. Bob: Will make a new stock of LVS in 10% sucrose for longer term storage
i. New stock harvested 1/19/2007 in 10% sucrose. Wayne Conlan had poor
results with glycerol and suggested 10% sucrose
ii. Action: Bob- Titer of LVS stock in 10% sucrose will be available next week
(~2/12)
2. Julie will check which macaque is older and whether one set could be controls from
one group and tox primed from the other group, rather than all being from control
groups (this one has been completed)
3. Bob: will bring the LVS growth SOP into final format- sent to Barbara 2/5/07
i. SOP was formatted per LBERI format
ii. Draft of SOP and traveler (cover page) provided
iii. Working Group: implementation of SOPs may be unique per institution
iv. Action: Barbara- contact Bob regarding last few edits (emailed Bob on
2/6/07)
B. Goals for Milestone #3 – Bioaerosol Development
1. Characterize the LVS bioaerosol using the Collison nebulizer
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2.
3.
4.
5.
6.
7.
i. Determine optimum medium for aerosol dispersal (protein conc. & antifoam)
ii. Determine optimum medium for aerosol recovery (AGI)
iii. Determine spray factors at various challenge concentrations
iv. Determine lowest spray concentration & how to quantitate
v. Determine differences in spray factor for reconstituted, vs. thawed, vs. fresh
Compare Collison to sparging generator
Compare Collison to micropump generator
Consider additional bioaerosol generators
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
C. MS#3 – Flow Diagram
1. Bioaerosol Development
i. Collison Nebulizer
1. Order & receive instrument
2. Set up instrument
a. Frozen LVS
b. Fresh LVS
c. Lyophilized LVS
3. Almost done with Collison testing
ii. Sparging Generator
1. Order & receive instrument
2. Set up instrument
a. Frozen LVS
b. Fresh LVS
3. ready to start sprays
iii. Micropump
1. Ordered & receive instrument
2. Set up instrument
a. Frozen LVS
b. Fresh LVS
iv. Down Select for Schu 3 Generator
a. Frozen LVS
b. Fresh LVS
v. Prepare bioaerosol SOP
D. MS#3 Schedule
1. Planned completion date for MS#3 is 3/1/2007;
2. This date will probably slip at least 6 weeks
i. Delay due to limited access to the bioaerosol suite
ii. Amount of delay may be affected by vaccination status
iii. If vaccination not required, may complete by 4/15/2007
iv. Don’t feel that this delay will adversely impact other milestones
v. Action: Barbara- update MS#3 timeline in MS Project file; revised end date is
~ 4/15/07
vi. The sprays are generated in an all enclosed environment and performed in
biosafety cabinet, so hopefully can complete the spray testing without the
vaccination.
vii. Rick: LVS vaccination unlikely available in this time frame but can complete
this milestone w/o vaccination if necessary
E. Milestone #3 – Bioaerosol Development & Accomplishments
1. The purpose of the sprays conducted in January was to fill in data gaps for the spray
factors for sprays performed with the Collison generator and frozen or fresh LVS
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2.
Performed 3 sprays in January
i. January 16 – frozen LVS
ii. January 17 – fresh LVS (48 hr Chamberlain’s broth)
iii. January 24 – fresh LVS (48 hr CB) – replicate spray of 1/17 experiment
F. Frozen LVS – Target vs. Actual Titers
1. The following slide demonstrates the ability to accurately predict the actual LVS titer
from frozen stock; each day is a different color. Accurate prediction is critical later for
giving the correct dose to the animals, as the confirmation of the dose delivered is
determined after the primate is exposed.
2. For each spray, the target cfu/ml is selected and then following the spray, the sample
is serially diluted and plated on CHAB to determine the actual titer.
3. The figure demonstrates the results to date which indicate that with more experience
LBERI is getting better at predicting the actual titer. 1/16/07 spray was on the curve
and thus the prediction worked well.
4. For testing, the LVS frozen vial, LBERI: Thaws vial, mixes in spray buffer, sprays,
returns to lab, dilutes and plates on CHAB to determine the actual dose in the spray.
G. Fresh LVS – Target vs. Actual Titers
1. The following slide demonstrates the ability to accurately predict the actual LVS titer
from fresh 48 hr culture
2. For each spray, the target cfu/ml is selected and then following the spray, the sample
is serially diluted and plated on CHAB
3. The figure demonstrates the results to date which indicate that the optical density
curve which we are using to predict actual concentration is quite accurate allowing for
good predictions of actual titers
4. Process: Fresh culture of LVS, spun down, spray, return to lab to dilute and
quantitate on CHAB.
