UNM TVDC Team Year 5: What to Finish? What resources to use?
Discussion lead by Rick Lyons
Minutes prepared by Barbara Griffith 10/6/09
Sent to UNM and LBERI scientists for review: 10/9/09
Edits back on 10/12/09
Action Items:
 LBERI: Primate protection is a strong goal for the last year of the TVD contract.
 UNM: Do titration of vaccination in rat to show some LVS doses are protective and some are not
protective.
 UNM: will develop a plan for rescreening the peptide libraries with fresh cells from vaccinated
NHP (spleen/LN/blood?)
 UNM TVDC: needs a comparison of the antibody titers across species
 Freyja/Patrick: Determine NIAID contact for Felgner arrays and coordinate shipment of UNM
TVDC sera samples to the Felgner array lab.
 UNM TVDC: Needs a big table of the highest priority items to complete on the contract: a table to
reference and look for patterns. Get the data organized for the vaccine development, for model
development, for assay development, for immune response analysis.
 LBERI: needs “lot to lot” variation of all the Chamberlains media used for the Ft growth.
 LBERI: needs to attain more consistent presented doses in the NHP model.
 LBERI: needs to know the sources of the variability in the presented doses and know where it is
coming from. Freyja offered a statistician to help LBERI with the analysis of existing data.
 Trevor :will analyze all the FT growth from the past statistically
 Trevor: A remaining variable is the plate to plate variability. Have used 3-4 lots of plates and
could analyze data.
 LBERI: consider using flow cytometry or other instrumentation for particle analysis to reduce
variability in the presented doses
Goal: demonstrating LVS protection in NHP model
Action: Clearly primate protection is a strong goal for the last year of the TVD contract.
USAMRIID discussion: the LVS lot #4 is a concern, as an IND, in NHP. LBERI will compare 4 lots of LVS
“head to head “(DVC lot 16, DVC lot 17, DVC lot 20 and USAMMDA IND 157 lot 4). UNM is growing the
SCHU S4 in Chamberlains, as the 2 contracts and NIAID agreed at the beginning of the contract.
However, DVC is growing in Mueller Hinton now
George Know in Pittsburgh: depending on how grow the SCHU S4 (human /mouse), Muller Hinton
developed a better innate response than if grown in Chamberlains.
USAMRIID grows in Mueller Hinton and UNM in Chamberlains: both determined essentially same LD50 in
cynos with SCHU S4, regardless of the growth media used for the SCHU S4.
Is there a difference in bacterial burdens in the organs at a distance in USAMRIID vs. UNM TVDC NHP
studies? If there is a huge difference, then the innate virulence would be suspect since both are using
cyno macaques model.
We have to bridge the models, with a correlate of protection assay. Need the protection “gold standard”
LVS to work in NHP.
Action: UNM Do titration of vaccination in rat to show some LVS doses are protective and some are not
protective.
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Vaccines: new/candidates- for usage in rats and NHP?
Action: UNM will develop a plan for rescreening the peptide libraries with fresh cells from vaccinated NHP
(spleen/LN/blood?)
Transcriptome: can we identify the peptides and look at their transcription pattern in the transcriptome?
Kathy agrees that we can.
Kathy: take Listeria information from Cerus /Aduro and proteome from UNM/ASU – integrate this data
Rick: use subcutaneous route rather than iv in Justin’s work. Could these Lm candidates work in the rat?
Malu, noted that tetramers and trackers to use in peptides in the rat model. Look at populations of T cells
that are responding. Rat has many immunological tools like monoclonal antibodies, similar to mouse.
Phillip: helpful to track antibody response? Just to know if there is a population of antibodies that are
present in responders vs. non-responders to vaccination and challenge?
Rick: hope to use the Felgner arrays, and gathering the information now from NIAID.
Rick: rat model with some protected and some were not protected, in the same group?
Mary: in the vaccinated primates, are the levels of antibodies the same as in the protected rats?
Terry: rats and NHP are similar
Julie W: titers are similar in NHP, to whole organism as the antigen.
Gopi: has around 10,000 limit of detection in rats with HK LVS
Heather: F novicida titers are higher than LVS
Freyja: conjugates are different
Action: UNM TVDC needs a comparison of the antibody titers across species
Bernard: Felgner array use status?
Freyja: Felgner arrays are in NIAID hands- Felgner project officer/subcontractor changed positions, so
NIAID hasn’t chased down the place to send the samples, which the UNM TVDC team has identified.
Action: Freyja/Patrick- Determine NIAID contact for Felgner arrays and coordinate shipment of UNM
TVDC sera samples to the Felgner array lab.
Karl / Heather: vaccinated with Fn – some lived and some died. Heather has pre-bleeds from all animals.
Rick: Felgner array data may be very useful and another models category of data to compare across
species models.
Rick: Action: need a big table of the highest priority items to complete on the contract: a table to reference
and look for patterns. Get the data organized for the vaccine development, for model development, for
assay development, for immune response analysis.
Bernard: has provided a table of comparison of all the mutants form Karl’s lab.
