LBERI- UNM Tech Call Minutes: 3/06/07
Prepared by: Barbara Griffith 3/6/07
Sent to LBERI: 3/10/07
Reviewed and Edited by: Bob Sherwood (3/15/07), Julie Wilder (3/12/07)
Distributed to NIAID: 3/15/07
Present: Bob Sherwood, Barbara Griffith, Julie Wilder, Rick Lyons, Ed Barr, Vicki Pierson,
,Freyja Lynn, Marlene Hammer,
Absent: Kristin DeBord
3/6/07 Action Items
 Barbara- Send Bob Terry’s data on the relative impact of selective vs non-selective
plates on LVS colony size and vendor information for purchasing selective plates
 Bob prepare an action /qc plan to prevent bacterial contamination on plates and broth
Bob prepare plan for QC check on media preparation and who reviews the QC
 Bob will check some random vials of LVS and assure no contamination is in the original
LVS frozen vials
 Bob won’t perform fresh cultures for sprays until the root cause of the contamination is
understood and eliminated.
 Bob will be titering the frozen LVS sucrose stocks monthly
 Bob/Ed will be utilizing additional trained staff and testing approximately 14 sprays in the
next 5-6 weeks to complete testing of the collision and sparging aerosol generators
 Julie Full details of the NHP B cell staining in whole blood and PBMC will be shown at
next tech call
 Julie will ask BD vendor for assistance, as vendor had said that antiCD20 works in
cynomologous primates
 Julie: could also try to get a spleen from primate to test CD20 staining, although this
primate would have been sacrificed for another reason.
 Julie: will work with ABSL 3 team to perform intradermal injection of 100,000 formalinfixed LVS on the upper back to test whether a DTH reaction will develop in the previously
LVS vaccinated primate
 Freyja: will email coating optimization protocol to Julie, and may explain the difference
between heat killed and formatlin fixed. (completed 3/7/07)
 From Feb Tech call: 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
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B. Goals for Milestone #3 – Bioaerosol Development
1. Characterize the LVS bioaerosol using the Collison nebulizer
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
2. Compare Collison to sparging generator
3. Compare Collison to micropump generator
4. Consider additional bioaerosol generators
5. Determine optimum method for LVS bioaerosol generation
6. Perform bioaerosol studies with Schu4 as described above to determine if LVS data
are predictive
7. 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-completed (updated from Feb call)
b. Fresh LVS- in progress
c. Lyophilized LVS-completed
ii. Sparging Generator
1. Order & receive instrument-completed
2. Set up instrument- completed
a. Frozen LVS- in progress
b. Fresh LVS- in progress
iii. Micropump
1. Ordered & receive instrument-completed
2. Set up instrument- in progress
a. Frozen LVS- in progress
b. Fresh LVS- in progress
iv. Down Select for Schu 3 Generator
a. Frozen LVS
b. Fresh LVS
v. Prepare bioaerosol SOP
D. Milestone #3 – Bioaerosol Development & Accomplishments
1. Determined titer of LVS stored in 20% sucrose to be about 2x109 cfu/mL – repeating
titer to confirm
2. The purpose of the sprays conducted in February was to fill in data gaps for the spray
factors for sprays performed with the Collison generator and frozen or fresh LVS
3. Performed 3 sprays in January
i. 2/14 – fresh LVS
ii. 2/21 – fresh LVS
iii. 2/23 – frozen LVS
E. Fresh LVS – 2/14
1. Performed 9 spray
i. 3 sprays at target concentration of 1x105 cfu/mL
ii. 3 sprays at target concentration of 1x106 cfu/mL
iii. 3 sprays at target concentration of 1x107 cfu/mL
2. All plates were contaminated
3. Marlene: will you do QC preps in advance of sprays, from this point onward?
4. Bob: Yes, will have to prepare CHAB 1 week ahead of sprays,
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i. Will need more planning for media prep and QC, before the sprays
Rick: is the media prep and inoculation phase the most likely time for contamination?
If so, could you use antibiotic selective plates?
Bob: could use selective plates, will knock it down, but may knock down the titer also.
Rick: In Terry Wu’s experience, selective plates decreased the size of colony but
didn’t decrease the titer. UNM is using selective plates with organ depositions, when
have high suspicion of natural contaminants that need to keep under control. UNM
purchases the plates.