5. Rick: sprays on 1/16 and 1/17 look very good, except high target outlier on 1/17
sprays.
6. Rick: have we predicted the range that we will need to use to give the correct low
dose to the NHP?
7. Bob: Yes. We expect SCHU S4 to have similar curve to LVS. Expect a fairly low
infectivity dose. So when the NHP are infected, the dose is low as needed. LBERI
has determined the lowest dose that allows detection of viable colonies in the all
glass impinger. As LBERI goes from low to the highest dose, LBERI has determined
what the spray factor will be and what the predictable dose will be in the spray. So
whether the dose is at 1 LD50 or 100 LD50, LBERI will be able to predict the dose
and reproduce the spray.
H. Spray Conc. Vs. Spray Factor (Frozen LVS)
1. The Spray Factor is a calculated value determined for each spray
i. Recovered cfu/L / spray conc. (cfu/mL) x 1000
ii. All aerosol scientists use the spray factor
iii. 10-6 to 10-7: standard vegetative spray factors and are good spray factors
2. The following figure is a compilation of the spray factors determined to date for LVS
sprays performed with thawed LVS
3. The preceding figure demonstrates that frozen LVS has very good spray factors at
10-6 to 10-7 up to 8 logs of LVS (over the log 4 to log 8 range)
4. LBERI would still like to see a little more data in the 5 to 7 log range; didn’t get
bacteria to the AGI in prior experiments. Expect that log 5, 6,& 7 will be linear
(horizontal) but need to perform the tests empirically.
5. The following figure is a compilation of the spray factors determined to date for LVS
sprays performed with fresh LVS (48 hr CB culture)
6. The preceding figure demonstrates that fresh LVS has very good spray factors at 10-6
to 10-7 up to 7 logs of LVS
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7.
Experiment on 1/24/07 has uncharacteristically low spray factors, in the 10-8 to 10-9
range. Rick: This represents a lack of recovered bacteria. Bob says they are under
recovering in the AGI. This indicated a problem with the AGI. (see below I.1.vii) Still
need more data in the 5 to 8 log range
I.
Milestone #3 – Bioaerosol Development
1. Data Analysis
i. Spray set ups are working OK
ii. Can accurately predict actual CFU/mL, but
iii. Spray 1/16/07 had problem with AGI recovery
iv. Spray 1/17/07 had problem with contamination
v. Spray 1/24/07 had problem with AGI recovery
vi. Took Collison unit completely apart and cleaned everything
vii. Had material buildup on the AGI sampler coupling (probably causing
increased shear forces with lower spray factors). Bob said Ed had set up a 3
way valve on the sampler in case he wanted to bypass the AGI sampler.
Sample is being pulled through all glass impinger. Ed set up the ability to
bypass AGI via coupler, if needed. So material was impacting on the top of
the coupler and small gap was becoming almost closed. By narrowing the
gap, they massively increased the shear force. Bob has removed coupler /3
way valve and Bob’s team is changing the tubing with every spray to prevent
future buildup.
viii. Need to repeat a few sprays
2. Plans for this month: Action items below: Bob
i. Repeating the last Collison sprays
1. Want to fill in the data gaps for spray factors
a. Frozen – 5 to 7 logs (1 day)
b. Fresh – 5 to 8 logs (2 days)
ii. Have scheduled 5 sprays in February to finish Collison and start sparging.
1. Will continue frozen and fresh comparisons with sparging and
micropump, but not do lyophilized, which worked so poorly with the
Collison
2. Rick: frozen would probably be preferable, if no virulence issues
arise. Less problems anticipated with frozen stocks than a fresh
culture. Rick: harder to QC each batch if needed to grow fresh each
time. Bob: frozen does not work for pestis, but hope that frozen will
work for LVS
3. Collison kills about 99% of viable organism in the original spray.
iii. Starting Sparging generator
1. Have already done some preliminary work on set up in the BSC
iv. Hoping micropump will have spray factors in 10-4 to 10-5 range.
J.