Ed: tables, summaries are important in the final contract reports so start thinking about it early!
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LBERI aerosol model and its variability-Options to improve consistency?
Bob: FT is most finicky organism that LBERI has tried to grow and have been working with it like YPestis
which behaves well. Action: Perhaps LBERI needs “lot to lot” variation of all the Chamberlains media
used for the Ft growth. This is a strong QA/QC, to show that a lot recovers a percentage of the FT. Ft
doesn’t grow as easily as any other bacteria that LBERI works with.
Rick: is the variation seen at the end of the spray, is it due to difference in the number of organisms into
the nebulizer (OD) or is it based on the survivability of the organism in the spray?
Trevor: Based on the back end. Could also be animal to animal variation?
Rick: Action: LBERI needs to attain more consistent presented doses in the NHP model. NHP variability
is built into the model and can’t be overcome.
Trevor: milestone 9- is qualifying the bioaerosol with a tight 1000 CFU dose for each and every delivery of
sprays
Rick: Was Mueller Hinton more reproducible spray factor than in Chamberlains (based on growth, LD50 in
mice and not based on aerosol)?
Bob: Mueller Hinton may not affect aerosols at all. Chamberlains is a great media to keep extraneous
proteins from affecting the responses (Chamberlains is a defined media rather than a lysate). Ft growth
is finicky by the media and adjustment by OD should have taken care of variability. If put 100 CFU on a
plate do I grow 100 on the plate?
Julie: If don’t qualify the media plates, are they a factor impacting recovery?
Bob: That is right. Cysteine supplemented- have shelf lives and keep refrigerated etc. Even fresh media
may be lot to lot variability.
Freyja: spread plate enumeration has been assessed with other bacteria – how much variability vs. spray
variability?
Bob: LBERI needs a set titer
Freyja: Action: LBERI needs to know the sources of the variability in the presented doses and know
where it is coming from! Freyja offered a statistician to help LBERI with the analysis of existing data.
Trevor: Action: Trevor will analyze all the FT growth from the past statistically
Michelle: pre- sprays were consistent; most variable was amount of bacteria coming through AGI (like
0.1% recovery), looked at growth across many cultures. LBERI had tighter results with current nebulizer
concentration. Action: Trevor- A remaining variable is the plate to plate variability. Have used 3-4 lots of
plates and could analyze data.
Ed: do you look at pre and post sprays?
Trevor: LBERI does an OD for the overnight growth, so the culture is in linear part of the vegetative
growth cycle. LBERI can’t use spray factor to normalize pre-sprays because the spray factor isn’t
determined until after bacteria grows for a couple of days post spray.
Bob: compounding errors: error in spray, plating, recovery etc so being within 10-20% is phenomenal
especially in 250 CFU/liter of air when targeting 1000 CFU. Presented doses are from 100 CFU to 7000
Ed: Does LBERI follow trending in the data?
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Bob/Trevor: have a spreadsheet for all data to look for trends. Major factor is the predicted dose when
you start the experiment. OD indicates 1x10e8, and then LBERI finds out later that they have lower than
planned presented doses.
Freyja: Is uncomfortable to taking an OD and adjusting it. Need a range of .55 to 1.0, knowing the
variability, better than growing to a specific OD. Vegetative growth introduces more error when targeting
one specific OD.
Trevor: look at the tightest data, with a range.
Freyja: data is not correlative, as she has seen. This is her suggestion.
Rick: slight changes in OD, cause huge changes in the concentration. Viability is less variable than
particle numbers.
Bob: can use dark field counter of particles to count Ft bacteria. Dilution factors are not perfect either.
Action: LBERI consider using flow cytometry or other instrumentation for particle analysis to reduce
variability in the presented doses
Rick: thinking of a secondary verification, other than OD which is crude.
Freyja: Don’t know the LBERI data for the particle size etc.
Trevor: particles out of nebulizer are the solution and not the bacteria itself.
Rick: suggesting particle count well before the spray. Presented dose can be doubled if the initial CFU
count is more accurate from the pre spray fluid. Need to tighten. Order equipment if needed.
Exposure Duration is determined by real time plethysmography
Ed: Is the impinger- nebulizer messing this Ft bacteria up?
Trevor: LBERI tested up to 6 types of nebulizers, and settled on Collison because wasn’t very different
from others that claimed to be more gentle. The “more gentle” nebulizers weren’t better. Collison outperformed all the other 6 “more gentle” nebulizers.
Julie W: The presented dose has a wide range that kills the NHP (50-3000 SCHU S4). The NHP still die
across a broad range of presented doses.
Bob: time to death correlates with dose, if dose is over 100,000. 1e6 is 2-3 days, etc.
Rick: An underlying concern is that the window of protection so tight in NHP that we have to be below a
threshold.
Ed: appreciates the variability issues, as seen in Anthrax. As models get defined, Ed asks that we do all
that we can to decrease variability in the model.
Rick: LBERI and UNM discuss the variability often and could try incorporating a particle counter.
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