Action: Barbara- Send Bob Terry’s data on the relative impact of selective vs nonselective plates on LVS colony size and vendor information for purchasing selective
plates
Rick: huge work loss when spray performed and the data is lost to media/plate
contamination
Bob: needs to know the real titer, and doesn’t want antibiotics to diminish the titer
Rick: could antibiotics make bacteria leaky and more fragile in sprays?
Vicki: if add antibiotics to spray, don’t want to hide a contaminate in the spray
Freyja: known equipment failure, so this is unlikely an ongoing issue; If media is
more stable, could make stocks?
Bob: CHAB agar lasts approx 1 month. Also get condensation problem in the plates
with refrigeration. Can result in contamination thereafter, though the plates pass the
initial QC.
Freyja: Pour plates under sterile conditions to let them dry out for a while. Make sure
the poured plates cool to room temperature before you close them, and can change
the lids out especially with plates at bottom of the bag.
Trevor: For pouring the plates, LBERI autoclaves media and cools it in waterbath
over night and then pours plates on the next day
Bob: pour in hood aseptically and leave cracked a bit to dry.
Vicki: expecting a good qc program so this doesn’t happen again.
Action: Bob prepare an action /qc plan and present at next technical call
CHAB QC failed – media was prepared the day before use and data were not
available at time of experiment
i. Refurbished the autoclaved entirely- had hard time holding 121 pressure and
then timer held till pressure hit 121. Took 1 hr for autoclave times in past.
After refurbished, is immediately going to 121 pressure for 20 min and 10
minutes
ii. Autoclave repairs necessitated recalibration of autoclave times
F. Fresh LVS – 2/21
1. Performed 9 sprays
i. 3 sprays at target concentration of 1x105 cfu/mL
ii. 3 sprays at target concentration of 1x106 cfu/mL
iii. 3 sprays at target concentration of 1x107 cfu/mL
2. Most plates were contaminated
i. CB broth was contaminated
1. was filter sterilized and not autoclaved
2. 48 hr growth in chamberlains
3. Innoculated broth with LVS and grew for 48 hrs, and got
contaminated during the LVS growth.
4. Media and components were made sterile
a. QC broke down as there was no QC check on the media
b. Action: Bob prepare plan for QC check on media preparation
and who reviews the QC
5. Trevor conveyed that it was likely due to user error when started the
LVS culture in the broth
6. Action: Bob will check some random vials of LVS and assure no
contamination is in the original LVS frozen vials
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Vicki: what is your environmental monitoring?
Trever: regular checks on the hoods semi annually
Freyja: have you identified the contaminant?
Bob: Don’t know. LBERI will research the cause, but this is the first
time seen this. Either from the inoculation or during the inoculation
process.
11. Vicki: won’t do further work until root cause is understood and
eliminated.
12. Action: Bob won’t perform fresh cultures for sprays until the root
cause of the contamination is understood and eliminated.
G. Frozen LVS – 2/23
1. Performed 9 spray
i. 3 sprays at target concentration of 1x105 cfu/mL
ii. 3 sprays at target concentration of 1x106 cfu/mL
iii. 3 sprays at target concentration of 1x107 cfu/mL
H. Frozen LVS – Target vs. Actual Titers
1. The following slide shows the predicted vs. the actual titers for all studies to date
performed with frozen LVS
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 most recent study
data fall on the line indicating that our predicted concentrations for the LVS stock are
correct
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Frozen LVS- CFU/ml vs Spray Factor
1. The following slide shows the data to date of all sprays performed with frozen LVS
and the calculated spray factors
2. The most recent data (2/23) are slightly lower than would have been predicted based
upon earlier sprays, 10-8 range, which is a little lower than desired.
3. Rick: what could be the cause?
4. Bob: perhaps the glycerol stocks are starting to degrade
5. Barbara: How long have the glycerol stocks been frozen?
6. Bob: approx 8 months (frozen in August) and do not freeze/thaw, Wayne said that
glycerol didn’t last long in the freezer and Bob has switched to sucrose.
7. Bob: spray set up is easier from frozen stocks
8. Barbara: how is LBERI testing the sucrose frozen stocks of LVS?
9. Bob : will intermix testing of the glycerol and sucrose stocks of LVS and titer over
time.
10. Vicki: What is the storage temperature for the frozen LVS stocks?
11. Bob: at -80F
12. Vicki: has the freezer been mapped and is it consistent in temperature across the
freezer?