Milestone #12 – Immune Responses in Animals and Humans
1. Immunoassay Development
i. Choose PBMC Purification Method
1. Method chosen: Purdue ListServ
2. Phenotype Blood and PBMCs to examine flow cytometrically what
subsets of T cells and B cells are present
3. Test whether method results in loss of B cells- artifact or true loss of
B cells
ii. Choose PBMC Freezing Method
1. Testing 3 protocols: Cerus, CTL, Lyons
iii. Develop Immunoassay methodologies
1. Proliferation assay: Works for Con A and LVS
2. IFNg, ELISPOT
a. Works for Con A; not yet working for LVS
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3. Plasma IgG ELISA
a. Works for Con A; working on LVS
b. Just starting these assays
iv. These assays will be valuable in MS 13 as well
K. Update on Test of Freezing PBMCs
1. Goal: To select the freezing protocol which provides the best recovery of cell viability
and function when thawed
2. Plan to compare 3 NHP freezing protocols which differ slightly in % and type of
serum used: Cerus, CTL and Lyons
i. Cerus: Frozen in 80% FBS/20% DMSO at 5 x 106/ml
ii. CTL: Frozen in 90% human A/B serum/10% DMSO at 10 x 106/ml
iii. Lyons: Frozen human cells in Gibco Recovery Cell Culture Freezing Media
(contains optimal ratio of fetal bovine serum:bovine serum (90% overall) 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 . Allows cells to apoptose
and thus the viability of the resulting cells should be higher.
L. Test of Cerus Protocol
1. Used TUL 8, Day 0 prebleed PBMCs which had been frozen down on 11/16/06 using
the CERUS protocol; thawed on 1/11/07 = 8 weeks later
2. We tested the proliferative capacity of these cells to Con A only as the Day 0 cells
were not expected to, and did not originally, proliferate in response to LVS
3. Two vials were thawed for each animal; originally froze 5 x 106 cells/vial
4. Cells from each vial were plated and tested for proliferation to Con A (i.e. the cells
from the duplicate vials were not combined)
5. Two cell concentrations/vial were tested and compared: 1 x 10 6 cells/ml and 0.25 x
106 cells/ml
6. Con A: 5 mg/ml
7. Can compare directly with the prior results with the fresh cells.
M. Viable Cell Recovery/%Viability
1. Each vial originally contained 5 x 106 cells/ 100% viable at the time of freezing
2. A00896 Vial 1: 3.125 x 106= 62.5% recovered/87.4% viable
3. A00896 Vial 2: 3.25 x 106= 65% recovered/93.8% viable
4. A00908 Vial 1: 3.24 x 106= 64.8% recovered/96.3% viable
5. A00908 Vial 2: 2.85 x 106= 57% recovered/97.4% viable
6. A00937 Vial 1: 2.49 x 106= 49.8% recovered/93.3% viable
7. A00937 Vial 2: 2.4 x 106= 48% recovered/91.4% viable
8. Got back 48-60% of the cells back and had high viability
N. Proliferation to Con A (as compared to “Fresh” values originally obtained)
1. Conclusion: Frozen cells proliferated well to Con A
2. Used plate reading luminometer
3. Comparing fresh cells and frozen/thawed cells, using 2 cell concentrations and media
versus ConA treatment
4. Saw higher background with frozen than with fresh which may be due to usage of two
different instruments (fresh done with UNM’s Victor Luminometer and frozen done
with LBERI Victor Light)
O. Potential caveat to proliferation results
1. “Fresh” results were collected on the Victor luminometer instrument at UNM; “Frozen”
results were collected on the Victor Light instrument at LRRI
i. Potentially the setting which adjusts how much light is let through the
aperture, and thus the “relative light units/well” may have been different
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ii. Action: Julie is contacting the Victor service rep to clarify this
iii. In the meantime, comparing stimulation index should give an accurate
comparison: Ratio of Con A:Media = Stimulation Index
P. Comparison of Stimulation Indices: Fresh vs. Frozen PBMCs
1. 1 x 106/ml plated Fresh: 1.08 x 106/0.046 x 106 = 23.5 (twice as high as frozen)
2. 1 x 106/ml plated Frozen: 2.19 x 106/0.21 x 106 = 10.4 ( frozen had higher bkg than
fresh)
3. 0.25 x 106/ml plated Fresh: 0.66 x 106/0.043 x 106 = 15.3
4. 0.25 x 106/ml plated Frozen: 1.49 x 106/0.16 x 106 = 9.31 ( frozen had higher bkg
than fresh)
5. Conclusion: Using the Cerus protocol, we can recover at least 50% of the cells and
approximately 45% of the activity
Q. Proliferation response by individual NHPs
1. Conclusion: Frozen PBMCs from each NHP proliferated to Con A
2. Frozen results for individuals matches the summary on prior slides
R. MS 12: Upcoming Experiments Action items below: Julie
1. Monday, 2/12: Will thaw cells from TUL 8, Day 28 (12/18/06), at 8 weeks post
freezing using the CTL protocol; and Wednesday, 2/21 will thaw cells from TUL 9,
Day 28 at 8 weeks post freezing using the CERUS protocol. By Day 28, cells should
have an LVS response.
