LBERI- UNM Tech Call Minutes: 12/13/06
Prepared by: 12/20-30/2006 Barbara Griffith
Sent to LBERI: 12/30/06
Reviewed and Edited by: J Wilder 1/3/2007
Distributed to NIAID: 1/8/2007
Present: Bob Sherwood, Barbara Griffith, Marlene Hammer, Vicki Pierson, Julie Wilder, Rick
Lyons
Absent: Joe Breen, Kristin DeBord, Freyja Lynn, Ed Barr,
Action Items:
 Bob/Trevor: will titer 3-5 random vials of their frozen LVS stocks to see if the vials are
consistent or if there is variation related to the storage location in their freezer boxes.
 Bob: will make new frozen stocks of LVS in 10% sucrose.
 Julie will communicate with Carol and the handlers regarding handling the drawn monkey
cells gently.
 Vicki: When it becomes available, Vicki will share data on the Baylor and USAMRIID on
the revival and response of the frozen cells from LVS vaccinees.
 Terry Wu: The SCHU S4 growth kinetics will be reported in the future
LBERI Update on Animal Model Development
Milestones
#3
#2
#4
#7
#8
#9
Active InactiveActive Inactive Inactive Inactive -
#12
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
A. Milestone 3- Optimization of bioaerosol methods
1. Overall Goals
a. 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
b. Compare Collison to sparging generator
c. Compare Collison to micropump generator
d. Consider additional bioaerosol generators
e. Determine optimum method for LVS bioaerosol generation
f. Perform bioaerosol studies with Schu4 as described above to determine if LVS
data are predictive
g. Compile SOP for Schu4 bioaerosol studies.
2. Accomplishments
a. No sprays this month
3. Plans for this month
a. Aerosol lab is scheduled all December
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b. Next LVS aerosols scheduled in early January- 6 sprays are scheduled in early
January
B. Milestone #12 – Immune Responses in Animals and Humans
1. Received materials necessary for freezing and testing the activity of frozen PBMC
preparations
i. One batch of PBMCs (from Day-4 naive bleed of Tul 8) was frozen down on
11/16/06 using the Cerus protocol; fresh cells from the same batch were
used in a proliferation assay ; Will be comparing Cerus, CTL and Lyons
protocols for freezing macaque cells
ii. Julie has the AB serum necessary for the CTL freezing protocol and will use
this protocol with the next batch of PBMC for freezing.
2. Received materials required for intracellular staining of IFNg in whole blood and
PBMC preparations
3. Received anti-human CD19 and anti-human IgM antibodies required for testing
whether B cells (CD 20+) are being lost in the PBMC preparation
4. Plans for Upcoming Month
i. Thaw cells frozen down on 11/16/06 and set up in a proliferation assay to
test functionality; Will compare in parallel to fresh, unfrozen cells. Will also
determine what fraction of the cell number can be recovered post thawing.
ii. Test whether NHP PBMCs stain positively for anti-human CD19 and antihuman IgM
–
If had stained with CD20 on whole blood, then detected B cells in the peripheral blood; however,
post PBMC isolation, LBERI couldn’t detect the CD20+ cells in the PBMC. They appeared lost.
This could be an antibody issue. No other scientists have seen this and is likely a reagent issue.
iii. Also, stimulate a fraction of PBMCs with Con A, followed by PMA+ionomycin
in the presence of GolgiPlug to measure intracellular IFNg by flow cytometry
C. Milestone #13 – Compare Assays in Animal Models for Sensitivity
1. Initiated inoculations of cynomolgous macaques with LVS
i. General study design: 3 NHPs received LVS by intradermal inoculation; 3
NHPs received LVS by sub-cutaneous inoculation
ii. NHPs were/will be bled at some timepoint before the vaccination to get the
baseline and at days 7, 14, 21 and 28 post-vaccination
iii. Plasma will be collected for future anti-LVS antibody ELISA and will be
stored for future work
2. Compare Assays in Animal Models for Sensitivity
i. Planned analysis and assays with PBMCs from blood draws
1. PBMCs are enumerated and an aliquot stained on a cytospin for
differential cell typing (mononuclear and granulocytes)
2. PBMCs are set up at various cell concentrations (1, 0.25 and 0.0625
x 106/ml) and stimulated to proliferate with Con A (positive control),
heat-killed and formalin-fixed LVS (at 1 (Hi), 0.0625 (Mid) and 0.0156
(Lo) x 105/ml).
