LBERI- UNM Tech Call Minutes: 11/07/06
Prepared by: 11/7/2006 Barbara Griffith
Sent to LBERI: 11/7/06
Reviewed and Edited by: Julie Wilder and Ed Barr
Distributed to NIAID: 11/16/2006
Revised:
Revised sent to NIAID:
Present: Bob Sherwood, Ed Barr, Barbara Griffith, Marlene Hammer, Vicki Pierson, Julie Wilder,
Trevor Brasel
Absent: Joe Breen, Kristin DeBord, Freyja Lynn, Rick Lyons
Action items:

Bob/Ed add dates of calibrations in January Quality Assurance report for the period of
July 06 to December 06. QA reports will be provided every 6 months, in Jan and in July.
 Bob/Ed: report on more sprays with Collison and on initial sprays with Sparging generator
 Julie: starting primate id and sc immunizations with LVS, with scheduled prebleeds and
multiple post bleeds. Fresh PBMC for proliferation and possibly ELISPOT assays.
 Julie: will be testing naïve primate PBMCs for proliferative responses, before and after
freezing using CTL and possibly other freezing protocol.
 Julie: investigating possible B cell loss artifact in PBMC preparation
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; setting up the 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. Began evaluation of fresh LVS bioaerosol with Collison
b. Began initial set up of sparging generator
3. Spray Factor vs. Spray Conc (Fresh LVS)
a. Used LVS after 48 hrs growth in shaker with Chamberlains
b. Spray 4.5 logs, which give spray factors of -6 to -7 log which is good and where
they wish to see the spray factors. -7 spray factors are seen as good for Y.
pestis studies
4. Fresh vs. Frozen- plot of spray concentration vs. spray factor
a. Log 4 range, fresh has slightly lower spray factor than frozen material
b. -6.5 to -5 for frozen which is slightly better than fresh at -7.5 to -7
c. So with this study, frozen is better than fresh, but LBERI will be performing more
fresh sprays in the next month.
5. Target vs. Actual LVS Counts
a. For some target CFU/liter, the actual CFU/liter is higher and for other target
CFU/liter, the actual CFU/liter is lower
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b. Didn’t have this problem with the frozen material; frozen target and actual
CFU/liter were closer to each other
c. Fresh are more variable even though a standard curve was done; the fresh target
and actual are not as close as LBERI would like
d. The target vs. actual is being measured for the generator solution which is used
to make the spray
6. Spray Count vs. AGI Recovery
a. Mathematically, the number of bacteria that should be aerosolized is based on
volume of generator solution put into the spray and the concentration of the
bacteria in the volume of the generator solution. This values allow the calculation
of the number of bacteria that should be in the spray.
b. Though the trend is upward, the CFU/liter in the AGI is not increasing as much as
the CFU in spray. At 200CFU/liter in the spray, LBERI detects 4 CFU/liter in the
AGI impinger
c. LBERI observes some variability in the bacterial counts which maybe due to
clumping. Bob will look at the stained bacteria to examine for clumps.
d. The spray is quantitated after it has been through the Collison generator.
e. Barbara: asked what is the optimal CFU/liter upon collection in impinger? Bob:
how low can you go is the purpose of this study? LBERI doesn’t want to go to
very large volumes or capturing bacteria onto filters and plating the filters. This
experiment indicates with a spray around 5x104 to 105, they are flying 8 CFU/liter
of air and plate counts are 5-6 CFU per plate. LBERI doesn’t want to go lower on
the CFU on the plates because they can’t quantitate a lower number of CFU.
With flying 8 viable cells per liter, the dose should be adequate to infect primates.