13. Bob: no, but they are on the bottom shelf which should be the coldest shelf.
14. Rick: Wayne ‘s observations on glycerol vs sucrose maybe the cause of the loss of
spray factor.
15. Freyja/Vicki: could LBERI used lyophilized LVS stocks for sprays?
16. Rick/Bob: lyophilized LVS didn’t spray well, they were killed 100% by the spray.
17. Bob: must solve frozen stability issue
18. Freyja: 6 months would be a reasonable stability and LBERI could make new frozen
stocks on a 6 month basis if necessary.
19. Vicki: how old are the sucrose stocks?
20. Bob: 10 days and have excellent titer.
21. Action: Bob will be titering the frozen LVS sucrose stocks monthly
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Milestone #3 – Bioaerosol Development
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Data Analysis
Frozen spray set up worked fine
Having problems with contamination for fresh LVS studies
i. Have had contamination in CB medium
ii. Have had contamination on CHAB plates
Have been trying to do studies right after media prep
Need to perform media QC prior to study which puts longer timeframe on each
experiment
Plans for this month: Action items below: Bob
i. Bob: need 14 days of sprays in the next 5-6 weeks, may need to work
weekends
ii. Ed: trying to get more help on the aerosols, including adding a 3rd Tech
iii. Complete Collison sprays with fresh LVS
1. Want to fill in the data gaps for spray factors
a. Fresh – 5 to 8 logs (2 days)
iv. Starting Sparging generator
1. Have already done some preliminary work on set up in the BSC
K. 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. IFN, ELISPOT
a. Works for Con A; not yet working for LVS
3. Plasma IgG ELISA: in development
iv. These assays in use on MS 13
L. 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 . (will be done in next month)
M. Test of Cerus Protocol ( review of previously presented data)
1. Used TUL 8, Day 0 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
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from the duplicate vials were not combined)
Two cell concentrations/vial were tested and compared: 1 x 10 6 cells/ml and 0.25 x
106 cells/ml; Con A: 5 g/ml
Summary: we can recover at least 50% of the cells (range of 48 – 65% yield and
87.4 – 97.4% viable) and approximately 45% of the activity
New data with Cerus protocol: Recovery of cells thawed on 2/28/07 (TUL 9, Day 28)
was 53.8 – 78.8%; 94.4 – 96.9% viable; Data is reproducible with Cerus freezing
protocol.
N. Proliferation to Con A (as compared to “Fresh” values originally obtained)
1. Conclusion: Frozen cells proliferated well to Con A
O. 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
6. Freyja: what are the stimulation indices?
7. Julie: relative light units with stimulus divided by media only relative light units. Two
Victor units used necessitated this comparison and now calibrated on both
instruments to be the same.
8. Freyja: caution using this type of correction factor, relative linearity may not be
consistent
9. Barbara: LBERI will be using only the one LBERI instrument from this point forward
10. Julie: only used 2 instruments due to recent acquisition of LBERI instrument
P. Test of CTL Freezing Protocol
1. Used TUL 8, Day 28 (12/18/06), at 8 weeks post freezing; thawed on 2/12/07= 8
weeks later
2. We tested the proliferative capacity of these cells to both Con A and LVS; also tested
their ability to secrete IFN by ELISPOT in response to ConA
3. Two vials were thawed from each of two NHPs; originally froze 5 x 10 6 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; Con A: 5 g/ml, LVS at Hi (1 x 105/ml) and Mid (0.25 x 105/ml) doses
6. Cell recovery ranged from 21.1 – 102.8% and viability ranged from 77.5 – 91.9%
i. One vial was very poorly recovered (21.1%), but others ranged from 56.8 –
102.8%
Q. Proliferation of Fresh vs Frozen cells- CTL protocol- day 28 post vaccination cells
1. Note: deficiency of frozen cells to proliferate in response to LVS
2. ConA response is quite good
3. Frozen cells didn’t respond well to the LVS
4. With Cerus protocol, have only tested ConA to date and will be testing the frozen
Cerus cells with LVS antigens
5. Freyja: different scales on the two graphs?
6. Julie: yes, it is one log higher. From this point forward, will be one Victor and can
compare directly in the future.