3. PBMCs (if extra are available) are set up at 20,000 cells/well for
Elispot and stimulated to secrete IFNg with Con A (positive control),
heat-killed and formalin-fixed LVS (at 1, 0.0625 and 0.0156 x
105/ml)
4. Vicki: what factors impact quantity of extra cells available?
Julie/Rick: trying to figure out why cell counts vary widely. The yield
from the blood draws is varying widely. Rick: Internally we have
discussed this variability. It may be a stress response- like length of
stay in new room before they are bled. Julie: receipt/processing the
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blood in the laboratory is standardized.
LVS Vaccination Schedule
11/20/06 vaccination date: Tul 8: i.d. route
11/29/06 vaccination date:Tul 9: s.c. route
4. Dosing of LVS
i. LVS is prepared from a frozen vial which has previously been tittered at
LBERI
ii. After dilution from the vial, the inoculum preparation is plated to calculate
the actual dose of LVS given, which is determined retrospectively per
Bob. They have an estimate of the dose but the actual dose is
retrospective.
iii. The intent was to deliver a dose i.d. of 1 x 105 LVS CFU/NHP, however
on 11/20, the titer was 10-fold higher than expected from the vial and a
10-fold dilution error was made due to a miscommunication, thus the
dose actually delivered was 1 x 107; Trevor diluted the vial to deliver
1x106 organisms but Julie had indicated that she wanted 105 delivered.
iv. On 11/29, we attempted to replicate the 1 x 107 dose in order to compare
the 2 inoculation routes, but the vial titered out lower than expected and
the dose delivered s.c. was 3 x 106 rather than the 1 x 107 desired for the
comparison.
v. Bob: LVS frozen vials were prepared before discussions at Work
Grouping; all are in glycerol and not in sucrose; Wayne has seen glycerol
cause titer drops over time. The lot of vials was the same.
vi. Rick: Action: LBERI will titer 3-5 random vials of their frozen LVS stocks
to see if the vials are consistent or if there is variation related to the
storage location in their freezer boxes.
vii. Vicki: didn’t see this type of variability in the clinical trial from lyophilized
vials so this may be a storage phenomenon
viii. Julie: should we make a new stock in 10% sucrose? Bob agrees that a
new stock in sucrose should be made. Action: LBERI will make new
frozen stocks of LVS in 10% sucrose.
5. LVS Vaccination results
i. Tul 8 NHPs are at Day23 post-vaccination; Tul 9 NHPs are at Day14
currently.
ii. No immediate skin reaction to LVS inoculation by either route seen in
NHP at LBERI. At Woods Hole meeting, it was reported by another
group that they saw an immediate reaction in humans after scarification
with LVS
iii. One Tul 8 monkey (A00937) noted to have 3 cm lump at the injection site
(I.D.) when anesthetized for Day 14 bleed (12/4/06). All monkeys this
week also have no inoculation reaction.
iv. Monkeys are healthy, normal activity with either 1x107 or lower
innoculum.
6. LVS i.d. vaccination: Show increased blood macrophages (probably more correctly
called activated monocytes) and PMNs on D7 and D14
i. had very high level of cells on day -4 in one monkey and this has only
been seen once. The difference may be due to drawing by only male
handler; all other blood drawings were done by female handler; 2 male
and 1 female macaque NHP in each group. Perhaps all 3 macaques are
responding differently to a male handler? Julie has access to records on
the handlers over the summer when PBMC isolation protocols were
being developed. Note: Later on 12/13, Julie learned that the male
handler did not draw the blood on TUL 8 day-4, but was in the room
when the animals were being bled.
ii. All are being bled by the same handler now and Tul 9 had no high
blood cell counts. Didn’t change percentage of cell type in the blood.
3.