Primates would get 5 liters of air which would calculate to about 40 bacteria
delivered to the lungs.
f. Ed additionally explained that the AGI is a high efficiency collection device and
that they culture viable cells. The optimum would be 100% viable cells in the
impinger; however, with the collision, 1% of the aerosolized cells are actually
viable. So Ed is looking at different generators to try to get better viability of the
bacteria reaching the impinger.
g. Vicki: answers her question regarding volume of air for primates and she agrees
with Ed that it is disappointing that the Collison kills 99% of bacteria.
h. Julie: asked whether bacteria impacting on the lung might increase the number of
bacteria that may survive, relative to hitting the impinger containing
Chamberlain’s media.
i. Bob: responded that some bacteria can be viable but not culturable; some
resuscitated microbes may be infective in animal lung but not survive in the
impinger.
j. Julie: maybe hitting lung and surviving better than just in Chamberlains; however,
Bob thinks that the lung is more hostile environment than Chamberlains in the
impinger. Bob thinks the LVS grow very well in Chamberlains.
k. Ed added that LBERI is not trying to determine what fraction of inhaled LVS are
deposited in the lungs. At 200 CFU /liter of air, they anticipate that the animals
will get an infection and LBERI will look at survival of the animal. LBERI doesn’t
determine the bacterial survival in the lung which parallels all the other labs doing
primate experiments with aerosols.
l. Bob: With mice, the actual deposition in the lung maybe closer to 15-30%;
however, Ed added that the deposition is lower, at about 7% for 1 micron
particles.
m. For primates, at 8 CFU/liter of air and breathing in 5 liters, the primate is exposed
to 40 CFU total which is probably lethal.
n. Marlene: will these challenges be done whole body or jut head? Bob said that the
small animal nose only and the primate is head only exposure.
o. Bob added that both exposure methods reduce the oral component. In whole
body exposure, the animals tend to groom themselves and tend to infect
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themselves orally. Nose or head only exposures reduce that oral infection
component.
7. Bob provided the Bioaersol check list, the Bioaersol protocol and the LVS bioaersol plate
count form in response to Barbara’s request for more details on the Bioaersols
a. LVS Bioaerosol Checklist
b. LVS Bioaerosol Plate Count Form
c. Bioaerosol Protocol
i. Running 10 min spray, with ramp up and ramp down for a total of 12
minutes; purging chamber for 2 minutes to clear for next spray.
ii. There’s a steep rise and steep drop off in the sprays but these transitions
are not vertical. When ramping to the max concentration in the spray,
there is exposure when quantitating both ends of the ramp up and ramp
down too. The spray time is computed at the top of the graph when the
animals are getting the maximum exposure. Animal sprays will be 10-20
minutes long.
8. Bioaerosol Spreadsheet
a. How getting volume sprayed- by weight of vessels looking at volume put up into
the spray into the air.
b. Impinger is the recovery into agi, which is very low. Need to go up to half of a log
to a log.
c. Vicki: There is a low LD50 for F. tularensis, so this method is not limiting for this
disease; however, if a higher LD50 would be needed, this may be harder with
this method. Bob responded that they would need to plate higher volumes and
put higher concentrations in generator solution. They would get higher viable
counts in the AGI, but also killing in spray can go even higher if the bacterial
concentration gets too high.
d. Vicki: What is the upper limit before the Collison gets clogged up? Ed- don’t know
that can reach the level of a concentration to allow clogging. The Collison
doesn’t have solution that goes through a small orifice. The orifice is much
bigger than the particle size. Clogging is not likely because LBER has sprayed
up to 5x109 which is much higher than currently being used.
9. Plans for November:
a. Perform additional bioaerosol experiments on vegetative LVS with Collison
generator
i. Repeat of studies performed on frozen LVS, but now with fresh LVS
ii. Plan to grow LVS in CB
iii. Plan to quantitate LVS on CHAB
b. Perform bioaerosol experiments on frozen LVS with sparging generator
i. Repeat of studies performed on Collison
ii. Plan to quantitate LVS on CHAB
iii. Will continue doing frozen and fresh, not lyophilized. Either frozen or
fresh may be needed in animals. Bob indicated that they may see other
differences with fresh and frozen with other aerosol generators.
B. Milestone 4- Validation of aerosol in primates (newly active to vaccinate primates)
1. Completed IACUC approval for LVS vaccination of NHP
2. Obtained 6 NHP for the protocol
3. Scheduled pre-bleeds and LVS vaccination- Julie is working on this
4. Plans for November
a. Obtain pre-bleed samples week of 11/13: for assay validation for milestone 12.