2. If cell recovery numbers permit, we can test their proliferative capacity to both Con A
and LVS and also by ELISPOT (Con A only, as we have still seen no response to
LVS by ELISPOT using the cell numbers we originally tested)
3. We will begin to develop the serum immunoglobulin ELISA for LVS.
4. We will use blood draws from the LVS-vaccinated NHPs (see MS 13) to test whether
B cells are being lost during the PBMC preparation. Will be using new antibodies
and strategies for detecting B cells
S. Milestone #13 – Compare Assays in Animal Models for Sensitivity
1. MS 13: LVS Vaccination of NHPs
i. Compare ID vs. SC vaccination
1. IFNg ELISPOT ( didn’t plate enough cells in prior experiment)
2. Proliferative response to LVS (presented in prior tech call)
3. Plasma IgG ELISA
T. Update on Vaccinated NHPs
1. NHPs underwent their semi-annual TB test and all were negative for TB. Gave the
NHPs a 2 week rest after TB test.
2. We wrote an IACUC amendment to continue to bleed the NHPs in order to test:
i. Whether plating more cells in the IFNg ELISPOT assay will allow us to detect
a response to LVS (Con A responsiveness is seen using 20,000 cells/well;
we have not yet tested higher cell concentrations for LVS)
ii. Whether S.C. vaccinated NHPs have more T cells in their PBMC
preparations than do I.D. vaccinated NHPs as they (SC group) responded
better as a group to Con A stimulation
iii. Whether we can detect intracellular IFNg staining in T cell populations in the
blood or PBMCs by flow cytometric methods. This would be an alternative to
the Elispot.
U. Vaccinated NHPs – Details on Ages
1. A00937
2.33 kg 2/5/04
2. A00896
2.15 kg 2/10/04
3. A00908
2.4 kg 2/14/04
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A00659
2.36 kg 1/2/04
A00868
2.36 kg
2/20/04
A00902
2.55 kg 10/4/03
These NHPs were previously on a toxicology study and were grouped in a way that
does not explain why the S.C. monkeys would have behaved differently than the I.D.
monkeys per se
8. Previous study: Group A: A00915, A00937, A00868; Group B: A00886, A00959,
A00902 and Group C: A00908, A00659, A00896
9. Our study: I.D. group: A00937, A00896 and A00908; S.C. group: A00659, A00868
and A00902
10. All the NHPs had been injected with a substance (antibody to anthrax protein Julie
suspects) and their clearance of the antibody was determined. Rick thinks this prior
exposure would not impact their response to LVS vaccination SC or ID.
4.
5.
6.
7.
V. MS 13: Plans for the next month- Action items below: Julie
1. Bleed NHPs again and plate more cells in the IFNg ELISPOT assay to test whether
we can detect a response to LVS (Con A responsiveness is seen using 20,000
cells/well; we have not yet tested higher cell concentrations for LVS)
2. Test whether S.C. vaccinated NHPs have more T cells in their PBMC preparations
than do I.D. vaccinated NHPs as they responded better as a group to Con A
stimulation
3. Test whether we can detect intracellular IFNg staining in T cell populations in the
blood or PBMCs by flow cytometric methods. Because IFNg Elispot will only tell
whether if there are cells secreted IFNg but it won’t tell us the phenotypes of the
cells, such as CD4 or CD 8 cell types or NK cells.
4. Begin to develop the serum immunglobulin anti-LVS ELISA using the frozen serum
from these NHPs
5. Rick: when draw the blood, determine whether the ConA responsiveness occurs
again (SC more responsive than ID) to determine if the SC ConA responsiveness is
reproducible. Set up proliferation assay with just ConA. Rick- acute vaccination
response should have dissipated by now.
6. Julie: will also mix up the SC and ID blood draws, rather than perform all SC and all
ID on single days each. Julie’s lab can work with 3 blood draws per day.
7. Rick: in live viral vaccination, can get lymphopenia in the blood at days 14-25. Since
LVS is a live intracellular vaccine, it may mimic a live viral cellular infection in some
ways. Maybe the kinetics are slightly different between the ID and SC.
8. Julie: If the phenomenon was due to the vaccine, we may have missed the window
with the ID. Action- Julie will do ConA test again, and if the SC/ID difference
disappears, then the next time LRRI vaccinates, will look at a different window for ID.
9. Julie: thinks Baylor study was SC and scarification.
10. Joe: ConA response was consistent and very unusual so good to follow up, as Rick
requested.
E. Next Meetings:
 March 6th, 2007: 12:00 pm – 1:00 pm MT, 2:00 pm – 3:00 pm ET
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