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iii. The amount of neutrophilia is quite low on day 0. So prevaccination, the
macaques don’t have many neutrophils. % macrophage (activated
monocytes) and neutrophils consistently go up in blood post vaccination
but no consistent change in the numbers of circulating cell counts.
iv: handling cells gently by drawers/handlers- Tony bled the monkey on the
day when got the huge response and numbers of cells (correction:
Learned later that Sheila O’Reilly bled these monkeys but Tony was
present in the room); either Sheila O’Reilly or Vicki White (learned this
on the afternoon of 12/13 after the tech call was over) has done the other
bleeds and she was expelling the blood through the needle and maybe
Tony wasn’t expelling through the needle. Bob said that they should not
be expelling the blood through the needle. Most of the handlers are
accustomed to the scientists using the serum or plasma and are not
accustomed to the blood cells being used. Action: Julie will communicate
with Carol and the handlers regarding handling the drawn monkey cells
gently. Julie would like more cells and more consistent cells numbers.
v. Bob- due concerns about monkey HepB, LBERI doesn’t want techs to
touch bloody needles but need to remove needle to expel cells at after
the draw. The handlers can be taught to safely remove the needles and
expel the blood not through a needle.
vi. Vicki- The one monkey which responded to LVS, may be an early
responder and the other monkeys may catch up. This is really fun to
see.
7. LVS s.c. vaccination results: Show increased blood PMNs on D7
i. Also show an increase in neutrophils and not a significant increase in
macrophages (activated monocytes) though their macrophages
(activated monocytes) were higher at the day 0.
8. TUL 8 day 0 Proliferation (naïve draw)- with high number of cells
i. ConA- big maroon bar. At day 0, just responding to Con A which is good.
We don’t expect the cells to respond to LVS when the animals have not
yet been vaccinated. Also see some high background to high dose
1x105 LVS formalin fix antigen which Rick mentioned has also been seen
in the mouse (orange bar). When the LVS antigen is at very high doses,
Rick saw some proliferation in the mouse too.
ii. The high dose is 1x105 and then goes down from there. Julie may drop
lowest dose of cells because can’t even see a ConA response with the
lowest dose. Rick thinks the accessory cells are depleted at the lowest
dose. If a Con A response is not detectable then an antigen response
won’t be either.
9. Average TUL 8 Day 0 Proliferation- ave across 3 monkeys- same data presented
differently.
10. TUL 8 Day 7 Proliferation
i. Data is kept separate per monkey to show the variety of responses.
ii. One monkey # 937 is responding to LVS at one concentration and is a
very good response in one animal.
iii. #937 is the animal with a high cell count and also the animal which had
the skin reaction.
11. TUL 9 Day 0 Proliferation
i. No response to LVS in naïve but get ConA response, as expected in
naïve monkeys.
ii. Not responding to the lowest dose of antigen and will drop this dose in
the future.
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12. LVS Vaccination: Plans for upcoming month
i. Continue preparation of PBMCs on days of scheduled bleeds
ii. Analyze all cell number, cell type, proliferation, and ELISPOT data
iii. Though not presented here, the Elispot data up to day 14 of both routes
of infection, is not showing significant IFN gamma secretion even in
#937.
D. Discussion on prior action items from prime tech call:
1. Vicki: looked up data on the LVS vaccinees in Baylor trial and USAMRIID study. In the
Baylor trial, the vaccinated human cells were frozen at 1x106 /vial and in the USAMRIID
protocol, the LVS vaccinated human cells were frozen at 50x106 cell/vial; Currently,
determining how well the cells revive and how well the cells respond. Action: When it
becomes available, Vicki will share data on the Baylor and USAMRIID on the revival and
response of the frozen cells from LVS vaccinees.
2. Rick: Looking at monkey responses to our LVS antigens in the current LBERI studies
before saying how many cells from LVS human vaccinees are needed. Julie: needed 1 to .25
million of PBMC/ml to detect the response; however, the response with the lower doses of
LVS is almost equivalent to the higher doses of LVS as antigen. The LVS antigens won’t be
limiting but the number of cells from the vaccinees will become limiting. Julie will continue to
test between 0.25 million/ml and 1 million /ml. As the animals become more immune, the
assay may take fewer cells. The two doses of cells are responding equivalently to Con A.
3. Terry is working on the growth curves and kinetics of SCHU S4 in culture. Action: The
SCHU S4 growth kinetics will be reported in the future by Terry.
E. Next Meetings:
 UTSA tech call on Tuesday 12/19
 ASU tech call on Wednesday 12/20 in place of prime tech call
 LBERI: next tech call is on 1/9 Tuesday (2nd Tuesday of January); in February, will
return to normal 1st Tuesday in the month for LBERI Tech call
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