Sub-NIAID Tech Call
7 August 2007
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
5797-1

#3
#2
#4
#7
#8
#9
#12/13
Active -
Inactive-
Active -
Inactive -
Inactive -
Inactive -
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
5797-2

Goals for Milestone #3 – Bioaerosol Development

Characterize the LVS bioaerosol using the Collison nebulizer

Determine optimum medium for aerosol dispersal (protein conc. & antifoam)

Determine optimum medium for aerosol recovery (AGI)

Determine spray factors at various challenge concentrations

Determine lowest spray concentration & how to quantitate

Determine differences in spray factor for reconstituted, vs. thawed, vs. fresh

Compare Collison to sparging generator

Compare Collison to micropump generator

Compare Collison to Aeromist generator

Consider additional bioaerosol generators (Aeroeclipse II, ultrasonic generators, others)

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
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MS 3: Bioaerosol Development
Collison Nebulizer
Order & receive instrument
Set up instrument
Frozen
LVS
Fresh
LVS
Lyophilized
LVS
Aeromist
Order & receive instrument
Set up instrument
Frozen
LVS
Fresh
LVS
Micropump
Order & receive instrument
Set up instrument
Frozen
LVS
Fresh
LVS
Down Select for
Schu 4 Generator
Red: completed
Green: in progress
Blue: steps in the milestone
Frozen
LVS
Fresh
LVS
Prepare bioaerosol SOP
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Milestone #3 – Bioaerosol Development
Accomplishments

Completed Collison LVS testing (fresh vs. frozen)

Completed sparging generator LVS testing; decided to halt testing based on difficult setup and poor spray factors

Micropump testing near completion (LVS, fresh vs. frozen and
BG spores); spray factors with LVS comparable to Collison

Tested Aeroeclipse II nebulizer with BG spores; spray factors equivalent to Collison

Tested ultrasonic generator system with BG spores; inefficient spray factors and difficult setup

Completed Aeromist testing with BG spores; spray factors better than any generator tested to date; initiated testing with frozen and fresh LVS
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
The following slides show target vs. actual concentrations and spray factors for fresh LVS cultures tested to date
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Aeromist: Target vs. Actual CFU/mL, Fresh LVS
Aeromist: Target vs. Actual CFU/mL (Fresh)
8.00
7.50
7.00
6.50
6.00
5.50
5.00
4.50
4.00
3.00
3.50
4.00
6.50
7.00
4.50
5.00
5.50
Target CFU/ml (Log10)
6.00
7/19/2007 (Aeromist) 7/19/2007 (Collison)
7.50
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Aeromist: Actual CFU/mL vs. Spray Factor, Fresh LVS
Aeromist: Actual CFU/mL vs. Spray Factor (Fresh)
-5.50
-5.70
-5.90
-6.10
-6.30
-6.50
-4.50
-4.70
0.00
-4.90
-5.10
-5.30
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Actual CFU/mL (Log10)
7/19/2007 (Aeromist) 7/19/2007 (Collison)
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
Early testing with fresh LVS demonstrate spray factors comparable to and exceeding those seen with the Collison

Initial tests demonstrated that Target vs. Actual CFU/mL is more accurate with the Collison
– 0.5-1.0 Log10 drop observed with the Aeromist
– Gentle nature of Aeromist (as compared to Collison) in question; further testing required

Easy setup

Cost effective
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Milestone #3 – Bioaerosol Development
Plans for this month


5797-10

Milestone #12/13 – Immune Responses in Animals and Humans
Immunoassay Development and Comparisons in Animal Models
Choose PBMC
Purification Method
Method chosen:
Purdue ListServ
Phenotype Blood and PBMCs
Test whether method results in loss of B cells
Red: completed
Green: in progress
Blue: steps in the milestone
Choose PBMC
Freezing Method
Develop
Immunoassay methodologies
Testing 3 protocols:
Cerus, CTL, Lyons
Proliferation assay:
Works for
Con A and
LVS
IFN g
ELISPOT
Plasma
IgG
ELISA
Works for
Con A; working on LVS
IFN g
Intracellular
Staining
Plasma
IgA
ELISA
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
Goal: To select the freezing protocol which provides the best recovery of cell viability and function when thawed

We are currently comparing 3 protocols which differ slightly in % and type of serum used: Cerus, CTL and Lyons
– Cerus: Frozen in 80% FBS/20% DMSO at 5 x 10 6 /ml
– CTL: Frozen in 90% human A/B serum/10% DMSO at 10 x
10 6 /ml
– Lyons: Frozen in Gibco Recovery Cell Culture Freezing
Media (contains optimal ratio of fetal bovine serum:bovine serum and 10% DMSO) at 5
– 10 x 10 6 /ml; also, thawed in presence of DNAse and left in 37 o incubator for 30 – 60 minutes before use
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Review of previously presented data regarding freeze/thaw testing

Original test of CERUS protocol spared about 50% of Con A proliferative capacity and 30% of antigen specific proliferation; 2 nd test of this protocol spared almost 100% of antigen-specific proliferation