7. Vicki: what day were cells harvested post vaccination?
8. Julie: used day 0 and day 28., so don’t have good LVS proliferative recovery
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R. Potential caveat to proliferation results
1. Fresh” results were collected on the Victor luminometer instrument at UNM;
2. “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
ii. Julie has met with the service representative and rectified this situation
(3/5/07); in the future, readings will be directly comparable
iii. In the meantime, comparing stimulation index should give an accurate
comparison: Ratio of Stimulus:Media = Stimulation Index
S. Proliferation expressed as stimulation index- CTL
1. Freyja: any rest for the cells post thawing?
2. Julie: only the Lyons lab protocol includes this resting step. The Cerus and CTL
freezing protocols didn’t call for a rest post thawing. So haven’t been doing it for the
Cerus or CTL frozen cells
3. Julie: need to bleed, plate, freeze in Lyons lab protocol and then 8 weeks later we will
thaw them.
4. Rick: proliferation maybe one of the harder, post thaw assays, based on the
literature.
5. Julie: don’t have gamma interferon elispot data from the prebleed
6. Rick: Emory uses elispot and intracellular gamma interferon, rather than proliferation
7. Julie: The Con A mitogen response may have mislead us relative to need for an
antigen specific response. CTL freezing protocol was not good for recovering
antigen specific proliferation response but Cerus freezing protocol hasn’t been tested
yet
8. Freyja: could you freeze cells from same animal for all 3 assays?
9. Julie: No, could bleed multiple animals and pool but then could get MLRs which
would complicate the analysis of the response
10. Vicki/Rick: don’t think should pool cells from different animals
T. Summary of Freezing Protocol Testing
1. Thus far, we have tested the recovery of cells after use of both Cerus and CTL
freezing protocols
2. Cell recovery seemed to be superior using the Cerus protocol
3. Proliferation response to Con A is well-preserved; however, recovery of the
proliferation response to LVS is poor (only tested with CTL protocol so far)
U. Update on B cell purification in PBMC preparation
1. Issue: CD20+ cells were abundant in naïve peripheral blood of NHPs by FACS
staining, but did not appear in naïve PBMC preparation (as if B cells were being
excluded by the Percoll gradient while T cells were being enriched – didn’t make
sense)
2. Possible artifact is being tested by staining B cells with other antibodies (specific for
CD19 and surface IgM). Used anti human CD19 antibodies that haven’t been tested
in primates previously by BD Biosciences and are not sold specifically as primatetested.
3. Blood drawn on 2/15/07 from 3 NHPs (1 from LVS ID group (TUL 8) and 2 from LVS
SC group (TUL 9))
4. Preliminary results suggest that anti-human CD19 does not bind NHP B cells in blood
or PBMC preparation so this antibody is not helpful; anti-IgM binds a small proportion
of cells in the whole blood and positive cells are enriched in PBMCs; anti-CD20 binds
a large proportion of cells in whole blood, but not in PBMCs (these cells do look
lymphocytic/monocytic). So anti-IgM data is the opposite of the antiCD20 data in
primates, but makes more sense.
5. Action: Julie Full details of the NHP B cell staining in whole blood and PBMC will be
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shown at next tech call
Freyja: do you know the viability?
Julie: have not used PI staining but are not looking at debris or dead cells.
Freyja: human antibodies in primates have sometimes very unreliable results
Action: Julie will ask BD vendor for assistance, as vendor had said that anti-CD20
works in cynomologous primates and is sold under the primate section in the catalog
Freyja: could rosette?
RicK: do miltenyi to purify and then stain
Julie: want to purify cells that proliferate in response to LVS and mostly want T cells,
which are enriched.
Freyja: think it is useful to know if you are selecting for an unusual population with the
PBMC preparations
Julie: could test with a B cell mitogen (e.g. Pokeweed or LPS)?
Julie: anti IgM data is more like truly staining B cells and is encouraging
Action: Julie: could also try to get a spleen from primate to test CD20 staining.