Want to know if the animals can be vaccinated and protected by an id or sc
route, before use they use aerosol route.
b. Vaccinate NHP on approximately Nov 20th and then post bleed on days 7, 14, 21,
28; will harvest bloods for cells and plasma; 3 animals will be immunized by id
and 3 will be immunized by subcutaneous routes. Both routes have been used in
literature.
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Julie will use a dose level 105 based on literature and is not trying scarification
because so scarification makes it so difficult to determine dose
d. Vicki: do you have literature supporting a 105 id dose in primates? Julie: yes, that
was the dose used in literature in primates and the literature reported some
protection from an aerosol challenge
e. Vicki: With a high dose id in rabbits, they saw necrosis, but the dose was higher
than 105.
f. Julie: there were skin reactions in the literature but they were fairly minor
g. Julie will test fresh PBMC cells for proliferation vs. heat killed or formalin fixed
LVS antigens from Terry. Terry is doing same comparison in the mice.
Previously, Julie had used mitogen stimulation of PBMCs from primates and will
use the mitogen as a control when they use LVS antigens. 1st priority for the
primate cells is the proliferation assay and the 2nd priority is ELISPOT for IFN
gamma.
c.
C. Milestone #12 – Immune Responses in Animals and Humans
1. Participated in the TVDC 2006 Annual Conference (Albuquerque, 9/27/06)
2. Ordered materials required for freezing and testing the activity of frozen PBMC
preparations. Julie will try the CTL protocol.
3. Ordered materials required for intracellular staining of IFNγ in whole blood and PBMC
preparations.
4. Investigated whether phenomenon of CD20+ B cells disappearing from PBMC fraction
could be confirmed using other B cell markers – answer, no. Neither CD19 nor surface
immunoglobulin antibodies specifically tested for NHPs are available for purchase. We
will try to use anti-human CD19 and surface Ig.
o In whole blood, could see CD20+ cells but couldn’t see CD20+ in the PBMCs.
o Julie doesn’t think they are really losing the CD20+, others don’t see this, so
maybe an artifact.
5. Test whether NHP PBMCs stain positively for anti-human CD19 and anti-human IgM.looking for B cells
6. Set up a proliferation assay and IFNγ ELISPOT assay with fresh PBMCs and reserve a
portion for freezing down. Thaw at a pre-determined timepoint (4 – 8 weeks later) and
set up in the same assays to see what fraction of function can be recovered. Perform
with non-immunized primate cells (naïve primate) for these studies. Trying to do in the
next month, though will be processing bloods twice per week for several weeks. They
will be staggering the primate vaccinations so that the number of bloods to process per
day is reasonable.
D. LBERI Quality Assurance Parameters
1. Balance calibration (annual)
2. Pipettor calibration (annual or as needed)
3. BSC recertification (annual)
4. Freezer, refrigerator, incubator temp monitoring (continuous with alarm)
5. QA review of protocols, SOPs, facilities, GLP studies & reports
6. ABSL3 airflow, temps, %RH monitoring (continuous)
7. Quarterly check on HEPA filters- looking for loading and pressure changes
8. Quarterly and Semiannual preventive maintenance of autoclaves
9. Validated operation of key equipment (spectrophotometer, centrifuges, flow cytometer,
10. ncubators)
11. Standard Operating Procedures (SOPs)
12. Flows and exposure systems: all calibrated vs. NIST traceable device for the flows
through and are calibrated with every usage. Flows into and out of exposure chamber
are calibrated annually vs. NIST traceable
13. Action: Bob/Ed- add dates of calibrations in January Quality Assurance report for July 06
to December 06.
14. Plesimography setup for measuring respirations to determine volume breathed in by
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animals. Ed is currently validating this instrument. Each time a monkey is in a box, there
is a calibration of the signal.
E. Next Meeting:
1. Next LBERI Tech call will be December 5th from 12pm-1pm MT and 2-3pm ET
2. Ed may not be available for 12/5 call.
3. January 2 is a holiday for UNM. Perhaps we can discuss shifting all the tech calls by one
week and having them on 1/9, 1/16, 1/23, and 1/30/07.
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