Original test of CTL protocol did not spare antigen-specific proliferation, but the 2 nd and 3 rd test did, nearly 100% of activity was recovered

Original test of Lyons protocol resulted in too few cells surviving the thaw to set them up for proliferation; 2 nd test of the Lyons protocol also did not spare many cells but the cells that were tested did proliferate well to LVS (nearly 100% of activity recovered)

In early June, aliquots of blood from 6 NHPs were frozen down using each protocol allowing us to directly compare them when thawed in early August (each NHP’s blood was frozen with each protocol)
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Proliferation of NHP PBMCs to LVS

Terry Wu (UNM) provided the LRRI team a new batch of heatkilled (HK) and formalin-fixed (FF) LVS to test

We tested whether these preparations would stimulate a proliferative response in PBMCs from LVS-vaccinated NHPs; all set up at 1 x 10 6 /ml

Set up at the same concentration that the old batches of HK- and
FF-LVS worked (1 x 10 5 /ml)
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Original batches of LVS are superior to the new batches in stimulating NHP PBMC proliferation
800000
600000
400000
200000
0
* x
* x
ID
SC
Two way ANOVA analysis shows that the ID group differs from the
SC group overall;
* indicates different than all other stimuli within vaccination route; x indicates different than all other stimuli within vacation route except each other (i.e. LVS ff
Hi = LVS hk Hi in the SC group)
Data interpretation: PBMCs from 6 vaccinated NHPs (3 ID, day 238; 3 SC, day 237) respond better to original LVS preparations than to the new ones; ID group seems to respond better to FF LVS than does the SC group
5797-15

Update on IFNg ELISPOT detection from LVSvaccinated NHPs

We had been having difficulty detecting IFNg secretion by
ELISPOT using PBMCs from previously vaccinated NHPs

Previously, using 200,000 cells/well gave a very high background response from which it was difficult to see an increase after LVS stimulation of the cells

Guido Ferrari, an ELISPOT expert that attended the CMI
Validation course in DC in June, suggested that our high background may be caused by platelet contamination of our
PBMCs; we thus plotted whether those assays which had high backgrounds also had high RBC contamination

We have also been titrating the number of cells required to see the IFN g spots; as well as titrating the coating antibody concentration
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High RBC Content Correlates with High Background of IFN g
Spots (Media only)
Experiment NHP Cells plated
/well
% RBC Media only LVS FF
(1 x 10 5 /ml)
LVS HK
(1 x 10 5 /ml)
TUL 10
TUL 10
TUL 11
TUL 11
A00868 500,000
A00902 500,000
A00659 200,000
A00902 200,000
18.6% 167,159,140 509,528,424 323,331,349
9.3% 144,135,107 307,330,278 320,283,244
5.5% 39,34 NT 63,70,25
13.8% 207,184,174 244,161,144 172,160,155
TUL 14
TUL 14
TUL 15
A00896 200,000
A00908 200,000
A00868 200,000
0.4%
0.8%
0.4%
0,5,0
2,3,3
16,14,13
10,14,5
15,18,4
27,22,39
4,3,4
4,3,1
20,13,16
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LVS stimulated IFN g secretion from LVS-vaccinated
NHPs increases with cell concentration
60
50
.33
.67
1
1.33
*
40
30
*
* *
20
10
*
*
0
Media LVS hk Hi LVS ff Hi
PBMCs (from 6 vaccinated NHPs) were plated at varying concentrations (x 10 6 cells/ml) and stimulated with either HK or FF
LVS (1 x 105 cells/ml; * = significantly different than media at that concentration
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100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
IFN g
Spot detection varies with cell number plated, but not with coating antibody concentration
.67
1
1.33
7.5
 g/ml
*
*
*
*
100
90
80
70
60
50
40
30
20
10
0
.67
1
1.33
15
 g/ml
*
*
*
*
*
Media LVS hk Hi LVS ff Hi
Media LVS hk Hi LVS ff Hi
.67
1
1.33
Media
30
 g/ml
*
LVS hk Hi
*
*
*
LVS ff Hi
*
PBMCs from 2 vaccinated NHPs, TUL 17) were plated at varying concentrations (x 10 cells/ml) on IFN g
ELISPOT plates that were
6 coated with varying concentrations of anti-
IFN g antibody and stimulated with either HK or FF LVS (1 x 10 5 cells/ml); * = significantly different than media at that concentration.
1.33 x 106/ml corresponds to 200,000 cells/well. Differing coating antibody concentrations make little difference in performance of the assay. 15
 g/ml is what the kit recommends.
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
Thaw frozen PBMCs in early August and test their proliferative capacity and their ability to produce IFN g as measured by
ELISPOT

Continue optimization of IFN g
ELISPOT by testing varying concentrations of anti-IFN g
-HRP antibody

Continue optimization of IgA anti-LVS ELISA on vaccinated NHP sera

Test higher concentrations of new HK- and FF-LVS on PBMCs to see in proliferation response increases
5797-20


Action: Julie will perform baseline IFN gamma Spot assays with naïve NHP bloods as a negative control. This provide confidence in the specificity of the response being detected with the vaccinated
NHP bloods.
5797-21