V. MS 12: Upcoming Experiments
1. Wednesday, 2/28; Thawed cells from TUL 9, Day 28 at 8 weeks post freezing using
the CERUS protocol; proliferation and ELISPOT data will be available for tech report
this month
2. We will fully analyze the B cell staining from blood and PBMCs stained on 2/15/07
3. We will also analyze the T cell phenotype of the NHPs bled on 2/15/07 to determine
whether S.C. (TUL 9) groups of NHPs have more T cells in their blood (suspected
due to their superior response to Con A as compared to TUL 8 NHPs)
W. Milestone #13 – Compare Assays in Animal Models for Sensitivity
1. MS 13: LVS Vaccination of NHPs
i. Compare ID vs. SC vaccination
1. IFN ELISPOT – in progress
2. Proliferative response to LVS -completed
3. Plasma IgG ELISA- in progress
X. Update on Vaccinated NHPs
1. We wrote an IACUC amendment to continue to bleed the NHPs in order to test:
i. Whether plating more cells in the IFN 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) – preliminary
results now available
ii. 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 – preliminary results suggest not
iii. Whether we can detect intracellular IFN staining in T cell populations in the
blood or PBMCs by flow cytometric methods
2. IACUC amendment also covered an intradermal injection of 100,000 formalin-fixed
LVS on the upper back to test whether a DTH reaction will develop; if 100,000 LVS
gives no reaction within 3 days, we proposed to wait 2 weeks and inject 1 million LVS
3. Action: Julie: will work with veterinary team to perform intradermal injection of
100,000 formalin-fixed LVS on the upper back to test whether a DTH reaction will
develop in the previously LVS vaccinated primates
Y. Update on IFN ELISPOT
1. Issue: Previoiusly we had plated only 20,000 cells/well and although this was
sufficient to show reactivity when stimulated with Con A, we were unable to detect
spots after ex vivo LVS stimulation in the vaccinated NHPs
2. Freyja: can you optimize the antibodies, is the antibody pair optimal for signal to
noise?
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Julie: will talk to rep about antibody pair and also if the reader is picking up cells that
are background ; hope can dial down the background
Using the blood drawn on 2/15/07 (Day 78 – 87 post-LVS vaccination), we plated
100,000 and 500,000 cells/well
i. 20,000 cells stimulated with Con A: average of 300 or 700 spots with 1 – 3
background (2 NHPs tested)
ii. 100,000 cells stimulated with LVS: 55 – 60 spots, but approximately 35
background
iii. 500,000 cells stimulated with LVS: 250 or 450 spots, with approximately 100
background. This is a high background
iv. Spots in unstimulated wells look very small and pale; with adjustment of
sensitivity I think they can be minimized
Z. Update on LVS ELISA
1. Coated ELISA plates with either formalin-fixed or heat-killed LVS at 3 different
concentrations (1 x 106/ml, 0.5 x 106/ml and 0.25 x 106/ml)
2. Tested d0 and d21 sera from vaccinated NHPs at dilutions of 1/100, 1/500, 1/2500
and 1/12500
3. Data are expressed as arbitrary unit = lowest OD > background x dilution (i.e. .100 x
12500 = 1250 units)
i. Freyja: hadn’t seen this method before?
ii. Julie has been using in an OVA assay in asthma model
4. Units of anit- HK-LVS
i. Freyja suggested a real coating optimization for the ELISA plates
ii. Julie: needs advice
iii. Action: Freyja: will email coating optimization protocol to Julie, and may
explain the difference between heat killed and formalin fixed. (completed
3/7/07)
5. Units of anti-HK vs FF LVS
i. Titer is a misnomer; should read units. Don’t have a primate antibody to use
as a positive control and for use as a standard curve
ii. Peak od was lower with the FF than the HK
6. Conclusions on LVS- ELISA
i. HK-LVS is superior to FF-LVS as a capture antigen
ii. HK-LVS can be used at any of the tested concentrations (will probably
choose 0.5 x 106/ml)
iii. Both sets of NHPs seroconverted, possibly the S.C. group have higher titers
iv. Day 7 and 14 sera are run and Day 28 will be run this week- did on heat
killed LVS at 0.5 millon per ml.
v. Plate are coated in PBS
vi. Data will be available at next tech call
AA. MS 13:
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Plans for the next monthFinish analysis of LVS-ELISA data
Schedule LVS-DTH test
Bleed another group of NHPs and continue to test their T cell phenotype to determine
whether S.C. vaccinated group have more T cells than the I.D. vaccinated group
Test whether we can detect intracellular IFN staining in T cell populations in the
blood or PBMCs by flow cytometric methods
Contact service representative to adjust ELISPOT reader settings
BB. Next Meetings:
 LBERI Technical call: May 1, 2007: 12:00 pm – 1:00 pm MT, 2:00 pm – 3:00 pm ET
 No Tech call in April, due to Subcontracting site visits and also the semi-annual
report is due on 4/7/07
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